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<strong>When</strong> <strong>citing</strong> <strong>an</strong> <strong>abstract</strong> <strong>from</strong> <strong>the</strong> <strong>2009</strong> <strong>Annual</strong> Meeting please use <strong>the</strong> <strong>for</strong>mat below.<br />
[Authors]. [Abstract Title]. Program No. XXX.XX. <strong>2009</strong> Neuroscience Meeting Pl<strong>an</strong>ner.<br />
Chicago, IL: <strong>Society</strong> <strong>for</strong> Neuroscience, <strong>2009</strong>. Online.<br />
<strong>2009</strong> Copyright by <strong>the</strong> <strong>Society</strong> <strong>for</strong> Neuroscience all rights reserved. Permission to republish <strong>an</strong>y<br />
<strong>abstract</strong> or part of <strong>an</strong>y <strong>abstract</strong> in <strong>an</strong>y <strong>for</strong>m must be obtained in writing by SfN office prior to<br />
publication.
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.1/A1<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH R01 EY012736<br />
NIH R01 EY015290<br />
NIH T32 EY013933<br />
HFSP<br />
Title: Gene expression directing retinal axon divergence in <strong>the</strong> albino mouse<br />
Authors: *P. BHANSALI 1 , A. REBSAM 2 , C. MASON 3 ;<br />
2 3 1<br />
Pathology & Cell Biol., Pathology & Cell Biology, Neurosci. <strong>an</strong>d Ophthalmology, Columbia<br />
Univ., New York, NY<br />
Abstract: At <strong>the</strong> optic chiasm, retinal g<strong>an</strong>glion cell (RGC) axons <strong>from</strong> <strong>the</strong> eye ei<strong>the</strong>r cross or<br />
remain uncrossed as <strong>the</strong>y project to targets in <strong>the</strong> brain, <strong>the</strong>reby establishing <strong>the</strong> circuits <strong>for</strong><br />
binocular vision. In oculocut<strong>an</strong>eous (OCA) <strong>an</strong>d ocular albinism (OA) in hum<strong>an</strong>s <strong>an</strong>d mice<br />
congenital reduction of mel<strong>an</strong>in syn<strong>the</strong>sis or aberr<strong>an</strong>t packaging into mel<strong>an</strong>osomes results in no<br />
or reduced pigment in <strong>the</strong> eye within <strong>the</strong> retinal pigment epi<strong>the</strong>lium (RPE) <strong>an</strong>d a concomit<strong>an</strong>t<br />
reduction in RGC fibers that project ipsilaterally. Consequently, binocular vision is impaired.<br />
Even though <strong>the</strong> genetic causes <strong>for</strong> albinism have been identified, <strong>the</strong> mech<strong>an</strong>ism of how<br />
disruptions in mel<strong>an</strong>in biogenesis <strong>an</strong>d processing in <strong>the</strong> RPE lead to perturbed neurogenesis <strong>an</strong>d<br />
retinal axon misrouting is not known. We have identified a program of gene expression within<br />
<strong>the</strong> ventrotemporal (VT) retina that directs <strong>the</strong> uncrossed projection, involving <strong>the</strong> tr<strong>an</strong>scription<br />
factors Foxd1, Zic2, <strong>an</strong>d <strong>the</strong> guid<strong>an</strong>ce receptor EphB1 that interacts with ephrinB2 at <strong>the</strong> chiasm<br />
midline. In albino mice, fewer RGCs express Zic2 <strong>an</strong>d EphB1 in a proportion mirroring <strong>the</strong><br />
reduced ipsilateral projection. Anterograde labeling of RGC projections to <strong>the</strong> dorsal lateral<br />
geniculate nucleus (dLGN) indicates that misrouted VT RGCs seem to be specified differently<br />
compared to normal VT RGCs. We fur<strong>the</strong>r investigated a possible temporal <strong>an</strong>d spatial shift in<br />
albino RGC specification, by comparing tr<strong>an</strong>scription factor <strong>an</strong>d guid<strong>an</strong>ce receptor expression<br />
associated with both <strong>the</strong> crossed (e.g., Foxg1, NrCAM, PlexinA1) <strong>an</strong>d uncrossed projections in<br />
pigmented <strong>an</strong>d albino retina. Differential expression patterns in <strong>the</strong> retina <strong>an</strong>d RGCs provide a<br />
basis <strong>for</strong> experimental <strong>an</strong>alysis of factors in <strong>the</strong> RPE that direct RGC proliferation <strong>an</strong>d
specification. Supported by <strong>the</strong> NIH (R01 EY012736, R01 EY015290, <strong>an</strong>d T32 EY013933) <strong>an</strong>d<br />
<strong>the</strong> HFSP.<br />
Disclosures: P. Bh<strong>an</strong>sali, None; A. Rebsam, None; C. Mason, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.2/A2<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: M103KV010007-06K2201-00710 <strong>from</strong> Brain Research Center of <strong>the</strong> 21st Century<br />
Frontier Research Program<br />
Title: Identification <strong>an</strong>d functional characterization of direct regulatory targets of <strong>the</strong> HMG-box<br />
tr<strong>an</strong>scription factor Sox2<br />
Authors: *J. KIM 1 , K. LEE 2 , H. JEONG 2 , E. JI 2 ;<br />
1 2<br />
Div. of Mol. Life Sci., Div. of Life <strong>an</strong>d Pharmaceut. Sci., Ewha Wom<strong>an</strong>s Univ., Seoul,<br />
Republic of Korea<br />
Abstract: Sox2 is a member of <strong>the</strong> Sox tr<strong>an</strong>scription factor family whose defining characteristics<br />
is <strong>the</strong> presence of HMG-box DNA binding domain. The role of Sox2 in neural tissues is to<br />
prevent premature neuronal differentiation <strong>an</strong>d maintain <strong>the</strong> growth of precursor cells. We have<br />
isolated Sox6 as a target of Sox2 by ChIP-on-chip assay using hum<strong>an</strong> neural stem cell line ReN<br />
cells which expresses Sox2 at a high level. Comparative genomic <strong>an</strong>alysis showed that five<br />
highly conserved potential Sox2 binding sites are present within a 200-base-pair cluster of Sox6<br />
promoter. We confirmed a strong <strong>an</strong>d specific binding between Sox2 <strong>an</strong>d <strong>the</strong>se sites by ChIP<br />
assay. Fur<strong>the</strong>rmore, we designed various luciferase based reporter constructs with Sox6 promoter<br />
<strong>an</strong>d demonstrated that 4 of <strong>the</strong> 5 sites strongly affect Sox2-driven Sox6 expression. A series of<br />
experiments to confirm our finding in vivo has been initiated using in ovo chicken<br />
electroporation system. Expression vectors encoding Sox2 were introduced to developing neural<br />
tube of chicken embryos. After electroporation, <strong>the</strong> effect of <strong>the</strong> overexpression was assessed by<br />
examining <strong>the</strong> neural tube by RNA in situ hybridization <strong>for</strong> Sox6. Indeed, we confirmed that<br />
Sox2 promotes <strong>the</strong> expression of Sox6 in vivo as well. Thus, our studies revealed that Sox6 is a<br />
bonafide regulatory target of Sox2. It is envisioned that defining regulatory relationship between<br />
Sox2 <strong>an</strong>d target genes <strong>an</strong>d determining <strong>the</strong> role of target genes will reveal <strong>the</strong> functional network<br />
of genes that govern neural stem cells <strong>an</strong>d neural development.
Disclosures: J. Kim, None; K. Lee, None; H. Jeong, None; E. Ji, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.3/A3<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NS031768<br />
Title: MEK/ERK signaling is required <strong>for</strong> glial progenitor specification during mammali<strong>an</strong> brain<br />
development<br />
Authors: *X. LI 1 , J. ZHONG 1 , J. CHARRON 2 , W. SNIDER 1 ;<br />
1 Neurosci. Ctr., 8131Neuroscience Res. bldg, Chapel Hill, NC; 2 Ctr. de recherche en<br />
c<strong>an</strong>cérologie de l'Université Laval, Quebec, QC, C<strong>an</strong>ada<br />
Abstract: Despite intensive study of RAF/MEK/ERK pathway, <strong>the</strong> essential functions mediated<br />
by this cascade in mammali<strong>an</strong> brain development remain largely unknown. Import<strong>an</strong>t functions<br />
related to neural progenitor proliferation, neurogenesis <strong>an</strong>d gliogenesis have all been proposed<br />
based on studies of upstream receptor tyrosine kinases <strong>an</strong>d growth factors. To define <strong>the</strong> function<br />
of MEK/ERK signaling during brain development in vivo, we combined conditional deletion of<br />
Mek1 using nestin promoter driven Cre recombination with complete elimination of Mek2 to<br />
generate a Mek1/2 double mut<strong>an</strong>t. This strategy resulted in almost complete elimination of<br />
MEK/ERK function in <strong>the</strong> developing brain. Surprisingly we found no deficit in radial<br />
progenitor proliferation or in <strong>the</strong> early phases of neurogenesis. In contrast, glial development was<br />
severely disrupted. Specialized midline glial populations failed to appear <strong>an</strong>d <strong>the</strong> corpus<br />
callosum did not <strong>for</strong>m. Fur<strong>the</strong>r, in vitro differentiation of E17.5 cortical progenitors to astrocytes<br />
under Ciliary neurotrophic factor (CNTF) stimulation was markedly impaired in Mek1/2 double<br />
mut<strong>an</strong>t. Western blots <strong>from</strong> <strong>the</strong>se cultures revealed that <strong>the</strong> JAK-STAT pathway was inhibited<br />
profoundly in <strong>the</strong> Mek1/2 mut<strong>an</strong>t, <strong>an</strong> effect due to reduced progenitor expression of <strong>the</strong> common<br />
cytokine receptor, gp130. Finally Notch signaling was reduced <strong>an</strong>d properties of radial glial<br />
cells, which serve as glial progenitors at late embryonic stages, were not maintained. We<br />
conclude that MEK/ERK signaling is critical <strong>for</strong> generating a glial progenitor population <strong>an</strong>d <strong>for</strong><br />
proper modulation of cytokine signaling in <strong>the</strong> regulation of astrocyte differentiation.<br />
Disclosures: X. Li, None; J. Zhong, None; J. Charron, None; W. Snider, None.
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.4/A4<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Title: Coordinated control of self-renewal <strong>an</strong>d differentiation of neural stem cells by Myc <strong>an</strong>d<br />
<strong>the</strong> p19ARF-p53 pathway<br />
Authors: *M. NAKAFUKU 1 , M. NAGAO 1 , K. CAMPBELL 1 , K. BURNS 1 , C.-Y. KUAN 1 , A.<br />
TRUMPP 2 ;<br />
1 Cincinnati Children's Hosp. Med. Ctr., Cincinnati, OH; 2 Deutsches Krebs<strong>for</strong>schungszentrum<br />
(DKFZ), DKFZ-ZMBH Alli<strong>an</strong>ce, Heidelberg, Germ<strong>an</strong>y<br />
Abstract: The modes of proliferation <strong>an</strong>d differentiation of neural stem cells (NSCs) are<br />
coordinately controlled during development, but <strong>the</strong> underlying mech<strong>an</strong>isms remain largely<br />
unknown. Here we provide evidence that <strong>the</strong> protooncoprotein Myc <strong>an</strong>d <strong>the</strong> tumor suppressor<br />
p19ARF regulate both NSC self-renewal <strong>an</strong>d <strong>the</strong>ir neuronal <strong>an</strong>d glial fate in a developmental<br />
stage-dependent m<strong>an</strong>ner. Early-stage NSCs have low p19ARF expression <strong>an</strong>d retain a high selfrenewal<br />
<strong>an</strong>d neurogenic capacity, whereas late-stage NSCs with higher p19ARF expression<br />
possess a lower self-renewal capacity <strong>an</strong>d predomin<strong>an</strong>tly generate glia. Overexpression of Myc<br />
or inactivation of p19ARF reverts <strong>the</strong> properties of late-stage NSCs to those of early-stage cells.<br />
Conversely, inactivation of Myc or <strong>for</strong>ced p19ARF expression attenuates self-renewal <strong>an</strong>d<br />
induces precocious gliogenesis through modulation of <strong>the</strong> responsiveness to STAT3-mediated<br />
gliogenic signals. These actions of p19ARF in NSCs are mainly mediated by p53. We propose<br />
that opposing actions of Myc <strong>an</strong>d <strong>the</strong> p19ARF-p53 pathway have import<strong>an</strong>t functions in<br />
coordinated developmental control of self-renewal <strong>an</strong>d neuronal <strong>an</strong>d glial fate choices in NSCs.<br />
Disclosures: M. Nakafuku, None; M. Nagao, None; K. Campbell, None; K. Burns, None; C.<br />
Ku<strong>an</strong>, None; A. Trumpp, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.5/A5<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: CIHR<br />
HSFO<br />
Centre <strong>for</strong> Stroke Recovery<br />
OGS<br />
OGSST<br />
Title: Eip63E, <strong>the</strong> Drosophila PFTAIRE, is required <strong>for</strong> <strong>the</strong> proper development of <strong>the</strong> central<br />
nervous system<br />
Authors: *Y. RODRIGUEZ GONZALEZ 1 , P. JAFARNEJAD 3 , D. HAWARI 2 , R. S.<br />
SLACK 2 , M. SONNENFELD 2 , D. S. PARK 2 ;<br />
1 Dept Neurosci, 2 Cell. <strong>an</strong>d Mol. Med., Univ. of Ottawa, Ottawa, ON, C<strong>an</strong>ada; 3 Dept. of Mol. <strong>an</strong>d<br />
Hum<strong>an</strong> Genet., Baylor Col. of Med., Houston, TX<br />
Abstract: The characterization of <strong>the</strong> processes that control brain development is critical <strong>for</strong><br />
developing <strong>the</strong>rapeutic strategies that rely on accurate differentiation <strong>an</strong>d targeting events.<br />
PFTAIRE is a novel member of <strong>the</strong> growing number of Cyclin Dependent Kinases (CDK) that is<br />
normally resident in neurons. CDK5, <strong>the</strong> best characterized of neuronal CDKs, has been shown<br />
to participate in brain development <strong>an</strong>d death. Although PFTAIRE biological function <strong>an</strong>d<br />
regulation are hardly known, its expression in neurons <strong>an</strong>d similarity to CDK5 suggest a<br />
potentially import<strong>an</strong>t role in <strong>the</strong> CNS. Using two different Eip63E Drosophila mut<strong>an</strong>t lines, we<br />
show here that PFTAIRE mut<strong>an</strong>t flies display defects as early as stage 9 of embryonic<br />
development, as revealed by immunostaining <strong>for</strong> CNS markers such as engrailed/invected (4D9),<br />
neurogli<strong>an</strong> (BP104), repo (8D12), fasciclinII (1D4) <strong>an</strong>d BP102 <strong>an</strong>tigen. Results show that <strong>the</strong><br />
defects include at least a striking disorg<strong>an</strong>ization of neuronal <strong>an</strong>d glia cell bodies as well as<br />
abnormal arr<strong>an</strong>gements of both commissural <strong>an</strong>d longitudinal axons of <strong>the</strong> ventral nerve cord.<br />
Disclosures: Y. Rodriguez Gonzalez, None; P. JafarNejad, None; D. Hawari, None; R.S.<br />
Slack, None; M. Sonnenfeld, None; D.S. Park, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.6/A6<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH Gr<strong>an</strong>t AG022555<br />
Title: Co-expression of VEGF <strong>an</strong>d Flk1 by neural stem cells in <strong>the</strong> adult dentate gyrus suggests<br />
<strong>an</strong> autocrine/paracrine role <strong>for</strong> VEGF in stem cell mainten<strong>an</strong>ce<br />
Authors: *G. M. BERNAL, D. A. PETERSON;<br />
Ctr. Stem Cell & Regen, Rosalind Fr<strong>an</strong>klin Univ., North Chicago, IL<br />
Abstract: The adult brain contains specialized <strong>an</strong>d spatially restricted germinal centers that<br />
support neural stem cell (NSC) self-renewal, differentiation, <strong>an</strong>d functional integration. These<br />
regions promote ongoing neurogenesis as a result of <strong>an</strong> enriched niche containing various cell<br />
components that express a variety of signaling molecules, m<strong>an</strong>y of which are shown to modulate<br />
proliferation <strong>an</strong>d differentiation. Underst<strong>an</strong>ding <strong>the</strong> potential interactions of neurogenic factors<br />
will not only elucidate regulation of neurogenesis in <strong>the</strong> germinal centers, but may also provide<br />
insights necessary to recruit stem cells to contribute to repair. Vascular endo<strong>the</strong>lial growth factor<br />
(VEGF) is a potent mitogen <strong>for</strong> NSCs when its levels are increased in <strong>the</strong> brain, such as<br />
following delivery to <strong>the</strong> hippocampal dentate gyrus or in response to injury. Fur<strong>the</strong>rmore,<br />
VEGF mediates <strong>the</strong> increase in hippocampal neurogenesis following <strong>an</strong>ti-depress<strong>an</strong>t medication,<br />
as well as modulating <strong>the</strong> survival of mature neurons. However, it is unknown whe<strong>the</strong>r basal<br />
levels of VEGF contribute to NSC mainten<strong>an</strong>ce or baseline neurogenesis in vivo. In <strong>the</strong> present<br />
study, we <strong>an</strong>alyzed VEGF distribution <strong>an</strong>d found complete colocalization with GFAP-positive<br />
cells in <strong>the</strong> dentate gyrus, including NSCs. As reported previously, <strong>the</strong> VEGF receptor Flk1 was<br />
found almost exclusively on both doublecortin-positive neuronal progenitor cells <strong>an</strong>d mature<br />
dentate gr<strong>an</strong>ule neurons. This relationship suggests that astrocytes may influence both<br />
developing <strong>an</strong>d mature neurons through VEGF expression. However, we demonstrate that Flk1<br />
is also expressed in a small population of GFAP-positive cells that also are known to express<br />
VEGF. This population of GFAP-positive cells is also Sox2-positive, indicating by this<br />
combination of expression that <strong>the</strong>se cells are Type 1 neural stem cells. Thus, neural stem cells<br />
are VEGF/Flk1-positive, but loose expression of VEGF with neuronal lineage progression. The<br />
coexpression of VEGF <strong>an</strong>d its receptor in this neural stem cell population suggests that stem cell<br />
mainten<strong>an</strong>ce, as distinct <strong>from</strong> proliferation of intermediate progenitor cells, could be<br />
accomplished through autocrine/paracrine mech<strong>an</strong>isms of VEGF signaling. VEGF expression is<br />
reduced with age suggesting that limited availability of VEGF may contribute to <strong>the</strong> age-related<br />
decline in neurogenesis.<br />
Disclosures: G.M. Bernal, None; D.A. Peterson, None.
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.7/A7<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: INSERM<br />
Retina Fr<strong>an</strong>ce<br />
EVI-GENORET LSHG-CT-2005-512036<br />
French National Ministry of Research (ACI - Young Researchers)<br />
Title: Role of Ptf1a <strong>an</strong>d modulation of its activity by cofactors during cell differentiation in <strong>the</strong><br />
chick neural retina<br />
Authors: *E. LELIEVRE 1,2,3 , L. HOUILLE 1,2 , V. BRAJEUL 1 , A. SLEMBROUCK 1,2 , S.<br />
THOMASSEAU 1,2 , J.-A. SAHEL 1,2,4 , O. GOUREAU 1,2 , X. GUILLONNEAU 1,2,3 ;<br />
1 Inst. De La Vision, PARIS, Fr<strong>an</strong>ce; 2 UPMC Univ. Paris 06, UMRS_968, Paris, Fr<strong>an</strong>ce; 3 Ctr. de<br />
Recherche des Cordeliers INSERM UMRS_872 Team 21, Paris, Fr<strong>an</strong>ce; 4 Ctr. Hospitalier Natl.<br />
d'Ophtalmologie des Quinze-Vingts, Paris, Fr<strong>an</strong>ce<br />
Abstract: During development, retinal progenitor cells give birth to <strong>the</strong> different cell subtypes of<br />
<strong>the</strong> adult neural retina in <strong>an</strong> orderly fashion. Cell-fate choices require interplay between extrinsic<br />
regulators <strong>an</strong>d a combinaison of intrinsic tr<strong>an</strong>scription factors.<br />
The p<strong>an</strong>creas tr<strong>an</strong>scription factor 1a (also known as p48) encodes a basic-loop-helix tr<strong>an</strong>scription<br />
factor which was originally isolated as a p<strong>an</strong>creatic specific gene. In <strong>the</strong> mouse retina Ptf1a is<br />
necessary <strong>for</strong> <strong>the</strong> specification of horizontal cells <strong>an</strong>d <strong>the</strong> vast majority of amacrine cell subtypes.<br />
However, overexpression of this tr<strong>an</strong>scription factor by itself in mouse is not sufficient to<br />
promote horizontal <strong>an</strong>d amacrine cell fates.<br />
Here we report that retroviral-mediated misexpression of Ptf1a in chick eye at embryonic day 2<br />
resulted in aberr<strong>an</strong>t retinal lamination <strong>an</strong>d cell differentiation. Indeed, ectopic Ptf1a induced a<br />
strong decrease in <strong>the</strong> number of brn3a-positive g<strong>an</strong>glion cells <strong>an</strong>d visinin-positive<br />
photoreceptors at E12, whereas it increased <strong>the</strong> number of amacrine <strong>an</strong>d horizontal cells at <strong>the</strong><br />
same embryonic stage. During early neurogenesis, endogenous Ptf1a was found to be mostly<br />
expressed in post-mitotic neurons but also detected in some mitotic PCNA-positive <strong>an</strong>d BrdUlabeled<br />
progenitors. However its misexpression did not induce precocious cell cycle exit as<br />
assayed by scoring <strong>the</strong> mitotic index by flow cytometry.<br />
Moreover misexpression of various mut<strong>an</strong>t <strong>for</strong>ms of Ptf1a that are unable to interact with
mammali<strong>an</strong> Suppressor of Hairless (RBP-J, a component of <strong>the</strong> Notch signaling pathway) or/<strong>an</strong>d<br />
its paralogue RBP-L but retain <strong>the</strong>ir ability to <strong>for</strong>m dimeric complex with class-A bHLH<br />
proteins, had graded effects on retinal lamination <strong>an</strong>d cell specification. Notably <strong>the</strong> complete<br />
loss of interactions with RBP-J <strong>an</strong>d RBP-L abolished <strong>the</strong> activity of ectopic Ptf1a on amacrine<br />
<strong>an</strong>d horizontal cell differentiation.<br />
Toge<strong>the</strong>r, our results indicate that, in <strong>the</strong> chick retina, Ptf1a is sufficient to promote<br />
horizontal/amacrine cell genesis <strong>an</strong>d to inhibit g<strong>an</strong>glion cell differentiation while progenitors that<br />
misexpress Ptf1a retain <strong>the</strong>ir ability to progress through cell cycle. Our study demonstrates that<br />
Ptf1a acts as part of a tr<strong>an</strong>scription complex containing RBP cofactors, as previously suggested<br />
in <strong>the</strong> mouse retina.<br />
Disclosures: E. Lelievre, None; L. Houille, None; V. Brajeul, None; A. Slembrouck,<br />
None; S. Thomasseau, None; J. Sahel, None; O. Goureau, None; X. Guillonneau, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.8/A8<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: Schram-Stiftung<br />
Wilhelm-S<strong>an</strong>der-Stiftung<br />
DFG<br />
Title: Hox genes in <strong>the</strong> pre-specification of sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic ciliary neuron<br />
progenitors<br />
Authors: L. HUBER, J. STUBBUSCH, M. FERDIN, *H. ROHRER;<br />
Max-Pl<strong>an</strong>ck Inst. Brain Res., 60528 Fr<strong>an</strong>kfurt/M, Germ<strong>an</strong>y<br />
Abstract: Autonomic neuron development is initiated by BMPs acting on neural crest cells in<br />
<strong>the</strong> primordia of sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic g<strong>an</strong>glia. BMPs induce different patterns of<br />
tr<strong>an</strong>scription factors in sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic ciliary g<strong>an</strong>glia, which indicates intrinsic<br />
differences between BMP-responsive progenitors in different autonomic g<strong>an</strong>glia (Müller <strong>an</strong>d<br />
Rohrer, 2002).<br />
Here we have addressed <strong>the</strong> molecular basis of this intrinsic difference <strong>an</strong>d pre-specification
towards sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic neuron fate. Anterior-posterior patterning of neural<br />
crest by Hox-genes is a likely expl<strong>an</strong>ation as Hox genes are expressed in <strong>the</strong> trunk but not in <strong>the</strong><br />
mesencephalic region where ciliary g<strong>an</strong>glion progenitors originate. Screening <strong>for</strong> Hox genes that<br />
are expressed in chick sympa<strong>the</strong>tic g<strong>an</strong>glia by RT-PCR <strong>an</strong>d in situ hybridization identified a<br />
number of Hox genes observed in sympa<strong>the</strong>tic neuron progenitors at E4 (HH23). Interestingly,<br />
expression was maintained in differentiated sympa<strong>the</strong>tic neurons at least up to E8. To test<br />
whe<strong>the</strong>r <strong>the</strong> identity of parasympa<strong>the</strong>tic ciliary progenitors could be altered by Hox genes<br />
present in trunk sympa<strong>the</strong>tic g<strong>an</strong>glia, HoxB8 was ectopically expressed in ciliary progenitors,<br />
using RCAS vectors. Interestingly, <strong>for</strong>ced expression of this HoxB gene leads to a strong<br />
induction of <strong>the</strong> bHLH tr<strong>an</strong>scription factor H<strong>an</strong>d2 <strong>an</strong>d of <strong>the</strong> noradrenergic marker genes TH <strong>an</strong>d<br />
DBH. During normal development H<strong>an</strong>d2 is virtually absent <strong>an</strong>d TH <strong>an</strong>d DBH are expressed<br />
only tr<strong>an</strong>siently in ciliary neurons, whereas H<strong>an</strong>d2, TH <strong>an</strong>d DBH are continuously expressed in<br />
sympa<strong>the</strong>tic neurons. Our findings suggest that HoxB8, toge<strong>the</strong>r with o<strong>the</strong>r Hox genes expressed<br />
in trunk neural crest, is involved in <strong>the</strong> pre-specification of sympa<strong>the</strong>tic progenitors <strong>an</strong>d that<br />
parasympa<strong>the</strong>tic progenitors c<strong>an</strong> be re-specified by HoxB8 to noradrenergic, sympa<strong>the</strong>tic neuron<br />
differentiation.<br />
Müller, F., <strong>an</strong>d Rohrer, H., (2002) Molecular control of ciliary neuron development: BMPs <strong>an</strong>d<br />
downstream tr<strong>an</strong>scriptional control in <strong>the</strong> parasympa<strong>the</strong>tic lineage. Development, 129, 5707-<br />
5717.<br />
Disclosures: L. Huber, None; J. Stubbusch, None; M. Ferdin, None; H. Rohrer, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.9/A9<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: ANPCyT PICT-1064<br />
NIH-FIRCA R03TW008026<br />
Title: Cerebrospinal fluid-contacting neurons of <strong>the</strong> spinal cord are a subset of embryonic<br />
Gata2 + neurons<br />
Authors: M. SARTORETTI 1 , D. DI BELLA 1 , A. CARCAGNO 1 , Y. PETRACCA 1 , M.<br />
GOULDING 2 , *G. M. LANUZA 1 ;<br />
1 Fundacion Inst. Leloir, Buenos Aires, Argentina; 2 The Salk Inst., La Jolla, CA
Abstract: In spite of <strong>the</strong> considerable progress made in underst<strong>an</strong>ding early events in cell fate<br />
specification, <strong>the</strong> mech<strong>an</strong>isms that lead to <strong>the</strong> generation of diversity of mature neuronal cell<br />
types in <strong>the</strong> adult remain to be elucidated. In <strong>the</strong> postnatal spinal cord, <strong>the</strong>re is a population of<br />
cells with a unique morphology around <strong>the</strong> ependyma of <strong>the</strong> central c<strong>an</strong>al, which are known as<br />
cerebrospinal fluid-contacting neurons (CCNs). These cells extent a dendritic-like process<br />
through <strong>the</strong> ependymal cell layer into <strong>the</strong> central c<strong>an</strong>al, <strong>an</strong>d are selectively marked by <strong>the</strong> TRP<br />
ch<strong>an</strong>nel PKD2L1. Immunohistochemical <strong>an</strong>alysis showed that CCNs express neuronal β-tubulin,<br />
but <strong>the</strong>y are negative or are labeled at very low levels with a NeuN <strong>an</strong>tibody. We found that<br />
CCNs express <strong>the</strong> tr<strong>an</strong>scription factors Gata2/3, which are specific markers of spinal V2b<br />
embryonic interneurons. CCNs c<strong>an</strong> be efficiently labeled with GFP in <strong>the</strong> Gata2 GFP knockin<br />
mice. We have also per<strong>for</strong>med lineage-tracing <strong>an</strong>alysis using <strong>the</strong> Gata3 Cre line crossed with GFP<br />
reporter <strong>an</strong>imals. This system allowed <strong>the</strong> identification of <strong>the</strong> cluster of CCNs in lamina X,<br />
distinct to V2b-derived interneurons that locate lateral in lamina VII. In order to determine<br />
whe<strong>the</strong>r distinct subsets of Gata2/3 neurons correlate with <strong>the</strong> time of progenitor cell cycle exit,<br />
we per<strong>for</strong>med a series of BrdU pulse-labeling experiments. Interestingly we found that CCNs are<br />
born <strong>from</strong> ventral progenitors between E13 <strong>an</strong>d E14, a period when neurogenesis in <strong>the</strong> neural<br />
tube is almost finished. The late birthdate of CCNs contrasts with <strong>the</strong> earlier development of <strong>the</strong><br />
population of Gata2/3-V2b interneurons that settle in lamina VII. The generation of early-born<br />
V2b cells requires <strong>the</strong> activity of <strong>the</strong> tr<strong>an</strong>scription factor Foxn4 in <strong>the</strong>ir progenitors acting by a<br />
mech<strong>an</strong>ism that involves Delta4/Notch signaling pathway. On <strong>the</strong> contrary, we found that <strong>the</strong><br />
differentiation of CCNs appears normal in Foxn4 -/- <strong>an</strong>d in Presenilin1 mut<strong>an</strong>t mice, indicating<br />
that differential genetic mech<strong>an</strong>isms control <strong>the</strong> genesis of distinct subsets of Gata2/3 neurons.<br />
These results show that dorsoventrally restricted populations of precursors in <strong>the</strong> developing<br />
neural tube c<strong>an</strong> sequentially generate different neuronal subtypes, contributing to <strong>the</strong><br />
diversification of neuronal fates.<br />
Disclosures: M. Sartoretti, None; D. Di Bella, None; A. Carcagno, None; Y. Petracca,<br />
None; M. Goulding, None; G.M. L<strong>an</strong>uza, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.10/A10<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: Intramural Research Program of <strong>the</strong> NIH, NINDS<br />
Title: Functional <strong>an</strong>alysis of <strong>the</strong> castor neuroblast enh<strong>an</strong>cer
Authors: *J. ROSS, M. KUNDU, A. KUZIN, T. BRODY, W. ODENWALD;<br />
NINDS, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: We have examined <strong>the</strong> enh<strong>an</strong>cers of late temporal network determin<strong>an</strong>ts of<br />
Drosophila neuroblast lineage development. The late temporal determin<strong>an</strong>t castor (cas) requires<br />
<strong>the</strong> POU domain-containing tr<strong>an</strong>scription factors Pdm-1 <strong>an</strong>d Pdm-2 <strong>for</strong> full expression. The cas<br />
neuroblast enh<strong>an</strong>cer contains multiple conserved POU domain DNA-binding sites. These<br />
octamer sites also overlap bHLH <strong>an</strong>d Homeo-domain binding sites in a configuration we refer to<br />
as extended POU binding sites. We have sought additional late temporal enh<strong>an</strong>cers based on<br />
presence of multiple conserved POU domain DNA-binding sites. This search identified<br />
enh<strong>an</strong>cers associated with CG7229 <strong>an</strong>d CG6559 that direct reporter gene expression to NBs<br />
during late lineage development. Using <strong>the</strong> enh<strong>an</strong>cer alignment tool cis-Decoder, we have<br />
identified conserved sequences (cis-Decoder tags or cDTs) shared in a pairwise fashion or<br />
among all three late neuroblast enh<strong>an</strong>cers. Based on this <strong>an</strong>alysis, tr<strong>an</strong>sgenic reporter lines were<br />
created that systematically <strong>an</strong>alyzed cDT function; this <strong>an</strong>alysis shows that cDTs per<strong>for</strong>m<br />
identifiably distinct roles in enh<strong>an</strong>cer function. We have also identified a Gooseberry early<br />
temporal network enh<strong>an</strong>cer based on presence of conserved POU binding sites. Our <strong>an</strong>alysis,<br />
involving placement of POU binding-sites in <strong>the</strong> enh<strong>an</strong>cer of <strong>an</strong>o<strong>the</strong>r early temporal network<br />
gene, that lacks POU binding sites, suggests that <strong>the</strong> octamer sequence plays different functional<br />
roles in different contexts.<br />
Disclosures: J. Ross, None; M. Kundu, None; A. Kuzin, None; T. Brody, None; W.<br />
Odenwald, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.11/A11<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH Gr<strong>an</strong>t P20 RR-16460<br />
Title: Dorsal root g<strong>an</strong>glion-specific promoter elements of <strong>the</strong> mouse neurogenin1 gene<br />
Authors: A. M. BENNETT, B. S. KOSS, S. R. QADRI, *R. C. MURRAY;<br />
Hendrix Col., Conway, AR
Abstract: The dorsal root g<strong>an</strong>glia (DRG) contain <strong>the</strong> cell bodies of several types of sensory<br />
neurons that detect innocuous touch, body/limb position, <strong>an</strong>d painful stimuli. The subset of<br />
neurons that detect painful stimuli, <strong>the</strong> nociceptive neurons, require <strong>the</strong> neurogenin1 (neurog1)<br />
gene <strong>for</strong> <strong>the</strong>ir development since most of <strong>the</strong>se cells are missing in embryos lacking neurog1 (Ma<br />
et al., 1999, Genes Dev. 13: 1717). To better underst<strong>an</strong>d <strong>the</strong> factors that regulate <strong>the</strong><br />
development of nociceptive neurons, we are attempting to characterize <strong>the</strong> promoter elements of<br />
<strong>the</strong> neurogenin1 gene that direct its expression in <strong>the</strong> DRG. Previous work has identified a<br />
proximal promoter element located immediately upstream of <strong>the</strong> neurog1 coding sequence that is<br />
able to drive <strong>the</strong> expression of a reporter gene in a subset of <strong>the</strong> normal expression pattern that<br />
includes <strong>the</strong> DRG (Murray et al., 2000, Dev. Dyn. 218: 189). However, this proximal element is<br />
inefficient <strong>an</strong>d appears to be sensitive to site of integration effects. A non-overlapping distal<br />
enh<strong>an</strong>cer found fur<strong>the</strong>r upstream of <strong>the</strong> neurog1 gene has also been identified (Gow<strong>an</strong> et al.,<br />
2001, Neuron 31: 219). The distal enh<strong>an</strong>cer efficiently drives <strong>the</strong> expression of a reporter gene in<br />
a subset of <strong>the</strong> normal neurog1 expression pattern, but this element does not drive expression in<br />
<strong>the</strong> DRG. In <strong>the</strong> current experiment, we have combined <strong>the</strong> distal enh<strong>an</strong>cer <strong>an</strong>d <strong>the</strong> proximal<br />
promoter element into a single construct upstream of <strong>the</strong> bacterial lacZ reporter gene. We have<br />
used this construct to generate two independent lines of tr<strong>an</strong>sgenic mice <strong>an</strong>d we have <strong>an</strong>alyzed<br />
<strong>the</strong> pattern of tr<strong>an</strong>sgene expression during embryonic development. The combined construct<br />
efficiently drives <strong>the</strong> expression of <strong>the</strong> lacZ reporter gene in a subset of <strong>the</strong> normal neurog1<br />
expression pattern that includes <strong>the</strong> DRG. Expression of <strong>the</strong> reporter gene in <strong>the</strong> DRG is weak<br />
relative to o<strong>the</strong>r regions of <strong>the</strong> same embryos suggesting that additional enh<strong>an</strong>cer elements are<br />
required <strong>for</strong> <strong>the</strong> normal regulation of neurog1 expression in <strong>the</strong> DRG.<br />
Disclosures: A.M. Bennett, None; B.S. Koss, None; S.R. Qadri, None; R.C. Murray, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.12/A12<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Title: Lentiviral mediated siRNA “knock-down” of Zic2 blocks in-vitro neuronal differentiation<br />
of mouse ES cells<br />
Authors: *L. Y. BROWN 1 , S. BROWN 2 ;<br />
1 OB/GYN, 2 Obstetrics <strong>an</strong>d Gynecology, Univ. of Vermont, Burlington, VT
Abstract: Heterozygous mutations in <strong>the</strong> tr<strong>an</strong>scription factor gene, Zic2 result in <strong>the</strong> <strong>for</strong>ebrain<br />
mal<strong>for</strong>mation known as holoprosencephaly in both hum<strong>an</strong>s <strong>an</strong>d mice, demonstrating that early<br />
brain patterning is uniquely sensitive to <strong>the</strong> level of Zic2 protein. O<strong>the</strong>r work has shown that<br />
Zic2 is expressed in <strong>the</strong> dorsal midline in <strong>the</strong> developing fetal brain <strong>an</strong>d that it plays a central<br />
role in <strong>the</strong> development of binocular vision. More recently, we have shown that Zic2 is<br />
expressed in <strong>the</strong> inner-cell-mass of preimpl<strong>an</strong>tation embryos as well as in gastrulation stage<br />
embryos, suggesting <strong>an</strong> import<strong>an</strong>t role in pre-neural patterning. Consistent with this observation,<br />
Zic2 is also expressed in cultured embryonic stem (ES) cells, providing <strong>an</strong> in-vitro model system<br />
<strong>for</strong> studies aimed at underst<strong>an</strong>ding <strong>the</strong> molecular pathways through which Zic2 may act.<br />
With this in mind, we have used lentiviral mediated siRNA to establish ES cells in which Zic2<br />
expression has been reduced by about 90%. To accomplish this, we tr<strong>an</strong>sduced ES cells with<br />
Zic2-specific siRNA lentiviral constructs designed by <strong>the</strong> Broad Institute at MIT. Colonies<br />
surviving puromycin selection were exp<strong>an</strong>ded <strong>an</strong>d those with reduced Zic2 expression were<br />
ascertained with immunoblot <strong>an</strong>d rtPCR. Surprisingly, ES cells with reduced Zic2 expression<br />
could be maintained in <strong>an</strong> undifferentiated state <strong>for</strong> several passages without loosing <strong>the</strong>ir normal<br />
morphology or expression of pluripotentiality markers, suggesting that Zic2 expression is not<br />
essential <strong>for</strong> pluripotentiality. However, neural differentiation of ES cells with Zic2 knock-down<br />
was markedly different <strong>from</strong> that seen in control cells: In control cultures, in-vitro neural<br />
differentiation resulted in numerous cells with axonal projections <strong>an</strong>d abund<strong>an</strong>t expression of<br />
st<strong>an</strong>dard neural markers such as Tu-20, NeuN <strong>an</strong>d Nestin. In contrast, when ES cells with<br />
reduced Zic2 were allowed to differentiate, cells expressing neural markers were almost entirely<br />
absent. However, makers of early glial differentiation such as NG2 were more abund<strong>an</strong>t in Zic2<br />
knock-down cultures compared to controls, suggesting that diminished Zic2 prevents neuronal<br />
differentiation <strong>an</strong>d results in increased glial differentiation.<br />
Neuronal differentiation of ES cells is likely to be complex <strong>an</strong>d to require <strong>the</strong> coordinated<br />
expression of m<strong>an</strong>y genes. Thus it is surprising that altered level of a single gene product is<br />
sufficient to block <strong>the</strong> <strong>for</strong>mation of neurons. We conclude that Zic2 is likely to have multiple<br />
downstream targets involved in neuronal differentiation.<br />
Disclosures: L.Y. Brown, None; S. Brown, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.13/A13<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: CIHR MOP 62826
Genome C<strong>an</strong>ada<br />
Title: P<strong>an</strong>nexin 2, a gap junction-like protein, <strong>for</strong>ms rod-like structures in <strong>the</strong> endoplasmic<br />
reticulum of postnatal hippocampal neural stem <strong>an</strong>d progenitor cells<br />
Authors: *L. SWAYNE, C. D. SORBARA, S. A. L. BENNETT;<br />
Univ. Ottawa, Ottawa, ON, C<strong>an</strong>ada<br />
Abstract: Various ion ch<strong>an</strong>nels, including gap junction connexin proteins are established<br />
regulators of neural stem <strong>an</strong>d progenitor cell proliferation, migration, <strong>an</strong>d specification in<br />
embryonic <strong>an</strong>d postnatal brain. The p<strong>an</strong>nexins (P<strong>an</strong>x1, 2, <strong>an</strong>d 3) are a newly discovered family of<br />
gap junction-like proteins that exhibit gap-junction hemich<strong>an</strong>nel activity. Expression of P<strong>an</strong>x2 is<br />
constrained to <strong>the</strong> central nervous system. Analysis of hippocampal P<strong>an</strong>x2 expression over <strong>the</strong><br />
course of postnatal development revealed that maximal P<strong>an</strong>x2 expression is observed at postnatal<br />
day seven, corresponding with peak postnatal neurogenesis. Fur<strong>the</strong>rmore, two major protein<br />
iso<strong>for</strong>ms of approximately 70 kDa were detected at early postnatal timepoints with <strong>the</strong> lower<br />
species predominating in adult. To date, P<strong>an</strong>x2 expression has been reported in adult neurons <strong>an</strong>d<br />
activated astrocytes but expression in neural stem <strong>an</strong>d progenitor cells has not yet been<br />
investigated. Postnatal hippocampal neural progenitor cells (NPCs) were cultured as<br />
neurospheres <strong>from</strong> day 3 (P3) C57BL/6 mouse pups <strong>an</strong>d <strong>an</strong>alyzed by RTPCR, Western blot,<br />
immunocytochemistry <strong>an</strong>d flow cytometry <strong>for</strong> P<strong>an</strong>x2 expression. P<strong>an</strong>x2 was expressed in a small<br />
subset of NPCs (approx. 7%) <strong>an</strong>d was localized to specific cell subtypes <strong>an</strong>d intracellular<br />
org<strong>an</strong>elles using <strong>an</strong>tigenic markers in vitro <strong>an</strong>d in vivo. Here, we report that, under proliferating<br />
conditions, P<strong>an</strong>x2 <strong>for</strong>med distinctive rod-like structures in multipotential nestin+/GFAP+ Type I<br />
neural stem cells <strong>an</strong>d nestin+ Type IIa NPCs, in <strong>the</strong> endoplasmic reticulum <strong>an</strong>d <strong>the</strong> Golgi<br />
apparatus. Taken toge<strong>the</strong>r, <strong>the</strong>se data suggest a potential novel communication role <strong>for</strong> p<strong>an</strong>nexins<br />
in postnatal neurogensis.<br />
Disclosures: L. Swayne, None; C.D. Sorbara, None; S.A.L. Bennett, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.14/A14<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: Support was provided by <strong>an</strong> Independent Medical Gr<strong>an</strong>t <strong>from</strong> EMD Serono <strong>an</strong>d Pfizer
Title: Focal adhesion kinase, Fyn <strong>an</strong>d tubulin modulated by interferon beta 1a<br />
Authors: *P. LOPRESTI;<br />
Northwestern Univ. Med. Sch., Chicago, IL<br />
Abstract: The activation of Fyn non-receptor kinase is a crucial step along <strong>the</strong> developmental<br />
maturation of oligodendrocytes <strong>an</strong>d myelination. Signaling cascade coupling Fyn to Focal<br />
adhesion kinase (FAK) to cellular process outgrowth has been established in <strong>the</strong> oligodendrocyte<br />
cell line CG- 4. Drugs that modulate oligodendrocyte cytoskeleton <strong>an</strong>d signaling c<strong>an</strong> be proposed<br />
to regulate <strong>the</strong> morphological maturation of oligodendrocytes during myelination <strong>an</strong>d overcome<br />
myelin repair failure. Here we test whe<strong>the</strong>r interferon (IFN) beta 1a, widely used <strong>for</strong> <strong>the</strong><br />
treatment of demyelination diseases such as multiple sclerosis, alters cellular process<br />
morphology <strong>an</strong>d intracellular distribution of signaling known to have a role in cellular process<br />
outgrowth. In this study we used CG-4 cells. CG-4 cells are <strong>the</strong> equivalent of oligodendrocyte<br />
progenitors. In <strong>the</strong> presence of growth factors <strong>the</strong>se cells are kept as oligodendrocyte precursor<br />
cells; whereas when growth factors are withdrawn, <strong>the</strong>y differentiate into oligodendrocytes. The<br />
mitotic oligodendrocyte precursor CG-4 cells were treated with IFN beta 1a, switched to a media<br />
that promote differentiation, <strong>an</strong>d <strong>the</strong>n harvested <strong>an</strong>d stained <strong>for</strong> Fyn, FAK <strong>an</strong>d tubulin. We found<br />
that FAK, Fyn, tubulin intracellular distribution <strong>an</strong>d cellular process morphology are altered in<br />
IFN-treated cells i.e., Fyn <strong>an</strong>d FAK staining appear more robust in treated vs. untreated cells. In<br />
addition, <strong>the</strong> cellular processes of treated cells are longer <strong>an</strong>d thicker th<strong>an</strong> those of <strong>the</strong> untreated<br />
cells. Thus, we have developed <strong>an</strong> in vitro system where <strong>the</strong> treatment of oligodendrocyte<br />
precursor cells with IFN alters cytoskeleton <strong>an</strong>d signaling intracellular distribution, which might<br />
be adv<strong>an</strong>tageous in <strong>the</strong> events underlying cellular process outgrowth during developmental<br />
myelination <strong>an</strong>d myelin repair.<br />
Disclosures: P. LoPresti, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.15/A15<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH/NCRR P20 RR15636<br />
Title: Constitutive stabilization of HIF-1 alpha in neural stem/progenitor cells
Authors: *T. ROITBAK, L. A. CUNNINGHAM;<br />
Univ. New Mexico, Albuquerque, NM<br />
Abstract: Hypoxia-inducible factor-1 alpha subunit (HIF-1α) is a tr<strong>an</strong>scriptional activator<br />
mediating adaptive cellular response to hypoxia. HIF-1α is constitutively stabilized in adult brain<br />
neurogenic zones, as well as in neural stem/progenitor cells (NSPCs) isolated <strong>from</strong> <strong>the</strong><br />
embryonic <strong>an</strong>d postnatal mouse brain. Constitutive stabilization of HIF-1α may be import<strong>an</strong>t <strong>for</strong><br />
mainten<strong>an</strong>ce of <strong>the</strong> neural stem cell phenotype <strong>an</strong>d ability to withst<strong>an</strong>d chronic hypoxia. To<br />
determine <strong>the</strong> mech<strong>an</strong>isms of constitutive stabilization of HIF-1α in NSPCs under normoxic<br />
conditions, we per<strong>for</strong>med in vitro ubiquitination assays, <strong>an</strong>d immunoprecipitation <strong>an</strong>d<br />
immunofluorescence subcellular localization studies. Western blot <strong>an</strong>alyses <strong>an</strong>d in vitro<br />
ubiquitination experiments demonstrated that HIF-1α protein was not ubiquitinated, although all<br />
components of <strong>the</strong> ubiquitination pathway including Von Hippel-Lindau tumor suppressor<br />
protein (VHL) were present <strong>an</strong>d functional in NSPCs. Immunofluorescence <strong>an</strong>d cell fractionation<br />
experiments demonstrated that HIF-1α was localized primarily within <strong>the</strong> cytoplasm <strong>an</strong>d was<br />
associated with <strong>the</strong> membr<strong>an</strong>ous structures, suggesting that sequestration within membr<strong>an</strong>ous<br />
structures may protect HIF-1α <strong>from</strong> ubiquitination <strong>an</strong>d degradation. To determine <strong>the</strong> functional<br />
signific<strong>an</strong>ce of constitutive HIF-1α stabilization in NSPCs, shRNAi was used to knockdown<br />
HIF-1α protein levels by ~85%, resulting in a signific<strong>an</strong>t increase in <strong>the</strong> sensitivity of NSPCs to<br />
hypoxia. shRNAi HIF-1α knockdown also eliminated active Notch-1 protein expression,<br />
increased β-catenin protein by ~2-fold, <strong>an</strong>d decreased VEGF by ~80%. These findings suggest a<br />
unique mech<strong>an</strong>ism <strong>for</strong> HIF-1α stabilization in NSPCs, <strong>an</strong>d support a role <strong>for</strong> HIF-1α in<br />
maintaining survival <strong>an</strong>d stem cell properties of NSPCs.<br />
Supported by NIH R01 NS047373 <strong>an</strong>d NIH/NCRR P20 RR15636.<br />
Disclosures: T. Roitbak, None; L.A. Cunningham, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.16/A16<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Title: Regulation of Notch1 in radial glia<br />
Authors: *E. TZATZALOS, S. DOH, J. FORINO, M. GRUMET, L. CAI;<br />
Rutgers Univ., Piscataway, NJ
Abstract: Radial glia are a type of neural stem cell which serve as a scaffold <strong>for</strong> neuronal<br />
migration during <strong>the</strong> development of <strong>the</strong> central nervous system (CNS). During early<br />
development, radial glia are prevalent as neuronal progenitors <strong>an</strong>d later on as glial progenitors.<br />
Radial glial morphology <strong>an</strong>d markers is maintained through <strong>the</strong> activation of Notch1 (act-Notch).<br />
However, <strong>the</strong> mech<strong>an</strong>ism by which Notch1 maintains <strong>the</strong> radial glial state remains confounded.<br />
Progress has been made in underst<strong>an</strong>ding <strong>the</strong> regulation of Notch1 through a novel enh<strong>an</strong>cer.<br />
Using computational methods, we have predicted several enh<strong>an</strong>cer c<strong>an</strong>didates <strong>for</strong> <strong>the</strong> regulation<br />
of Notch1 <strong>an</strong>d have also provided evidence that Notch1 enh<strong>an</strong>cer regulates Notch1 through<br />
Nkx6-1. Nkx6-1 is a homeobox tr<strong>an</strong>scription factor that is expressed by progenitors in <strong>the</strong><br />
embryonic neuroepi<strong>the</strong>lium <strong>an</strong>d is involved in <strong>the</strong> specification of interneurons <strong>an</strong>d<br />
motoneurons. We have also begun to <strong>an</strong>alyze <strong>the</strong> role of Nkx6-1 in cell fate determination<br />
through its interaction with act-Notch1. Nkx6-1 is present in mouse embryonic stem cells that<br />
have stable expression of act-Notch1. Preliminary over-expression of Nkx6-1 demonstrated<br />
nuclear localization in mouse embryonic-derived neural stem cells. This research provides<br />
insight into <strong>the</strong> biology of <strong>the</strong> neural stem cell <strong>an</strong>d <strong>the</strong> complex regulation of gene expression<br />
during neural stem cell differentiation.<br />
Disclosures: E. Tzatzalos, None; S. Doh, None; J. Forino, None; M. Grumet, None; L. Cai,<br />
None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.17/A17<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH AG020047 to D.A.P.<br />
Title: Stimulation of neural precursor cells in <strong>the</strong> adult rat non-neurogenic entorhinal cortex<br />
using a BDNF <strong>an</strong>d DNOlig2 gene delivery approach<br />
Authors: *F. C. KLEMPIN, D. A. PETERSON;<br />
Rosalind Fr<strong>an</strong>klin Univ., North Chicago, IL<br />
Abstract: In <strong>the</strong> adult brain, neural stem cells reside in a particular niche <strong>for</strong>med by a<br />
microenvironment that promotes neuronal development. Outside of <strong>the</strong>se spatially restricted<br />
neurogenic regions, <strong>the</strong> subventricular zone (SVZ)/olfactory bulb <strong>an</strong>d subgr<strong>an</strong>ular zone (SGZ) in<br />
<strong>the</strong> hippocampal dentate gyrus, proliferating progenitor cells differentiate into glia cells <strong>an</strong>d
never express mature neuronal genes. In models of neurodegeneration, neurogenic niches are<br />
shown to exhibit <strong>an</strong> injury-induced increase in adult neurogenesis. However, in typically<br />
gliogenic cortical areas, such as <strong>the</strong> entorhinal cortex (ECX), no local proliferation or neuronal<br />
differentiation of endogenous precursor cells occurs. We conducted experiments to investigate if<br />
enh<strong>an</strong>cing <strong>the</strong> instructive niche with appropriate signals could stimulate progenitor cells in nonneurogenic<br />
regions. We found, that adenoviral gene delivery of neurotrophic factor BDNF into<br />
<strong>the</strong> ECX locally increased proliferation after 1 day <strong>an</strong>d up to 2 month. A signific<strong>an</strong>t proportion<br />
of <strong>the</strong> recruited cells expressed <strong>the</strong> glial progenitor marker NG2 <strong>an</strong>d only a small number of<br />
S100β or Sox2-positive cells were detected. Import<strong>an</strong>tly, <strong>the</strong> majority of BrdU-positive cells<br />
failed to express markers of immature or differentiated neurons or glia. Since BDNF alone was<br />
insufficient to stimulate neurogenesis, we utilized a combined gene delivery approach. Recent<br />
studies suggest that Olig2 directs SVZ precursor cells toward astrocytic <strong>an</strong>d oligodendrocytic<br />
fates. Inhibition of glial signals in committed cortical progenitor cells expressing NG2 <strong>an</strong>d Olig2<br />
may lead to a non-gliogenic cell fate decision. Accordingly, a retroviral vector carrying a<br />
domin<strong>an</strong>t-negative <strong>for</strong>m of Olig2 <strong>an</strong>d <strong>the</strong> reporter green fluorescent protein (GFP) was delivered<br />
into <strong>the</strong> ECX <strong>an</strong>d SGZ (Olig2VP16-GFP, construct is a kind gift <strong>from</strong> M. Goetz). Surprisingly,<br />
gene delivery of DNOlig2 ch<strong>an</strong>ged proliferation in <strong>the</strong> projection target SGZ after 1 day without<br />
altering proliferation in <strong>the</strong> ECX. However, <strong>the</strong> inhibition of Olig2 signaling within <strong>the</strong> ECX led<br />
to morphological ch<strong>an</strong>ges in tr<strong>an</strong>sduced cells with less oligodendrocytic features <strong>an</strong>d increased<br />
proportion of S100β+ expression among GFP expressing cells, suggesting that cell fate has been<br />
pushed to <strong>an</strong>o<strong>the</strong>r lineage. These experiments will add to our underst<strong>an</strong>ding of <strong>the</strong> molecular<br />
regulators of cell genesis <strong>an</strong>d differentiation <strong>an</strong>d tissue repair in non-neurogenic regions that are<br />
subject to neurodegeneration, such as Alzheimer’s disease.<br />
Disclosures: F.C. Klempin, None; D.A. Peterson, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.18/A18<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NIH Gr<strong>an</strong>t DA019674 (ANM)<br />
NIH Gr<strong>an</strong>t MH076145 (JC)<br />
Title: Mosaic <strong>an</strong>euploidy alters cell fate in <strong>the</strong> developing brain
Authors: *A. N. MOSLEY 1 , S. E. PETERSON 2 , D. BUSHMAN 3 , J. CHUN 2 ;<br />
1 Neurosciences Grad. Program, UCSD, La Jolla, CA; 2 The Scripps Res. Inst., La Jolla, CA;<br />
3 Biomed. Sci. Grad. Program, UCSD, La Jolla, CA<br />
Abstract: The developing <strong>an</strong>d adult brain is a genetic mosaic made up of both <strong>an</strong>euploid <strong>an</strong>d<br />
euploid cells. Aneuploidy seen in <strong>the</strong> normal brain has been termed “mosaic <strong>an</strong>euploidy”<br />
whereby cells exhibit stochastic losses <strong>an</strong>d gains of whole chromosomes. The majority of this<br />
mosaic <strong>an</strong>euploidy is due to chromosomal loss, producing “loss of heterozygosity” (LOH) that<br />
c<strong>an</strong> unmask recessive traits at loci on <strong>the</strong> lost chromosome. This could lead to functional ch<strong>an</strong>ges<br />
in neural cells. Here we asked whe<strong>the</strong>r mosaic <strong>an</strong>euploidy influences astrocytic differentiation of<br />
neural progenitor cells (NPCs). Leukemia inhibitory factor receptor (LIFR)-mediated astrocytic<br />
differentiation was assessed in <strong>the</strong> developing mouse CNS. The LIFR allele, which resides on<br />
chromosome 15, was tracked by crossing LIFR+/- mice with mice hemizygous <strong>for</strong> a ubiquitously<br />
expressed, enh<strong>an</strong>ced green fluorescent protein (eGFP) gene on <strong>the</strong> same chromosome. NPCs<br />
<strong>from</strong> <strong>the</strong>se embryos were cultured with ciliary neurotrophic factor (CNTF) <strong>for</strong> 6 days to induce<br />
astrocytic differentiation through LIFR. Cells were <strong>the</strong>n separated into eGFP negative <strong>an</strong>d<br />
positive populations by fluorescence-activated cell sorting (FACS), <strong>an</strong>d fluorescent in situ<br />
hybridization (FISH) <strong>an</strong>alysis demonstrated that <strong>the</strong> GFP negative population was highly<br />
enriched <strong>for</strong> <strong>an</strong>euploid cells missing one copy of chromosome 15. Expression of <strong>the</strong> astrocytic<br />
marker GLAST was compared in <strong>the</strong> eGFP negative populations <strong>from</strong> LIFR(+/-)eGFP(+/0)<br />
embryos <strong>an</strong>d LIFR(+/+)eGFP(+/0) embryos using flow cytometry. GLAST expression in<br />
LIFR(+/-)eGFP(+/0) embryos was signific<strong>an</strong>tly reduced as compared to LIFR(+/+)eGFP(+/0)<br />
embryos, indicating that mosaic <strong>an</strong>euploidy c<strong>an</strong> alter <strong>the</strong> differentiation of NPCs. Similar<br />
reductions were not seen when GFP positive populations were compared. These results<br />
demonstrate that mosaic <strong>an</strong>euploidy, through LOH, c<strong>an</strong> functionally alter astrocyte cell fate <strong>an</strong>d<br />
may possibly affect o<strong>the</strong>r lineages, contributing to <strong>the</strong> generation of phenotypic <strong>an</strong>d functional<br />
diversity seen within <strong>the</strong> brain.<br />
Disclosures: A.N. Mosley, None; S.E. Peterson, None; D. Bushm<strong>an</strong>, None; J. Chun, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.19/A19<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: NINDS contract# HHSN27120072301C
NIH Neuroscience Blueprint<br />
Title: A developmental tr<strong>an</strong>slating ribosome affinity purification (TRAP) <strong>an</strong>alysis of Bergm<strong>an</strong>n<br />
glial cell lineage<br />
Authors: *A. MILOSEVIC, J. P. DOYLE, N. HEINTZ;<br />
Rockefeller Univ., New York, NY<br />
Abstract: Bergm<strong>an</strong>n glial cells are a specific subtype of astrocyte that reside in <strong>the</strong> Purkinje cell<br />
layer <strong>an</strong>d have <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> development <strong>an</strong>d function of <strong>the</strong> cerebellum. These cells<br />
derive <strong>from</strong> radial glia <strong>an</strong>d cycling progenitors during early postnatal life, but it is not known<br />
whe<strong>the</strong>r <strong>the</strong>y originate <strong>from</strong> restricted or multipotent astrocyte progenitors. Results of<br />
tr<strong>an</strong>spl<strong>an</strong>tation studies support <strong>the</strong> multipotent progenitor hypo<strong>the</strong>sis in which final<br />
differentiation into Bergm<strong>an</strong>n glial cells depends upon environmental cues. To investigate <strong>the</strong><br />
molecular basis of this process we utilized <strong>the</strong> recently described TRAP methodology (Heim<strong>an</strong><br />
et al, 2008; Doyle et al, 2008) to <strong>an</strong>alyze <strong>the</strong> tr<strong>an</strong>slational profiles of Bergm<strong>an</strong>n glial cells <strong>from</strong><br />
postnatal day 7, postnatal day 18, <strong>an</strong>d adult mice using Septin 4 bacTRAP tr<strong>an</strong>sgenic mice. This<br />
<strong>an</strong>alysis reveals a number of differentially regulated mRNAs, m<strong>an</strong>y of which are specifically<br />
enriched in <strong>the</strong> Bergm<strong>an</strong>n glial cells in postnatal day 7. As expected, a number of <strong>the</strong>se<br />
developmentally regulated tr<strong>an</strong>scripts include those known to be involved in cell cycle<br />
regulation. Import<strong>an</strong>tly, we also identified a number of differentially regulated tr<strong>an</strong>scripts known<br />
to be involved in signal tr<strong>an</strong>sduction, including Gper, Rlbp1, <strong>an</strong>d Sucnr1, as well as several<br />
known tr<strong>an</strong>scription factors such as Lbx2, Med4, Mycbp, Polr3k, <strong>an</strong>d Prrx1. In addition to those<br />
genes with known function, TRAP <strong>an</strong>alysis of Bergm<strong>an</strong>n glial development identifies m<strong>an</strong>y<br />
differentially regulated genes that have not been well characterized, but potentially play a role in<br />
differentiation. Since genes involved in signal tr<strong>an</strong>sduction <strong>an</strong>d tr<strong>an</strong>scription are good c<strong>an</strong>didates<br />
<strong>for</strong> <strong>the</strong> control of cellular differentiation, we concentrated on <strong>the</strong>se genes. Screening of <strong>the</strong> gene<br />
expression nervous system (GENSAT) database reveals that m<strong>an</strong>y of <strong>the</strong> genes identified with<br />
developmental TRAP <strong>an</strong>alysis are indeed enriched in white matter progenitors <strong>an</strong>d Bergm<strong>an</strong>n<br />
glia. Our developmental TRAP <strong>an</strong>alysis <strong>the</strong>re<strong>for</strong>e identifies numerous genes likely to be<br />
involved in Bergm<strong>an</strong>n glial cell differentiation.<br />
Disclosures: A. Milosevic, None; J.P. Doyle, None; N. Heintz, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.20/A20
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Support: Fight For Sight<br />
C<strong>an</strong>cer Research UK<br />
Title: Elucidating <strong>the</strong> mech<strong>an</strong>ism of p27Xic1-mediated gliogenesis in <strong>the</strong> retina being<br />
suppressed by Cyclin/Cdks <strong>an</strong>d NM23 members<br />
Authors: A. BILITOU 1 , T. MOCHIZUKI 3 , *S.-I. OHNUMA 2 ;<br />
1 2 3<br />
Inst. of Ophthalmology, Univ. Col. London, London, United Kingdom; Oncology, Univ. of<br />
Cambridge, Cambridge, United Kingdom<br />
Abstract: Events of cell proliferation, differentiation <strong>an</strong>d maturation overlap in <strong>the</strong> Xenopus<br />
retina during eye development. A cell cycle inhibitor, p27Xic1, has been shown to promote<br />
Müller glial cell fate <strong>an</strong>d this activity is independent of its cell cycle regulatory function. We<br />
have found that NM23 nucleoside diphosphate kinases are expressed in <strong>the</strong> vertebrate retina<br />
during development <strong>an</strong>d distributed in <strong>the</strong> ciliary marginal zone (CMZ) in a pattern overlapping<br />
that of p27Xic1. NM23s c<strong>an</strong> suppress p27Xic1-mediated gliogenesis in <strong>the</strong> retina through <strong>the</strong>ir<br />
direct interaction. Here we report that cell cycle components, that are also expressed in <strong>the</strong> retina<br />
in different spatio-temporal patterns, functionally interact with p27Xic1 to affect cell fate<br />
determination. We show that Cyclin D1/cdk4, Cyclin E1/cdk2 or Cyclin A2/cdk2 c<strong>an</strong> inhibit<br />
p27Xic1-mediated gliogenesis <strong>an</strong>d we elucidate <strong>the</strong> mech<strong>an</strong>ism. We have also <strong>an</strong>alysed <strong>the</strong><br />
effects of this interaction on cell proliferation versus differentiation <strong>an</strong>d report a competition<br />
between NM23 members <strong>an</strong>d cell cycle components <strong>for</strong> binding with p27Xic1.<br />
Disclosures: A. Bilitou, None; T. Mochizuki, None; S. Ohnuma, None.<br />
Poster<br />
505. Cell Lineage <strong>an</strong>d Cell Fate Specification<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 505.21/A21<br />
Topic: A.02.b. Cell lineage <strong>an</strong>d cell fate specification<br />
Title: E-box regulates <strong>the</strong> nestin gene expression in neural precursor cells<br />
Authors: S. KIM 1 , R.-H. PARK 1 , S.-T. YOU 1 , J.-S. PARK 1 , S.-S. KIM 1,2 , S.-C. PARK 3 , *Y.-D.<br />
LEE 4,1,5,2 , H. SUH-KIM 1,6,7 ;
1 Dept. of Anatomy, Ajou Univ. Sch. of medicine, Suwon, Republic of Korea; 2 Ctr. <strong>for</strong> Cell death<br />
Regulating Biodrug, Ajou Univ, Sch. of medicie, Suwon, Republic of Korea; 3 Dept. of Life<br />
Science, Sookmyung Univ., Seoul, Republic of Korea; 4 Dept of Anat, Sch. of Med., Paldal-Gu,<br />
Suwon, Republic of Korea; 5 Dept. of molecular science <strong>an</strong>d technology, Ajou Univ., Suwon,<br />
Republic of Korea; 6 Interdisciplinary course <strong>for</strong> neuroscience <strong>an</strong>d technology, Ajou Univ.,<br />
Suwon, Republic of Korea; 7 BK21, Div. of cell tr<strong>an</strong>s<strong>for</strong>mation <strong>an</strong>d restoration, Ajou Univ, Shool<br />
of medicine, Suwon, Republic of Korea<br />
Abstract: Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) tr<strong>an</strong>scription factor that is<br />
expressed in neuronal precursors during <strong>an</strong> early development of <strong>the</strong> nervous system. Nestin,<br />
which is expressed during <strong>the</strong> embryogenesis like Ngn1, is a characteristic intermediate filament<br />
protein expressed in neural stem cells. The posterior 637bp of <strong>the</strong> 2 nd intron of <strong>the</strong> rat Nestin<br />
gene has been known to sufficient <strong>for</strong> <strong>the</strong> CNS-specific Nestin expression. In <strong>the</strong> present work,<br />
we found that Ngn1 c<strong>an</strong> promote Nestin expression <strong>an</strong>d majority of Ngn1 positive cells showed<br />
simult<strong>an</strong>eous expression with Nestin gene. Ngn1, Ngn2 <strong>an</strong>d Mash1, proneural bHLHs, increased<br />
2 nd E-box in 637bp of <strong>the</strong> Nestin 2 nd intron mediated reporter gene activity. Fur<strong>the</strong>rmore, direct<br />
interactions of Ngn1, Ngn2 <strong>an</strong>d Mash1 proteins <strong>an</strong>d E-box were observed by chromatin<br />
immunoprecipitation (ChIP) assay in E11.5 mouse embryo brain. Tr<strong>an</strong>sgenic (Tg) mouse<br />
containing 637bp of Nestin 2 nd intron containing wild type E-box <strong>an</strong>d mutated E-box<br />
demonstrated <strong>the</strong> functional import<strong>an</strong>ce of <strong>the</strong> E-box (1407-1412) in vivo. These results indicate<br />
that proneural Ngn1 promotes Nestin expression via a binding of <strong>an</strong> E-box of <strong>the</strong> Nestin 2 nd<br />
intron.<br />
Disclosures: S. Kim, None; R. Park, None; S. You, None; J. Park, None; S. Kim, None; S.<br />
Park, None; Y. Lee, None; H. Suh-Kim, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.1/A22<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NICHD, Intramural Research Program<br />
NIH gr<strong>an</strong>t R01EY004864<br />
NIH gr<strong>an</strong>t P30EY006360
Research to Prevent Blindness<br />
Title: Neurod1 control of gene expression in <strong>the</strong> mammali<strong>an</strong> pineal <strong>an</strong>d retina: Results <strong>from</strong> in<br />
vivo cre/loxp-mediated gene inactivation<br />
Authors: *M. J. OCHOCINSKA 1 , E. M. MUÑOZ 2 , N. POZDEYEV 3 , S. GOEBBELS 4 , P. M.<br />
IUVONE 3 , T. FURUKAWA 5 , K.-A. NAVE 4 , D. C. KLEIN 1 ;<br />
1 NIH, Be<strong>the</strong>sda, MD; 2 Inst. of Histology <strong>an</strong>d Embryology, Sch. of Medicine, Natl. Univ. of<br />
Cuyo, Natl. Council of Research, Sci. <strong>an</strong>d Technol. (CONICET), Mendoza, Argentina; 3 Dept. of<br />
Ophthalmology <strong>an</strong>d Pharmacol., Emory Univ. Sch. of Med., Atl<strong>an</strong>ta, GA; 4 Dept. of<br />
Neurogenetics, Max-Pl<strong>an</strong>ck-Institute of Exptl. Med., Göttingen, Germ<strong>an</strong>y; 5 Osaka Biosci. Inst.,<br />
Osaka, Jap<strong>an</strong><br />
Abstract: NeuroD1 encodes a basic helix-loop-helix (bHLH) tr<strong>an</strong>scription factor involved in<br />
cell cycle regulation, retinal cell genesis, <strong>an</strong>d in development of neural <strong>an</strong>d endocrine structures.<br />
NeuroD1 mRNA is highly abund<strong>an</strong>t in <strong>the</strong> adult mammali<strong>an</strong> pineal gl<strong>an</strong>d <strong>an</strong>d exhibits a<br />
developmental expression pattern similar to that in <strong>the</strong> retina, consistent with <strong>the</strong> common<br />
evolutionary origin of pinealocytes <strong>an</strong>d retinal photoreceptors. While both tissues express a<br />
common set of tr<strong>an</strong>scription <strong>an</strong>d phototr<strong>an</strong>sduction genes, <strong>the</strong> pineal gl<strong>an</strong>d is composed 95% of<br />
pinealocytes, whereas <strong>the</strong> retina is far more heterogeneous. The greater degree of cellular<br />
homogeneity makes <strong>the</strong> pineal gl<strong>an</strong>d a useful model to use to underst<strong>an</strong>d developmental<br />
mech<strong>an</strong>isms shared by both tissues. Previous attempts to study <strong>the</strong> role of NeuroD1 in <strong>the</strong> retina<br />
<strong>an</strong>d pineal gl<strong>an</strong>d have been limited by <strong>the</strong> fact that NeuroD1 -/- <strong>an</strong>imals die shortly after birth. To<br />
overcome this limitation, we used a Cre/loxP approach, in which Cre recombinase was expressed<br />
in pinealocytes <strong>an</strong>d retinal photoreceptors, under control of <strong>the</strong> promoter <strong>for</strong> Crx, which is<br />
selectively expressed at high levels in <strong>the</strong>se cells. NeuroD1 flox/+ /Crx-cre +/- mice were crossed<br />
with NeuroD1 flox/flox mice to obtain <strong>the</strong> conditional NeuroD1 knockout (cKO,<br />
NeuroD1 flox/flox /Crx-cre +/- ) <strong>an</strong>d control (NeuroD1 flox/flox /Crx-cre -/- ) mice. NeuroD1 expression was<br />
undetectable in <strong>the</strong> pineal gl<strong>an</strong>d <strong>an</strong>d retinas of <strong>the</strong> cKO mice, but was expressed at normal levels<br />
in o<strong>the</strong>r tissues tested. The cKO <strong>an</strong>imals were visually blind by 4 months of age, based on rod<br />
<strong>an</strong>d cone-related electroretinogram responses; however <strong>the</strong> nonvisual mel<strong>an</strong>opsin system<br />
appeared to be functional based on evidence of entrained rhythmic gene expression in <strong>the</strong> pineal<br />
gl<strong>an</strong>d. Gene expression in <strong>the</strong> cKO <strong>an</strong>d control adult pineal gl<strong>an</strong>d <strong>an</strong>d retina was assessed by<br />
Affymetrix microarray <strong>an</strong>alysis using <strong>the</strong> GeneChip Mouse Genome Array 430_2.0 <strong>an</strong>d<br />
GeneSpring GX10 software; qRT-PCR was used to confirm results. The microarray results<br />
identified several potential Neurod1-regulated genes. In <strong>the</strong> cKO retina, <strong>the</strong> expression of 20<br />
genes was altered > 2-fold, including genes linked to phototr<strong>an</strong>sduction <strong>an</strong>d <strong>the</strong> immune<br />
response. In <strong>the</strong> cKO pineal gl<strong>an</strong>d, <strong>the</strong> expression of 10 genes was altered > 2-fold. The rhythmic<br />
expression of several genes was also eliminated. These findings provide clear evidence of <strong>the</strong><br />
role NeuroD1 plays in controlling gene expression in <strong>the</strong> mammali<strong>an</strong> pinealocyte <strong>an</strong>d retina.<br />
Moreover, strategies designed to produce retinal photoreceptors following photoreceptor<br />
degeneration must consider <strong>the</strong> import<strong>an</strong>ce of NeuroD1.<br />
Disclosures: M.J. Ochocinska, None; E.M. Muñoz, None; N. Pozdeyev, None; S. Goebbels,<br />
None; P.M. Iuvone, None; T. Furukawa, None; K. Nave, None; D.C. Klein, None.
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.2/A23<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: EU FPVI 512012, PROMEMORIA<br />
Deutsche Krebshilfe<br />
Title: Polysialic acid deficiency affects distinct interneuron populations of <strong>the</strong> mouse <strong>for</strong>ebrain<br />
Authors: I. RÖCKLE 1 , B. WEINHOLD 1 , H. BURKHARDT 1 , R. GERARDY-SCHAHN 1,2 ,<br />
*H. HILDEBRANDT 1,2 ;<br />
1 2<br />
H<strong>an</strong>nover Med. School, Cell. Chem., H<strong>an</strong>nover, Germ<strong>an</strong>y; Ctr. <strong>for</strong> Systems Neurosci.<br />
H<strong>an</strong>nover, H<strong>an</strong>nover, Germ<strong>an</strong>y<br />
Abstract: The neural cell adhesion molecule NCAM <strong>an</strong>d its posttr<strong>an</strong>slational modification with<br />
polysialic acid (polySia) are tightly linked to neural development <strong>an</strong>d plasticity. Genetic ablation<br />
of <strong>the</strong> two polySia syn<strong>the</strong>sizing enzymes ST8SiaII <strong>an</strong>d ST8SiaIV generates polySia-negative but<br />
NCAM-positive mice that are characterized by severe defects of major brain fiber tracts<br />
(Weinhold et al. 2005, J Biol Chem 280:429711; Hildebr<strong>an</strong>dt et al. Brain, in press). In addition,<br />
loss of polySia impairs migration of subventricular zone (SVZ) derived interneuron precursors<br />
towards <strong>the</strong> olfactory bulb <strong>an</strong>d affects t<strong>an</strong>gential <strong>an</strong>d radial migration of undefined precursors<br />
during cortical development (Weinhold et al. 2005, J Biol Chem 280:429711; Angata et al. 2007,<br />
Mol Cell Biol 27:6659). Independent <strong>from</strong> migration, removal of polySia promotes<br />
differentiation of SVZ precursors in vitro <strong>an</strong>d selectively enh<strong>an</strong>ces <strong>the</strong>ir maturation towards <strong>the</strong><br />
calretinin-positive type of olfactory bulb interneurons (Röckle et al. 2008, Dev Neurobiol<br />
68:1170). In <strong>the</strong> current study, we comparatively <strong>an</strong>alyzed <strong>the</strong> composition of selected<br />
interneuron populations in prefrontal cortex, hippocampus <strong>an</strong>d olfactory bulb of St8sia2 <strong>an</strong>d<br />
St8sia4 single <strong>an</strong>d double knockout mice. Immunofluorescence <strong>an</strong>d co-localization studies of<br />
major interneuron markers (parvalbumin, calbindin, calretinin, tyrosine hydroxylase) revealed<br />
pronounced but distinct alterations of different prefrontal cortex <strong>an</strong>d olfactory bulb interneuron<br />
subtypes in <strong>the</strong>se mouse lines with different degrees of polySia-deficiency during brain<br />
development. These results indicate that polysialic acid is essential <strong>for</strong> <strong>the</strong> development of<br />
specific interneuron populations of <strong>the</strong> mouse <strong>for</strong>ebrain.
Disclosures: I. Röckle, None; B. Weinhold, None; H. Burkhardt, None; R. Gerardy-Schahn,<br />
None; H. Hildebr<strong>an</strong>dt, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.3/A24<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: McLone Professorship Fund<br />
Kiw<strong>an</strong>is<br />
Gr<strong>an</strong>t <strong>from</strong> Spastic Paralysis Research Foundation of Illinois-Eastern Iowa District of<br />
Title: Epigenetic regulation of neurogenesis via histone H3 methylation <strong>an</strong>d acetylation<br />
Authors: *V. BOSHNJAKU 1 , S. ICHI 1 , Y.-W. SHEN 1 , B. MANIA-FARNELL 2 , D.<br />
MCLONE 1 , T. TADANORI 1 , C. MAYANIL 1 ;<br />
1 2<br />
Developmental Biol., Children's Mem. Res. Ctr., Chicago, IL; Dept. of Biol., Purdue Univ. at<br />
Calumet, Hammond, IN<br />
Abstract: During neurogenesis stem cells differentiate into neurons, which no longer proliferate<br />
but extend nerve fibers. Although several molecules are known to inhibit cell proliferation during<br />
neuronal differentiation, <strong>the</strong> pathways of cell-division suppression are not fully identified. In this<br />
study methylation <strong>an</strong>d acetylation marks on histone H3 were examined to determine what role<br />
<strong>the</strong>se epigenetic marks play in neurogenesis. Two model systems were used <strong>for</strong> <strong>the</strong>se studies.<br />
The first system was <strong>the</strong> Splotch (Pax3 mut<strong>an</strong>t) homozygous mouse embryos which display<br />
premature neurogenesis when compared to age-matched wild type controls. The second system<br />
was <strong>the</strong> ND7 cell line, a hybrid between rat neonatal dorsal root g<strong>an</strong>glion cells <strong>an</strong>d mouse<br />
neuroblastoma cells. ND7 cells differentiate into neurons in response to NGF <strong>an</strong>d cAMP. First,<br />
neural tube expl<strong>an</strong>ts were cultured <strong>from</strong> wild type <strong>an</strong>d Splotch homozygous mouse embryos <strong>an</strong>d<br />
migratory neural crest cells were allowed to differentiate. Using immunohistochemistry, a<br />
signific<strong>an</strong>t increase in H3K4me2 <strong>an</strong>d H3K27me2 was observed in <strong>the</strong> Splotch embryos<br />
compared to wild type. This increase was associated with a decreased expression of KDM6B<br />
(<strong>for</strong>merly known as JMJD3). Additionally, chromatin immunoprecipitation (ChIP) assays<br />
showed that on <strong>the</strong> Hes1 promoter, H3K4me2 <strong>an</strong>d H3K27me2 methylation marks were increased<br />
<strong>an</strong>d demethylases UTX <strong>an</strong>d KDM6B (JMJD3) were decreased. Overall, <strong>the</strong>se results suggest that<br />
H3K27 is methylated on Hes1 promoter to allow neurogenesis. Second, immunostaining data
<strong>from</strong> ND7 cells, showed <strong>an</strong> increase in <strong>the</strong> expression of <strong>the</strong> histone acetyltr<strong>an</strong>sferase GCN5, <strong>an</strong>d<br />
increased acetylation of H3K9 <strong>an</strong>d H3K18 in differentiated compared to undifferentiated cells.<br />
Fur<strong>the</strong>rmore, increased expression of NeuroD, Brn3a <strong>an</strong>d Ngn2 in differentiated compared to<br />
undifferentiated ND7 cells suggest that acetylation of H3K9 <strong>an</strong>d H3K18 on <strong>the</strong> promoters of<br />
genes such as NeuroD, Brn3a, <strong>an</strong>d Ngn2 may be involved in neurogenesis.<br />
Disclosures: V. Boshnjaku, Spastic Paralysis Research Foundation of Illinois-Eastern Iowa<br />
District of Kiw<strong>an</strong>is, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); McLone Professorship Fund, O<strong>the</strong>r; S. Ichi,<br />
None; Y. Shen, None; B. M<strong>an</strong>ia-Farnell, None; D. McLone, McLone Professorship Fund,<br />
O<strong>the</strong>r; T. Tad<strong>an</strong>ori, None; C. May<strong>an</strong>il, Spastic Paralysis Research Foundation of Illinois-<br />
Eastern Iowa District of Kiw<strong>an</strong>is, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); McLone Professorship Fund,<br />
O<strong>the</strong>r.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.4/A25<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: Fundación La Caixa Gr<strong>an</strong>t 2005X1045<br />
Sp<strong>an</strong>ish Ministerio de Ciencia e Innovación Gr<strong>an</strong>t BFU 2008-02950-C03-01<br />
Comunidad de Madrid Gr<strong>an</strong>t CCG08-CSIC/SAL-3617<br />
Title: Neurogenin3 undergoes nucleo-cytoplasmic shuttling <strong>an</strong>d associates with microtubules<br />
during <strong>the</strong> development of hippocampal neurons<br />
Authors: *M.-A. AREVALO, J. SIMON-ARECES, G. MEMBRIVE, C. GARCIA-<br />
FERNANDEZ, L. GARCIA-SEGURA;<br />
Inst. Cajal. CSIC, Madrid, Spain<br />
Abstract: Neurogenin3 (Ngn3), a proneural gene controlled by Notch receptor, is implicated in<br />
<strong>the</strong> control of dendrite morphology <strong>an</strong>d synaptic plasticity of cultured hippocampal neurons.<br />
Here we report <strong>the</strong> localization <strong>an</strong>d subcellular distribution of Ngn3 in <strong>the</strong> CD1 mouse<br />
hippocampus in vivo <strong>an</strong>d in neuronal cultures. In situ hybridization showed Ngn3 mRNA
expression in pyramidal layer <strong>an</strong>d dentate gyrus of adult mouse hippocampus.<br />
Immunohistochemistry studies revealed that Ngn3 localization is mostly cytoplasmic in <strong>the</strong><br />
hippocampal eminence at E17 <strong>an</strong>d P0. At P10 it is cytoplasmic in CA1-CA3 pyramidal neurons<br />
<strong>an</strong>d nuclear in gr<strong>an</strong>ule cells of <strong>the</strong> dentate gyrus. In <strong>the</strong> adult hippocampus Ngn3 is localized in<br />
<strong>the</strong> nucleus <strong>an</strong>d cytoplasm of both pyramidal neurons <strong>an</strong>d gr<strong>an</strong>ule cells. During development of<br />
cultured hippocampal neurons, Ngn3 mRNA expression is higher at stages of neuronal<br />
polarization, as judged by RT-PCR, <strong>an</strong>d it is mostly cytoplasmic. The tracking of <strong>the</strong> subcellular<br />
localization of Ngn3 in neurons infected with a virus expressing myc-Ngn3 suggests that <strong>the</strong><br />
protein is quickly tr<strong>an</strong>slocated to <strong>the</strong> cell nucleus after syn<strong>the</strong>sis <strong>an</strong>d <strong>the</strong>n re-exported to <strong>the</strong><br />
cytoplasm. Treatment with leptomycinB, a potent <strong>an</strong>d specific inhibitor of <strong>the</strong> exportin CRM1,<br />
induced its accumulation into <strong>the</strong> nucleus, suggesting that CRM1 mediates <strong>the</strong> nuclear export of<br />
Ngn3. Pharmacological perturbation of <strong>the</strong> growth cone cytoskeleton revealed that Ngn3 is<br />
associated with microtubules. This study reports <strong>for</strong> <strong>the</strong> first time <strong>the</strong> localization of Ngn3 in <strong>the</strong><br />
cytoplasm of hippocampal neurons <strong>an</strong>d addresses a new function <strong>for</strong> this protein out of <strong>the</strong><br />
nucleus. Altoge<strong>the</strong>r <strong>the</strong>se results suggest that, in response to <strong>an</strong> unidentified cell signal, Ngn3<br />
goes out of <strong>the</strong> nucleus <strong>an</strong>d associates to microtubules in <strong>the</strong> dendrite <strong>an</strong>d axon to support <strong>the</strong><br />
growth of neuritic processes, thus contributing to neuronal development.<br />
Disclosures: M. Arevalo, None; J. Simon-Areces, None; G. Membrive, None; C. Garcia-<br />
Fern<strong>an</strong>dez, None; L. Garcia-Segura, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.5/A26<br />
Topic: A.02.d. Neuronal differentiation<br />
Title: Drosophila neural zinc finger protein dNZF is required <strong>for</strong> differentiation of a subset of<br />
neurons<br />
Authors: *S. OHSAKO, T. YAMADA;<br />
Dept Neurosci Basic Tech., Tokyo Metropolit<strong>an</strong> Inst., Fuchu, Jap<strong>an</strong><br />
Abstract: Neural zinc finger factor (NZF)/myelin tr<strong>an</strong>scription factor (MyT) gene family is<br />
constituted of tr<strong>an</strong>scription factors with DNA-binding domains of a C2HC-type Zn finger<br />
conserved <strong>from</strong> C. eleg<strong>an</strong>s to hum<strong>an</strong>. Hum<strong>an</strong> MyT1 was first identified by virtue of its binding<br />
to cis-regulatory elements of a glia-specific gene, <strong>the</strong> myelin proteolipid protein gene. On <strong>the</strong><br />
o<strong>the</strong>r h<strong>an</strong>d, Xenopus MyT1 was shown to play a critical role in neuronal differentiation. The
functions of <strong>the</strong> NZF/MyTgene family, however, remain unsolved. To elucidate NZF/MyT<br />
function, we previously identified a Drosophila homolog dNZF. Although only one cDNA clone<br />
was isolated, RT-PCR <strong>an</strong>alysis showed that multiple dnzf tr<strong>an</strong>scripts are generated by alternative<br />
splicing of <strong>the</strong> gene. Western blot <strong>an</strong>alysis partially confirmed <strong>the</strong> presence of <strong>the</strong> iso<strong>for</strong>ms. We<br />
generated dnzf mut<strong>an</strong>ts that almost die at <strong>the</strong> pharate adult stage, by imprecise excision of a Pelement<br />
tr<strong>an</strong>sposon inserted near <strong>the</strong> tr<strong>an</strong>scriptional start site of <strong>the</strong> dnzf gene. A few adult<br />
escapers, however, showed <strong>an</strong> uncoordinated movement, <strong>an</strong>d died shortly <strong>the</strong>reafter.<br />
Immunochemical <strong>an</strong>alysis revealed that dNZF is expressed in a subset of neurons. We replaced<br />
P{SURor-P} with P{GAL4, w+} using targeted tr<strong>an</strong>sposition to make dnzf-GAL4 line that<br />
mimics <strong>the</strong> endogenous expression pattern. Blockage of neural tr<strong>an</strong>smission in adult flies by<br />
dnzf-GAL4 driven temperature-sensitive dynamin (shibire) expression at <strong>the</strong> non-permissive<br />
temperature, caused <strong>an</strong> uncoordinated movement as seen in dnzf mut<strong>an</strong>t escapers. This could be<br />
due to dysfunction of neurons expressing dNZF, suggesting that dNZF may be involved in<br />
neuronal differentiation.<br />
Disclosures: S. Ohsako, None; T. Yamada, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.6/A27<br />
Topic: A.02.d. Neuronal differentiation<br />
Title: Olfactomedin 1 interacts with VGF <strong>an</strong>d may regulate neuronal differentiation<br />
Authors: *N. NAKAYA 1 , A. CHENG 3 , J. P. MUNASHINGE 4 , Y. TAKADA 2 , T. V.<br />
JOHNSON 1 , M. P. MATTSON 3 , S. TOMAREV 1 ;<br />
1 MMG/LMDB, 2 Imaging Core Unit, Natl. Eye Institute/NIH, Be<strong>the</strong>sda, MD; 3 Lab. of Neurosci.,<br />
Natl. Inst. of Aging/ NIH, Baltimore, MD; 4 In Vivo NMR Ctr., Natl. Inst. of Neurolog. Disorders<br />
<strong>an</strong>d Stroke/NIH, Be<strong>the</strong>sda, MD<br />
Abstract: Purpose: Olfactomedin 1 (Olfm1), also known as noelin <strong>an</strong>d p<strong>an</strong>cortin, is a secreted<br />
glycoprotein belonging to <strong>the</strong> family of olfactomedin domain-containing proteins. It is highly<br />
conserved in vertebrates <strong>an</strong>d mainly expressed in <strong>the</strong> neural crest <strong>an</strong>d neuronal tissues. Published<br />
data suggest that Olfm1 regulates retinal g<strong>an</strong>glion cell differentiation <strong>an</strong>d/or axonal extension in<br />
zebrafish. Here, we study possible functions of mammali<strong>an</strong> Olfm1 in cultured neurons <strong>an</strong>d<br />
mouse brain in vivo. Methods: Olfm1 protein was purified <strong>from</strong> stably tr<strong>an</strong>sfected PC12 cells<br />
using lectin-agarose beads. E18 rat hippocampal neurons <strong>an</strong>d adult rat SVZ neuronal precursor
cells were cultured with purified Olfm1 or tr<strong>an</strong>sfected with Olfm1-expressing vector. Proteins<br />
interacting with Olfm1 were identified by a pull-down assay combined with mass-spectrometry.<br />
Immunocytochemistry was used to determine <strong>the</strong> cellular localization of Olfm1 <strong>an</strong>d o<strong>the</strong>r<br />
proteins. Magnetic reson<strong>an</strong>ce imaging (MRI) with a 14 Tesla Bruker Av<strong>an</strong>ce sc<strong>an</strong>ner was used<br />
to <strong>an</strong>alyze structural ch<strong>an</strong>ges in <strong>the</strong> brain of mice with a deletion in <strong>the</strong> central part of <strong>the</strong> Olfm1<br />
protein (Olfm1 KO). A three dimensional volume encompassing <strong>the</strong> whole brain was specified<br />
using three orthogonal scout images. Results: Olfm1 was detected in different regions of adult<br />
mouse brain with <strong>the</strong> highest levels in cerebral cortex, hippocampus <strong>an</strong>d olfactory bulb. In<br />
cultured hippocampal neurons, endogenous Olfm1 was detected in <strong>the</strong> cell soma, axonal bodies<br />
<strong>an</strong>d growth cones. Overexpression of full length Olfm1 in hippocampal neurons by tr<strong>an</strong>sient<br />
tr<strong>an</strong>sfection increased <strong>the</strong> length of growing axons by 20%. Similarly, treatment of neuronal<br />
precursor cells <strong>from</strong> adult rat SVZ with purified Olfm1 protein also facilitated axonal growth,<br />
suggesting that Olfm1 may function extracellularly. VGF, a neurotrophin-inducible protein, was<br />
identified as a protein interacting with Olfm1 by a pull-down assay. In neuronal cells, Olfm1 <strong>an</strong>d<br />
VGF co-localized along <strong>the</strong> axons <strong>an</strong>d axon terminals. Olfm1 KO mice are 30% smaller th<strong>an</strong><br />
<strong>the</strong>ir wild type littermates <strong>an</strong>d relatively infertile. A similar phenotype has been described <strong>for</strong><br />
VGF KO mice. The MRI <strong>an</strong>alysis showed that <strong>the</strong> brain volume, <strong>an</strong>d especially <strong>the</strong> volume of<br />
major nerve fibers located in <strong>the</strong> <strong>an</strong>terior commissure <strong>an</strong>d corpus callosum as well as optic nerve,<br />
was strongly reduced in Olfm1 KO. Conclusions: Olfm1 may have import<strong>an</strong>t functions in <strong>the</strong><br />
regulation of neuronal differentiation <strong>an</strong>d /or axonal growth in developing <strong>an</strong>d adult mammali<strong>an</strong><br />
brain. We suggest that, like VGF, Olfm1 may contribute to some psychiatric conditions<br />
including depression or schizophrenia.<br />
Disclosures: N. Nakaya, None; A. Cheng, None; J.P. Munashinge, None; Y. Takada,<br />
None; T.V. Johnson, None; M.P. Mattson, None; S. Tomarev, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.7/A28<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NIH R01 MH067715-01<br />
NIH R25 MH071584-04<br />
NIH R25 MH077823-03
NIH T32 MH018268-24<br />
Solnit Integrated Training Program at Yale Child Study Center<br />
Title: FGFR2 is required in radial glial cells <strong>for</strong> excitatory neurogenesis in prefrontal cortex <strong>an</strong>d<br />
normal per<strong>for</strong>m<strong>an</strong>ce on cognitive tasks<br />
Authors: *H. E. STEVENS 1 , K. M. SMITH 2 , M. E. MARAGNOLI 2 , Y. OHKUBO 2 , D.<br />
FAGEL 2 , M. SCHWARTZ 3 , T. HORVATH 4 , F. M. VACCARINO 5 ;<br />
2 3 4 5<br />
Yale Child Study Ctr., Dept. of Neurobio., Dept. of Comparative Med., Child Study Ctr.,<br />
1<br />
Yale Univ. Sch. Med., New Haven, CT<br />
Abstract: FGF lig<strong>an</strong>ds (FGF2, FGF8, <strong>an</strong>d FGF17) are expressed in <strong>an</strong>terior regions of <strong>the</strong><br />
developing cerebral cortex <strong>an</strong>d bind FGF receptor 2 (FGFR2) which is expressed in <strong>the</strong><br />
ventricular <strong>an</strong>d subventricular zones of <strong>the</strong> embryonic cortex, particularly in rostral regions. We<br />
generated conditional fgfr1, fgfr2 <strong>an</strong>d double fgfr1/fgfr2 knockout mice lacking FGF receptors in<br />
radial glial cells of <strong>the</strong> dorsal telencephalon <strong>from</strong> embryonic day 14 onward, by GFAPCremediated<br />
recombination. Animals were <strong>an</strong>alyzed by stereological morphometric <strong>an</strong>alyses to<br />
determine <strong>the</strong> number of different cell types in <strong>the</strong> cerebral cortex <strong>an</strong>d connected subcortical<br />
regions. Mice were also tested in a number of behavioral paradigms.<br />
Embryonic fgfr2 KO mice showed deficits in proliferative cells in <strong>the</strong> cortical subventricular<br />
zone as well as decreased numbers of TBR2+ neuronal precursors <strong>an</strong>d TBR1+ excitatory<br />
neurons. Adult fgfr2 KO mice had reduced TBR1+ excitatory neuron numbers <strong>an</strong>d cortical<br />
volumes, more marked in medial prefrontal regions. These excitatory neuron deficits were more<br />
marked in fgfr1/fgfr2 KO mice. Inhibitory interneurons of <strong>the</strong> parvalbumin type were<br />
signific<strong>an</strong>tly lower throughout <strong>the</strong> adult neocortex of mice lacking ei<strong>the</strong>r fgfr1 or fgfr2. In <strong>the</strong><br />
dorsal bed nuclei of <strong>the</strong> stria terminalis (BST), a projection area <strong>for</strong> neurons in medial prefrontal<br />
cortex, fewer <strong>an</strong>d smaller glutamate synapses were observed by electron microscopy in fgfr2 KO<br />
mice. Calretinin positive neurons were also deficient in <strong>the</strong> BST <strong>an</strong>d septum, <strong>an</strong>d this decrease<br />
was correlated in both wildtype <strong>an</strong>d mut<strong>an</strong>t mice with medial prefrontal excitatory neuron<br />
number. Mut<strong>an</strong>t <strong>an</strong>d wildtype mice showed no differences in stress reactivity but demonstrated<br />
locomotor hyperactivity <strong>an</strong>d impairments in object recognition. Mice also showed distinct<br />
patterns of behavior on social approach <strong>an</strong>d <strong>an</strong>xiety paradigms.<br />
We conclude that FGFR2 signaling in radial glial cells <strong>an</strong>d <strong>the</strong>ir progeny is necessary <strong>for</strong> <strong>the</strong><br />
normal development of <strong>the</strong> <strong>an</strong>terior regions of <strong>the</strong> cerebral cortex <strong>an</strong>d its connections within<br />
limbic circuits. Fgfr2 is also required <strong>for</strong> <strong>the</strong> development of distinct subtypes of inhibitory<br />
neurons within <strong>the</strong> neocortex <strong>an</strong>d in <strong>the</strong> BST <strong>an</strong>d septum, deficits which likely develop<br />
secondarily to <strong>the</strong> primary alteration in excitatory circuitry. The contribution of FGF to <strong>the</strong>se<br />
neural structures is not essential <strong>for</strong> <strong>the</strong>ir regulation of stress reactivity but does affect motor <strong>an</strong>d<br />
cognitive behavior.<br />
Disclosures: H.E. Stevens, None; K.M. Smith, None; M.E. Maragnoli, None; Y. Ohkubo,<br />
None; D. Fagel, None; M. Schwartz, None; T. Horvath, None; F.M. Vaccarino, None.
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.8/A29<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: Gr<strong>an</strong>ts-in-aid <strong>for</strong> scientific research No. 13035038<br />
Gr<strong>an</strong>ts-in-aid <strong>for</strong> scientific research No. 15659062<br />
Title: Histone deacetylase SIRT1 modulates neuronal differentiation<br />
Authors: *S. HISAHARA 1,2 , M. TANNO 3 , S. SHIMOHAMA 1 , M. SATO 4 , Y. HORIO 2 ;<br />
1 Neurol., Sapporo Med. Univ, Sch. Med., Sapporo, Jap<strong>an</strong>; 2 Pharmacol., 3 Second Dept. of Intrnl.<br />
Med., Sapporo Med. University, Sch. of Med., Sapporo, Jap<strong>an</strong>; 4 Morphological <strong>an</strong>d<br />
Physiological Sci., Univ. of Fukui, Fukui, Jap<strong>an</strong><br />
Abstract: SIRT1 is <strong>the</strong> mammali<strong>an</strong> homolog of yeast Sir2, <strong>an</strong> NAD+-dependent deacetylase<br />
involved in tr<strong>an</strong>scriptional silencing, genome stability, <strong>an</strong>d longevity. SIRT1 was found to<br />
deacetylate not only histones but also several tr<strong>an</strong>scriptional regulatory proteins involved in <strong>the</strong><br />
control of metabolism such as FOXO protein family, peroxisome proliferator-activated receptor<br />
gamma (PPARγ), coactivator 1α (PGC1α), <strong>an</strong>d <strong>the</strong> nuclear receptor LXR. Neural precursor cells<br />
(NPCs) differentiate into neurons, astrocytes <strong>an</strong>d oligodendrocytes in response to intrinsic <strong>an</strong>d<br />
extrinsic ch<strong>an</strong>ges. Notch signals maintain undifferentiated NPCs but <strong>the</strong> mech<strong>an</strong>isms underlying<br />
<strong>the</strong> neuronal differentiation are largely unknown. Previously, we have shown that SIRT1 was<br />
expressed in nestin-positive cells in <strong>the</strong> subventricular zone of adult mouse brain, indicating<br />
expression of SIRT1 in <strong>the</strong> NPCs. We show that SIRT1 modulates neuronal differentiation.<br />
SIRT1 was found in <strong>the</strong> cytoplasm of embryonic <strong>an</strong>d adult NPCs <strong>an</strong>d was tr<strong>an</strong>siently localized in<br />
<strong>the</strong> nucleus in response to differentiation stimulus. SIRT1 started to tr<strong>an</strong>slocate into <strong>the</strong> nucleus<br />
within 10 min after <strong>the</strong> tr<strong>an</strong>sfer of NPCs into differentiation conditions, stayed in <strong>the</strong> nucleus,<br />
<strong>an</strong>d <strong>the</strong>n gradually re-tr<strong>an</strong>slocated to <strong>the</strong> cytoplasm following several hours. The number of<br />
neurospheres that generated Tuj1+ neurons was signific<strong>an</strong>tly decreased by pharmacological<br />
inhibitors of SIRT1, domin<strong>an</strong>t-negative SIRT1 <strong>an</strong>d SIRT1-siRNA, whereas overexpression of<br />
SIRT1, but not that of cytoplasm-localized mut<strong>an</strong>t SIRT1, enh<strong>an</strong>ced neuronal differentiation <strong>an</strong>d<br />
decreased Hes1 expression. Expression of SIRT1-siRNA impaired neuronal differentiation <strong>an</strong>d<br />
migration of NPCs into <strong>the</strong> cortical plate in <strong>the</strong> embryonic brain. Nuclear receptor co-repressor<br />
(N-CoR), which has been reported to bind SIRT1, promoted neuronal differentiation <strong>an</strong>d<br />
synergistically increased <strong>the</strong> number of Tuj1+ neurons with SIRT1, <strong>an</strong>d both bound <strong>the</strong> Hes1<br />
promoter region in differentiating NPCs. Hes1 tr<strong>an</strong>sactivation by Notch1 was inhibited by SIRT1<br />
<strong>an</strong>d/or N-CoR. Our study indicated that SIRT1 is a player of repressing Notch1-Hes1 signaling
pathway <strong>an</strong>d its tr<strong>an</strong>sient tr<strong>an</strong>slocation into <strong>the</strong> nucleus may have a role in <strong>the</strong> differentiation of<br />
NPCs.<br />
Disclosures: S. Hisahara, None; M. T<strong>an</strong>no, None; S. Shimohama, None; M. Sato, None; Y.<br />
Horio, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.9/A30<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: EPICURE LSH-CT-2006-037315<br />
DFG Bl 421/1-2<br />
ForNeuroCell<br />
Title: Learning is related to <strong>the</strong> regenerative capacity of <strong>the</strong> hum<strong>an</strong> hippocampus<br />
Authors: *F. A. SIEBZEHNRUBL 1,2 , R. CORAS 2 , E. PAULI 3 , H. B. HUTTNER 3 , M.<br />
NJUNTING 2 , K. KOBOW 2 , C. VILLMANN 3 , E. HAHNEN 4 , W. NEUHUBER 3 , D. WEIGEL 3 ,<br />
M. BUCHFELDER 3 , H. STEFAN 3 , D. A. STEINDLER 1 , I. BLUMCKE 2 ;<br />
1 Dept Neurosci, Univ. Florida, Gainesville, FL; 2 Neuropathology, 3 Univ. Erl<strong>an</strong>gen-Nuremberg,<br />
Erl<strong>an</strong>gen, Germ<strong>an</strong>y; 4 Hum<strong>an</strong> Genet., Univ. Cologne, Cologne, Germ<strong>an</strong>y<br />
Abstract: The brain maintains its capacity to generate new neurons throughout life. Several<br />
<strong>an</strong>imal studies support neurogenesis in <strong>the</strong> hippocampus as a cellular substrate <strong>for</strong> learning.<br />
Using hippocampal resection tissue obtained <strong>from</strong> patients with focal seizures, we show that<br />
neurogenesis is decreased in hum<strong>an</strong>s with compromised learning. Using a novel exp<strong>an</strong>sion <strong>an</strong>d<br />
differentiation paradigm, hippocampal progenitor cells failed to differentiate into neurons in vitro<br />
when obtained <strong>from</strong> patients with learning impairment <strong>an</strong>d gr<strong>an</strong>ule cell loss in <strong>the</strong> hippocampus.<br />
Memory scores were assessed using intracarotid amobarbital injections (WADA test). Both<br />
neuronal differentiation in vitro <strong>an</strong>d gr<strong>an</strong>ule cell loss in vivo predicted memory scores in<br />
patients. We observed loss of BDNF <strong>an</strong>d cdk5 expression in patients with learning impairment.<br />
Neurons were successfully differentiated in vitro when obtained <strong>from</strong> patients with normal<br />
memory acquisition, suggesting that <strong>the</strong> regenerative capacity of <strong>the</strong> hippocampus is required <strong>for</strong><br />
encoding new memories in <strong>the</strong> hum<strong>an</strong> brain.
Disclosures: F.A. Siebzehnrubl, None; R. Coras, None; E. Pauli, None; H.B. Huttner,<br />
None; M. Njunting, None; K. Kobow, None; C. Villm<strong>an</strong>n, None; E. Hahnen, None; W.<br />
Neuhuber, None; D. Weigel, None; M. Buchfelder, None; H. Stef<strong>an</strong>, None; D.A. Steindler,<br />
None; I. Blumcke, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.10/A31<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NIH NS15918 to NCS<br />
Title: Odor<strong>an</strong>t stimulation causes neurotr<strong>an</strong>smitter respecification in interneurons of <strong>the</strong><br />
olfactory bulb<br />
Authors: *D. DULCIS, S. ENRIGHT, N. C. SPITZER;<br />
Neurobiol Section, Div. Biol Sci., UCSD, La Jolla, CA<br />
Abstract: Neurotr<strong>an</strong>smitter (NT) specification is regulated by calcium-dependent electrical<br />
activity in <strong>the</strong> developing Xenopus laevis embryo. This activity determines <strong>the</strong> numbers of<br />
neurons expressing classical neurotr<strong>an</strong>smitters (glutamate, acetylcholine, GABA, <strong>an</strong>d glycine) in<br />
<strong>the</strong> spinal cord <strong>an</strong>d <strong>the</strong> numbers of neurons expressing monoamines in <strong>the</strong> brain. Electrical<br />
activity triggered by selective sensory circuit activation in <strong>the</strong> brain c<strong>an</strong> also regulate <strong>the</strong> number<br />
of interneurons that express a specific NT, focusing on <strong>the</strong> dopaminergic VSC neurons following<br />
activation of <strong>the</strong> retino-hypothalamic projection. Regulation is achieved by co-expression of <strong>an</strong><br />
additional tr<strong>an</strong>smitter by reserve pool neurons that already innervate <strong>the</strong> correct target.<br />
To test whe<strong>the</strong>r circuit-specific recruitment of reserve pool neurons is a general phenomenon in<br />
<strong>the</strong> brain, we are investigating <strong>the</strong> effect of <strong>the</strong> activation of <strong>an</strong>o<strong>the</strong>r sensory modality, olfaction.<br />
Olfactory-mediated kin recognition in tadpoles is used to distinguish kin siblings <strong>from</strong> non-kin<br />
siblings via olfactory kinship cues, <strong>an</strong>d 4-day old larvae display aversion behavior to water-borne<br />
non-kin odor<strong>an</strong>ts. Different classes of interneurons in <strong>the</strong> glomerular/periglomerular regions of<br />
<strong>the</strong> olfactory bulb were identified by tyrosine hydroxylase, GABA, NPY, <strong>an</strong>d VGlut1,2<br />
immunocytochemistry <strong>an</strong>d fur<strong>the</strong>r characterized by expression of tr<strong>an</strong>scription factors Pax6 <strong>an</strong>d<br />
Lim1,2 <strong>an</strong>d nuclear markers DAPI/DRAQ5.<br />
We exposed isolated stage 39 tadpoles to ei<strong>the</strong>r kinship or non-kinship odor<strong>an</strong>ts <strong>for</strong> 24 hr <strong>an</strong>d<br />
compared NT expression in olfactory bulb interneurons at stage 42 to controls (exposed to bl<strong>an</strong>k<br />
saline <strong>for</strong> 24 hr). Our preliminary results show that non-kin exposure leads to <strong>an</strong> increase
(82±12%) in <strong>the</strong> number of GABAergic interneurons <strong>an</strong>d to a simult<strong>an</strong>eous reduction (34±9%)<br />
of NPY co-expression within GABAergic cells of <strong>the</strong> periglomerular region, while <strong>the</strong> number of<br />
dopaminergic (TH+Pax6+) cells remains const<strong>an</strong>t. Interestingly, exposure to kinship cues did not<br />
affect <strong>the</strong> number of GABAergic periglomerular interneurons, but caused a similar reduction<br />
(46±3%) in NPY expression. Future experiments will pharmacologically suppress electrical<br />
activity or selectively block glutamate receptors to test <strong>the</strong> activity-dependence of this<br />
phenomenon. Behavioral assays will be used to determine <strong>the</strong> effect of NT respecification in <strong>the</strong><br />
olfactory bulb on kin recognition. This knowledge is expected to help bridge <strong>the</strong> developmental<br />
complexity of NT specification with novel clinical approaches to selectively replenish NT<br />
expression in diseased brains via circuit activation by natural stimuli.<br />
Disclosures: D. Dulcis , None; S. Enright, None; N.C. Spitzer, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.11/A32<br />
Topic: A.02.d. Neuronal differentiation<br />
Title: Polysialic acid in dopaminergic system development<br />
Authors: *M. SCHIFF 1,3 , B. WEINHOLD 1 , C. GROTHE 2,3 , H. HILDEBRANDT 1,3 ;<br />
1 Inst. <strong>for</strong> Cell. Chem., 2 Inst. <strong>for</strong> Neuro<strong>an</strong>atomy, H<strong>an</strong>nover Med. Sch., H<strong>an</strong>nover, Germ<strong>an</strong>y; 3 Ctr.<br />
<strong>for</strong> Systems Neurosci., H<strong>an</strong>nover, Germ<strong>an</strong>y<br />
Abstract: Polysialic acid, a posttr<strong>an</strong>slational modification of <strong>the</strong> neural cell adhesion molecule<br />
NCAM is <strong>an</strong> import<strong>an</strong>t regulator of neurogenesis. It is essential <strong>for</strong> proper brain development<br />
<strong>an</strong>d involved in regulating neuroblast migration <strong>an</strong>d differentiation. In <strong>the</strong> current study,<br />
expression <strong>an</strong>d function of polysialic acid in <strong>the</strong> development of <strong>the</strong> dopaminergic system of <strong>the</strong><br />
ventral midbrain of mice were investigated. Using immunohistochemistry, polysialic acid was<br />
detected on nestin-positive radial glia processes <strong>an</strong>d on cell somata in <strong>the</strong> pial zone of <strong>the</strong><br />
midbrain at embryonic day E11.5 <strong>an</strong>d E14.5. Real-time RT-PCR <strong>an</strong>alyses established a detailed<br />
picture of <strong>the</strong> mRNA expression profiles of NCAM, <strong>the</strong> major carrier of polySia, <strong>an</strong>d of <strong>the</strong> two<br />
key enzymes of polySia syn<strong>the</strong>sis, ST8SiaII <strong>an</strong>d ST8SiaIV. These profiles provide a predictive<br />
signature <strong>for</strong> <strong>the</strong> course of NCAM polysialylation during brain development. Their comparison<br />
with <strong>the</strong> combined expression patterns of <strong>the</strong> dopaminergic marker genes tyrosine hydroxylase,<br />
dopamine tr<strong>an</strong>sporter, Nurr1 <strong>an</strong>d Pitx3 revealed a marked correlation. Asking <strong>for</strong> a possible role<br />
of polysialylation in dopaminergic differentiation, mice lacking polysialic acid due to ablation of
oth St8siaII <strong>an</strong>d St8siaIV were <strong>an</strong>alyzed at selected time points by tyrosine hydroxylase<br />
immunohistochemistry <strong>an</strong>d by real-time RT-PCR of dopaminergic markers. Surprisingly, no<br />
differences between wild type <strong>an</strong>d mut<strong>an</strong>t mice could be detected. Likewise, enzymatic removal<br />
of polysialic acid <strong>from</strong> cultured neurons of <strong>the</strong> ventral embryonic midbrain had no effect on <strong>the</strong><br />
expression of dopaminergic marker genes. We conclude that, despite its abund<strong>an</strong>ce, polysialic<br />
acid is not required <strong>for</strong> migration, differentiation, axonal projection or postnatal mainten<strong>an</strong>ce of<br />
mDA neurons.<br />
Disclosures: M. Schiff, None; B. Weinhold, None; C. Gro<strong>the</strong>, None; H. Hildebr<strong>an</strong>dt, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.12/A33<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NIH MH074702 to NCS<br />
Title: Calcium-spike-driven specification of neurotr<strong>an</strong>smitters in embryonic development: A<br />
cell-autonomous process?<br />
Authors: L. XU, *N. C. SPITZER;<br />
Neurobio. Section, Div. Biol. Sci, Kavli Inst. <strong>for</strong> Brain <strong>an</strong>d Mind, UCSD, La Jolla, CA<br />
Abstract: Calcium-dependent electrical activity in Xenopus spinal neurons homeostatically<br />
regulates specification of <strong>the</strong> neurotr<strong>an</strong>smitters that neurons express without affecting cell<br />
identity. However, <strong>the</strong> mech<strong>an</strong>ism underlying this plasticity remains elusive. Several major<br />
cognitive disorders of hum<strong>an</strong> development, such as autism <strong>an</strong>d schizophrenia, involve<br />
dysregulation of <strong>the</strong> metabolism of neurotr<strong>an</strong>smitters or neurotr<strong>an</strong>smitter receptors. Determining<br />
whe<strong>the</strong>r <strong>the</strong> regulation of neurotr<strong>an</strong>smitter specification driven by calcium spike activity is<br />
achieved by a cell-autonomous mech<strong>an</strong>ism has <strong>the</strong> potential to make a valuable contribution to<br />
our underst<strong>an</strong>ding of <strong>the</strong>se disorders.<br />
We have developed a targeting system to alter calcium spike activity in isolated Xenopus spinal<br />
neurons in vivo. Two strategies were employed. First, DNA injection was utilized, instead of<br />
RNA injection, to achieve mosaic expression. Second, injections were per<strong>for</strong>med at <strong>the</strong> 16-cell<br />
stage, after neuronal lineages are determined, to facilitate targeting different subgroups of spinal<br />
neurons by injecting specific blastomeres. DNA constructs of hKir2.1 or rNav2aαβ were injected<br />
into blastomeres to suppress or enh<strong>an</strong>ce calcium spike activity in isolated spinal neurons. Both
constructs were tagged with or coinjected with fluorescent proteins to allow visualization of <strong>the</strong>ir<br />
expression. Immunocytochemistry in spinal cord cryostat sections was used to detect ch<strong>an</strong>ges in<br />
tr<strong>an</strong>smitter expression in isolated activity-altered neurons.<br />
Calcium imaging revealed that expression of a hKir2.1-mCherry plasmid signific<strong>an</strong>tly decreased<br />
<strong>the</strong> incidence of calcium spike activity in isolated spinal neurons, while mCherry alone had no<br />
effect on calcium spike patterns. We hypo<strong>the</strong>sized that <strong>the</strong> suppression of calcium spike activity<br />
would lead to increased expression of glutamate <strong>an</strong>d decreased expression of GABA. However,<br />
cell-autonomous acquisition of glutamate expression in motoneurons or interneurons expressing<br />
hKir2.1-mCherry was not detected. In addition, a cell-autonomous decrease in <strong>the</strong> incidence of<br />
GABAergic interneurons expressing hKir2.1-mCherry was not observed. These results suggest<br />
that activity-dependent neurotr<strong>an</strong>smitter specification is not cell-autonomous. Fur<strong>the</strong>r study will<br />
be focused on testing <strong>the</strong> threshold <strong>for</strong> rNav2aαβ to increase <strong>the</strong> calcium spike activity in isolated<br />
neurons <strong>an</strong>d determining <strong>the</strong> role of activity-dependent trophic factors in activity-dependent<br />
neurotr<strong>an</strong>smitter specification.<br />
Disclosures: L. Xu, None; N.C. Spitzer, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.13/A34<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: SORST<br />
JSPS Research Fellowships <strong>for</strong> Young Scientists<br />
Title: Sox21, a regulator of adult neurogenesis in mouse hippocampus<br />
Authors: *S. MATSUDA 1,2 , H. J. OKANO 2 , S. TSUTSUMI 3 , H. ABURATANI 3 , Y. SAGA 4 ,<br />
H. SUGIMOTO 1 , H. OKANO 2 ;<br />
1 Kyoto Univ., Kyoto, Jap<strong>an</strong>; 2 Physiol., Keio University, Sch. of Med., Tokyo, Jap<strong>an</strong>; 3 Genome<br />
Sci. Div., Univ. of Tokyo, RCAST, Tokyo, Jap<strong>an</strong>; 4 Div. of Mammali<strong>an</strong> Develop., Natl. Inst. of<br />
Genet., Shizuoka, Jap<strong>an</strong><br />
Abstract: Endogenous neural stem cells (NSCs) continuously give rise to neurons in <strong>the</strong> adult<br />
hippocampus in vivo, <strong>an</strong>d <strong>the</strong> phenomenon "adult neurogenesis" is strictly regulated both by<br />
intrinsic <strong>an</strong>d extrinsic factors. However, precise mech<strong>an</strong>isms during a series of neurogenesis
emain to be fully identified.<br />
In present study, we focused on Sox21, a tr<strong>an</strong>scription factor with HMG box DNA-binding<br />
domain, whose expression is restricted in neurogenic regions. Recently, specific auto<strong>an</strong>tibody<br />
against Sox21 was identified in a limbic par<strong>an</strong>eoplastic syndrome patient, who exhibited<br />
neurological symptoms, such as loss of consciousness <strong>an</strong>d seizure. These facts imply <strong>the</strong><br />
involvement of Sox21 in <strong>the</strong> CNS physiology via <strong>the</strong> regulation of neurogenesis.<br />
Here, we generated Sox21-knockout (KO) mice to elucidate its role in vivo. First of all, we<br />
investigated adult neurogenesis in hippocampal dentate gyrus (DG). In <strong>the</strong> adult mice, majority<br />
of Sox21-positive cells localized in neurogenic subgr<strong>an</strong>ular zone of DG, <strong>an</strong>d were turned out to<br />
be type-1 (stem) <strong>an</strong>d -2a/b (tr<strong>an</strong>siently amplifying: TA) cells. BrdU labeling <strong>an</strong>d <strong>the</strong> followed<br />
chasing studies revealed a dramatic decrease of newly generated neurons <strong>an</strong>d type-1 stem cells in<br />
KO-mice DG. Also, <strong>the</strong>re was <strong>an</strong> abnormal exp<strong>an</strong>sion of TA cells in a prior period (type-2a<br />
cells). Toge<strong>the</strong>r with <strong>the</strong> result that Sox21-null cells tended to be more proliferative <strong>an</strong>d lacked<br />
Dcx expression (type-2b marker), Sox21 ablation seems to cause <strong>the</strong> accelerated proliferation<br />
<strong>an</strong>d <strong>the</strong> differentiation arrest of type-2a cells. In-vitro/In vivo <strong>an</strong>alyses by <strong>the</strong> retrovirus-mediated<br />
overexpression of Sox21 <strong>an</strong>d domin<strong>an</strong>t-repressing <strong>for</strong>m of Sox21 also resulted in similar<br />
phenotypes: Sox21-<strong>for</strong>ced expression lowered <strong>the</strong> proliferative status of hippocampal<br />
progenitors, downregulated stemness markers, <strong>an</strong>d induced expression of <strong>the</strong> neuronal<br />
differentiation markers. These results imply that Sox21 functions as a tr<strong>an</strong>scriptional repressor<br />
<strong>an</strong>d regulates adult neurogenesis by promoting tr<strong>an</strong>sition of TA cells into later differentiation<br />
stages.<br />
Fur<strong>the</strong>rmore, we addressed <strong>the</strong> molecular mech<strong>an</strong>ism of <strong>the</strong> role of Sox21 in adult neurogenesis.<br />
To <strong>an</strong>alyze <strong>the</strong> Sox21-target genes globally, we per<strong>for</strong>med ChIP-sequencing using cultured<br />
neural stem cells <strong>from</strong> embryos. Now some c<strong>an</strong>didate targets are being validated by expression-<br />
<strong>an</strong>d function-based screening. Findings <strong>from</strong> this study may disclose <strong>the</strong> intrinsic molecular<br />
mech<strong>an</strong>isms which govern adult neurogenesis.<br />
Disclosures: S. Matsuda, JSPS research fellow, A. Employment (full or part-time); H.J.<br />
Ok<strong>an</strong>o, None; S. Tsutsumi, None; H. Aburat<strong>an</strong>i, None; Y. Saga, None; H. Sugimoto,<br />
None; H. Ok<strong>an</strong>o, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.14/A35<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: Jap<strong>an</strong>-C<strong>an</strong>ada Joint Health Research Program
Title: How do GFAP-expressing neural progenitors divide <strong>an</strong>d generate neuron-committed<br />
progeny in <strong>the</strong> postnatal hippocampus?<br />
Authors: *T. SEKI 1 , T. NAMBA 2 , Y. LIU 3 , S. SHIODA 4 ;<br />
1 Div. of Develop. Neurosci, Tohoku Univ. Sch. Med., Sendai, Jap<strong>an</strong>; 2 Dept. of Neurochemistry,<br />
Natl. Inst. of Neurosci., Tokyo, Jap<strong>an</strong>; 3 Dept. of Psychiatry, Juntendo Univ. Sch. Med., Tokyo,<br />
Jap<strong>an</strong>; 4 Dept. of Anat., Showa Univ. Sch. Med., Tokyo, Jap<strong>an</strong><br />
Abstract: Neurons continue to be generated in <strong>the</strong> dentate gyrus of <strong>the</strong> adult hippocampus.<br />
Several lines of evidence have demonstrated that <strong>the</strong> primary progenitors of dentate gr<strong>an</strong>ule cells<br />
share some astrocytic features <strong>an</strong>d express glial fibrillary acidic protein (GFAP). Here we<br />
examined how <strong>the</strong> astrocyte-like progenitors divide <strong>an</strong>d differentiate into <strong>the</strong>ir derivative neuroncommitted<br />
progeny by using tr<strong>an</strong>sgenic mice with mouse GFAP promoter-controlled enh<strong>an</strong>ced<br />
green fluorescent protein (mGFAP-EGFP Tg mice). BrdU-labeling <strong>an</strong>alysis shows that one third<br />
of BrdU-labeled cells in <strong>the</strong> subgr<strong>an</strong>ular zone tr<strong>an</strong>siently express EGFP, <strong>an</strong>d <strong>the</strong>n mainly develop<br />
into neurons, confirming that <strong>the</strong> EGFP+ proliferating cells give rise to neurons. Characterization<br />
of <strong>the</strong> EGFP+/Ki67+ proliferating cells shows that <strong>the</strong>y consist of three types of putative<br />
progenitors; 1) primary progenitors expressing GFAP <strong>an</strong>d a stem cell marker (Sox2), 2) early<br />
intermediate progenitor cells expressing GFAP, Sox2 <strong>an</strong>d a neuronal marker (Hu), <strong>an</strong>d 3) late<br />
intermediate progenitors expressing Sox2 <strong>an</strong>d neuronal markers (Hu, polysialic acid-neural cell<br />
adhesion molecules <strong>an</strong>d doublecortin). To directly observe <strong>the</strong> cell division pattern <strong>an</strong>d neuronal<br />
differentiation process of <strong>the</strong> EGFP+ cells, we developed a new postnatal brain slice culture<br />
system, <strong>an</strong>d per<strong>for</strong>med a time-lapse imaging <strong>an</strong>alysis in hippocampal slices <strong>from</strong> mGFAP-eGFP<br />
Tg mice at postnatal day 4-9. The preliminary experiments indicate that most dividing cells in<br />
<strong>the</strong> early postnatal dentate gyrus express GFAP, <strong>an</strong>d <strong>the</strong>y finally develop into neurons in vitro.<br />
The time-lapse observation demonstrates that <strong>the</strong> EGFP+ cells predomin<strong>an</strong>tly undergo<br />
symmetric division to produce new pairs of neurons via intermediate state in which <strong>the</strong>y express<br />
both GFAP <strong>an</strong>d Hu. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that <strong>the</strong> GFAP+ primary neural<br />
progenitors give rise to early intermediate progenitors expressing both GFAP <strong>an</strong>d neuronal<br />
markers, <strong>an</strong>d <strong>the</strong>n become late intermediate progenitors expressing only neuronal markers, <strong>an</strong>d<br />
that at least in <strong>the</strong> early postnatal dentate gyrus, a subpopulation of <strong>the</strong> GFAP+ proliferating cells<br />
functions as <strong>an</strong> intermediate progenitor that is defined as a cell dividing symmetrically <strong>an</strong>d<br />
generating a pair of neurons.<br />
Disclosures: T. Seki , Jap<strong>an</strong>-C<strong>an</strong>ada Joint Health Research Program, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); T. Namba, None; Y. Liu, None; S. Shioda, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.15/A36<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: Fondazione CARIFE<br />
PRIN 2007<br />
Title: Properties <strong>an</strong>d maturation of adult-generated dopaminergic neurons in <strong>the</strong> mammali<strong>an</strong><br />
olfactory bulb<br />
Authors: *A. PIGNATELLI 1,2 , J. ACKMAN 4 , A. P. BELTRAMI 5 , S. ZUCCHINI 3 , C.<br />
GAMBARDELLA 2 , D. C. MARTINS 2 , O. BELLUZZI 2 ;<br />
2 Dept. di Biologia ed Evoluzione, Sezione di Fisiologia e Biofisica, 3 Dept. of Clin. <strong>an</strong>d Exptl.<br />
Medicine, Section of Pharmacol., 1 Univ. of Ferrara, Ferrara, Italy; 4 Yale Univ. Sch. of Med.,<br />
New Haven, CT; 5 Ctr. Interdipartimentale Medicina Rigenerativa (CIME), Univ. of Udine,<br />
Udine, Italy<br />
Abstract: The olfactory bulb (OB) of mammals contains a large population of dopaminergic<br />
interneurons (DA) within <strong>the</strong> glomerular layer (GL). Dopamine has been shown both in vivo <strong>an</strong>d<br />
in vitro to modulate several aspects of olfactory in<strong>for</strong>mation processing, but <strong>the</strong> functional<br />
properties of DA neurons have rarely been described due to <strong>the</strong> inability to recognize <strong>the</strong>se cells<br />
in living preparations. We succeeded in this by using a tr<strong>an</strong>sgenic mouse strain harboring <strong>an</strong><br />
eGFP reporter construct under <strong>the</strong> promoter of tyrosine hydroxylase (TH), <strong>the</strong> rate-limiting<br />
enzyme <strong>for</strong> ca<strong>the</strong>colamine syn<strong>the</strong>sis. As a result, we could identify dopaminergic neurons (TH-<br />
GFP cells) in living preparations <strong>an</strong>d study <strong>the</strong> functional properties of such neurons in <strong>the</strong> OB,<br />
in both slices <strong>an</strong>d dissociated cells. The most prominent feature of DA neurons was <strong>the</strong><br />
autorhythmicity. In <strong>the</strong>se cells we identified five main voltage-dependent conduct<strong>an</strong>ces: <strong>the</strong> two<br />
having largest amplitude were a fast tr<strong>an</strong>sient Na+ current <strong>an</strong>d a delayed rectifier K+ current. In<br />
addition, we observed three smaller inward currents, sustained by Na+ ions (persistent type) <strong>an</strong>d<br />
by Ca2+ ions (low- <strong>an</strong>d high-voltage activated). Using pharmacological tools <strong>an</strong>d ion<br />
substitution methods we showed that <strong>the</strong> pacemaking process is supported by <strong>the</strong> interplay of <strong>the</strong><br />
persistent Na+ current <strong>an</strong>d of a T-type Ca2+current.<br />
A signific<strong>an</strong>t fraction of <strong>the</strong> interneurons added in adulthood to <strong>the</strong> GL of <strong>the</strong> OB are<br />
dopaminergic. Typically, bulbar DA neurons are restricted to <strong>the</strong> GL, but we also detected <strong>the</strong><br />
presence of TH-GFP+ cells in <strong>the</strong> mitral <strong>an</strong>d external plexi<strong>for</strong>m layers. We hypo<strong>the</strong>sized that<br />
<strong>the</strong>se could be adult-generated neurons committed to become DA but not yet entirely<br />
differentiated. Accordingly, TH-GFP+ cells outside <strong>the</strong> GL exhibit functional properties<br />
(appear<strong>an</strong>ce of pacemaker currents, synaptic connection with <strong>the</strong> olfactory nerve, intracellular<br />
chloride concentration, <strong>an</strong>d o<strong>the</strong>r), marking a gradient of maturity toward <strong>the</strong> dopaminergic<br />
phenotype along <strong>the</strong> mitral-glomerular axis. Finally, we propose that <strong>the</strong> establishment of a<br />
synaptic contact with <strong>the</strong> olfactory nerve is <strong>the</strong> key event allowing <strong>the</strong>se cells to complete <strong>the</strong>ir<br />
differentiation toward <strong>the</strong> DA phenotype <strong>an</strong>d to reach <strong>the</strong>ir final destination.
Disclosures: A. Pignatelli, None; J. Ackm<strong>an</strong>, None; A.P. Beltrami, None; S. Zucchini,<br />
None; C. Gambardella, None; D.C. Martins, None; O. Belluzzi, None.<br />
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.16/A37<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NIH Gr<strong>an</strong>t NS045933<br />
Title: Growth/differentiation factor 11 facilitates temporal progression of neurogenesis in <strong>the</strong><br />
developing spinal cord<br />
Authors: *J.-P. LIU 1 , Y. SHI 2 ;<br />
1 Univ. Virginia, Charlottesville, VA; 2 Univ. of Virginia, Charlottesville, VA<br />
Abstract: Various types of neurons <strong>an</strong>d glia are generated following a precise spatial <strong>an</strong>d<br />
temporal order during neurogenesis. The mech<strong>an</strong>isms that control this sequential generation of<br />
neuronal <strong>an</strong>d glial cell types <strong>from</strong> <strong>the</strong> same progenitor population are not well understood.<br />
Growth differentiation factor 11 (Gdf11) belongs to <strong>the</strong> TGF-β family of proteins, <strong>an</strong>d is<br />
expressed tr<strong>an</strong>siently in cells adjacent to <strong>the</strong> progenitor domain in <strong>the</strong> developing spinal cord.<br />
We have examined <strong>the</strong> phenotypes of Gdf11 -/- mouse embryos <strong>an</strong>d found that without Gdf11,<br />
neuronal differentiation in <strong>the</strong> spinal cord progresses at a slower rate. Higher numbers of<br />
progenitor cells, along with a delay in gliogenesis are also observed in Gdf11 -/- spinal cord but<br />
only after <strong>the</strong> peak of Gdf11 expression, indicating that Gdf11 c<strong>an</strong> cause long-lasting ch<strong>an</strong>ges in<br />
progenitor properties. These ch<strong>an</strong>ges c<strong>an</strong> be preserved in vitro, as neurospheres derived <strong>from</strong><br />
Gdf11 -/- <strong>an</strong>d wild-type littermates at a stage after, but not be<strong>for</strong>e Gdf11 expression exhibit<br />
differences in proliferation <strong>an</strong>d differentiation potential. Moreover, <strong>the</strong>se ch<strong>an</strong>ges in progenitor<br />
properties c<strong>an</strong> be induced in vitro by <strong>the</strong> addition of Gdf11. We fur<strong>the</strong>r demonstrate that Gdf11’s<br />
effects on progenitor cells, at least in part, are mediated by its ability to up-regulate p57 kip2 <strong>an</strong>d<br />
down-regulate Pax6 expression. These results support a model in which Gdf11 secreted by<br />
newly-born neurons in <strong>the</strong> developing spinal cord facilitates <strong>the</strong> temporal progression of<br />
neurogenesis by acting as a feed-back signal on <strong>the</strong> progenitor cells to enh<strong>an</strong>ce cell cycle exit,<br />
<strong>an</strong>d to ch<strong>an</strong>ge <strong>the</strong>ir proliferation <strong>an</strong>d differentiation ability.<br />
Disclosures: J. Liu, None; Y. Shi, None.
Poster<br />
506. Neurogenesis II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.17/A38<br />
Topic: A.02.d. Neuronal differentiation<br />
Title: Analysis of mice with disruption of gamma protocadherin genes in excitatory neurons<br />
Authors: *Y. ITOGA 1 , S. HAMADA 2 , T. HIRABAYASHI 1 , T. YAGI 1 ;<br />
1 Kokoro Biol. Group, Grad. Sch. of Frontier Biosci., OSAKA UNIVERSITY, Suita / Osaka,<br />
Jap<strong>an</strong>; 2 Dev. Nutr. <strong>an</strong>d Hlth. Sci., Fukuoka Women's Univ., Fukuoka, Jap<strong>an</strong><br />
Abstract: Clustered protocadherins (Pcdhs) are diverse cell adhesion molecules. Genes encoding<br />
about 60 clustered Pcdhs are arr<strong>an</strong>ged in three clusters (Pcdhα, Pcdhβ, <strong>an</strong>d Pcdhγ) at a single<br />
chromosomal allele in mammali<strong>an</strong> genome. Among <strong>the</strong>m, Pcdhα <strong>an</strong>d Pcdhγ have multiple<br />
variable exons <strong>an</strong>d three const<strong>an</strong>t exons. Single exon of <strong>the</strong> multiple variable exons c<strong>an</strong> combine<br />
with three const<strong>an</strong>t exons by cis splicing of <strong>the</strong> mRNA. In <strong>the</strong> clustered Pcdh family, totally <strong>the</strong>re<br />
are about 60 distinct iso<strong>for</strong>ms. Each neuron expresses distinct combination of two or three each<br />
clustered Pcdh iso<strong>for</strong>ms, suggesting that clustered Pcdhs have a role <strong>for</strong> generating neural<br />
diversity <strong>an</strong>d org<strong>an</strong>ization in <strong>the</strong> brain. According to <strong>the</strong> previous reports, gene targeted mice<br />
lacking <strong>the</strong> Pcdhγ gene cluster showed neuronal loss by apoptotic cell death in <strong>the</strong> spinal cord<br />
<strong>an</strong>d died shortly after birth. To <strong>an</strong>alyze <strong>the</strong> function of Pcdhγ in neural development more detail,<br />
we generated mice with conditional alleles in which Pcdhγ proteins c<strong>an</strong> be null <strong>from</strong> specific cell<br />
types by Cre recombination. We crossed <strong>the</strong> conditional Pcdhγ mice with α-calmodulin kinase 2<br />
Cre (CaMK2Cre) tr<strong>an</strong>sgenic mice to selectively inactivate Pcdhγ proteins in excitatory neurons<br />
in <strong>the</strong> <strong>for</strong>ebrain. In <strong>the</strong> mut<strong>an</strong>ts, we detected apoptotic cells in <strong>the</strong>ir hippocampus, amygdala, <strong>an</strong>d<br />
piri<strong>for</strong>m cortex. Interestingly, apoptotic cells were neurons in which CaMK2 promoter would not<br />
work. These results suggest that Pcdhγ protiens regulate survival of neurons non-cell<br />
autonomously.<br />
Disclosures: Y. Itoga, None; S. Hamada, None; T. Hirabayashi, None; T. Yagi, None.<br />
Poster<br />
506. Neurogenesis II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 506.18/A39<br />
Topic: A.02.d. Neuronal differentiation<br />
Support: NIH Gr<strong>an</strong>t R01 MH067715 (FMV)<br />
NRSA T32 MH018268 (BGR)<br />
Brown-Coxe Postdoctoral Fellowship (BGR)<br />
Title: Role of Fgfs in radial glia development of <strong>the</strong> cerebral cortex<br />
Authors: *B. G. RASH, K. M. SMITH, Y. OHKUBO, F. M. VACCARINO;<br />
Child Study Ctr., Yale Univ., New Haven, CT<br />
Abstract: Cortical neurons are generated by radial glia <strong>an</strong>d intermediate precursor cells (IPC)s<br />
within two germinal zones, <strong>the</strong> ventricular zone (VZ) <strong>an</strong>d subventricular zone (SVZ),<br />
respectively. Evidence points to various roles <strong>for</strong> fibroblast growth factors (Fgf)s in cortical<br />
development, including <strong>the</strong> regulation of proliferation in <strong>the</strong> VZ, establishment of cortical neuron<br />
number, <strong>an</strong>d controlling cortical area patterning. We are investigating development of <strong>the</strong> early<br />
cerebral cortex, focusing on <strong>the</strong> role of Fgfs in <strong>the</strong> birth <strong>an</strong>d differentiation of cortical neurons.<br />
All Fgf signaling in <strong>the</strong> brain is thought to occur through three Fgf receptors, FgfR1-3. Our<br />
examination of embryos that conditionally lack FgfR1-3 function within <strong>the</strong> dorsal telencephalon<br />
shows a subst<strong>an</strong>tial reduction in proliferation in <strong>the</strong> VZ. Conditional Fgf receptor triple deletion<br />
using ei<strong>the</strong>r Emx1 or hGFAP CRE lines caused a decrease in radial glial density <strong>an</strong>d severe<br />
reduction in overall cortical surface area. In contrast with bone development, <strong>an</strong>alysis of <strong>an</strong><br />
allelic series of FgfR1-3 compound mut<strong>an</strong>ts revealed that FgfR3 functions redund<strong>an</strong>tly with<br />
FgfR1 <strong>an</strong>d FgfR2 with respect to cortical progenitor proliferation, radial unit production, <strong>an</strong>d<br />
overall cortical size.<br />
In tr<strong>an</strong>sgenic mice engineered to overexpress Fgf2 within <strong>the</strong> Emx1 lineage of <strong>the</strong> dorsal<br />
telencephalon, we found a subst<strong>an</strong>tial increase in VZ proliferation, as expected. Surprisingly,<br />
however, similar to <strong>the</strong> triple FgfR mut<strong>an</strong>ts we observed that radial glia number <strong>an</strong>d density were<br />
dramatically reduced <strong>an</strong>d radial glial structure was disorg<strong>an</strong>ized, correlating with subst<strong>an</strong>tial<br />
defects in <strong>the</strong> laminar structure of <strong>the</strong> cortical plate. Overall cortical size was markedly reduced,<br />
yet <strong>the</strong>se mice live into adulthood. Toge<strong>the</strong>r, <strong>the</strong>se results will lead to <strong>an</strong> enh<strong>an</strong>ced model of Fgf<br />
function in cortical stem cell biology.<br />
Disclosures: B.G. Rash, None; K.M. Smith, None; Y. Ohkubo, None; F.M. Vaccarino, None.<br />
Poster
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.1/A40<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: CCMTR<br />
Title: Effects of extremely low frequency electric fields on fetal porcine neural progenitor cells<br />
Authors: *J. H. LIM 1 , S. D. MCCULLEN 2 , E. G. LOBOA 2 , N. J. OLBY 1 ;<br />
1 2<br />
Dept. of Clin. Sci., Col. of Vet. Medicine, NCSU, Raleigh, NC; Joint Dept. of Biomed. Engin.,<br />
UNC-CH <strong>an</strong>d NCSU, Raleigh, NC<br />
Abstract: Application of sinusoidal electric fields of physiological strength have been observed<br />
to affect cellular processes including alignment, proliferation, differentiation <strong>an</strong>d apoptosis by<br />
causing a redistribution of charged cell surface receptors, altering cell shape, <strong>an</strong>d increasing<br />
release of signaling molecules such as intracellular calcium. However, <strong>the</strong>re has been limited <strong>an</strong>d<br />
contradictory research in this area. In <strong>the</strong> present study, we applied different, well-qu<strong>an</strong>tified,<br />
extremely low frequency electric fields (ELFEF) to porcine neural progenitor cells (NPCs) <strong>an</strong>d<br />
investigated <strong>the</strong> effects of electric stimulation on cell patterning, proliferation <strong>an</strong>d differentiation.<br />
Porcine NPCs were grown directly on custom fabricated interdigitated electrodes <strong>the</strong>reby<br />
utilizing <strong>the</strong> best qualities of capacitive coupling (a non-invasive, primarily electric field) but<br />
localizing <strong>the</strong> field to a 1cm x1cm x100µm region accessible visually <strong>for</strong> fluorescence-based<br />
assays. Neurospheres were isolated <strong>from</strong> porcine fetal cerebral tissue, <strong>an</strong>d seeded at a density of<br />
5x10 4 cells per electrode. Cultures of NPCs were exposed to ELFEF of magnitude 1V/cm at<br />
frequencies of 1Hz, 10Hz <strong>an</strong>d 50Hz <strong>for</strong> 3, 7 <strong>an</strong>d 14 days. Immunofluorochemistry was<br />
per<strong>for</strong>med to evaluate <strong>the</strong> expression of neural markers, specifically: nestin <strong>for</strong> neural<br />
progenitors, GFAP <strong>for</strong> astroglia, Tuj1 <strong>for</strong> neurons, <strong>an</strong>d NG2 <strong>for</strong> immature oligodendroglial<br />
differentiation. Ten images of immunoreactive cells were counted at 20x magnification <strong>an</strong>d<br />
expressed as percentages of <strong>the</strong> total number of cells within <strong>the</strong> same field.<br />
Porcine NPCs grew well in all electric field treatments, producing both differentiated cells with<br />
morphology typical of neurons <strong>an</strong>d astrocytes, <strong>an</strong>d maintaining neurospheres in spite of<br />
withdrawal of growth factors. There were no neurospheres in <strong>the</strong> control groups <strong>an</strong>d no<br />
signific<strong>an</strong>t differences were found in total cell counts between groups. Nestin was expressed by<br />
<strong>the</strong> majority of cells in all groups at 3 <strong>an</strong>d 7 days, but not at 14 days. NG2 expression was low in<br />
all groups. GFAP expression rate was signific<strong>an</strong>tly increased in all groups at 14 days, however<br />
GFAP expression in both <strong>the</strong> 10 <strong>an</strong>d 50Hz stimulation groups was signific<strong>an</strong>tly lower th<strong>an</strong> <strong>the</strong> 1<br />
Hz <strong>an</strong>d control groups. GFAP <strong>an</strong>d Tuj1 co-expression rate was signific<strong>an</strong>tly higher in <strong>the</strong> 10 <strong>an</strong>d<br />
50Hz stimulation groups th<strong>an</strong> <strong>the</strong> 1Hz <strong>an</strong>d control groups at 14 days. In summary, exposure of<br />
porcine NPCs to ELFEF c<strong>an</strong> potentially control <strong>the</strong>ir differentiation potential <strong>an</strong>d could be used<br />
as a tool <strong>for</strong> m<strong>an</strong>ipulating cell fate.
Disclosures: J.H. Lim, None; S.D. McCullen, None; E.G. Loboa, None; N.J. Olby, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.2/A41<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: BK21 Project <strong>for</strong> Medical Sciences, Yonsei Univ College of Medicine<br />
Title: Identification of unknown differentially expressed genes that promote neuronal <strong>an</strong>d<br />
oligodendroglial differentiation of hum<strong>an</strong> neural stem <strong>an</strong>d progenitor cells<br />
Authors: *K. HWANG 1 , M. KIM 1 , K. PARK 1,2 ;<br />
1 Yonsei Univ. Col. of Med., Bk21 Project For Med. Sciences, Yonsei Univ. Col. of Med., Seoul,<br />
Republic of Korea; 2 Dept. of pediatrics, Yonsei Univ. Col. of Med., Seoul, Republic of Korea<br />
Abstract: Individual cells were isolated <strong>from</strong> <strong>the</strong> telencephalon of a hum<strong>an</strong> fetal cadaver at 13<br />
weeks of gestation <strong>an</strong>d grown as neurospheres in long-term cultures. Cultured neurospheres are<br />
mainly composed of multipotent neural stem cells (NSCs) <strong>an</strong>d progenitors. To identify <strong>the</strong> genes<br />
involved in <strong>the</strong> proliferation <strong>an</strong>d differentiation of hum<strong>an</strong> NSCs/progenitors in cultured<br />
neurospheres, we employed a novel method of RT-PCR using <strong>an</strong>nealing control primers. RT-<br />
PCR <strong>an</strong>d real-time PCR were used to validate each of <strong>the</strong> differentially expressed genes (DEGs).<br />
We identified <strong>an</strong>d sequenced 34 DEGs. A Basic Local Alignment Search Tool (BLAST) search<br />
was conducted to identify regions of local similarity <strong>for</strong> <strong>the</strong> DEG sequences. Our results indicate<br />
that <strong>the</strong> expression of two known <strong>an</strong>d two unknown genes was enh<strong>an</strong>ced during neurosphere<br />
proliferation, <strong>an</strong>d <strong>the</strong> expression of 27 known <strong>an</strong>d three unknown genes was enh<strong>an</strong>ced during<br />
neurosphere differentiation. Among unknown DEGs highly expressed during neurosphere<br />
differentiation, overexpression of NM_015329 or AK_023048 by lentiviral vector in<br />
neurospheres decrease cell proliferation rate, NM_015329 overexpressed cells predomin<strong>an</strong>tly<br />
differentiate into neurons at <strong>the</strong> expense of glial differentiation, <strong>an</strong>d AK_023048 overexpressed<br />
cells largely differentiate into neurons <strong>an</strong>d oligodendrocytes. This work provide insight into <strong>the</strong><br />
molecular events involved in <strong>the</strong> differentiation of hum<strong>an</strong> NSCs/progenitors <strong>an</strong>d tr<strong>an</strong>spl<strong>an</strong>tation<br />
<strong>the</strong>rapy.<br />
Disclosures: K. Hw<strong>an</strong>g, Brain Korea 21 Project <strong>for</strong> Medical Science, C. O<strong>the</strong>r Research<br />
Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); M. kim, None; K.<br />
Park, None.
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.3/A42<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research, Gr<strong>an</strong>t number 86600<br />
Scottish Rite Charitable Foundation of C<strong>an</strong>ada, Gr<strong>an</strong>t number 07114<br />
Title: Characterization of <strong>the</strong> spinal cord ependymal layer, a potential neural stem cell niche<br />
Authors: *L. HAMILTON, M. K. V. TRUONG, M. R. BEDNARCZYK, A. AUMONT, K. J.<br />
L. FERNANDES;<br />
Dept. de Pathologie et de Biologie Cellulaire, Univ. De Montreal, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Be<strong>for</strong>e attempting to modulate stem cell activity <strong>for</strong> <strong>the</strong>rapeutic purposes, it is<br />
necessary to underst<strong>an</strong>d <strong>the</strong> cellular components <strong>an</strong>d regulatory factors present in <strong>the</strong> stem cell’s<br />
microenvironment or niche. In <strong>the</strong> adult mammali<strong>an</strong> spinal cord, ependymal cells are normally<br />
relatively quiescent but display typical neural stem cell properties in vitro <strong>an</strong>d following spinal<br />
cord injury in vivo. Using <strong>the</strong> well-defined <strong>for</strong>ebrain stem cell niche as a model, we characterized<br />
<strong>the</strong> spinal cord ependymal cell niche. The central c<strong>an</strong>al was found to possess a rudimentary subependymal<br />
layer composed of small numbers of astrocytes, oligodendrocyte progenitors <strong>an</strong>d<br />
mature neurons that are closely apposed to <strong>the</strong> ependymal cells. Unlike in <strong>the</strong> <strong>for</strong>ebrain SVZ, <strong>the</strong><br />
majority of proliferative activity occurs within ependymal cells ra<strong>the</strong>r th<strong>an</strong> sub-ependymal cells.<br />
Proliferating ependymal cells divide in <strong>the</strong> rostro-caudal pl<strong>an</strong>e, are associated with <strong>the</strong> surface of<br />
blood vessels, <strong>an</strong>d normally self-renew ra<strong>the</strong>r th<strong>an</strong> produce tr<strong>an</strong>sit-amplifying progenitors.<br />
Unexpectedly, properties indicative of neural precursor activity were found to be concentrated<br />
dorsally within <strong>the</strong> ependymal layer: <strong>the</strong>re was a dorsal-to-ventral gradient of proliferating<br />
ependymal cells, <strong>an</strong>d sub-populations of dorsal t<strong>an</strong>ycytic ependymal cells were highly<br />
immunoreactive <strong>for</strong> <strong>the</strong> neural precursor markers Nestin <strong>an</strong>d GFAP. Thus, <strong>the</strong> key features of <strong>the</strong><br />
<strong>for</strong>ebrain stem cell niche are also present in <strong>the</strong> adult spinal cord, but with a distinct org<strong>an</strong>ization.<br />
These findings represent a first description of <strong>the</strong> ependymal cell niche in <strong>the</strong> adult spinal cord.<br />
Disclosures: L. Hamilton, None; M.K.V. Truong, None; M.R. Bednarczyk, None; A.<br />
Aumont, None; K.J.L. Fern<strong>an</strong>des, None.
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.4/A43<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: PDT Gr<strong>an</strong>t 63/135<br />
NIH Gr<strong>an</strong>t R01NS048255<br />
Title: Precursor-like cells in <strong>the</strong> ependyma of <strong>the</strong> neonatal rat spinal cord<br />
Authors: *N. MARICHAL 1 , G. GARCÍA 2 , M. RADMILOVICH 3 , O. TRUJILLO-CENÓZ 2 , R.<br />
E. RUSSO 2 ;<br />
2 Neurofisiología Celular y Mol., 1 Inst. Clemente Estable, Montevideo, Uruguay; 3 Histología y<br />
Embriología, Facultad de Medicina, Montevideo, Uruguay<br />
Abstract: The ependyma of <strong>the</strong> spinal cord contains cells that react to injury with active<br />
proliferation. These cells c<strong>an</strong> migrate <strong>an</strong>d differentiate mainly into glial cells but under some<br />
conditions <strong>the</strong>y also become neurons. This endogenous response may be due to <strong>the</strong> preservation<br />
of progenitor cells similar to those in <strong>the</strong> embryo. During development, dividing precursors in<br />
<strong>the</strong> cortical ventricular zone have characteristic molecular <strong>an</strong>d functional features. However, <strong>the</strong><br />
characteristics of post-natal spinal precursor-like cells remain poorly understood.<br />
The aim of this study was to characterize <strong>the</strong> morphological, molecular <strong>an</strong>d functional properties<br />
of putative precursor cells in neonatal rat (P0-P5) spinal cord. For this, we per<strong>for</strong>med<br />
immunohistochemistry <strong>for</strong> markers expressed in neural stem cells such nestin, vimentin, 3CB2 ,<br />
brain lipid binding protein <strong>an</strong>d glial fibrillary acidic protein (GFAP). We found that nestin <strong>an</strong>d<br />
vimentin expressed mostly in <strong>the</strong> ventral <strong>an</strong>d dorsal midline raphe of <strong>the</strong> central c<strong>an</strong>al (CC),<br />
whereas ependymal cells located laterally expressed little or no nestin or vimentin. The cells in<br />
<strong>the</strong> dorsal <strong>an</strong>d ventral raphes had <strong>the</strong> typical bipolar morphology of radial glia, with <strong>an</strong> endfoot<br />
contacting <strong>the</strong> CC <strong>an</strong>d a process stretching to <strong>the</strong> pial surface. Their somas located at different<br />
dist<strong>an</strong>ces <strong>from</strong> <strong>the</strong> CC lumen. We next explored <strong>the</strong> electrophysiological properties of <strong>the</strong>se cells<br />
in tr<strong>an</strong>sverse spinal cord slices (300 µm) using <strong>the</strong> whole cell patch clamp technique.<br />
Fluorophores or biocytin were used to reveal <strong>the</strong> morphology of recorded cells. Some displayed<br />
physiological properties typical of cycling precursors in <strong>the</strong> embryonic cerebral cortex: passive<br />
response properties, low input resist<strong>an</strong>ces <strong>an</strong>d dye coupling with neighboring cells.<br />
Carbenoxolone (100 µM) subst<strong>an</strong>tially increased <strong>the</strong> input resist<strong>an</strong>ce indicating that dye coupling<br />
was mediated via gap junctions. Clustered precursor-like cells were molecularly heterogenous<br />
because some cells expressed nestin whereas o<strong>the</strong>rs did not. In addition, some cells appeared<br />
uncoupled <strong>an</strong>d had voltage gated outward currents with sustained <strong>an</strong>d tr<strong>an</strong>sient components that
were blocked by tetraethylammonium (10 mM) <strong>an</strong>d 4-aminopiridine (1-4 mM) respectively,<br />
suggesting <strong>the</strong> presence of delayed rectifier <strong>an</strong>d A type K + currents. This electrophysiological<br />
phenotype resembles that of early oligodendrocyte progenitors. We conclude that in <strong>the</strong> early<br />
post-natal life, <strong>the</strong> ventral <strong>an</strong>d dorsal midline raphe of <strong>the</strong> spinal cord retain different kinds of<br />
precursor-like cells. Underst<strong>an</strong>ding <strong>the</strong> biology of post-natal endogenous spinal stem cells will<br />
help developing of new <strong>the</strong>rapeutic strategies to treat spinal cord injury.<br />
Disclosures: N. Marichal, None; G. García, None; M. Radmilovich, None; O. Trujillo-<br />
Cenóz, None; R.E. Russo, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.5/A44<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: KSEF R13-2008-023-01002<br />
Intramural Research Gr<strong>an</strong>t Program<br />
Title: Tr<strong>an</strong>scriptional profiling of autophagic pathway in adult neural stem cells<br />
Authors: H. LEE 1 , H. NAM 1 , S. PARK 1 , S.-H. BAEK 3 , E.-K. KIM 3 , S.-W. YU 3 , *S. KIM 2 ;<br />
1 Biochem. & Mol. Biol, Univ. of Uls<strong>an</strong> Col. of Med., Seoul, Republic of Korea; 2 Biochem. &<br />
Mol. Biol, Univ. of Uls<strong>an</strong> Col. of Med., Seoul, ; 3 Departments of Neurol. <strong>an</strong>d Ophthalmology,<br />
Col. of Natural Sciences, Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Adult hippocampal neural stem (HCN) cells, like o<strong>the</strong>r stem cells, retain both selfrenewal<br />
<strong>an</strong>d multilineage differentiation capabilities. As stem cells have been studied <strong>for</strong> its<br />
<strong>the</strong>rapeutic potential <strong>for</strong> neurodegenerative disorders, one of major concerns is how to augment<br />
<strong>the</strong> survival of engrafted neural stem cells (NSCs) in vivo. The molecular mech<strong>an</strong>isms that<br />
regulate <strong>the</strong> viability of NSCs have not been elucidated yet. Based on our recent report indicating<br />
that autophagy is a default death pathway in HCN cells following insulin withdrawal, this study<br />
is designed to fur<strong>the</strong>r investigate <strong>the</strong> molecular network by which autophagy governs <strong>the</strong><br />
viability following insulin withdrawal in HCN cells. For this, we obtained <strong>the</strong> differential gene<br />
expression profile of HCN cells ei<strong>the</strong>r with or without insulin <strong>for</strong> 24hrs or 48hrs by using of<br />
whole genome rat microarray <strong>an</strong>alysis. The 412 genes showed signific<strong>an</strong>t ch<strong>an</strong>ges in expression<br />
levels in <strong>the</strong> initial insulin-deprived 24hrs. In prolonged insulin withdrawal, 136 genes showed
signific<strong>an</strong>t ch<strong>an</strong>ges in gene expression. The differentially-expressed genes are involved in<br />
biological processes including tr<strong>an</strong>sport, tr<strong>an</strong>scriptional regulation, cellular metabolism <strong>an</strong>d cell<br />
death. Ch<strong>an</strong>ges in expression levels of r<strong>an</strong>domly selected genes were validated by real time RT-<br />
PCR. Selected genes showed a reproducible pattern of gene expression in real time RT-PCR<br />
compared with those <strong>from</strong> microarray experiments. This is <strong>the</strong> first report of gene expression<br />
profiling in HCN cells following a single neurogenic growth factor withdrawal. Fur<strong>the</strong>rmore, this<br />
profile may lead to future studies to develop strategies to enh<strong>an</strong>ce viability of engrafted neural<br />
stem cells in cell-based <strong>the</strong>rapy targeting a variety of hum<strong>an</strong> disorders including<br />
neurodegenerative disease <strong>an</strong>d c<strong>an</strong>cer.<br />
Disclosures: H. Lee, None; H. Nam, None; S. Park, None; S. Baek, None; E. Kim, None; S.<br />
Yu, None; S. Kim, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.6/A45<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: P30 DA-018310<br />
Title: Spatial <strong>an</strong>d temporal <strong>an</strong>alyses of peptides during regeneration in <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> Schmidtea<br />
mediterr<strong>an</strong>ea by mass spectrometry<br />
Authors: *E. V. ROMANOVA 1 , J. J. COLLINS 2,3 , P. A. NEWMARK 2,3 , J. V. SWEEDLER 1 ;<br />
1 Univ. Illinois & Beckm<strong>an</strong> Inst., Urb<strong>an</strong>a, IL; 2 Howard Hughes Med. Inst., Chevy Chase, MD;<br />
3 Dept. of Cell <strong>an</strong>d Developmental Biol., Univ. of Illinois, Urb<strong>an</strong>a, IL<br />
Abstract: The nervous system is <strong>an</strong> import<strong>an</strong>t regulator of tissue regeneration in a number of<br />
vertebrate <strong>an</strong>d invertebrate models, yet little is known about <strong>the</strong> neural signals that act to<br />
influence regeneration. Pl<strong>an</strong>ari<strong>an</strong>s are a powerful model to investigate <strong>the</strong> molecular basis of<br />
regeneration; a number of immunocytochemical, ultrastructural, <strong>an</strong>d pharmacological reports<br />
indicate that neuropeptides influence regenerative processes in pl<strong>an</strong>ari<strong>an</strong>s. To study <strong>the</strong> peptide<br />
complement associated with regeneration in pl<strong>an</strong>ari<strong>an</strong>s, we are using <strong>the</strong> model pl<strong>an</strong>ari<strong>an</strong> species<br />
Schmidtea mediterr<strong>an</strong>ea. This species is this ideal model <strong>for</strong> such studies because a number of<br />
genomic tools are available: clonal lines with defined genetic backgrounds, <strong>an</strong> extensive<br />
collection of Expressed Sequence Tags (ESTs), <strong>an</strong>d a fully-sequenced genome. Relating genetic<br />
in<strong>for</strong>mation to actual biological functions of peptides is complicated by a lack of in<strong>for</strong>mation on
prohormones in this model, <strong>the</strong> multitude of final peptide <strong>for</strong>ms arising <strong>from</strong> alternative splicing,<br />
<strong>an</strong>d extensive posttr<strong>an</strong>slational processing of <strong>the</strong> final peptides. Mass spectrometric<br />
measurements at <strong>the</strong> peptide level offer signific<strong>an</strong>t adv<strong>an</strong>tage as <strong>the</strong>y allow detailed high<br />
throughput characterization of structurally similar gene products without <strong>the</strong> requirement of a<br />
priori knowledge of <strong>the</strong> identity of relev<strong>an</strong>t peptides.<br />
In this study we use label-free mass spectrometry (MS) based workflow <strong>for</strong> spatial <strong>an</strong>d temporal<br />
<strong>an</strong>alysis of peptides <strong>from</strong> pl<strong>an</strong>ari<strong>an</strong>s regenerating <strong>an</strong>terior <strong>an</strong>d posterior tissues <strong>an</strong>d characterize a<br />
subset of peptides potentially associated with <strong>the</strong> regeneration process. Peptides were measured<br />
in individual samples of <strong>an</strong>terior <strong>an</strong>d posterior regenerates collected at various time points postamputation<br />
by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry<br />
(MALDI-TOF MS). Principle component <strong>an</strong>alyses (PCA) of complex peptide profiles in<br />
regenerating tissues revealed temporal trends in peptide ch<strong>an</strong>ges taking place during 72 h postamputation.<br />
A subset of novel putative peptides that exhibit time-dependent ch<strong>an</strong>ges were<br />
characterized by liquid chromatography <strong>an</strong>d t<strong>an</strong>dem mass spectrometry (LC/MS-MS) with <strong>the</strong><br />
aid of bioin<strong>for</strong>matic tools <strong>an</strong>d <strong>an</strong> in-house database of prohormones expressed in S.<br />
mediterr<strong>an</strong>ea. Differentially detected peptides have been found to originate <strong>from</strong> several novel<br />
neuropeptide prohormones.<br />
Disclosures: E.V. Rom<strong>an</strong>ova, None; J.J. Collins, None; P.A. Newmark, None; J.V.<br />
Sweedler, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.7/A46<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Division of Intramural Research, NINDS<br />
Title: Comprehensive phenotyping reveals <strong>the</strong> true identities of neural stem cells <strong>an</strong>d<br />
intermediate progenitors <strong>an</strong>d resolves <strong>the</strong>ir ontogenetically ch<strong>an</strong>ging lineage progressions<br />
underlying cortical neurogenesis<br />
Authors: *D. MARIC, Y. H. CHANG, J. L. BARKER;<br />
NINDS/NIH, Be<strong>the</strong>sda, MD<br />
Abstract: We used immunolabeling with a multitude of developmentally relev<strong>an</strong>t markers to<br />
phenotype neural stem cells (NSC) <strong>an</strong>d intermediate progenitors (IP) emerging during <strong>the</strong>
embryonic period of rat corticogenesis. At <strong>the</strong> onset of cortical neurogenesis, proliferating<br />
neuroepi<strong>the</strong>lial (NE) cells initially express integrin beta1 heterodimers, but lack o<strong>the</strong>r lineageselective<br />
markers. These lineage-negative (Lin-) NE cells exhibit <strong>the</strong> self-renewing <strong>an</strong>d<br />
differentiating seminal properties attributed to NSCs. While self-renewing by symmetrical<br />
divisions, Lin- NSCs express tr<strong>an</strong>scription factors sox1 <strong>an</strong>d sox2, but not pax6. These cells also<br />
divide asymmetrically to generate <strong>the</strong> first-born pioneer neurons. Up-regulation of pax6 in Lin-<br />
NSCs diversifies <strong>the</strong>ir seminal properties, which include generation of self-renewing <strong>an</strong>d<br />
differentiating neuronal <strong>an</strong>d glial progenitors sequentially expressing integrin beta1 <strong>an</strong>d multiple<br />
Lin+ surface markers (CD15, CD57, CDw60, CD24, A2B5 <strong>an</strong>d PSA-NCAM). M<strong>an</strong>y of <strong>the</strong>se<br />
progenitors up-regulate <strong>the</strong> IP-specific tr<strong>an</strong>scription factor Eomes/Tbr2, which promotes<br />
neurogenesis <strong>from</strong> <strong>the</strong>se cells to generate ei<strong>the</strong>r pioneer, Cajal-Retzius or pyramidal neurons,<br />
with <strong>the</strong> <strong>for</strong>mer two phenotypes gradually disappearing <strong>an</strong>d <strong>the</strong> latter persisting throughout<br />
cortical development. Lin- pax6+ NSCs rapidly decline during cortical neurogenesis as <strong>the</strong>y give<br />
rise to Lin+ IPs <strong>an</strong>d Lin- pax6+ integrin beta1+ radial glial progenitors (RGPs), which also selfrenew<br />
<strong>an</strong>d initially differentiate into pyramidal neurons <strong>an</strong>d later into astrocytes. Thus, Lin-<br />
NSCs, RGPs <strong>an</strong>d Lin+ IPs each exhibit self-renewing <strong>an</strong>d differentiating seminal properties,<br />
which may explain how <strong>the</strong>se properties have become widely used in <strong>the</strong> NSC field to<br />
collectively identify “NSC/progenitor” cells. Because <strong>the</strong>se generic functional attributes are<br />
currently accepted as norm in this field, <strong>the</strong> true identities of NSC <strong>an</strong>d progenitor phenotypes<br />
have thus become obscured, if not reduced to “sem<strong>an</strong>tics”. Our results demonstrate that Lin-<br />
sox1+ sox2+ pax6- NSCs ontogenetically precede <strong>an</strong>d give rise to both Lin- sox1+ sox2+ pax6+<br />
RGPs <strong>an</strong>d Lin+ sox1+ sox2+ pax6+ tbr2+ IPs. Each of <strong>the</strong>se NE phenotypes <strong>for</strong>ms a separate<br />
<strong>an</strong>d distinct neurogenic compartment. Our findings <strong>the</strong>re<strong>for</strong>e unequivocally delineate <strong>the</strong><br />
phenotypic identity <strong>an</strong>d <strong>the</strong> seminal properties of NSCs <strong>from</strong> those of <strong>the</strong>ir progenitor progeny<br />
<strong>an</strong>d outline <strong>the</strong>ir ontogenetically ch<strong>an</strong>ging contributions to corticogenesis. In addition, this study<br />
provides a conceptual blueprint <strong>an</strong>d experimental strategy to more precisely elucidate <strong>the</strong> cellular<br />
<strong>an</strong>d molecular circuits underlying neural lineage progressions during corticogenesis.<br />
Disclosures: D. Maric, None; Y.H. Ch<strong>an</strong>g, None; J.L. Barker, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.8/A47<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Title: Identifying epigenetic regulatory mech<strong>an</strong>isms in neural restricted precursors derived <strong>from</strong><br />
<strong>the</strong> cortex <strong>an</strong>d g<strong>an</strong>glionic eminence
Authors: *C. L. RICUPERO, H. LI, M. GRUMET, R. P. HART;<br />
WM Keck Ctr. Collab Neurosci, Rutgers Univ., Piscataway, NJ<br />
Abstract: Epigenetic regulation is essential <strong>for</strong> differentiating neuronal <strong>an</strong>d glial precursors.<br />
Epigenetic modifications such as histone methylation have been linked to cell fate; however <strong>the</strong><br />
underlying mech<strong>an</strong>isms controlling <strong>the</strong> placement of epigenetic marks are still largely unknown.<br />
We predict that specific epigenetic marks are reliable indicators <strong>for</strong> potential gene regulation,<br />
<strong>the</strong>reby revealing <strong>the</strong> programming of ultimate cell phenotype. To test this hypo<strong>the</strong>sis, 19 neural<br />
precursor clones were isolated <strong>an</strong>d immortalized <strong>from</strong> <strong>the</strong> E14.5 rat cortex <strong>an</strong>d g<strong>an</strong>glionic<br />
eminence. Isolation at this key developmental stage provided multipotential, neuronal <strong>an</strong>d glial<br />
restricted precursors. Gene expression <strong>an</strong>alysis of a differentiated time course was per<strong>for</strong>med<br />
using 5 clones representing each restricted phenotype. Results show clear separation of<br />
phenotype at key neuronal <strong>an</strong>d glial genes such as Mash1, Vgat <strong>an</strong>d Cystathionine Beta<br />
Synthase. However, when focusing on mRNA levels in <strong>an</strong> undifferentiated state, separation of<br />
phenotype is not as clear. To test our prediction that chromatin state in undifferentiated<br />
precursors may reveal downstream gene expression, we focused on trimethyl histone<br />
modifications linked to gene activation or repression. Clones exhibit distinct marks in specific<br />
promoters related to neural differentiation. For example, in neuronal clone Ge6, promoter<br />
sequences upstream of <strong>the</strong> neuron-specific tr<strong>an</strong>scription factor MASH1 (Ascl1) <strong>an</strong>d <strong>the</strong><br />
Neuregulin receptor Erbb4 displayed high levels <strong>for</strong> a marker associated with gene expression<br />
(H3K4me3) <strong>an</strong>d lower levels <strong>for</strong> a marker associated with gene repression (H3K27me3). In<br />
contrast, glial-restricted clone Ge2 showed <strong>an</strong> inverse relationship displaying increased levels of<br />
H3K27me3 <strong>an</strong>d low H3K4me3. This suggests that cell fate may already be predetermined<br />
through chromatin modifications in undifferentiated cells. Results <strong>from</strong> assaying o<strong>the</strong>r<br />
tr<strong>an</strong>scription factor promoters, including members of <strong>the</strong> DLX <strong>an</strong>d Sox families, provide<br />
additional clues <strong>for</strong> decoding differentiation potential. Our goal is to discover predictive histone<br />
modifications at key regulatory genes responsible <strong>for</strong> downstream cell fate decisions.<br />
Disclosures: C.L. Ricupero, None; H. Li, None; M. Grumet, None; R.P. Hart, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.9/A48<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Montecatone Foundation 2005-09
PRIN 2005<br />
Title: Death resist<strong>an</strong>t neural progenitors yield mostly neurons: An erythropoietin-dependent<br />
process<br />
Authors: *A. GORIO, G. MARFIA, D. BOTTAI, L. MADASCHI, F. MARRA, S. CARELLI,<br />
A. M. DI GIULIO;<br />
Dept Med, Surgery & Odontoiatry, Pharmacol. Labs Fac Med., 20142 Mil<strong>an</strong>o, Italy<br />
Abstract: Traumatic lesions of <strong>the</strong> cord are characterized by a secondary process of<br />
degeneration, that is a complex condition of ischemia-like syndrome <strong>an</strong>d neuroinflammation. We<br />
reported that mouse adult neural stem cells, isolated <strong>from</strong> <strong>the</strong> subventricular zone (SVZ),<br />
accumulate at <strong>the</strong> site of injury <strong>an</strong>d improve <strong>the</strong> early rate of hind limb functional recovery,<br />
however, few weeks later <strong>the</strong> tr<strong>an</strong>spl<strong>an</strong>ted are totally phagocytated by macrophages. In view of<br />
such results we aimed at isolating adult neural stem cells after a prolonged ischemic insult, this<br />
could provide a population of cells capable of resisting at <strong>the</strong> unfavourable site of injury. Here<br />
we report <strong>the</strong> isolation <strong>from</strong> SVZ of neural precursors at several hours after death of <strong>the</strong> mouse.<br />
These cells differentiate mostly in neurons ( > 50% of total cells) showing high activation of<br />
HIF1α <strong>an</strong>d MAPK with <strong>the</strong> specific expression of both erythropoietin <strong>an</strong>d its receptor. The<br />
addition of <strong>an</strong>tibodies to erythropoietin to <strong>the</strong> specific medium inhibits <strong>the</strong> differentiation<br />
towards <strong>the</strong> neuronal phenotype <strong>an</strong>d obliterate <strong>the</strong> voltage sensitive calcium currents. These<br />
results suggest that <strong>the</strong> expression of ischemia-induced EPO signaling is <strong>an</strong> indispensable<br />
precondition to gain <strong>an</strong>d maintain <strong>the</strong> neuronal morphological <strong>an</strong>d functional phenotype by <strong>the</strong><br />
death resist<strong>an</strong>t neural precursors. The induction of EPO is of great import<strong>an</strong>ce in brain<br />
preconditioning <strong>an</strong>d suggest that <strong>the</strong>se cells may represent a possible successful c<strong>an</strong>didate <strong>for</strong><br />
tr<strong>an</strong>spl<strong>an</strong>tation into <strong>the</strong> central nervous system after injury.<br />
Disclosures: A. Gorio , None; G. Marfia, None; D. Bottai, None; L. Madaschi, None; F.<br />
Marra, None; S. Carelli, None; A.M. Di Giulio, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.10/A49<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: NEI Gr<strong>an</strong>t EY016790
The Glaucoma Foundation<br />
NEI P30 EY014801<br />
Research to Prevent Blindness<br />
Title: Tr<strong>an</strong>scriptional control of retina g<strong>an</strong>glion cell fate<br />
Authors: *J. HERTZ, J. L. GOLDBERG;<br />
Bascom Palmer Eye Inst., Univ. Miami Miller Sch. of Med., Miami, FL<br />
Abstract: What are <strong>the</strong> molecular signals that regulate how stem cells differentiate into<br />
particular neural cell fates? For example, <strong>the</strong> differentiation of retinal g<strong>an</strong>glion cells (RGCs)<br />
<strong>from</strong> retinal progenitors is regulated by a combination of intrinsic <strong>an</strong>d extrinsic signals. Among<br />
<strong>the</strong>se, cell fate-specifying signals have been shown to be strongly dependent on tr<strong>an</strong>scriptional<br />
control. For example, <strong>the</strong> basic helix-loop-helix (bHLH) tr<strong>an</strong>scription factor Math5 is necessary<br />
<strong>for</strong> RGC fate, although its expression is not exclusive to RGCs in <strong>the</strong> retina. It is not known what<br />
mech<strong>an</strong>isms control RGC fate decisions upstream or downstream of Math5, or whe<strong>the</strong>r<br />
simult<strong>an</strong>eous expression of o<strong>the</strong>r tr<strong>an</strong>scription factors is also required. Fur<strong>the</strong>rmore, it is not yet<br />
known whe<strong>the</strong>r a fate-choice tr<strong>an</strong>scription factor c<strong>an</strong> specify RGCs in <strong>the</strong> absence of prior or<br />
simult<strong>an</strong>eous expression of Math5. Here, we hypo<strong>the</strong>size that o<strong>the</strong>r developmental tr<strong>an</strong>scription<br />
factors are sufficient <strong>for</strong> RGC specification even without Math5 expression, but that preceding<br />
Math5 expression will fur<strong>the</strong>r enh<strong>an</strong>ce RGC cell fate specification. To address this hypo<strong>the</strong>sis,<br />
we have cultured retinal progenitors derived <strong>from</strong> Math5-/- <strong>an</strong>d wildtype control mice in a<br />
neurosphere assay to determine whe<strong>the</strong>r <strong>the</strong> demonstrated dependence on Math5 in vivo <strong>for</strong><br />
RGC differentiation is observed outside of <strong>the</strong> in vivo environment. We have found that Math5-<br />
/- embryonic retinas are able to generate neurosphere cultures, <strong>an</strong>d that <strong>the</strong>se neurospheres c<strong>an</strong><br />
be tr<strong>an</strong>sfected with exogenous genes using electroporation. Here we present <strong>the</strong> results of a<br />
screen of specific tr<strong>an</strong>scription factors, including NeuroD, Brn-3b, Islet1, BarH1, <strong>an</strong>d Pax6, to<br />
rescue RGC cell fate specification in Math5-/- progenitors, or to increase RGC specification in<br />
wild type progenitors. Taken toge<strong>the</strong>r, <strong>the</strong>se data address <strong>the</strong> role of Math5 as a necessary,<br />
permissive tr<strong>an</strong>scription factor in cell fate specification of RGCs.<br />
Disclosures: J. Hertz, None; J.L. Goldberg, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.11/A50
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Howard Hughes Medical Institute<br />
NIH Gr<strong>an</strong>t HD007333<br />
Title: Identification <strong>an</strong>d characterization of neuropeptides in <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> Schmidtea<br />
mediterr<strong>an</strong>ea<br />
Authors: *J. J. COLLINS 1 , X. HOU 2 , E. V. ROMANOVA 2 , F. XIE 2 , J. V. SWEEDLER 2 , P.<br />
A. NEWMARK 1 ;<br />
1 Howard Hughes Med. Inst. <strong>an</strong>d Dept. of Cell <strong>an</strong>d Developmental Biol., 2 Dept. of Chem., Univ.<br />
of Illinois at Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a, IL<br />
Abstract: Freshwater pl<strong>an</strong>ari<strong>an</strong>s possess a virtually unlimited capacity to regenerate lost or<br />
damaged tissues. Following amputation, pluripotent stem cells called neoblasts proliferate <strong>an</strong>d<br />
differentiate to give rise to every tissue in <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> including muscles, intestine, germ line,<br />
<strong>an</strong>d a functional central nervous system. The nervous system is <strong>an</strong> import<strong>an</strong>t regulator of<br />
regeneration in a variety of <strong>an</strong>imals, but few studies have identified specific neural signals that<br />
influence regeneration. Neuropeptides are secreted peptide hormones (typically 3-40 amino acids<br />
in length) that represent <strong>the</strong> most diverse class of cell to cell signaling molecules in metazo<strong>an</strong>s.<br />
Classic <strong>an</strong>d contemporary studies of pl<strong>an</strong>ari<strong>an</strong>s have suggested that neuroendocrine activity may<br />
influence regeneration <strong>an</strong>d neoblasts, yet little is known about <strong>the</strong> diversity <strong>an</strong>d types of<br />
neuroendocrine hormones that are present in pl<strong>an</strong>ari<strong>an</strong>s. There<strong>for</strong>e, we used a combination of<br />
bioin<strong>for</strong>matics, mass spectrometry, <strong>an</strong>d molecular approaches to identify <strong>an</strong>d validate >40 fulllength<br />
neuropeptide prohormone genes in <strong>the</strong> model pl<strong>an</strong>ari<strong>an</strong> Schmidtea mediterr<strong>an</strong>ea. In total,<br />
<strong>the</strong>se prohormones are predicted to be processed into 200 distinct peptides, of which nearly 100<br />
have been confirmed with mass spectrometry. In addition to encoding peptides similar to wellcharacterized<br />
vertebrate <strong>an</strong>d invertebrate neuropeptides (myomodulins, neuropeptide Y-like<br />
family members, <strong>an</strong>d FMRF-like peptides), <strong>the</strong> newly indentified prohormones encode m<strong>an</strong>y<br />
novel neuropeptides. To characterize <strong>the</strong>se genes we per<strong>for</strong>med whole-mount in situ<br />
hybridization on our entire collection of neuropeptide prohormone genes in both sexual <strong>an</strong>d<br />
asexual pl<strong>an</strong>ari<strong>an</strong>s. We find that that neuropeptide prohormones are expressed in a variety of<br />
discrete cell populations in <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> central <strong>an</strong>d peripheral nervous systems in asexual<br />
<strong>an</strong>imals <strong>an</strong>d, in some cases, are expressed in reproductive structures in sexual <strong>an</strong>imals. To<br />
<strong>an</strong>alyze <strong>the</strong> extent to which prohormones are expressed in overlapping cell populations in <strong>the</strong><br />
nervous system, we per<strong>for</strong>med multi-color fluorescent in situ hybridization <strong>an</strong>alyses. While some<br />
prohormones share common expression patterns, we observe that a majority of prohormone<br />
genes are expressed in non-overlapping populations of cells in <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> nervous system.<br />
Toge<strong>the</strong>r, our observations suggest a high level of complexity in <strong>the</strong> patterning of <strong>an</strong>d chemistry<br />
within <strong>the</strong> pl<strong>an</strong>ari<strong>an</strong> nervous system. Future studies aimed at <strong>the</strong> characterization of <strong>the</strong>se<br />
prohormone genes in S. mediterr<strong>an</strong>ea may identify novel neuroendocrine regulators of pl<strong>an</strong>ari<strong>an</strong><br />
behavior, reproduction, physiology, regeneration, <strong>an</strong>d stem cells.<br />
Disclosures: J.J. Collins, None; X. Hou, None; E.V. Rom<strong>an</strong>ova, None; F. Xie, None; J.V.<br />
Sweedler, None; P.A. Newmark, None.
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.12/A51<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Swedish research council<br />
KMA<br />
Title: Alterations in expression <strong>an</strong>d mRNA editing of GABAA receptor subunits during chick<br />
retinal development <strong>an</strong>d retinal progenitor cell differentiation in vitro<br />
Authors: *H. J. RING 1 , H. BOIJE 2 , J. OHLSON 3 , C. DANIEL 3 , A. RUNDBERG NILSSON 2 ,<br />
M. ÖHMAN 3 , F. HALLBÖÖK 2 ;<br />
2 1 3<br />
Neurosci., Uppsala Univ., Uppsala, Sweden; Mol. biology <strong>an</strong>d functional genomics,<br />
Stockholm Univ., Stockholm, Sweden<br />
Abstract: GABA has multiple functions during <strong>the</strong> development of <strong>the</strong> nervous system, <strong>from</strong><br />
effects on proliferation <strong>an</strong>d migration to synaptogenesis <strong>an</strong>d circuits <strong>for</strong>mation. The functions<br />
vary during <strong>the</strong> course of development <strong>an</strong>d in this study <strong>the</strong> aim was to relate alterations in<br />
GABAA receptor subunit expression to <strong>the</strong> retinal development.<br />
The GABAA receptor is <strong>an</strong> ion-gated tr<strong>an</strong>smembr<strong>an</strong>e receptor consisting of five subunits<br />
arr<strong>an</strong>ged around a central pore. There are sixteen different GABAA subunits (α1-6, β1-4, γ1-3, δ,<br />
ε, π) <strong>an</strong>d <strong>the</strong> combination of <strong>the</strong> subunits results in different receptor functions. Alterations in <strong>the</strong><br />
receptor subtype repertoire over time will ch<strong>an</strong>ge GABA function. Fur<strong>the</strong>rmore, we have<br />
discovered a novel site of selective editing of <strong>the</strong> tr<strong>an</strong>script coding <strong>for</strong> <strong>the</strong> GABAA receptor α3<br />
subunit which may also contribute to <strong>the</strong> ch<strong>an</strong>ges in receptor functions.<br />
We examined; 1) alteration of subunit expression in cultured retinal progenitor cells (RPC)<br />
be<strong>for</strong>e <strong>an</strong>d after in vitro differention 2) temporal expression of <strong>the</strong> subunits in developing retina<br />
<strong>from</strong> E4 to 6 months post hatch <strong>an</strong>d 3) extent of mRNA editing of <strong>the</strong> tr<strong>an</strong>script encoding <strong>the</strong> α3<br />
subunit during development. mRNA expression was <strong>an</strong>alyzed using qRT-PCR. RPCs were<br />
prepared <strong>from</strong> CMZ of chick E6.5 <strong>an</strong>d E12 retina respectively. By growing <strong>the</strong> RPCs in DMEM<br />
based medium including EGF, FGF-2, insulin <strong>an</strong>d B-27 <strong>the</strong> cells proliferated, <strong>for</strong>med spheres<br />
<strong>an</strong>d remained undifferentiated. The cells were differentiated by excluding growth factors <strong>an</strong>d<br />
supplementing with 10 % serum. For <strong>the</strong> temporal studies <strong>an</strong>d <strong>for</strong> examination of <strong>the</strong> extent of<br />
Gabra-3 editing, total RNA <strong>from</strong> retina with increasing age was isolated.<br />
The results showed that <strong>the</strong> mRNA expression of several subunits was signific<strong>an</strong>tly altered
during development as well as between undifferentiated <strong>an</strong>d differentiated cultured RPCs. For<br />
example, <strong>the</strong> α6 subunit expression was upregulated 11 times in differentiated E12 cells. α6<br />
expression was also upregulated during retinal development. The specific mRNA editing of <strong>the</strong><br />
α3 tr<strong>an</strong>script increased <strong>from</strong> low levels to over 95 % in <strong>the</strong> adult chick retina.<br />
In conclusion, this study shows that <strong>the</strong> mRNA expressions of several of <strong>the</strong> GABAA subunits<br />
are specifically regulated during retinal development. This implies that specific subunits are<br />
import<strong>an</strong>t during late retinal development whereas o<strong>the</strong>rs are import<strong>an</strong>t during early<br />
development. The increase of <strong>the</strong> extent of <strong>the</strong> mRNA editing of <strong>the</strong> α3 subunit during <strong>the</strong> course<br />
of development indicates that mRNA editing contributes to <strong>the</strong> ch<strong>an</strong>ging functional roles <strong>for</strong><br />
GABA by modified GABAA receptor assembly.<br />
Disclosures: H.J. Ring, None; H. Boije, None; J. Ohlson, None; C. D<strong>an</strong>iel, None; A.<br />
Rundberg Nilsson, None; M. Öhm<strong>an</strong>, None; F. Hallböök, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.13/A52<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Shriners Hospital Gr<strong>an</strong>ts 8610 <strong>an</strong>d 8750<br />
Shriners Postdoctoral Fellowship<br />
National Multiple Sclerosis <strong>Society</strong> Gr<strong>an</strong>t 1458<br />
UC Davis Health System Research Award<br />
Title: C<strong>an</strong>onical Wnt signaling as a niche signal <strong>for</strong> <strong>the</strong> self-renewal <strong>an</strong>d neuronal differentiation<br />
of olfactory neural stem cells during development<br />
Authors: *Y. WANG 1,3 , P. LOTT 2 , Y. LI 1,3 , L. SONG 1,3 , T. ZHAO 1,3 , T. YAMAGAMI 1,3 , C.<br />
ZHOU 1,3 ;<br />
1 Univ. of Cali<strong>for</strong>nia-Davis, Sacramento, CA; 2 Genet. Grad. Group, Univ. of Cali<strong>for</strong>nia-Davis,<br />
Davis, CA; 3 Inst. <strong>for</strong> Pediatric Regenerative Medicine, Shriners Hosp. <strong>for</strong> Children, 2425<br />
Stockton Blvd, Sacrament, CA
Abstract: The mammali<strong>an</strong> olfactory epi<strong>the</strong>lium (OE) has a unique stem cell/progenitor niche,<br />
which includes horizontal basal cells (HBCs) <strong>an</strong>d globose basal cells (GBCs). These cells are<br />
responsible <strong>for</strong> <strong>the</strong> const<strong>an</strong>t neurogenesis under normal physiological condition <strong>an</strong>d rapid<br />
neuronal regeneration after injuries. However, <strong>the</strong> signal pathway that regulates <strong>the</strong> behavior of<br />
<strong>the</strong>se olfactory neural stem/progenitor cells remains little understood. Using our newly generated<br />
Wnt signaling reporter tr<strong>an</strong>sgenic mice, we have found that <strong>the</strong> c<strong>an</strong>onical Wnt signaling pathway<br />
is mainly activated in GBCs <strong>an</strong>d weakly in HBCs during olfactory development. The dynamics<br />
of Wnt signaling activation in <strong>the</strong> developing OE coincides well with <strong>the</strong> location <strong>an</strong>d timing of<br />
<strong>the</strong> expression of multiple c<strong>an</strong>onical Wnt signaling components <strong>an</strong>d neurogenesis. In <strong>the</strong><br />
olfactory neurosphere culture, Wnt3a <strong>an</strong>d related activators signific<strong>an</strong>tly increased <strong>the</strong> <strong>for</strong>mation<br />
of both <strong>the</strong> primary <strong>an</strong>d secondary neurospheres. Conversely, <strong>the</strong> Wnt <strong>an</strong>tagonist Dkk1<br />
signific<strong>an</strong>tly suppressed <strong>the</strong> neurosphere <strong>for</strong>mation. Fur<strong>the</strong>rmore, <strong>the</strong> Wnt activators<br />
signific<strong>an</strong>tly promoted neuronal differentiation <strong>from</strong> <strong>the</strong> OE neurospheres under <strong>the</strong><br />
differentiation condition. Our study has revealed <strong>an</strong> import<strong>an</strong>t role of <strong>the</strong> Wnt signaling pathway<br />
in both self-renewal <strong>an</strong>d neuronal differentiation of olfactory neural stem cells.<br />
Disclosures: Y. W<strong>an</strong>g, None; P. Lott, None; Y. Li, None; L. Song, None; T. Zhao, None; T.<br />
Yamagami, None; C. Zhou, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.14/A53<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Frazier Research Institute<br />
Title: Morphological characterization of adult bone marrow stem cells after intr<strong>an</strong>asal delivery<br />
to mice with striatal lesions<br />
Authors: E. H. CHARTOFF, D. DAMEZ-WERNO, J. PETERSON, L. HASSINGER, D.<br />
POTTER, V. PEEV, A. M. CATALDO, *B. M. COHEN;<br />
McLe<strong>an</strong> Hosp, Belmont, MA<br />
Abstract: Bone marrow derived adult progenitor cells (MAPCs) are thought to hold promise <strong>for</strong><br />
<strong>the</strong> treatment of neurodegenerative disorders <strong>an</strong>d neurotoxicity. However, <strong>the</strong>re are several<br />
potential limitations to <strong>the</strong>ir use: invasive methods of delivery (e.g. intracr<strong>an</strong>ial impl<strong>an</strong>tation) are<br />
impractical <strong>an</strong>d risky, <strong>the</strong> long-term functional consequences are not well characterized, <strong>an</strong>d <strong>the</strong>
fate of MAPCs within <strong>the</strong> CNS is not understood. We have shown that intr<strong>an</strong>asal (IN) delivery<br />
of mouse-derived MAPCs expressing green fluorescent protein (GFP) to recipient mice with<br />
intracerebral stab lesions results in subst<strong>an</strong>tial migration of MAPCs to CSF <strong>an</strong>d brain<br />
parenchyma. In this study, we examined <strong>the</strong> temporal <strong>an</strong>d morphological fate of IN-delivered<br />
MAPCs in mice that received ei<strong>the</strong>r unilateral sham or 6-OHDA striatal lesions. In addition, we<br />
assessed <strong>the</strong> behavioral effects of MAPCs on amphetamine-induced rotational behavior in sham<br />
<strong>an</strong>d 6-OHDA-lesioned mice. We observed GFP-positive MAPCs in <strong>the</strong> olfactory epi<strong>the</strong>lium <strong>an</strong>d<br />
ipsilateral striatum at 7d, 14d, 1mos, <strong>an</strong>d 2 mos post-MAPC IN delivery in sham-lesioned mice.<br />
At 7d, massive GFP-positive immunoreactivity in <strong>the</strong> striatum primarily identified cell fragments<br />
with no clear cellular morphology. Ultrastructural examination of <strong>the</strong> MAPCs confirmed that <strong>the</strong><br />
majority of MAPCs at this time point were in various states of degradation. However, a small<br />
number of intact GFP-positive cells were observed <strong>an</strong>d some of <strong>the</strong>se <strong>for</strong>med clear synaptic<br />
contacts with adjacent cells. At later time points <strong>the</strong>re was comparably less GFP-positive<br />
immunoreactivity in <strong>the</strong> striatum, but those remaining cells appeared healthier. Qualitatively<br />
<strong>the</strong>re were no observable differences at 2 mos post-MAPC delivery in <strong>the</strong> location or<br />
morphology of GFP-positive cells in mice that received unilateral 6-OHDA lesions compared to<br />
sham-lesioned mice. Finally, in both sham- <strong>an</strong>d 6-OHDA-lesioned mice, MAPCs had similar<br />
effects on amphetamine-induced rotational behavior: amphetamine-induced ipsilateral turns were<br />
decreased in 6-OHDA-lesioned mice <strong>an</strong>d amphetamine elicited contralateral turns in shamlesioned<br />
mice. Toge<strong>the</strong>r <strong>the</strong>se results suggest that intr<strong>an</strong>asal delivery of MAPCs is a viable route<br />
of administration but raise caution that MAPCs c<strong>an</strong> have non-specific effects on behavior.<br />
Disclosures: E.H. Chartoff, None; D. Damez-Werno, None; J. Peterson, None; L.<br />
Hassinger, None; D. Potter, None; V. Peev, None; A.M. Cataldo, None; B.M. Cohen, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.15/A54<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: AOSpine<br />
Title: Efficient production of neural cells <strong>from</strong> hum<strong>an</strong> bone marrow-derived mesenchymal stem<br />
cells<br />
Authors: *A. R. ALEXANIAN, J. GROSEK, C. STADIG;<br />
Neurosci Rese Labs 151, Med. Coll Wisconsin-Neurosurgery, Milwaukee, WI
Abstract: Stem cell-based <strong>the</strong>rapies to repair <strong>an</strong>d replace lost neural cells are a potential<br />
treatment <strong>for</strong> central nervous system (CNS) diseases. Bone marrow (BM)-derived mesenchymal<br />
stem cells (MSCs) have great potential as <strong>the</strong>rapeutic agents against neurological maladies since<br />
<strong>the</strong>y have <strong>the</strong> ability to differentiate into neural phenotypes <strong>an</strong>d c<strong>an</strong> be readily isolated <strong>an</strong>d<br />
exp<strong>an</strong>ded <strong>for</strong> auto-tr<strong>an</strong>spl<strong>an</strong>tation with no risk of rejection. In our previous studies we have<br />
demonstrated that neural cells c<strong>an</strong> be efficiently generated <strong>from</strong> mouse BM-derived MSCs by<br />
exposing cells to epigenetic modifiers <strong>an</strong>d neural inducing factors. These neurally induced MSCs<br />
(NI-MSCs) differentiate into neuronal- <strong>an</strong>d glial-like cells in vitro, release neurotrophic factors<br />
NGF <strong>an</strong>d BDNF, survive <strong>an</strong>d integrate after tr<strong>an</strong>spl<strong>an</strong>tation in intact <strong>an</strong>d injured spinal cords<br />
(ISC), <strong>an</strong>d improve functional recovery of rats with moderate spinal cord contusion.<br />
Recently, to elucidate whe<strong>the</strong>r we c<strong>an</strong> apply this approach to induce hum<strong>an</strong> bone marrow<br />
derived MSCs into neural cells, several neural induction protocols have been tested. In <strong>the</strong>se<br />
studies hMSCs were exposed to epigenetic modifiers such as inhibitors of DNA methylation <strong>an</strong>d<br />
histone deacetylation <strong>an</strong>d to neural environment (NSC condition medium <strong>an</strong>d fixed NSCs) or<br />
neural inducing factors such as N<strong>an</strong>nog, Chordin, neurotrophines, mitogens, sonic hedgehog,<br />
retinoic acid, ascorbic acid, pharmacological agents that increase intracellular cAMP level <strong>an</strong>d to<br />
different combinations of <strong>the</strong>se agents. The results demonstrated that <strong>the</strong> experiments in which<br />
cells treated simult<strong>an</strong>eously with inhibitors of DNA methylation, hitone deacetylation <strong>an</strong>d<br />
pharmacological agents that increase cAMP level were <strong>the</strong> most effective method <strong>for</strong> production<br />
of cell with neural morphology <strong>an</strong>d neural expression profile. Expression of neural markers was<br />
confirmed with immunocytochemistry, Western blot <strong>an</strong>d PCR. ELISA studies showed that <strong>the</strong>se<br />
neurally induced cells released neurotrophic factors GDNF <strong>an</strong>d BDNF. Thus, hMSCs neurally<br />
modified with this methodology c<strong>an</strong> be a suitable source of cells not only to replace damaged<br />
neurons <strong>an</strong>d glia in ISC but also to provide support to CNS tissue (promoting cell survival <strong>an</strong>d<br />
axonal growth) by producing neurotrophic factors.<br />
Disclosures: A.R. Alex<strong>an</strong>i<strong>an</strong>, None; J. Grosek, None; C. Stadig, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.16/A55<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Swedish Research Council 11548<br />
ALF Göteborg
Torsten <strong>an</strong>d Ragnar Söderberg’s Foundations<br />
Title: The intermediate filament protein synemin is expressed in neural stem/progenitor cells<br />
Authors: *U. WILHELMSSON 1 , L. LANDQVIST 1 , Y. PAN 2 , A. STÅHLBERG 1 , M. FAIZ 1 ,<br />
K. MALMGREN 1 , B. RYDENHAG 1 , M. CURTIS 1 , O. SKALLI 2 , M. PEKNY 1 ;<br />
1 Univ. of Go<strong>the</strong>nburg, Go<strong>the</strong>nburg, Sweden; 2 Louisi<strong>an</strong>a State Univ., Shreveport, LA<br />
Abstract: Similar to <strong>the</strong> intermediate filament (n<strong>an</strong>ofilament) protein nestin, synemin is widely<br />
expressed in <strong>the</strong> developing central nervous system. Here we characterized <strong>the</strong> expression of<br />
synemin iso<strong>for</strong>ms in mouse <strong>an</strong>d rat neural stem cells in vitro, in mouse <strong>an</strong>d hum<strong>an</strong> neurospheres<br />
<strong>an</strong>d in mouse <strong>an</strong>d hum<strong>an</strong> brain. We found that synemin mRNA <strong>an</strong>d protein were present in<br />
neural progenitor cells derived <strong>from</strong> adult rat hippocampus <strong>an</strong>d <strong>from</strong> adult mouse brain, in<br />
primary neurospheres prepared <strong>from</strong> postnatal day 6 mouse brain or <strong>from</strong> hum<strong>an</strong> hippocampus.<br />
In both mouse <strong>an</strong>d rat neural stem cells, synemin was present as unpolymerized protein while<br />
nestin <strong>an</strong>d o<strong>the</strong>r intermediate filament (n<strong>an</strong>ofilament) proteins normally polymerize into <strong>the</strong><br />
intermediate filament (n<strong>an</strong>ofilament) network. In mouse neurospheres, which contain both<br />
immature <strong>an</strong>d more differentiated cells, we found equally large pools of unpolymerized <strong>an</strong>d<br />
polymerized synemin. Single cell qu<strong>an</strong>titative rtPCR showed that about 20% of cells <strong>from</strong><br />
disassociated primary neurospheres expressed synemin mRNA <strong>an</strong>d that most of synemin positive<br />
cells also expressed Sox2 <strong>an</strong>d nestin. In <strong>the</strong> ventricle wall of <strong>the</strong> adult mouse <strong>an</strong>d hum<strong>an</strong> brain<br />
synemin-positive cells were found both in <strong>the</strong> ependymal cell layer <strong>an</strong>d in some cells within <strong>the</strong><br />
subventricular zone. Our data show that mouse <strong>an</strong>d rat neural progenitor cells in culture as well<br />
as cells of mouse <strong>an</strong>d hum<strong>an</strong> neurospheres contains intermediate filament protein synemin. Most<br />
interestingly, in neural progenitor cells synemin is found almost exclusively in <strong>an</strong> unpolymerized<br />
<strong>for</strong>m which might imply a novel non-structural function <strong>for</strong> this intermediate filament protein<br />
Disclosures: U. Wilhelmsson, None; L. L<strong>an</strong>dqvist, None; Y. P<strong>an</strong>, None; A. Ståhlberg,<br />
None; M. Faiz, None; K. Malmgren, None; B. Rydenhag, None; M. Curtis, None; O. Skalli,<br />
None; M. Pekny, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.17/A56<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: Americ<strong>an</strong> Heart Association 0810071Z
NIH R01 NS047373<br />
Title: Regulation of metabolic phenotype in neural stem/progenitor cells (NSPCs) by hypoxia<br />
inducible factor-1α (HIF-1α)<br />
Authors: *K. M. HARMS, L. A. CUNNINGHAM;<br />
Neurosciences, Univ. of New Mexico Sch. of Med., Albuquerque, NM<br />
Abstract: HIF-1α is a tr<strong>an</strong>scription factor that regulates <strong>an</strong>giogenic <strong>an</strong>d metabolic gene<br />
expression. Embryonic <strong>an</strong>d adult NSPCs display constitutive stabilization of HIF-1α under nonhypoxic<br />
conditions (Roitbak et al., 2008.) <strong>an</strong>d rely primarily on glycolysis <strong>for</strong> ATP syn<strong>the</strong>sis.<br />
Glucose deprivation in <strong>the</strong> presence of pyruvate results in 98% cell death of NSPCs within 24<br />
hrs, whereas 24 hr of complete <strong>an</strong>oxia in <strong>the</strong> presence of glucose does not compromise NSPC<br />
viability. To probe mitochondrial function, we exposed NSPCs in culture to various<br />
mitochondrial inhibitors <strong>an</strong>d found that NSPCs were relatively resist<strong>an</strong>t compared to neurons<br />
(cell death in response to 24hr exposure to 1 mM cy<strong>an</strong>ide: 0% vs. 75%, 100 nM rotenone: 40%<br />
vs. 70%, 500 nM oligomycin: 30% vs. 90% , NSPCs vs. neurons, respectively). To determine<br />
whe<strong>the</strong>r <strong>the</strong> glycolytic phenotype of NSPCs is due to constitutive stabilization of HIF-1α, we<br />
utilized a Cre/loxP system to knockout HIF-1α gene expression in NSPCs in culture. NSPCs<br />
were isolated <strong>from</strong> <strong>the</strong> telencephalon of embryonic day 14 mice homozygous <strong>for</strong> a floxed allele<br />
of HIF-1α, in which exon 2 is fl<strong>an</strong>ked by loxP sites (HIF-1α fl/fl ; Ry<strong>an</strong> et al., 2000, C<strong>an</strong>cer Res).<br />
HIF-1α fl/fl NSPCs were exp<strong>an</strong>ded <strong>for</strong> 2 weeks (4 passages) <strong>an</strong>d subsequently tr<strong>an</strong>sduced with<br />
adenovirus that conferred Cre-recombinase expression (48 hr incubation at 50 MOI). Cremediated<br />
recombination was ~100% with no apparent toxicity <strong>an</strong>d resulted in complete genomic<br />
deletion of <strong>the</strong> floxed sequence as assessed by PCR <strong>an</strong>alysis. HIF-1α protein decreased by 53%<br />
at 48 hrs following infection with Cre adenovirus, which was associated with a 30% decrease in<br />
resist<strong>an</strong>ce to 24 hr of <strong>an</strong>oxia. At later passages (P8) 2 weeks following Cre-mediated<br />
recombination, active HIF-1α was completely eliminated as assessed by <strong>an</strong> in vitro<br />
tr<strong>an</strong>scriptional HRE binding assay. Current studies will determine whe<strong>the</strong>r HIF-1α gene deletion<br />
leads to a metabolic shift in NSPCs <strong>from</strong> glycolysis to mitochondrial oxidative phosphorylation.<br />
Disclosures: K.M. Harms, None; L.A. Cunningham, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.18/B1<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses
Support: FNRS - Belgium<br />
Title: Probing <strong>the</strong> role of gap-junction coupling in neuroprotective effect of Neural precursor<br />
cells<br />
Authors: A. RAVELLA 1 , S. CHINTAWAR 2 , *M. PANDOLFO 2 ;<br />
1 Lab. of Exptl. Neurol., 2 Univ. Libre de Bruxelles, Brussels, Belgium<br />
Abstract: We have previously shown that adult Neural precursor cells (NPCs) tr<strong>an</strong>spl<strong>an</strong>tation<br />
rescued <strong>the</strong> ataxic phenotype in Spinocerebellar ataxia type 1 mouse model characterized by<br />
ataxia due to <strong>the</strong> loss of cerebellar Purkinje cells (PCs). Immunohistochemical <strong>an</strong>alysis <strong>an</strong>d<br />
confocal microscopy <strong>an</strong>d profiling revealed that connexin 43 (Cx43) is expressed at <strong>the</strong> interface<br />
of grafted cells <strong>an</strong>d <strong>the</strong> rescued host purkinje cells. We are currently investigating <strong>the</strong> expression<br />
of different connexin proteins on undifferentiated <strong>an</strong>d differentiated NPCs derived <strong>from</strong> <strong>the</strong> SVZ<br />
of adult FVB/N mice. Cx43 is abund<strong>an</strong>tly expressed be<strong>for</strong>e <strong>an</strong>d after differentiation. Cx26 is<br />
expressed only by differentiated cells. Cx36, 37 <strong>an</strong>d 45 are currently being tested on<br />
undifferentiated <strong>an</strong>d differentiated glial <strong>an</strong>d neuronal progeny. We wished to study <strong>the</strong> effect of<br />
inflammatory environment on <strong>the</strong> expression of different connexin proteins on NPCs. They were<br />
treated separately with TNF-α + IFN- γ <strong>an</strong>d TNF-α + IL-1β. No signific<strong>an</strong>t difference was<br />
observed between treated <strong>an</strong>d control cells at mRNA level <strong>for</strong> Cx43 <strong>an</strong>d more connexins are<br />
being followed up.<br />
Disclosures: A. Ravella, None; S. Chintawar, None; M. P<strong>an</strong>dolfo, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.19/B2<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: NIGMS RO1 GM072005-01<br />
The Stem Cell Biology Fund<br />
Title: Astrocyte-derived interleukin-6 promotes specific neuronal differentiation of neural<br />
progenitor cells <strong>from</strong> adult hippocampus
Authors: *J. OH 1,2 , M. A. MCCLOSKEY 2 , D. S. SAKAGUCHI 2,1 ;<br />
1 Neuroscience; Biomed. Sci., 2 Genetics, Develop. <strong>an</strong>d Cell Biol., Iowa State Univ., Ames, IA<br />
Abstract: The purpose of this study was to investigate <strong>the</strong> ability of <strong>an</strong> astrocyte-derived factor<br />
to influence neural progenitor cell differentiation. Adult rat hippocampal progenitor cells<br />
(AHPCs; a gift <strong>from</strong> F.H. Gage, Salk Institute, La Jolla, CA) are self-renewing multipotent<br />
neural precursors that have <strong>the</strong> ability to differentiate into neurons <strong>an</strong>d glia (astrocytes <strong>an</strong>d<br />
oligodendrocytes). We previously demonstrated a signific<strong>an</strong>t increase in neuronal differentiation<br />
of AHPCs immunoreactive <strong>for</strong> <strong>the</strong> neuronal marker, class III β-tubulin (TUJ1), when cultured in<br />
<strong>the</strong> presence of soluble factors derived <strong>from</strong> astrocytes isolated <strong>from</strong> neonatal rat brains (Oh et<br />
al., 2008, J Biomed Mater Res A). Interleukin-6 (IL-6) is a c<strong>an</strong>didate <strong>for</strong> <strong>an</strong> astrocyte-derived<br />
soluble factor that c<strong>an</strong> modulate AHPC differentiation. To test <strong>the</strong> ability of IL-6 to promote or<br />
enh<strong>an</strong>ce neuronal differentiation of neural precursors, we cultured AHPCs in <strong>the</strong> absence or<br />
presence of purified rat recombin<strong>an</strong>t IL-6 <strong>for</strong> 6-7 days. In <strong>the</strong> presence of IL-6, we observed that<br />
(1) <strong>the</strong> percentage of TUJ1-immunoreactivity was signific<strong>an</strong>tly higher <strong>an</strong>d (2) <strong>the</strong> average<br />
neurite length per TUJ1-immunoreactive AHPC was signific<strong>an</strong>tly greater, when compared to<br />
those cultured in <strong>the</strong> absence of IL-6. In addition, in <strong>the</strong> presence of IL-6, (3) tr<strong>an</strong>sient inward<br />
current (tetrodotoxin-sensitive) density was increased with no signific<strong>an</strong>t differences in sustained<br />
outward current (tetraethyl ammonium-sensitive) density, apparent resting membr<strong>an</strong>e potential<br />
or cell capacit<strong>an</strong>ce. These results demonstrate that astrocyte-derived IL-6 enh<strong>an</strong>ces neuronal<br />
differentiation of AHPCs, as supported by <strong>the</strong> expression of phenotypic markers, neurite<br />
outgrowth <strong>an</strong>d electrical membr<strong>an</strong>e properties. Our findings have import<strong>an</strong>t implications <strong>for</strong><br />
underst<strong>an</strong>ding injury-induced neurogenesis <strong>an</strong>d <strong>the</strong> development of <strong>the</strong>rapeutic strategies using<br />
neural progenitor cells <strong>for</strong> possible replacement of damaged neurons.<br />
Supported by NIGMS (RO1 GM072005-01) <strong>an</strong>d <strong>the</strong> Stem Cell Biology Fund<br />
Keywords: Neural stem cells, Interleukin-6, Neuronal differentiation, Electrophysiology<br />
Disclosures: J. Oh, None; M.A. McCloskey, None; D.S. Sakaguchi, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.20/B3<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Support: IRME fundation<br />
AFM fundation
Inserm Osteoarticular research gr<strong>an</strong>t (PRO-A)<br />
Agence Nationale de la Recherche (ATOS)<br />
EFS 2005-11<br />
Title: Hum<strong>an</strong> olfactory nasal stem cells: A new subtype of mesenchymal stem cells <strong>an</strong>d a<br />
promising tool <strong>for</strong> repairing <strong>the</strong> central nervous system<br />
Authors: E. NIVET 1,2 , B. DELORME 3 , M. VIGNES 4 , J. GAILLARD 3 , T. HAUPL 5 , C.<br />
PIERRISNARD 1 , J. RINGE 5 , N. BARIL 1 , A. DEVÈZE 6 , J. MAGNAN 6 , J. SOHIER 7 , F.<br />
LANTÉ 4 , P. CHARBORD 3 , L. SENSEBÉ 3 , P. LAYROLLE 7 , M. KHRESTCHATISKY 2 , F.<br />
FÉRON 2 , *F. S. ROMAN 8 ;<br />
1 Cnrs-umr6149, Univ. de Provence, Marseilles, Fr<strong>an</strong>ce; 2 Cnrs-umr6184, Univ. de la<br />
Méditerr<strong>an</strong>ée, Marseilles, Fr<strong>an</strong>ce; 3 Inserm ESPRI-EA3855, Univ. Fr<strong>an</strong>çois Rabelais, EFS, Tours,<br />
Fr<strong>an</strong>ce; 4 Cnrs umr-5247, Univ. de Montpellier 1 et 2, Montpellier, Fr<strong>an</strong>ce; 5 Rheumatology <strong>an</strong>d<br />
Clin. Immunol., Charité-Universitätsmedizin, Berlin, Germ<strong>an</strong>y; 6 ORL, Univ. de la Méditerr<strong>an</strong>ée,<br />
AP-HM, Marseilles, Fr<strong>an</strong>ce; 7 Inserm U791, Faculté de chirurgie dentaire, N<strong>an</strong>tes, Fr<strong>an</strong>ce; 8 Univ.<br />
Marseilles, Marseilles, Fr<strong>an</strong>ce<br />
Abstract: Worldwide, we are one of <strong>the</strong> three laboratories that have developed <strong>an</strong> efficient<br />
method <strong>for</strong> cultivating <strong>an</strong>d purifying hum<strong>an</strong> nasal olfactory stem cells. From <strong>the</strong> point of view of<br />
clinical applicability, <strong>the</strong> nasal epi<strong>the</strong>lium is ideally suited <strong>for</strong> simple, relatively non-invasive<br />
autologous harvest of stem cells <strong>from</strong> adult patients. One c<strong>an</strong> envisage simply collecting a biopsy<br />
<strong>from</strong> <strong>the</strong> nose of patients, culturing olfactory stem cells <strong>from</strong> this tissue <strong>an</strong>d relocating <strong>the</strong>m in<br />
damaged areas of <strong>the</strong> same patients, eventually after orientating <strong>the</strong> cells towards a given specific<br />
phenotype.<br />
After having shown that <strong>the</strong>se cells are multipotent <strong>an</strong>d c<strong>an</strong> be grown in large numbers in vitro,<br />
we demonstrated that <strong>the</strong>y belong to <strong>the</strong> Mesenchymal Stem Cell superfamily by a combination<br />
of cellular <strong>an</strong>d molecular techniques. However, Affymetrix microarray <strong>an</strong>alyses revealed some<br />
gene expression differences between hum<strong>an</strong> olfactory <strong>an</strong>d bone marrow mesenchymal stem cells.<br />
Accordingly, we confirmed that hum<strong>an</strong> olfactory nasal stem cells are rapidly dividing <strong>an</strong>d prone<br />
to give rise to osteocytes, neurons <strong>an</strong>d glial cells. As a consequence, we used <strong>the</strong>m <strong>for</strong><br />
tr<strong>an</strong>spl<strong>an</strong>tation in <strong>an</strong>imal models of brain diseases.<br />
In a mouse model of excitotoxically-induced loss of memory, we grafted hum<strong>an</strong> nasal olfactory<br />
stem cells into <strong>the</strong> hippocampus. We showed that exogenous cells migrate towards <strong>the</strong> damaged<br />
hippocampal cell layers where <strong>the</strong>y differentiate into neurons <strong>an</strong>d never into astrocytes. We also<br />
provided evidence that cell grafting stimulates endogenous neurogenesis in <strong>the</strong> dentate gyrus.<br />
Using ex vivo electrophysiological recordings, we observe that olfactory stem cells re-establishe<br />
hippocampal synaptic tr<strong>an</strong>smission <strong>an</strong>d enh<strong>an</strong>ce long term potentiation. Finally, using <strong>an</strong><br />
associative memory test (<strong>the</strong> olfactory tubing maze) based on a hippocampal-depend<strong>an</strong>t task, we<br />
demonstrated that grafted olfactory stem cells restore learning <strong>an</strong>d memory abilities. Noticeably,<br />
a similar result was obtained when stem cells were grafted into <strong>the</strong> cerebrospinal fluid of<br />
lesioned mice.<br />
These studies pave <strong>the</strong> way <strong>for</strong> clinical trials based on autologous impl<strong>an</strong>tation of olfactory stem
cells after a memory loss consecutive to hypoxic injury or in patients with neurodegenerative<br />
diseases, similarly to what has been done with olfactory ensheathing cells in paraplegic patients.<br />
Disclosures: E. Nivet, None; B. Delorme, None; M. Vignes, None; J. Gaillard, None; T.<br />
Haupl, None; C. Pierrisnard, None; J. Ringe, None; N. Baril, None; A. Devèze, None; J.<br />
Magn<strong>an</strong>, None; J. Sohier, None; F. L<strong>an</strong>té, None; P. Charbord, None; L. Sensebé, None; P.<br />
Layrolle, None; M. Khrestchatisky, None; F. Féron, None; F.S. Rom<strong>an</strong>, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.21/B4<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Title: Non-cell line induced adult adipose-tissue derived stem cells promote erectile function<br />
after bilateral cavernous nerve crush injury in aged rats<br />
Authors: *T. M. FANDEL 1 , A. J. BELLA 2 , G. LIN 1 , C.-S. LIN 1 , T. F. LUE 1 ;<br />
1 2<br />
Urology, Knuppe Mol. Urology Lab., S<strong>an</strong> Fr<strong>an</strong>cisco, CA; Urology, Dept. of Neurosci. <strong>an</strong>d Div.<br />
of Urology, Ottawa, ON, C<strong>an</strong>ada<br />
Abstract: Purpose: To determine <strong>the</strong> effect of non-cell line induced adult adipose tissue-derived<br />
stem cells (ADSC) in preserving erectile function after bilateral cavernous nerve crush injury in<br />
aged rats.<br />
Materials <strong>an</strong>d Methods: ADSC were isolated <strong>from</strong> adipose tissue specimen <strong>from</strong> 28 aged (18<br />
month old) male rats. Animals were divided into 4 groups: 1) Laparotomy with no nerve injury<br />
(n=9), 2) Laparotomy with cavernous nerve crush <strong>an</strong>d intracorporal injection of saline (n=4), 3)<br />
Laparotomy with cavernous nerve crush <strong>an</strong>d intracorporal injection of saline with PLGA (n=6)<br />
<strong>an</strong>d 4) Laparotomy with cavernous nerve crush <strong>an</strong>d intracorporal injection of ADSC with PLGA<br />
(n=9). Four weeks after injury all <strong>an</strong>imals underwent functional assessment of intracavernous<br />
pressure (ICP) during electrical stimulation of <strong>the</strong> cavernous nerve. Me<strong>an</strong> arterial pressure<br />
(MAP) was determined by aortic c<strong>an</strong>nulation. Immunohistochemistry was utilized to assess<br />
nNOS positivity in <strong>the</strong> dorsal nerve of <strong>the</strong> penis. The number of cells positive <strong>for</strong> nNOS was<br />
qu<strong>an</strong>titated by microscopy. One way <strong>an</strong>alysis of vari<strong>an</strong>ce with post-testing was used to assess<br />
statistical signific<strong>an</strong>ce.<br />
Results: Me<strong>an</strong> ICP/MAP ratio was 0.52 +/-0.2, 0.16+/-.021, 0.20+/-0.11, <strong>an</strong>d 0.43+/-0.14 <strong>for</strong><br />
groups 1, 2, 3, <strong>an</strong>d 4, respectively. ICP/MAP ratio was signific<strong>an</strong>tly greater in sham <strong>an</strong>imals<br />
relative to injured controls (p
<strong>an</strong>imals (p
neural differentiation by PBD-MAPCs, we find that extracellular matrix composition is key to<br />
initial development of neural morphology <strong>an</strong>d behavior, including extension of processes with<br />
growth cone-like endings <strong>an</strong>d leading process migration. Soluble factors or cytokines may be<br />
more import<strong>an</strong>t in adv<strong>an</strong>ced differentiation of specific neural subtypes after initial development<br />
of a neural lineage. PBD-MAPCs also survive <strong>an</strong>d develop neural phenotypes when introduced<br />
into rat brains. We <strong>the</strong>re<strong>for</strong>e tr<strong>an</strong>spl<strong>an</strong>ted PBD-MAPCs into <strong>the</strong> striatum of a rodent model of<br />
Parkinson’s disease <strong>an</strong>d observed improvement of symptoms. Nigrostriatal dopamine neurons in<br />
rats were unilaterally lesioned with 6-hydroxydopamine injection to <strong>the</strong> medial <strong>for</strong>ebrain bundle<br />
<strong>an</strong>d rats were tested weekly until <strong>the</strong>y achieved a const<strong>an</strong>t rate of turns in 30 minutes in response<br />
to <strong>an</strong> apomorphine challenge. Primordial PBD-MAPC spheroids were delivered to <strong>the</strong> striatum<br />
on <strong>the</strong> lesioned side <strong>an</strong>d <strong>an</strong>imals were again tested <strong>for</strong> apomorphine-induced turning once a<br />
week. Within two weeks of tr<strong>an</strong>spl<strong>an</strong>t, rats receiving PBD-MAPCs made signific<strong>an</strong>tly fewer<br />
turns in response to apomorphine th<strong>an</strong> be<strong>for</strong>e tr<strong>an</strong>spl<strong>an</strong>t. Because PBD-MAPCs inherently<br />
express GFP, we were able to assess <strong>the</strong> degree of survival <strong>an</strong>d integration of tr<strong>an</strong>spl<strong>an</strong>ted cells<br />
in <strong>the</strong> host tissue <strong>an</strong>d correlate this with behavioral improvements. The physiological similarity<br />
of swine <strong>an</strong>d hum<strong>an</strong>s suggests that a similar population of stem cells may be present in hum<strong>an</strong><br />
peripheral blood. Such cells would hold great promise <strong>for</strong> development of autologous tr<strong>an</strong>spl<strong>an</strong>t<br />
<strong>the</strong>rapies of disorders such as Parkinson’s, <strong>for</strong> which stem cell <strong>the</strong>rapies have been proposed.<br />
Alternatively, swine are recognized as ideal xenotr<strong>an</strong>spl<strong>an</strong>t donors <strong>for</strong> hum<strong>an</strong>s, <strong>an</strong>d PBD-<br />
MAPCs <strong>from</strong> this source may also prove useful in <strong>the</strong> development of effective <strong>the</strong>rapies.<br />
Disclosures: N. Spitzer, None; G.S. Sammons, None; E.M. Price, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.23/B6<br />
Topic: A.03.b. Stem cells: Basic biology II: Expression patterns, properties, <strong>an</strong>d responses<br />
Title: Soluble factors expressed by mesenchymal stem cells isolated <strong>from</strong> <strong>the</strong> bone marrow,<br />
adipose tissue <strong>an</strong>d umbilical cord act differently on central nervous system derived cell<br />
populations<br />
Authors: *A. J. SALGADO 1 , C. RIBEIRO 1 , J. FRAGA 1 , B. GOH 2 , K. HAMEL 2 , N. NEVES 3 ,<br />
J. GIMBLE 2 , R. REIS 3 , N. SOUSA 1 ;<br />
1 Univ. Minho, Braga, Portugal; 2 2- Stem Cell Laboratory, Pennington Biomed. Res. Ctr., Baton<br />
Rouge, LA; 3 3- 3B’s Res. Group – Biomaterials, Biodegradable <strong>an</strong>d Biomimetics, Braga,<br />
Portugal
Abstract: Currently <strong>the</strong>re are no effective treatments <strong>for</strong> major hum<strong>an</strong> brain <strong>an</strong>d spinal cord<br />
injuries/disorders, fact that commonly leads to serious social <strong>an</strong>d personal costs <strong>from</strong> both <strong>the</strong><br />
afflicted victims <strong>an</strong>d <strong>the</strong>ir families. In <strong>the</strong> last decade stem/progenitor cells have been put<br />
<strong>for</strong>ward as a possible tr<strong>an</strong>spl<strong>an</strong>tation <strong>an</strong>d <strong>the</strong>rapeutically source <strong>for</strong> CNS related applications.<br />
Several precursor <strong>an</strong>d stem cell populations have been tr<strong>an</strong>spl<strong>an</strong>ted into <strong>the</strong> injured/degenerating<br />
CNS of <strong>an</strong>imal models including mesenchymal like stem cells obtained <strong>from</strong> <strong>the</strong> bone marrow<br />
(MSCs), adipose tissue (ASCs) <strong>an</strong>d <strong>the</strong> Wharton jelly matrix of <strong>the</strong> umbilical cord<br />
(UCSCs/HUCPVCs). However it is still not clear whe<strong>the</strong>r <strong>the</strong>y have similar or opposite effects<br />
on <strong>the</strong> different CNS derived cell populations. In this sense <strong>the</strong> objective of <strong>the</strong> present work was<br />
to underst<strong>an</strong>d if MSCs, ASCs <strong>an</strong>d HUCPVCs preferentially act, or not, on specific cell<br />
populations within <strong>the</strong> CNS. Hippocampal neurons <strong>an</strong>d cortical glial cells were exposed to MSCs<br />
ASCs <strong>an</strong>d HUCPVCs conditioned media (CM) (obtained 24, 48, 72 <strong>an</strong>d 96 hours after 3 days of<br />
culture) <strong>for</strong> 1 week. Cell viability experiments (MTS test) revealed that CM obtained <strong>for</strong> both<br />
cell populations at all time points did not cause <strong>an</strong>y deleterious effects on neurons <strong>an</strong>d glial cells.<br />
Immunocytochemistry <strong>an</strong>d total cell counts revealed that hippocampal cultures incubated with<br />
ASCs <strong>an</strong>d HUCPVCs CM displayed higher numbers of neurons (MAP-2 positive cells) when<br />
compared to <strong>the</strong> control. However this effect was higher <strong>for</strong> ASCs CM (up to 6 fold) when<br />
compared to HUCPVCs CM (up to 2 fold), whenever FGF-2 <strong>an</strong>d B27 supllement were present in<br />
<strong>the</strong> media. In <strong>the</strong> absence of <strong>the</strong> latter HUCPVCs CM incubated cultures disclosed higher<br />
number of differentiated neurons (up to two fold). Fur<strong>the</strong>rmore it was also observed in glial cell<br />
cultures that MSCs <strong>an</strong>d ASCs CM preferentially stimulated astrocytes/oligodendrocytes <strong>an</strong>d<br />
microglia proliferation, respectivelly, while <strong>the</strong> numbers of microglial cells were reduced<br />
whenever incubated with HUCPVCs CM. Similar effects on hippocampal neurons populations<br />
were observed in direct contact co-culture systems. The work here in presented suggests that<br />
MSCs, ASCs <strong>an</strong>d HUCPVCs disclose a different secretome profile that specifically targets<br />
different CNS derived cell populations. We are currently <strong>an</strong>alysing <strong>the</strong> levels of expression of<br />
import<strong>an</strong>t trophic factors such as NGF, BDNF, GDNF, VEGF, HFG <strong>an</strong>d o<strong>the</strong>rs in order to<br />
underst<strong>an</strong>d <strong>the</strong> observed differences.<br />
Disclosures: A.J. Salgado, None; C. Ribeiro, None; J. Fraga, None; B. Goh, None; K.<br />
Hamel, None; N. Neves, None; J. Gimble, None; R. Reis, None; N. Sousa, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.24/B7<br />
Topic: A.03.e. Stem cells: Postnatal neurogenesis I: Regulatory mech<strong>an</strong>isms <strong>an</strong>d genetic models
Support: USPHS gr<strong>an</strong>t NS038281<br />
Nor<strong>the</strong>ast Engl<strong>an</strong>d Stem Cell Institute<br />
Title: Deregulation of neurogenic signaling pathways in <strong>the</strong> absence of <strong>the</strong> norepinephrine<br />
tr<strong>an</strong>sporter (NET)<br />
Authors: *M. SIEBER-BLUM 1 , Y. F. HU 2 ;<br />
1 Nor<strong>the</strong>ast Engl<strong>an</strong>d Stem Cell Inst., Newcastle Univ., Newcastle upon Tyne, United Kingdom;<br />
2 Cell Biology, Neurobio. <strong>an</strong>d Anat., Med. Col. of Wisconsin, Milwaukee, WI<br />
Abstract: By comparing <strong>the</strong> tr<strong>an</strong>scriptome of NET-deficient <strong>an</strong>d wild type mouse neural crest<br />
cells, we have identified a profile of differentially expressed genes. NET is <strong>the</strong> target of<br />
psychotropic subst<strong>an</strong>ces, such as tricyclic <strong>an</strong>tidepress<strong>an</strong>ts <strong>an</strong>d <strong>the</strong> drug of abuse, cocaine. NET<br />
mutations have been implicated in depression, <strong>an</strong>xiety, orthostatic intoler<strong>an</strong>ce <strong>an</strong>d attention<br />
deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of <strong>the</strong><br />
peripheral <strong>an</strong>d central nervous systems is to internalize norepinephrine <strong>from</strong> <strong>the</strong> synaptic cleft.<br />
By contrast, during embryogenesis norepinephrine (NE) tr<strong>an</strong>sport promotes differentiation of<br />
neural crest stem cells <strong>an</strong>d locus ceruleus progenitors into noradrenergic neurons, whereas NET<br />
inhibitors block noradrenergic differentiation. Comparison <strong>an</strong>alyses have identified a number of<br />
import<strong>an</strong>t differentially expressed genes, including genes relev<strong>an</strong>t to neural crest <strong>for</strong>mation,<br />
noradrenergic neuron differentiation <strong>an</strong>d <strong>the</strong> phenotype of NETKO mice. Examples of<br />
differentially expressed genes that affect noradrenergic cell differentiation include bone<br />
morphogenetic protein signaling pathways, <strong>the</strong> Phox2b binding partner Tlx2, <strong>the</strong> ubiquitin ligase<br />
Praja2, <strong>an</strong>d <strong>the</strong> inhibitor of Notch signaling, Numbl. Differentially expressed genes that are<br />
likely to contribute to <strong>the</strong> NETKO mouse phenotype include dopamine-β-hydroxylase (Dbh),<br />
tyrosine hydroxylase (Th), <strong>the</strong> peptide tr<strong>an</strong>smitter ‘cocaine <strong>an</strong>d amphetamine regulated<br />
tr<strong>an</strong>script’ (Cart), <strong>an</strong>d <strong>the</strong> serotonin receptor subunit Htr3a. Real-time PCR confirmed<br />
differential expression of key genes not only in neural crest cells, but also in <strong>the</strong> adult superior<br />
cervical g<strong>an</strong>glion <strong>an</strong>d locus ceruleus. In addition to known genes we have identified novel<br />
differentially expressed genes <strong>an</strong>d thus provide a valuable database <strong>for</strong> future studies. We<br />
conclude that loss of NET function during mouse embryonic development deregulates signaling<br />
pathways that are critically involved in neural crest <strong>for</strong>mation <strong>an</strong>d noradrenergic cell<br />
differentiation. The data fur<strong>the</strong>r suggest deregulation of signaling pathways in <strong>the</strong> development<br />
<strong>an</strong>d/or function of <strong>the</strong> NET-deficient peripheral, central <strong>an</strong>d enteric nervous systems.<br />
Disclosures: M. Sieber-Blum, None; Y.F. Hu, None.<br />
Poster<br />
507. Stem Cells: Basic Biology II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 507.25/B8<br />
Topic: A.03.g. Embryonic Stem Cells: Neural differentiation<br />
Title: Microarray-based profiling of gene expression in neural stem cells<br />
Authors: *Y. KIM 1 , C. SOO YOUNG 2 , K. SEUNG UP 2 , L. YOUNG SIK 2 , L. MYUNG AE 2 ;<br />
1 2<br />
ajou univercity school of medicine, Suwon, Republic of Korea; Ajou Univ. Sch. of Med.,<br />
suwon, Republic of Korea<br />
Abstract: Microarray-based Profiling of Gene Expression in Neural Stem Cells.<br />
Neurogenesis <strong>an</strong>d neural precursor/stem cell biology in adult CNS are import<strong>an</strong>t both toward<br />
potential future <strong>the</strong>rapeutic applications <strong>for</strong> CNS repair, <strong>an</strong>d regarding <strong>the</strong> fundamental function<br />
of <strong>the</strong> CNS. Nurr1, a tr<strong>an</strong>scription factor belonging to <strong>the</strong> orph<strong>an</strong> nuclear receptor superfamily, is<br />
critical in <strong>the</strong> development <strong>an</strong>d mainten<strong>an</strong>ce of <strong>the</strong> dopaminergic system. Experimental studies in<br />
Nurr1 knock-out mice indicate that Nurr1 deficiency results in impaired dopaminergic function<br />
<strong>an</strong>d increased vulnerability of those midbrain dopaminergic neurons that degenerate in PD. To<br />
investigate possible molecular cascades that are under <strong>the</strong> control of Nurr1 in dopaminergic<br />
neurogenesis, we compared <strong>the</strong> gene-expression profiles of hum<strong>an</strong> NSC lines <strong>an</strong>d Nurr1overexpressing<br />
hum<strong>an</strong> NSC lines using Affymetrix GeneChip Hum<strong>an</strong> Genome U133 Plus 2.0<br />
arrays. We identified that 189 genes have Nurr1-binding site in <strong>the</strong>ir promoter. However, Only<br />
29 genes were found to be up- or down-regulated by Nurr1 in all 3 Nurr1-overexpressing NSCs.<br />
To determine <strong>the</strong> validity of results obtained by <strong>the</strong> microarray <strong>an</strong>alysis, twelve selected genes<br />
that are up-regulated including Pleiotrophin, Neuromedin U, Calponin1, etc. <strong>an</strong>d are downregulated<br />
including G0S2, Gal<strong>an</strong>in were subjected to semi-qu<strong>an</strong>titative real-time RT-PCR<br />
<strong>an</strong>alysis. These expression pattern observed by real-time RT-PCR showed a good concord<strong>an</strong>ce<br />
with <strong>the</strong> pattern <strong>an</strong>alyzed by microarray. To confirm if genes identified in this survey are direct<br />
targets of Nurr1, we per<strong>for</strong>med luciferase reporter gene <strong>an</strong>alysis <strong>an</strong>d EMSA of <strong>the</strong> NMU <strong>an</strong>d<br />
G0S2 promoters. To <strong>an</strong>alyze whe<strong>the</strong>r Nurr1 gene targets identified in NSCs were also targets in<br />
o<strong>the</strong>r cell type, NMU, PTN, BAALC <strong>an</strong>d G0S2 mRNA levels were assessed in HEK293 cells<br />
tr<strong>an</strong>sduced with a Nurr1 expression vector. Genes regulated by Nurr1 that we identified in this<br />
study may play essential roles in dopaminergic neurogenesis. In addition, identification of genes<br />
controlling <strong>the</strong> development of central DA neurons may provide new insight into <strong>the</strong> molecular<br />
mech<strong>an</strong>isms leading to midbrain DA neurogenesis <strong>an</strong>d <strong>the</strong> etiology of DA neuronal cell deaths in<br />
PD.<br />
Disclosures: Y. Kim, None; C. Soo Young, None; K. Seung Up, None; L. Young Sik,<br />
None; L. Myung Ae, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.1/B9<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Title: Morphologic effect of dexamethasone on dendritic length of CA3 hippocampus neurons<br />
Authors: *A. B. SILVA, L. F. LEÓN, A. L. DE GANTE;<br />
Escuela de Biología, Benemérita Univ. Autónoma De Puebla, Puebla, Mexico<br />
Abstract: Glucocorticoids are steroids hormones that c<strong>an</strong> cross <strong>the</strong> blood barrier <strong>an</strong>d <strong>the</strong>n c<strong>an</strong> be<br />
in all <strong>the</strong> brain’s structures in few minutes since <strong>the</strong>ir secretion by adrenal cortex. (S<strong>an</strong>di, 2003).<br />
The hippocampus is a brain’s structure, express high concentrations of glucocorticoid’s receptors<br />
that have been implicated with behavioral response to stress (Me<strong>an</strong>ey y cols., 1988). However,<br />
high concentration of glucocorticoids c<strong>an</strong> to cause hipocampal impairment with hormonal,<br />
cognitive alterations <strong>an</strong>d affective disorders (Valencia-Alfonso et al., 2004).<br />
In adults rats, chronic corticosterone administration results in reduced apical dendritic length of<br />
CA3 neurons (Woolley y cols., 1990) <strong>an</strong>d reorg<strong>an</strong>ization of apical dendrites in medial prefrontal<br />
cortex (Wellm<strong>an</strong>, 2001).<br />
Dexamethasone is a syn<strong>the</strong>tic glucocorticoid that provokes ch<strong>an</strong>ges in learning <strong>an</strong>d memory in<br />
rats <strong>an</strong>d profound ultrastructural ch<strong>an</strong>ges in some pyramidal neurons in <strong>the</strong> region CA3<br />
(D<strong>an</strong>ilczuk, et al., 2006, Haynes, et al., 2004). In order to determine whe<strong>the</strong>r treatment with<br />
dexamethasone cause morphologic alterations in CA3 hippocampus neurons, male Wistar rats<br />
(21 postnatal days) received 0.2 mg/Kg by five or fourteen days. Control rats received 1 ml/Kg<br />
vehicle. One week after, each <strong>an</strong>imal was tr<strong>an</strong>scardially perfused under barbiturate <strong>an</strong>aes<strong>the</strong>sia<br />
(70 mg/Kg sodium pentobarbital) using phosphate buffer (pH 7.4). The brains were removed <strong>an</strong>d<br />
processed <strong>for</strong> Golgi-Cox staining (Gibb <strong>an</strong>d Kolb, 1998). After, <strong>the</strong> brains were <strong>the</strong>n blocked <strong>an</strong>d<br />
sliced coronally in 200µm thick sections at level of hippocampus. The tissue was placed in<br />
ammonium hydroxide <strong>an</strong>d Kodak film fixer <strong>an</strong>d <strong>the</strong>n dehydrated with alcohol <strong>an</strong>d xylene.<br />
Hippocampal neurons were drawn using camera lucida at magnification of 400x. On <strong>the</strong> draws,<br />
<strong>the</strong> neurons were <strong>an</strong>alyzed with Sholl <strong>an</strong>alysis (Sholl, 1953) to qu<strong>an</strong>tify <strong>the</strong> basal <strong>an</strong>d apical<br />
dendritic length. Too, <strong>the</strong> density of spines dendritic was estimated on tracings of a 10 µm long<br />
terminal segment of basal <strong>an</strong>d apical dendritic br<strong>an</strong>ch (1000x magnification).<br />
The short treatment with dexamethasone (5 days) did not effect on <strong>the</strong> dendritic length of CA3<br />
neurons, while <strong>the</strong> long treatment (14 days) did provoke less dendritic arborization. In relation to<br />
density of spine dendritic, we observed reduced density in short treatment but <strong>the</strong> spine dendritic<br />
of neurons treated along 14 day don’t have a good staining impregnation, possibly owing to high<br />
degree of exposure to dexamethasone, inhibit glucose utilization in <strong>the</strong> hippocampus (Me<strong>an</strong>ey y<br />
cols., 1988).<br />
Disclosures: A.B. Silva, None; L.F. León, None; A.L. de G<strong>an</strong>te, None.
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.2/B10<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: NIH gr<strong>an</strong>t NS21184<br />
NIH gr<strong>an</strong>t NS33574<br />
NIH gr<strong>an</strong>t EB00217<br />
Title: Revisiting <strong>the</strong> developmental onset of long-term potentiation in rat hippocampal area CA1<br />
Authors: *G. CAO, K. M. HARRIS;<br />
Ctr. <strong>for</strong> Learning <strong>an</strong>d Memory, Univ. Texas, Austin, Austin, TX<br />
Abstract: Long-term potentiation (LTP), <strong>the</strong> persistent increase in synaptic efficacy induced by<br />
high-frequency stimulation, is one of <strong>the</strong> most import<strong>an</strong>t models <strong>for</strong> studying cellular<br />
mech<strong>an</strong>isms of learning <strong>an</strong>d memory. We previously reported that enduring LTP, lasting more<br />
th<strong>an</strong> 3 hr, emerges by postnatal day 15 in rat hippocampal area CA1 when LTP was induced<br />
using a tet<strong>an</strong>us stimulation protocol of 2 trains of 100 pulses at 100Hz with a 20s interval (Harris<br />
<strong>an</strong>d Teyler, 1985; Jackson et al., 1995). At younger ages, PTP lasting less th<strong>an</strong> 1 minute was first<br />
induced at day 4 <strong>an</strong>d short-term potentiation lasting 1-1.5 hr was first induced at day 11. It has<br />
been our hypo<strong>the</strong>sis that enduring LTP requires dendritic spines because mature dendritic spines<br />
are first prevalent at day 15 (Harris et al., 1992). However, no systematic <strong>an</strong>alysis of spine<br />
<strong>for</strong>mation or alternative LTP induction protocols have been conducted between day 11 <strong>an</strong>d day<br />
15 when spines are presumably generated as well. Here we used <strong>the</strong>ta-burst stimulation (8 bursts<br />
with 30s interval, each burst containing 10 trains of 4 pulses at 100Hz, with 200ms intervals<br />
between trains) to study LTP endur<strong>an</strong>ce at postnatal days 8-15. We confirmed that STP emerges<br />
at postnatal day 11, <strong>an</strong>d lasts less th<strong>an</strong> 2 hr, while enduring LTP lasting at least 4 hr first occurs<br />
at postnatal day 13. In order to test whe<strong>the</strong>r mature dendritic spines are needed <strong>for</strong> enduring LTP,<br />
we are using confocal fluorescence <strong>an</strong>d electron microscopy to investigate dendrites in more<br />
detail at all of <strong>the</strong> ages beginning at day 8 <strong>an</strong>d leading up to <strong>the</strong> threshold age day 13 <strong>for</strong><br />
enduring LTP.<br />
Disclosures: G. Cao, None; K.M. Harris, None.
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.3/B11<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: Wellcome Trust PhD Studentship<br />
MRC Gr<strong>an</strong>t<br />
Title: Imaging local calcium signals <strong>an</strong>d chloride regulation during dendritic development<br />
Authors: *W. WEFELMEYER, C. J. AKERMAN;<br />
Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom<br />
Abstract: Intracellular chloride concentration is tightly regulated throughout development,<br />
which is most notable in <strong>the</strong> switch of <strong>the</strong> gamma-Aminobutyric acid type A (GABA-A) receptor<br />
system <strong>from</strong> depolarising to hyperpolarising. However, we still know very little about <strong>the</strong> role of<br />
chloride regulation in activity-dependent neuronal development. Dendritic growth in particular is<br />
a key step in <strong>the</strong> establishment of neuronal circuits <strong>an</strong>d <strong>for</strong> proper neuronal integration <strong>an</strong>d<br />
function. The morphological development of neurons has been shown to be affected by calciummediated<br />
signalling events (Lohm<strong>an</strong>n et al. 2005; Nat Neurosci) <strong>an</strong>d we have developed methods<br />
that allow <strong>for</strong> precise spatial <strong>an</strong>alysis of local calcium signals within <strong>the</strong> dendrites of Cornu<br />
Ammonis area one (CA1) hippocampal pyramidal neurons. In org<strong>an</strong>otypic hippocampal slices of<br />
postnatal-day one (P1) Wistar rats, we observed a strong preference <strong>for</strong> calcium signals to occur<br />
at established dendritic br<strong>an</strong>chpoints <strong>an</strong>d at filopodia, which are highly dynamic parts of <strong>the</strong><br />
growing dendrite. GABA-A receptor <strong>an</strong>tagonists caused a selective reduction in <strong>the</strong> frequency of<br />
local calcium signals at filopodia, but not at o<strong>the</strong>r locations within <strong>the</strong> dendrite. This is consistent<br />
with a spatially restricted role <strong>for</strong> depolarising GABA signals during dendritic growth. To<br />
underst<strong>an</strong>d whe<strong>the</strong>r this selective GABA-A receptor effect is underpinned by a tight spatial<br />
regulation of chloride concentration, we have employed a chloride-sensitive fluorescent protein<br />
to optically measure intracellular chloride. Immature CA1 neurons in hippocampal slices were<br />
tr<strong>an</strong>sfected with <strong>the</strong> ratiometric, chloride-sensitive fluorescence reson<strong>an</strong>ce energy tr<strong>an</strong>sfer<br />
(FRET) protein, Cl-Sensor (Markova et al. 2008; J Neurosci Methods). This enables us to<br />
per<strong>for</strong>m long-term, time-lapse two-photon imaging experiments <strong>an</strong>d has revealed spatial<br />
differences in individual dendrites. We are currently relating <strong>the</strong>se differences to dendritic<br />
growth dynamics. Toge<strong>the</strong>r, this work fur<strong>the</strong>rs our underst<strong>an</strong>ding of <strong>the</strong> local regulation of<br />
signalling mech<strong>an</strong>isms that may underlie dendritic development.<br />
Disclosures: W. Wefelmeyer, None; C.J. Akerm<strong>an</strong>, None.
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.4/B12<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: NSC-96-2628-B-002-053-MY3<br />
Title: Membr<strong>an</strong>e <strong>an</strong>d morphological properties of subplate neurons in <strong>the</strong> developing<br />
somatosensory neocortex of rat<br />
Authors: *C.-C. LIAO 1 , L.-J. LEE 1,2 ;<br />
1 Anat. <strong>an</strong>d Cell Biol., 2 Neurobio. <strong>an</strong>d Cognitive Sci. Ctr., Natl. Taiw<strong>an</strong> Univ., Taipei, Taiw<strong>an</strong><br />
Abstract: Subplate neurons are import<strong>an</strong>t <strong>for</strong> <strong>the</strong> development of <strong>the</strong> neocortex. However, <strong>the</strong><br />
structural <strong>an</strong>d functional maturation of <strong>the</strong>se cells are not fully understood. Using brain slices<br />
whole-cell patch clamp recording <strong>an</strong>d intracellular neurobiotin labeling techniques, we aimed to<br />
study <strong>the</strong> electrophysiological <strong>an</strong>d morphological features of subplate neurons in <strong>the</strong><br />
somatosensory neocortex of rat during <strong>the</strong> first postnatal month. In <strong>the</strong> physiological aspect,<br />
dramatic ch<strong>an</strong>ges occurred in <strong>the</strong> early postnatal days but reached <strong>the</strong> plateau by <strong>the</strong> end of <strong>the</strong><br />
second week. The resting membr<strong>an</strong>e potential became more negative while <strong>the</strong> input resist<strong>an</strong>t<br />
<strong>an</strong>d time const<strong>an</strong>t were reduced. The threshold <strong>for</strong> generating action potential was getting lower<br />
<strong>an</strong>d <strong>the</strong> spike amplitude was increased. Accordingly, <strong>the</strong> half amplitude duration <strong>an</strong>d <strong>the</strong><br />
maximum rise <strong>an</strong>d decay rate of action potential were decreased. The recorded neurons were<br />
simult<strong>an</strong>eously labeled with neurobiotin <strong>an</strong>d <strong>the</strong>n reconstructed <strong>for</strong> morphological <strong>an</strong>alysis. The<br />
dendrosomatic parameters also exhibited developmental ch<strong>an</strong>ges. Exuber<strong>an</strong>t yet uni<strong>for</strong>m<br />
dendritic extension <strong>an</strong>d arborization mainly occurred during <strong>the</strong> first week, whereas prominent<br />
dendritic remodeling including terminal pruning <strong>an</strong>d elongation took place in <strong>the</strong> second week.<br />
Taken toge<strong>the</strong>r, <strong>the</strong>se results suggested that <strong>the</strong> subplate neurons in <strong>the</strong> somatosensory neocortex<br />
mature rapidly within two weeks. This developmental profile of subplate corresponds with <strong>the</strong><br />
maturation of thalamocortical projection <strong>an</strong>d may play import<strong>an</strong>t role <strong>for</strong> <strong>the</strong> critical period<br />
plasticity.<br />
Disclosures: C. Liao, None; L. Lee, None.<br />
Poster
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.5/B13<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Title: The age-dependent ch<strong>an</strong>ges of three-dimensional org<strong>an</strong>ization of rats’ star-shaped neurons<br />
in <strong>the</strong> cerebral cortex<br />
Authors: *M. NEBIERIDZE, N. MITAGVARIA;<br />
Beritashvili Inst. of Physiol., Tbilisi, Georgia<br />
Abstract: Introduction. Revelation of aging caused structural-morphological ch<strong>an</strong>ges in <strong>the</strong><br />
brain is <strong>the</strong> major <strong>the</strong>oretical <strong>an</strong>d practical problem of modern biology. Among <strong>the</strong> various<br />
morphological ch<strong>an</strong>ges having place in <strong>the</strong> course of aging <strong>the</strong> damage of dendrites has a high<br />
functional value.<br />
The goal of <strong>the</strong> present study was <strong>an</strong>alysis of star-shaped neurons three-dimensional org<strong>an</strong>ization<br />
<strong>an</strong>d geometry of dendritic br<strong>an</strong>ching in <strong>the</strong> IV layer of sensorymotory <strong>an</strong>d auditory cortex of<br />
young (3-4 months) <strong>an</strong>d old (32-34 months) rats.<br />
Methods. The neurons revealed by Golgi method in 180-200 mkm thick frontal, sagittal <strong>an</strong>d<br />
horizontal sections were studied by me<strong>an</strong>s of a light microscope. The neurons, which dendrites<br />
were most intact in all directions were sketched by me<strong>an</strong>s of drawing apparatus. The origin of<br />
coordinates was located in <strong>the</strong> center of neurons’ body. Y axis was perpendicular to <strong>the</strong> cerebral<br />
surface, <strong>an</strong>d Y coordinates were defined <strong>from</strong> a sketch <strong>an</strong>d Z - <strong>from</strong> <strong>the</strong> microscope micro-screw<br />
indicator.<br />
The coordinates of all characteristic points, necessary <strong>for</strong> estimation of dendrites’ geometry were<br />
defined also <strong>from</strong> <strong>the</strong> drawing. Received data in <strong>the</strong> <strong>for</strong>m of matrixes were <strong>an</strong>alyzed <strong>for</strong> every<br />
selected neuron by me<strong>an</strong>s of special software <strong>an</strong>d <strong>the</strong> results were statistically evaluated<br />
(Students’ criteria).<br />
Results. In old rats’ brain reduction of <strong>the</strong> number of free br<strong>an</strong>ch terminals of star-shaped<br />
neurons, as well as <strong>the</strong> br<strong>an</strong>ching of dendrites, <strong>the</strong> maximal length of dendrites, <strong>the</strong> area of<br />
dendrites field, <strong>the</strong> total length of dendrites <strong>an</strong>d <strong>the</strong> volume, occupied by <strong>the</strong> dendritic fields of<br />
<strong>the</strong> neurons has been established.<br />
In <strong>the</strong> process of comparison qu<strong>an</strong>titative data received <strong>from</strong> frontal, sagittal <strong>an</strong>d horizontal<br />
sections, have to be taken in <strong>the</strong> account that to <strong>the</strong> X, Y <strong>an</strong>d Z axes on frontal sections<br />
corresponds Z, Y, X axis on sagittal sections <strong>an</strong>d Z, X, Y axis - on horizontal sections.<br />
Conclusion. It is established, that at aging dendritic projections of <strong>the</strong> star-shaped neurons<br />
decrease in all directions, except <strong>an</strong> axis “- Y” in sensorymotory cortex , <strong>an</strong>d in “-Z” axis<br />
direction in auditory cortex.<br />
Noted ch<strong>an</strong>ges specify that in process of aging <strong>the</strong> dendrites of star-shaped neurons partially<br />
undergo to atrophy <strong>an</strong>d/or degeneration of high order br<strong>an</strong>ches.
Disclosures: M. Nebieridze , None; N. Mitagvaria, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.6/B14<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: fellowship of <strong>the</strong> Interdisciplinary Neuroscience Graduate Program at Arizona State<br />
University<br />
Title: Dendritic tree <strong>an</strong>d dendritic territory org<strong>an</strong>ization of <strong>an</strong> adult Drosophila motoneuron<br />
Authors: *F. J. VONHOFF, C. DUCH;<br />
Arizona State Univ., Phoenix, AZ<br />
Abstract: Dendritic structure lays <strong>the</strong> bedrock <strong>for</strong> synaptic connections within a neural network.<br />
Dendritic shape is regulated by intrinsic factors, external molecular cues, humoral cues, neural<br />
activity, <strong>an</strong>d a variety of neuronal processes, such as dendritic tiling, pruning <strong>an</strong>d<br />
synaptogenesis. Structural abnormalities have been described in several <strong>for</strong>ms of mental<br />
retardation, e.g. Rett Syndrome. We use Drosophila mel<strong>an</strong>ogaster to identify molecular cues<br />
which regulate dendritic structure during <strong>the</strong> development of <strong>an</strong> identified adult motoneuron,<br />
MN5. This model offers <strong>the</strong> adv<strong>an</strong>tage to identify <strong>the</strong> same single neuron across different<br />
<strong>an</strong>imals, <strong>an</strong>d to target genetic m<strong>an</strong>ipulations without ch<strong>an</strong>ging <strong>the</strong> surrounding network. To study<br />
mech<strong>an</strong>isms controlling motoneuron dendritic architecture, we first established a qu<strong>an</strong>titative<br />
database of <strong>the</strong> neuron’s average morphology <strong>an</strong>d <strong>the</strong> natural variability across <strong>an</strong>imals. For this,<br />
MN5 was dye labeled <strong>an</strong>d reconstructed in 3 dimensions <strong>from</strong> confocal image stacks.<br />
Morphometric parameters such as total dendritic length, number of dendrites, dendritic radii,<br />
br<strong>an</strong>ch order distribution in 3 dimensional space show a coefficient of variation below 11%.<br />
Along <strong>the</strong> dendritic field, a fixed number of dendritic sub-trees c<strong>an</strong> be identified. Across <strong>an</strong>imals<br />
<strong>the</strong>se sub-trees differ in projection areas, size, <strong>an</strong>d shapes, but <strong>the</strong>y do not overlap with each<br />
o<strong>the</strong>r in 3-dimensional space, suggesting competitive interactions of dendritic trees <strong>from</strong> <strong>the</strong><br />
same neuron during development. Interactions between identified sub-trees of <strong>the</strong> same neuron<br />
would require individual sub-tree specific molecular cues. There<strong>for</strong>e, genetic m<strong>an</strong>ipulations<br />
should target sub-sets of dendrites only. We have identified one such c<strong>an</strong>didate regulator, <strong>the</strong><br />
tr<strong>an</strong>scription factor AP-1 (heterodimer of Fos <strong>an</strong>d Jun). Over-expressing AP-1 in adult MN5<br />
selectively inhibits dendritic growth of posterior sub-trees but also causes structural ch<strong>an</strong>ges in<br />
<strong>an</strong>terior sub-trees. This suggests that different parts of <strong>the</strong> dendritic field of one neuron may be
egulated by different tr<strong>an</strong>scription factors. This study provides impetus <strong>for</strong> <strong>the</strong> underst<strong>an</strong>ding of<br />
<strong>the</strong> mech<strong>an</strong>isms underlying <strong>the</strong> development of motoneuron dendritic architectures.<br />
Disclosures: F.J. Vonhoff, None; C. Duch, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.7/B15<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Title: A geometric expl<strong>an</strong>ation of dendritic local pl<strong>an</strong>arity<br />
Authors: *Y. KIM 1 , R. SINCLAIR 1 , E. DE SCHUTTER 1,2 ;<br />
1 Okinawa Inst. of Sci. <strong>an</strong>d Technol., Okinawa, Jap<strong>an</strong>; 2 Univ. of Antwerp, Antwerpen, Belgium<br />
Abstract: Pl<strong>an</strong>ar dendrites are observed in Purkinje cells <strong>an</strong>d retinal g<strong>an</strong>glion cells. Moreover,<br />
Uylings <strong>an</strong>d Smit (Uylings & Smit 1975) discovered that in pyramidal cells of rabbit visual<br />
cortex, dendritic br<strong>an</strong>ches are locally pl<strong>an</strong>ar, shown by a peak at 180° in <strong>the</strong> cone <strong>an</strong>gle<br />
distribution. In a later paper (Uylings & V<strong>an</strong> Pelt 2002), tension or <strong>for</strong>ced equilibria during <strong>the</strong><br />
growth process were suggested to be a potential cause of this phenomenon.<br />
We investigated whe<strong>the</strong>r local pl<strong>an</strong>arity was a general trait of most neuron types. Using eight<br />
different cell types we measured cone <strong>an</strong>gles of local br<strong>an</strong>ches <strong>an</strong>d confirmed that <strong>the</strong> local<br />
pl<strong>an</strong>arity was a general property of <strong>the</strong> neurons studied.<br />
Next, we examined whe<strong>the</strong>r local pl<strong>an</strong>arity was a consequence of physical constraints on<br />
biological neurons.<br />
To this end, we constructed a model with artificial geometrical entities based on r<strong>an</strong>dom<br />
sampling of points in space, but without including <strong>an</strong>y physical <strong>for</strong>ces such as tension. Local<br />
pl<strong>an</strong>ar structure, characterized by a peak at 180° in <strong>the</strong> cone <strong>an</strong>gle histogram, was observed in<br />
this study.<br />
However, <strong>the</strong> model cone <strong>an</strong>gle distribution had a longer tail, compared to that of real cells. We<br />
attempted to bring <strong>the</strong> model distribution closer to that of real cells by adding <strong>the</strong> constraint that<br />
<strong>the</strong> parent <strong>an</strong>d children segments lie in <strong>the</strong> same direction. This is a reasonable assumption, since<br />
dendritic growth is often toward a chemo-attract<strong>an</strong>t or a target area. The resulting <strong>an</strong>gle<br />
distribution had a shorter tail, comparable to that of real cells.<br />
We conclude that <strong>the</strong> local pl<strong>an</strong>ar structure of dendrites c<strong>an</strong> be largely explained without <strong>an</strong>y<br />
physical constraints.
Disclosures: Y. Kim, None; R. Sinclair, None; E. De Schutter, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.8/B16<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: NIAAA Gr<strong>an</strong>t AA017354<br />
Title: Dendritic remodeling over <strong>the</strong> adolescent period in <strong>the</strong> basolateral amygdala of male <strong>an</strong>d<br />
female rats<br />
Authors: *W. A. KOSS 1 , C. E. BELDEN 2 , S. K. DECKER 2 , J. M. JURASKA 2 ;<br />
2 Dept. of Psychology, 1 Univ. Illinois, Champaign, IL<br />
Abstract: Studies in hum<strong>an</strong>s <strong>an</strong>d rats indicate that <strong>the</strong> amygdala continues to develop throughout<br />
adolescence. Previous data <strong>from</strong> our laboratory has shown signific<strong>an</strong>t cell losses (both neurons<br />
<strong>an</strong>d glia) occurring between day 35 <strong>an</strong>d 90 in <strong>the</strong> basolateral amygdala (BLA) (Rubinow <strong>an</strong>d<br />
Juraska, JCN, <strong>2009</strong>). Interestingly, <strong>the</strong> volume of <strong>the</strong> BLA did not ch<strong>an</strong>ge to reflect <strong>the</strong> cell loss.<br />
Fur<strong>the</strong>rmore <strong>the</strong>re was a signific<strong>an</strong>t increase in volume without ch<strong>an</strong>ge in cell number <strong>from</strong> day<br />
20 to 35. These findings have led us to <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> volume ch<strong>an</strong>ges may be due to<br />
signific<strong>an</strong>t dendritic growth <strong>an</strong>d remodeling. In <strong>the</strong> current study, male <strong>an</strong>d female Long Ev<strong>an</strong><br />
hooded rats were sacrificed at days 20, 35, <strong>an</strong>d 90, <strong>an</strong>d <strong>the</strong>ir brains were stained with Golgi-Cox.<br />
Both dendritic br<strong>an</strong>ching <strong>an</strong>d spine density are being measured. We have found a signific<strong>an</strong>t<br />
effect of age in spine density caused primarily by a signific<strong>an</strong>t increase <strong>from</strong> day 20 to day 35.<br />
Dendritic br<strong>an</strong>ching measurements are currently ongoing. A complete <strong>an</strong>alysis of dendritic tree<br />
length <strong>an</strong>d arborization will be presented. The continued growth of <strong>the</strong> dendritic tree in <strong>the</strong> BLA<br />
may contribute to <strong>the</strong> high sensitivity of adolescents to environmental influences.<br />
Disclosures: W.A. Koss, None; C.E. Belden, None; S.K. Decker, None; J.M. Juraska, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.9/B17<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: NIH Gr<strong>an</strong>t NS21184<br />
NIH Gr<strong>an</strong>t NS33574<br />
NIH Gr<strong>an</strong>t EB002170<br />
Title: Do spines <strong>for</strong>m first on apical or oblique dendrites during development in <strong>the</strong> rat<br />
hippocampus?<br />
Authors: *A. HAESSLY 1 , B. SHI 1 , J. N. BOURNE 1 , J. M. MENDENHALL 1 , J. SPACEK 2 , K.<br />
M. HARRIS 1 ;<br />
1 Ctr. <strong>for</strong> Learning <strong>an</strong>d Memory, Inst. Neurosci, Austin, TX; 2 The Fingerl<strong>an</strong>d Dept of Pathology,<br />
Charles Univ. Hosp., Hradec Kralove, Czech Republic<br />
Abstract: To address this question spine density was qu<strong>an</strong>tified using 3D reconstruction <strong>from</strong><br />
serial section tr<strong>an</strong>smission electron microscopy. Dendrites in stratum radiatum of CA1 pyramidal<br />
cells <strong>from</strong> perfusion fixed rat brain at mature ages (days 65-75) were compared to <strong>an</strong> immature<br />
age (day 21). The dendrites were grouped into two categories with smaller caliber dendrites<br />
r<strong>an</strong>ging <strong>from</strong> 0.3-0.8 µm <strong>an</strong>d larger caliber dendrites r<strong>an</strong>ging <strong>from</strong> 0.85-2.27 microns in<br />
diameter. In mature area CA1, <strong>the</strong> smaller caliber dendrites had a lower linear spine density<br />
(2.6±1.5 spines/µm, (me<strong>an</strong> ± s.d.)) th<strong>an</strong> <strong>the</strong> larger caliber dendrites (10.5 ± 3.2 spines/µm,<br />
p
Disclosures: A. Haessly, None; B. Shi, None; J.N. Bourne, None; J.M. Mendenhall, None; J.<br />
Spacek, None; K.M. Harris, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.10/B18<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: DFG DU 331/4-1<br />
Title: Sub-dendritic targeting of GABAergic <strong>an</strong>d cholinergic synapses in a Drosophila motor<br />
neuron<br />
Authors: C. KUEHN, *C. DUCH;<br />
Arizona State Univ., Tempe, AZ<br />
Abstract: For efficient synaptic tr<strong>an</strong>smission, postsynaptic neuronal receptors need to be<br />
localized in precise apposition to presynaptic terminals that release <strong>the</strong> neurotr<strong>an</strong>smitter.<br />
Modelling <strong>an</strong>d electrophysiological studies have shown that <strong>the</strong> location of input synapses in<br />
complex dendritic trees c<strong>an</strong> affect <strong>the</strong> computational properties of <strong>the</strong> postsynaptic neuron. Here<br />
we investigate whe<strong>the</strong>r sub-dendritic targeting <strong>an</strong>d clustering occurs in a tr<strong>an</strong>smitter specific<br />
m<strong>an</strong>ner in complex dendritic trees of <strong>the</strong> identified flight motor neuron MN5, located in a nonlayered<br />
neuropil in Drosophila mel<strong>an</strong>ogaster. Nicotinic acetylcholine receptors containing α7<br />
subunits mediate fast excitatory signaling at most synapses in <strong>the</strong> Drosophila escape circuit,<br />
including synapses to MN5. The most abund<strong>an</strong>t fast inhibitory receptors in <strong>the</strong> insect CNS are<br />
ionotropic GABA receptors. We use immunocytochemistry <strong>an</strong>d targeted expression of tagged<br />
receptors to map <strong>the</strong> expression of <strong>the</strong> RDL subunit of <strong>the</strong> GABAAR <strong>an</strong>d <strong>the</strong> α7 subunit of <strong>the</strong><br />
nicotinic AChR onto 3 dimensional geometric reconstructions of <strong>the</strong> MN5. We find a distinct<br />
spatial pattern <strong>for</strong> <strong>the</strong> distribution of GABAARs. Interestingly, <strong>the</strong> majority are clustered on<br />
identified dendritic sub-trees in <strong>the</strong> <strong>an</strong>terior distal region of <strong>the</strong> dendritic tree, near <strong>the</strong> spike<br />
initiating zone. There<strong>for</strong>e, specific sub-dendritic recognition mech<strong>an</strong>isms must exist <strong>for</strong> <strong>the</strong><br />
targeting <strong>an</strong>d mainten<strong>an</strong>ce of GABAergic synapses. In contrast, α7 subunit containing AChRs<br />
are evenly distributed over <strong>the</strong> whole dendritic tree. Previous studies have predicted that during<br />
flight MN5 integrates tonic excitatory cholinergic drive into steady firing frequencies, <strong>an</strong>d that<br />
specific sequences of motor neuron firing are ensured by sharp inhibitory feedback within <strong>the</strong><br />
central pattern generating network. We now test in multi-compartment models whe<strong>the</strong>r
experimentally derived sub-dendritic distribution rules <strong>for</strong> cholinergic <strong>an</strong>d GABAergic synapses<br />
aid behaviorally relev<strong>an</strong>t input-output computations of this motor neuron.<br />
Disclosures: C. Kuehn, None; C. Duch, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.11/B19<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: VIEP-BUAP No. FLAG/SAL08/G<br />
PROMEP, BUAP-CA-120<br />
Title: Maternal separation disrupts dendritic morphology of neurons in prefrontal cortex,<br />
hippocampus, <strong>an</strong>d nucleus accumbens in rat offspring<br />
Authors: *G. FLORES 1 , E. MONROY 2 , E. HERNÁNDEZ-TORRES 2 ;<br />
1 2<br />
Univ. Autonoma de Puebla / Inst. de Fisiologia, Puebla, Mexico; Facultad de Medicina, Univ.<br />
Autonoma de Puebla, Puebla, Mexico<br />
Abstract: Neonatal maternal separation in rats has widely been used as a neurodevelopmental<br />
model that mimics <strong>an</strong>xiety disorders. The behavioral ch<strong>an</strong>ges resulting <strong>from</strong> maternal separation<br />
persist throughout adulthood. We investigated here <strong>the</strong> effect of maternal separation <strong>an</strong>d<br />
substitute maternal h<strong>an</strong>dling on <strong>the</strong> dendritic morphology of neurons <strong>from</strong> <strong>the</strong> prefrontal cortex<br />
(PFC), CA1 ventral hippocampus <strong>an</strong>d nucleus accumbens (NAcc) in pre-pubertal (postnatal day<br />
35, PND35) <strong>an</strong>d post-pubertal (PND60) rats. The morphological characteristics of dendrites were<br />
studied by using <strong>the</strong> Golgi-Cox procedure followed by Sholl <strong>an</strong>alysis. Maternal separation<br />
induced decreases in total dendritic length <strong>an</strong>d dendritic spine density in <strong>the</strong> neurons of PFC,<br />
CA1 ventral hippocampus <strong>an</strong>d NAcc mainly at postpubertal age. Conversely, h<strong>an</strong>dling produced<br />
<strong>an</strong> increase in dendritic spine density in PFC <strong>an</strong>d hipocampal pyramidal neurons at prepubertal<br />
age. In addition, this paradigm also increases <strong>the</strong> dendritic length in prefrontal cortex neurons at<br />
both ages. Ra<strong>the</strong>r interestingly h<strong>an</strong>dling rats showed a decrease in <strong>the</strong> dendritic length of NAcc<br />
neurons at post-pubertal age. These results suggest that a reduction in <strong>the</strong> connection concerning<br />
dendritic spine number <strong>an</strong>d dendritic arbor occur at <strong>the</strong> level of <strong>the</strong> NAcc, ventral hippocampus<br />
<strong>an</strong>d PFC in early development, in <strong>an</strong>imals maternally separated, whereas h<strong>an</strong>dling only decreases
<strong>the</strong> connection at <strong>the</strong> level of <strong>the</strong> NAcc. These <strong>an</strong>atomical modifications may be relev<strong>an</strong>t to<br />
altered behaviors observed in this <strong>an</strong>imal model.<br />
Disclosures: G. Flores, None; E. Monroy, None; E. Hernández-Torres, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.12/B20<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> JSPS Fellows19.1838<br />
Title: Prenatal stress attenuates <strong>the</strong> dendritic maturation of dentate gr<strong>an</strong>ule cells by decreasing<br />
<strong>the</strong> expression of mineralocorticoid receptor<br />
Authors: *M. TAMURA 1 , A. KAKITA 2 , N. MATSUKI 1 , R. KOYAMA 1 ;<br />
1 2<br />
Lab. Chem Pharmacol, Grad Sch. Pharmaceut. Sci., Univ. Tokyo, Tokyo, Jap<strong>an</strong>; Brain Res.<br />
Inst., Niigata Univ., Niigata, Jap<strong>an</strong><br />
Abstract: Stress during embryonic stages increases <strong>the</strong> risk of psychiatric disorders including<br />
depressive disorders in <strong>the</strong> adulthood. However, <strong>the</strong> mech<strong>an</strong>isms underlying this phenomenon<br />
remain to be elucidated. Here, we investigated whe<strong>the</strong>r <strong>the</strong> prenatal stress affects <strong>the</strong><br />
morphological maturation of dentate gr<strong>an</strong>ule cells in <strong>the</strong> hippocampal <strong>for</strong>mation, whose<br />
malfunction is likely to be involved in <strong>the</strong> development of depressive disorders. Pregn<strong>an</strong>t dams<br />
(Wistar/ST rats) were subjected to <strong>the</strong> restraint stress <strong>for</strong> 15-21 d postcoitum. First, <strong>the</strong> <strong>for</strong>cedswimming<br />
test revealed that <strong>the</strong> prenatal stress induced depressive-like behaviors at postnatal<br />
day 60 (P60). To <strong>an</strong>alyze <strong>the</strong> maturation of gr<strong>an</strong>ule cells, new-born gr<strong>an</strong>ule cells in <strong>the</strong> offspring<br />
were visualized by <strong>the</strong> expression of membr<strong>an</strong>e-targeted yellow fluorescent protein using<br />
retrovirus-mediated gene delivery at P0. At P14, <strong>the</strong> br<strong>an</strong>ch number <strong>an</strong>d <strong>the</strong> total length of<br />
gr<strong>an</strong>ule cell dendrites signific<strong>an</strong>tly decreased in <strong>the</strong> prenatally-stressed rats. Import<strong>an</strong>tly, <strong>the</strong>se<br />
abnormalities were sustained even in <strong>the</strong> adulthood (P60). Finally, we found that <strong>the</strong> expression<br />
level of mineralocorticoid receptor, which is a high-affinity receptor of corticosterone, decreased<br />
in gr<strong>an</strong>ule cells by <strong>the</strong> prenatal stress, <strong>an</strong>d that <strong>an</strong> <strong>an</strong>tagonist of mineralocorticoid receptors<br />
impaired <strong>the</strong> dendritic maturation of gr<strong>an</strong>ule cells in cultured hippocampal slices. These results<br />
suggest that prenatal stress inhibits <strong>the</strong> dendritic maturation of gr<strong>an</strong>ule cells by decreasing <strong>the</strong><br />
expression of mineralocorticoid receptor.
Disclosures: M. Tamura, None; A. Kakita, None; N. Matsuki, None; R. Koyama, None.<br />
Poster<br />
508. Development <strong>an</strong>d Org<strong>an</strong>ization of Dendritic Arbors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 508.13/B21<br />
Topic: A.04.i. Dendritic growth <strong>an</strong>d br<strong>an</strong>ching<br />
Support: NIH gr<strong>an</strong>t MH068433<br />
Title: Cellular distribution of Dab2IP iso<strong>for</strong>ms in <strong>the</strong> developing cerebellum<br />
Authors: S. QIAO 1 , *R. HOMAYOUNI 2 ;<br />
1 Biol., Univ. of Memphis, Memphis, TN; 2 Biol. / Bioin<strong>for</strong>matics, Univ. Memphis, Memphis, TN<br />
Abstract: Dab2IP (DOC-2/DAB2 interacting protein) is a member of <strong>the</strong> Ras GTPase-activating<br />
protein family which was previously identified by virtue of its interaction with Disabled-1 <strong>an</strong>d -2<br />
family proteins. The Dab2IP gene contains 18 exons associated with two different promoters.<br />
Western blot <strong>an</strong>alysis revealed multiple iso<strong>for</strong>ms of Dab2IP (r<strong>an</strong>ging between 110KDa <strong>an</strong>d<br />
130KDa) that appear at different times during cerebellar development. Dab2IP knockout (KO)<br />
mice which contained a retroviral gene trap cassette downstream of <strong>the</strong> first promoter still<br />
expressed <strong>the</strong> largest Dab2IP iso<strong>for</strong>m, which appeared only after post-natal day 14 in <strong>the</strong><br />
cerebellum. This suggests that <strong>the</strong> largest Dab2IP iso<strong>for</strong>m is produced by <strong>the</strong> second promoter.<br />
To determine if <strong>the</strong> cellular distribution of <strong>the</strong> different Dab2IP iso<strong>for</strong>ms, we examined Dab2IP<br />
gene expression using <strong>the</strong> LacZ reporter in <strong>the</strong> gene trap cassette in <strong>the</strong> KO mice <strong>an</strong>d Dab2IP<br />
protein expression using immunofluoresence microscopy in WT mice. We found that <strong>the</strong> first<br />
Dab2IP promoter is active in gr<strong>an</strong>ule cells (GCs) <strong>an</strong>d Purkinje cells (PCs) early during cerebellar<br />
development, reached a peak activity at P14, <strong>an</strong>d decreased beyond P30. Consistent with this<br />
observation, Dab2IP protein was detected, using <strong>an</strong> <strong>an</strong>tibody which recognizes all Dab2IP<br />
iso<strong>for</strong>ms, in GC <strong>an</strong>d PC populations throughout cerebellar development. Interestingly, Dab2IP<br />
protein was still detected in PCs in <strong>the</strong> Dab2IP KO mice, but was absent in <strong>the</strong> GC population.<br />
This result suggests that <strong>the</strong> larger Dab2IP iso<strong>for</strong>m is expressed in PCs. Previously, we reported<br />
that PCs in <strong>the</strong> Dab2IP KO mice lacked normal complex spikes but occasionally exhibited<br />
unusually large sodium spikes. In addition, Dab2IP KO contained signific<strong>an</strong>tly more climbing<br />
fiber synapses as measured by VGluT2 immunoreactivity. However, in <strong>the</strong> present study, we<br />
found that Dab2IP does not co-localize with VGluT2 or PSD95 in <strong>the</strong> molecular layer. These<br />
results suggest that <strong>the</strong> differences in <strong>the</strong> PC firing patterns in <strong>the</strong> Dab2IP KO mice may not be
due to a per- or post-synaptic function of Dab2IP. The mech<strong>an</strong>ism by which Dab2IP regulates<br />
PC firing remains to be determined.<br />
Disclosures: S. Qiao, None; R. Homayouni, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.1/B22<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: NS048249<br />
Title: Satellite glial cell precursors clear neuronal corpses during dorsal root g<strong>an</strong>glia<br />
development via <strong>the</strong> novel engulfment receptor Jedi-1<br />
Authors: J. L. SCHEIB 1 , H.-H. WU 1 , E. BELLMUNT 2 , V. VENEGAS 3 , C. BURKERT 1 , L. F.<br />
REICHARDT 4 , Z. ZHOU 3 , I. FARINAS 2 , *B. D. CARTER 1 ;<br />
1 Ctr. Mol Neurosci Dept Biochem, V<strong>an</strong>derbilt Univ. Med. Sch., Nashville, TN; 2 2Dept. de<br />
Biología Celular <strong>an</strong>d CIBERNED, Univ. de Valencia, Burjassot, Spain; 3 3Verna <strong>an</strong>d Marrs<br />
McLe<strong>an</strong> Dept. of Biochem. <strong>an</strong>d Mol. Biol., Baylor Col. of Med., Houston, TX; 4 4Dept. of<br />
Physiol., Univ. of Cali<strong>for</strong>nia, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: During <strong>the</strong> development of peripheral g<strong>an</strong>glia approximately 50 % of <strong>the</strong> neurons<br />
generated undergo apoptosis as part of a normal pruning process; however, how <strong>the</strong> massive<br />
numbers of corpses are efficiently removed is largely unknown. We report that satellite glial cell<br />
precursors are <strong>the</strong> primary phagocytic cells <strong>for</strong> apoptotic corpse removal in developing dorsal<br />
root g<strong>an</strong>glia (DRG). Confocal <strong>an</strong>d electron microscopic <strong>an</strong>alysis revealed that glial precursors,<br />
not macrophages, are responsible <strong>for</strong> clearing dead DRG neurons. Even in neurotrophin 3 null<br />
<strong>an</strong>imals, with signific<strong>an</strong>tly more apoptotic neurons, only 3% of <strong>the</strong> corpses appeared to be<br />
phagocytosed by macrophages while 75% were engulfed by satellite glial precursors. Moreover,<br />
we identified Jedi-1, as a novel engulfment receptor, homologous to <strong>the</strong> invertebrate engulfment<br />
receptors Draper <strong>an</strong>d CED-1, expressed in <strong>the</strong> glial precursor cells. Ectopic expression of Jedi-1<br />
in fibroblasts facilitated binding to dead neurons <strong>an</strong>d knocking down Jedi in glial cells or over<br />
expressing a truncated <strong>for</strong>m lacking <strong>the</strong> intracellular domain (Jedi-deltaICD) inhibited<br />
engulfment of apoptotic neurons. Expression of Jedi-deltaICD in C. eleg<strong>an</strong>s resulted in<br />
accumulation of <strong>the</strong> truncated receptor around apoptotic bodies <strong>an</strong>d inhibited <strong>the</strong>ir clear<strong>an</strong>ce,<br />
suggesting this receptor c<strong>an</strong> also bind dead cells in <strong>the</strong> nematode. Taken toge<strong>the</strong>r, <strong>the</strong>se results
eveal <strong>the</strong> cellular <strong>an</strong>d molecular mech<strong>an</strong>ism by which neuronal corpses are culled during<br />
development, which may be crucial <strong>for</strong> preventing autoimmune attack later in life.<br />
Disclosures: J.L. Scheib, None; H. Wu, None; E. Bellmunt, None; V. Venegas, None; C.<br />
Burkert, None; L.F. Reichardt, None; Z. Zhou, None; I. Farinas, None; B.D. Carter, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.2/B23<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: HD 37100<br />
DA 05072<br />
DA 07261<br />
Title: Hypo<strong>the</strong>rmia is protective against PCP-induced apoptosis in <strong>the</strong> developing mouse brain<br />
Authors: *C. M. YUEDE, S. WANG, Y.-Q. QIN, C. E. CREELEY, J. W. OLNEY;<br />
Dept Psychiatry, Washington Univ., St. Louis, MO<br />
Abstract: Administration of <strong>an</strong> NMDA <strong>an</strong>tagonist to rodents during synaptogenesis c<strong>an</strong> trigger<br />
widespread apoptotic neurodegeneration (Ikonomidou et al., 1999) <strong>an</strong>d result in long-term<br />
behavioral disturb<strong>an</strong>ces (W<strong>an</strong>g et al., 2001). Hypo<strong>the</strong>rmia has been shown to inhibit apoptosis in<br />
neuronal cell cultures (Bossenmeyer-Pourie et al., 2000), <strong>an</strong>d to be neuroprotective in studies of<br />
hypoxia-ischemia during <strong>the</strong> postnatal period (Laptook et al., 1994; Thoresen et al., 1995).<br />
There<strong>for</strong>e, we conducted experiments designed to determine whe<strong>the</strong>r lowering ambient<br />
temperature would be protective against neuroapoptosis induced by <strong>the</strong> NMDA <strong>an</strong>tagonist,<br />
phencyclidine (PCP). Postnatal day 4 ICR mouse pups were treated with ei<strong>the</strong>r PCP (35 mg/kg)<br />
or saline <strong>an</strong>d placed in temperature-controlled chambers at 35°, 30° or 24° (C). After 5 hours at<br />
<strong>the</strong> assigned temperature, pups were perfused <strong>an</strong>d brains removed <strong>for</strong> immunohistochemical<br />
evaluation of caspase-3 activation. Results indicate that ambient temperature has a signific<strong>an</strong>t<br />
effect on amount of neuroapoptosis occurring after PCP exposure, with <strong>the</strong> most damage<br />
observed at 35°, <strong>an</strong>d brains of <strong>an</strong>imals kept at 24° showing no evidence of neuroapoptotic<br />
damage. The degree of damage observed in <strong>the</strong> brains of <strong>an</strong>imals kept at 30° was less th<strong>an</strong> that<br />
observed at 35°, suggesting partial protection against PCP-induced neuroapoptosis. These results
suggest that ambient temperature may signific<strong>an</strong>tly influence <strong>the</strong> severity of damage observed<br />
after PCP exposure, <strong>an</strong>d that hypo<strong>the</strong>rmia is protective against PCP-induced neuroapoptotic<br />
damage.<br />
Disclosures: C.M. Yuede, None; S. W<strong>an</strong>g, None; Y. Qin, None; C.E. Creeley, None; J.W.<br />
Olney, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.3/B24<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: NSERC Discovery Gr<strong>an</strong>t<br />
Title: Heat shock protein 25 expression <strong>an</strong>d preferential Purkinje cell survival in <strong>the</strong> Lurcher<br />
mut<strong>an</strong>t mouse cerebellum<br />
Authors: C. DUFFIN 1 , R. MCFARLAND 2 , J. SARNA 3 , M. VOGEL 2 , *C. L. ARMSTRONG 1 ;<br />
1 2 3<br />
Univ. of Guelph, Guelph, ON, C<strong>an</strong>ada; Maryl<strong>an</strong>d Psychiatric Res. Ctr., Baltimore, MD; Univ.<br />
of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: The spatial org<strong>an</strong>ization of <strong>the</strong> mouse cerebellum into tr<strong>an</strong>sverse zones <strong>an</strong>d<br />
parasagittal stripes is reflected during <strong>the</strong> temporal progression of Purkinje cell death in <strong>the</strong><br />
Lurcher mut<strong>an</strong>t mouse (+/Lc). Neurodegeneration in <strong>the</strong> +/Lc mut<strong>an</strong>t is apparent by <strong>the</strong> second<br />
postnatal week <strong>an</strong>d is initially seen in all four tr<strong>an</strong>sverse zones: <strong>the</strong> <strong>an</strong>terior (lobules I-V), central<br />
(lobules VI, VII), posterior (lobules VIII, dorsal IX) <strong>an</strong>d nodular (ventral lobule IX <strong>an</strong>d lobule X)<br />
zone. However, <strong>from</strong> postnatal day (P)25-P36, Purkinje cell loss proceeds more rapidly in <strong>the</strong><br />
<strong>an</strong>terior zone, followed by <strong>the</strong> posterior <strong>an</strong>d central zones, <strong>an</strong>d is signific<strong>an</strong>tly delayed in <strong>the</strong><br />
nodular zone. Coronal sections through <strong>the</strong> +/Lc cerebellum reveal that surviving Purkinje cells<br />
are restricted to <strong>the</strong> paraflocculus/flocculus <strong>an</strong>d <strong>the</strong> nodular zone <strong>an</strong>d could be detected as late as<br />
P146 (~ 5 months). Within this region, <strong>the</strong> pattern of preferentially surviving calbindinimmunoreactive<br />
Purkinje cells reflects <strong>the</strong> expression of <strong>the</strong> constitutively-expressed small heat<br />
shock protein HSP25 in <strong>the</strong> wildtype cerebellum. While <strong>the</strong> role of constitutively-expressed<br />
HSP25 in <strong>the</strong> wildtype cerebellum is not clear, it appears to play a neuroprotective role in <strong>the</strong><br />
flocculonodular region of <strong>the</strong> +/Lc mut<strong>an</strong>t cerebellum as <strong>the</strong> percentage of surviving Purkinje<br />
cells that are HSP25-immunopositive signific<strong>an</strong>tly increases over time.
Disclosures: C. Duffin, None; R. McFarl<strong>an</strong>d, None; J. Sarna, None; M. Vogel, None; C.L.<br />
Armstrong, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.4/B25<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Title: The <strong>an</strong>tiapoptotic protein lifeguard plays a role in cerebellar cell survival<br />
Authors: *T. HURTADO DE MENDOZA, C. G. PEREZ-GARCIA, T. T. KROLL, N. C.<br />
HOONG, D. D. M. O'LEARY, I. M. VERMA;<br />
Salk Inst., La Jolla, CA<br />
Abstract: Lifeguard (LFG) is a membr<strong>an</strong>e protein originally isolated as <strong>an</strong> inhibitor of <strong>the</strong> Fas<br />
apoptotic pathway. LFG is highly expressed in <strong>the</strong> brain, especially in <strong>the</strong> cerebellum. In order to<br />
study <strong>the</strong> biological role of LFG in vivo, we generated mice with reduced LFG expression by<br />
siRNA lentiviral tr<strong>an</strong>sgenesis. siLFG mice showed up to 90% reduction of LFG expression in <strong>the</strong><br />
cerebellum affecting primarily gr<strong>an</strong>ular <strong>an</strong>d Purkinje cells. Compared to wild type littermates <strong>the</strong><br />
cerebellum of siLFG mice showed a reduction of <strong>the</strong> internal gr<strong>an</strong>ular layer. Downregulation of<br />
LFG expression severely affected Purkinje cells differentiation <strong>an</strong>d survival, with a late<br />
org<strong>an</strong>ization into a monolayer <strong>an</strong>d a delayed onset of Parvalbumin expression in parallel with<br />
aberr<strong>an</strong>t cell morphologies <strong>an</strong>d increased apoptosis. In addition to <strong>the</strong> siLFG, we also used <strong>the</strong><br />
LFG null mice that present a similar but less severe phenotype th<strong>an</strong> siLFG mice, where Purkinje<br />
cells are also affected but with <strong>an</strong> internal gr<strong>an</strong>ular layer apparently normal. Fas treated<br />
org<strong>an</strong>otypic cerebellar cultures <strong>from</strong> LFG null mice showed increased apoptosis in both Purkinje<br />
<strong>an</strong>d gr<strong>an</strong>ular cell fields compared to wild type cerebellar slices, implicating <strong>the</strong> Fas pathway in<br />
<strong>the</strong> death of those two main cerebellar cell types. These data suggest a role <strong>for</strong> LFG in <strong>the</strong><br />
development <strong>an</strong>d survival of Purkinje cells in <strong>the</strong> cerebellum.<br />
Disclosures: T. Hurtado de Mendoza, None; C.G. Perez-Garcia, None; T.T. Kroll,<br />
None; N.C. Hoong, None; D.D.M. O'Leary, None; I.M. Verma, None.<br />
Poster
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.5/B26<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: Johns Hopkins University startup funds<br />
Title: Developmental apoptosis regulates spacing <strong>an</strong>d rod/cone signaling through mel<strong>an</strong>opsinexpressing<br />
retinal g<strong>an</strong>glion cells<br />
Authors: *D. MCNEILL 1 , R. L. BROWN 2 , R. KURUVILLA 1 , W. GUIDO 3 , S. HATTAR 1 ;<br />
1 Johns Hopkins Univ., Baltimore, MD; 2 Dept. of Vet. <strong>an</strong>d Comparative Anatomy,<br />
Pharmacology, <strong>an</strong>d Physiol., Washington State Univ., Pullm<strong>an</strong>, WA; 3 Dept. of Anat. <strong>an</strong>d<br />
Neurobio., Virginia Commonwealth Univ., Richmond, VA<br />
Abstract: During normal development, about half of <strong>the</strong> neurons initially produced subsequently<br />
undergo apoptosis. The reasons <strong>for</strong> this programmed cell loss remain unclear, especially in <strong>the</strong><br />
central nervous system (CNS). Bax null mice provide a good model to study this phenomenon<br />
because <strong>the</strong>y lack apoptosis in m<strong>an</strong>y neuronal populations, including retinal g<strong>an</strong>glion cells<br />
(RGCs). To determine how developmental apoptosis contributes to <strong>the</strong> structure <strong>an</strong>d behavioral<br />
outputs of a specific type of neuron, we examined <strong>the</strong> morphology <strong>an</strong>d function of <strong>the</strong><br />
mel<strong>an</strong>opsin-expressing subset of RGCs (mRGCs) in bax knockout mice. The mRGCs provide a<br />
tractable model system within <strong>the</strong> CNS because of <strong>the</strong>ir distinct morphology, axonal targets, <strong>an</strong>d<br />
function in detecting <strong>an</strong>d relaying light in<strong>for</strong>mation to regulate circadi<strong>an</strong> rhythms <strong>an</strong>d pupil<br />
constriction. Antibody labeling of mRGCs in adult retinas revealed that bax null mice contain<br />
about twice as m<strong>an</strong>y mRGCs as wild type, similar to published data <strong>for</strong> <strong>the</strong> RGC population as a<br />
whole. In addition, about half of <strong>the</strong> mRGCs in <strong>the</strong> bax knockout are in clumps of two or more<br />
cells with t<strong>an</strong>gled dendrites, unlike <strong>the</strong> evenly spaced mosaic <strong>an</strong>d dendritic network of mRGCs<br />
in <strong>the</strong> wild type. Similar clumps are present at younger ages be<strong>for</strong>e <strong>the</strong> population undergoes<br />
apoptosis. Thus <strong>the</strong>se clumps of mRGCs are present during normal development but are<br />
subsequently removed via bax-mediated apoptosis. Genetic labeling of mRGC axons in bax<br />
knockout mice revealed grossly normal targeting of both <strong>the</strong> suprachiasmatic nucleus (SCN),<br />
which regulates circadi<strong>an</strong> rhythms, <strong>an</strong>d <strong>the</strong> olivary pretectal nucleus (OPN), which controls pupil<br />
constriction. Although bax <strong>an</strong>d mel<strong>an</strong>opsin single knockouts could entrain <strong>the</strong>ir wheel running<br />
activity to <strong>an</strong> external light cycle similar to wild type, bax/mel<strong>an</strong>opsin double knockout mice<br />
failed to entrain. This indicates that <strong>the</strong> rod/cone input to mRGCs is dysfunctional in <strong>the</strong> bax<br />
knockout. It remains to be determined if <strong>the</strong> cell spacing defect results in <strong>the</strong> loss of rod/cone<br />
input to mRGCs in <strong>the</strong> bax knockout. Our data reveal that bax-mediated apoptosis is not<br />
involved in mRGC targeting of <strong>the</strong> SCN <strong>an</strong>d OPN. Ra<strong>the</strong>r, it plays a crucial role in <strong>for</strong>ming input<br />
<strong>from</strong> rods <strong>an</strong>d cones as well as <strong>for</strong>ming <strong>the</strong> evenly spaced photoreceptive network of mRGCs
across <strong>the</strong> retina. This suggests a role <strong>for</strong> apoptosis during development in regulating cell spacing<br />
<strong>an</strong>d dendritic <strong>for</strong>mation.<br />
Disclosures: D. McNeill, None; R.L. Brown, None; R. Kuruvilla, None; W. Guido, None; S.<br />
Hattar, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.6/B27<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: CONACyT 47158-Q<br />
DGAPA-UNAM<br />
Title: Role of NADPH oxidase in <strong>the</strong> development of rat cerebellum<br />
Authors: *A. B. COYOY SALGADO, J. MORÁN;<br />
Inst. De Fisiología Celular, Univ. Nacional Autónoma De México, México, Mexico<br />
Abstract: Reactive oxygen species (ROS), such as superoxide <strong>an</strong>ion c<strong>an</strong> influence gene<br />
expression, cellular growth <strong>an</strong>d differentiation in a variety of neuronal <strong>an</strong>d non neuronal cells by<br />
modulating intracellular signaling pathways. Among several cell sources of ROS, NADPHoxidase<br />
is a major superoxide producing flavoenzyme. In neuronal tissues, <strong>the</strong> role of ROS<br />
derived <strong>from</strong> this enzyme has been documented in cellular dysfunction under various<br />
pathological conditions. The physiological function of NADPH oxidase in nerve tissue has not<br />
been fully elucidated. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, rat cerebellum represents <strong>an</strong> appropriate model <strong>for</strong> <strong>the</strong><br />
study of <strong>the</strong> cellular <strong>an</strong>d molecular mech<strong>an</strong>isms during development. Cerebellar development<br />
occurs postnatally <strong>an</strong>d most of <strong>the</strong> cellular events, such as proliferation, migration, cell death <strong>an</strong>d<br />
neuronal maturation take place during <strong>the</strong> first 21 post natal days (PND). In this study we<br />
characterized <strong>the</strong> levels of homologues of <strong>the</strong> NADPH oxidase catalytic subunits (NOX1, NOX2<br />
<strong>an</strong>d NOX4) in <strong>the</strong> developing rat cerebellum. We detected mRNA <strong>an</strong>d protein of all NADPHoxidase<br />
homologues at postnatal day (PND) 4, 8, 12, 16 <strong>an</strong>d 25. The mRNA levels of <strong>the</strong>se<br />
homologues did not ch<strong>an</strong>ge with time, except NOX4 that showed a reduction at PND 16 <strong>an</strong>d 25.<br />
The protein levels of NOX1 decrease at PND 12 <strong>an</strong>d 25, NOX2 increase at PND 8 <strong>an</strong>d 16, <strong>an</strong>d<br />
NOX4 decrease <strong>the</strong> protein level at PND 12, 16 <strong>an</strong>d 25. In <strong>an</strong> in vitro model of cerebellar gr<strong>an</strong>ule<br />
neurons migration, we found that migrating neurons showed low levels of ROS, in contrast to
stationary neurons, <strong>the</strong> migration speed ch<strong>an</strong>ges with <strong>an</strong> <strong>an</strong>tioxid<strong>an</strong>t <strong>an</strong>d NADPH-oxidase<br />
inhibitor. These results suggest that both ROS <strong>an</strong>d NADPH oxidase may have a role during<br />
cerebellar development.<br />
Disclosures: A.B. Coyoy Salgado, Dr. Mor<strong>an</strong> Andrade, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
CONACyT, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Morán, DCONACyT 47158-Q, C. O<strong>the</strong>r Research<br />
Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); DGAPA-UNAM<br />
IN223107, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind<br />
support).<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.7/B28<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: PRIN Anno 2006 - prot. 2006050725_00<br />
neurodegenerativo Ex. Art. 56 533F/B/1 to NC<br />
Title: Role of D-serine racemase during neuronal cell death<br />
Authors: *N. CANU 1,2,3 , F. FLORENZANO 5 , A. DI LUZIO 5 , T. BORSELLO 3 , M. CIOTTI 4 ;<br />
1 Dept. of Neurosci., Univ. of Tor Vergata, Roma, Italy; 2 Consiglio Nazionale delle Ricerche,<br />
Inst. of neurobiology <strong>an</strong>d molecular medicine, Rome, Italy; 3 Inst. di Ricerche Farmacologiche<br />
Mario Negri,, Mil<strong>an</strong>, Italy; 4 Consiglio Nazionale delle Ricerche, Inst. di Ricerche<br />
Farmacologiche Mario Negri,, Rome, Italy; 5 Consiglio Nazionale delle Ricerche, Inst. di<br />
Neurobiologia e Medicina Molecolare, Rome, Italy<br />
Abstract: N-Methyl-d-aspartate receptor (NMDAR) stimulation promotes neuronal survival<br />
under both in vitro <strong>an</strong>d in vivo conditions. D-serine racemase (D-SR) is a brain enriched enzyme<br />
that converts L-serine into D-serine <strong>the</strong> endogenous co-agonist at <strong>the</strong> "glycine site" of NMDAR<br />
that is required <strong>for</strong> <strong>the</strong> receptor activation. Factors regulating <strong>the</strong> levels of D-serine have<br />
implications <strong>for</strong> <strong>the</strong> NMDAR tr<strong>an</strong>smission, but little is known on <strong>the</strong> signal pathways <strong>an</strong>d events<br />
influencing D-SR expression. We have investigated <strong>the</strong> potential role of <strong>the</strong> D-SR in <strong>the</strong> death of<br />
cerebellar gr<strong>an</strong>ule neurons induced by reduction of extracellular potassium. We have found that
during this process <strong>the</strong> level of D-SR declines rapidly in <strong>the</strong> first six hour of apoptosis when only<br />
15% of neurons are apoptotic. The D-SR decrease is not prevented nei<strong>the</strong>r by caspases nor by<br />
ca<strong>the</strong>psin B inhibition. Interestingly, D-serine racemase levels is restored by inhibiting <strong>the</strong><br />
proteasome system that is known to be involved upstream caspase <strong>an</strong>d ca<strong>the</strong>psin activation in<br />
this model of neuronal apoptosis <strong>an</strong>d known to be a main regulator of D-SR <strong>an</strong>d D-serine levels.<br />
Moreover, c-Jun N-terminal kinases (c-JNKs) are also involved in regulation of D-SR level,<br />
since c-JNKs inhibition prevents apoptosis-induced D-SR reduction. shRNA plasmid DNA<br />
mediated inhibition of D-SR gene expression increseas CGN cell death after reduction of<br />
extracellular potassium. Both over expression of D-SR <strong>an</strong>d treatment with NMDA <strong>an</strong>d D-serine<br />
are able to protect CGN <strong>from</strong> apoptosis. D-SR is also differentially modulated during neuronal<br />
cell death caused by tau over-expression <strong>an</strong>d proteasome inhibition. Supported by PRIN Anno<br />
2006 - prot. 2006050725_002 <strong>an</strong>d neurodegenerativo Ex. Art. 56 533F/B/1 to NC<br />
Disclosures: N. C<strong>an</strong>u, None; F. Florenz<strong>an</strong>o, None; A. Di Luzio, None; T. Borsello, None; M.<br />
Ciotti, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.8/B29<br />
Topic: A.06.c. Developmental cell death: O<strong>the</strong>r trophic factors<br />
Title: The EBF2 tr<strong>an</strong>scription factor activates Igf1 gene expression, regulating Purkinje cell<br />
survival at birth<br />
Authors: *L. P. CROCI, M.S. 1 , V. BARILI 3 , D. CHIA 4 , R. VAN VUGT 2 , G.<br />
MASSERDOTTI 2 , P. ROTWEIN 4 , G. CONSALEZ 2 ;<br />
1 2<br />
S<strong>an</strong> Raffaele Scientific Inst., Mil<strong>an</strong>o, MI, Italy; S<strong>an</strong> Raffaele Scientific Inst., Mil<strong>an</strong>o, Italy;<br />
3 4<br />
Univ. Vita-Salute S<strong>an</strong> Raffaele, Mil<strong>an</strong>o, Italy; Dept. of Biochem. <strong>an</strong>d Mol. Biol., Oregon Hlth.<br />
<strong>an</strong>d Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: Ebf genes encode atypical HLH tr<strong>an</strong>scription factors (TFs) implicated in various<br />
aspects of neuronal development. The Ebf2 null cerebellum shows massive Purkinje cell (PC)<br />
apoptosis at birth. We r<strong>an</strong> <strong>an</strong> in silico search <strong>for</strong> genes containing putative EBF binding sites in<br />
<strong>the</strong>ir promoter. One of <strong>the</strong>m, Igf1, is coexpressed with Ebf2. IGF1 has been implicated in cell<br />
survival in vitro <strong>an</strong>d in vivo. In luciferase assays, Ebf2 overexpression tr<strong>an</strong>sactivates Igf1<br />
promoter. Accordingly, Igf1 expression is profoundly downregulated in Ebf2 null PCs. As<br />
regards <strong>the</strong> IGF1 signaling pathway, AKT (protein kinase B) is hypophosphorylated at both
Ser473 <strong>an</strong>d Thr308 in <strong>the</strong> null cerebellum on postnatal day 1. We cultured late embryonic<br />
cerebellar slices <strong>from</strong> Ebf2 null mut<strong>an</strong>ts <strong>an</strong>d controls in media containing purified IGF1 or<br />
vehicle. IGF1 rescued PC death to wt levels in mut<strong>an</strong>t slices, whereas it had no effect on control<br />
slices. Taken toge<strong>the</strong>r, our results indicate that EBF2 promotes PC survival by regulating Igf1<br />
gene expression. Our findings may have wider implications <strong>for</strong> <strong>the</strong> study of cell survival <strong>an</strong>d its<br />
local tr<strong>an</strong>scriptional regulation in neurons.<br />
Disclosures: L.P. Croci, None; V. Barili, None; D. Chia, None; R. V<strong>an</strong> Vugt, None; G.<br />
Masserdotti, None; P. Rotwein, None; G. Consalez, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.9/B30<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Title: Protogenin deficient mice exhibit abnormalities of neural crest derived cr<strong>an</strong>iofacial<br />
structure<br />
Authors: *M.-J. FANN 1 , Y.-C. WANG 2 , Y.-H. WONG 2 , W.-C. KUO 2 ;<br />
1 Dept of Life Sci., Natl. Y<strong>an</strong>g-Ming Univ., Taipei, Taiw<strong>an</strong>; 2 Y<strong>an</strong>g-Ming Univ., Taipei, Taiw<strong>an</strong><br />
Abstract: Mouse protogenin (Prtg) is a member of immunoglobulin superfamily. Strong<br />
expression of Prtg is in <strong>the</strong> somites, neural tube <strong>an</strong>d br<strong>an</strong>chial arches in E9.5 mouse embryos but<br />
disappear drastically as development proceeds. To investigate <strong>the</strong> function of Prtg, we generated<br />
<strong>the</strong> Prtg knockout (Prtg -/- ) mice. Number of Prtg -/- embryos exhibits <strong>the</strong> expected Mendeli<strong>an</strong> ratio<br />
<strong>an</strong>d Prtg -/- embryos do not have defects in size <strong>an</strong>d morphology at all embryonic stages<br />
examined. However, 44.4% of Prtg -/- mice die within 72 hours after birth. The Prtg -/- mice lack<br />
milk in <strong>the</strong> stomach. In addition, <strong>the</strong> survived Prtg -/- mice are growth retarded. Histological<br />
<strong>an</strong>alysis <strong>an</strong>d skeletal staining reveal <strong>the</strong> palatal bone, skull <strong>an</strong>d cr<strong>an</strong>ial nerve defects in Prtg -/-<br />
mice. As <strong>the</strong>se defective regions are developmentally derived <strong>from</strong> cr<strong>an</strong>ial neural crest cells, we<br />
thus <strong>an</strong>alyzed <strong>the</strong> expression of molecular markers that play key roles <strong>for</strong> <strong>the</strong> development of<br />
neural crest cells. Whole-mount in situ hybridization results demonstrated that expression of<br />
Msx1, Pax3, <strong>an</strong>d Twist1 are altered. To fur<strong>the</strong>r trace <strong>the</strong> defect of neural crest derived structure,<br />
we used Wnt1-Cre/R26R reporter mice crossed with Prtg -/- mice to examine <strong>the</strong> lineage of neural<br />
crest cells. We found that cr<strong>an</strong>ial neural crest cells are deficient in E9 Prtg -/- embryos. Moreover,<br />
abnormal cell death occurs in E8.5 prorhombomeres, suggesting that Prtg may play a role to
modulate cr<strong>an</strong>ial neural crest survival <strong>an</strong>d consequently influence patterning of <strong>the</strong>ir derived<br />
cells.<br />
Disclosures: M. F<strong>an</strong>n, None; Y. W<strong>an</strong>g, None; Y. Wong, None; W. Kuo, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.10/B31<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: CNRS <strong>an</strong>d UPMC gr<strong>an</strong>ts<br />
NIH gr<strong>an</strong>t NS34309<br />
Title: Protein kinase C (PKC) activity is involved both in developmental <strong>an</strong>d pathological<br />
Purkinje cell death in Lurcher mut<strong>an</strong>t mice<br />
Authors: *H. S. ZANJANI 1,2 , V. GAUTHERON 1 , M. W. VOGEL 2 , J. MARIANI 1 ;<br />
1 UMR 7102; Lab. DVSN, Univ. Paris 6; CNRS, Paris, Fr<strong>an</strong>ce; 2 Dept. of Psychiatry (MPRC),<br />
Univ. of Maryl<strong>an</strong>d, Sch. of Med., Baltimore, MD<br />
Abstract: An import<strong>an</strong>t model of pathological cell death in vertebrates is <strong>the</strong> Lurcher (gene<br />
symbol: Lc) mouse mut<strong>an</strong>t. The Lurcher mutation in <strong>the</strong> δ2 glutamate receptor (GluRδ2) ch<strong>an</strong>ges<br />
this orph<strong>an</strong> receptor into a constitutively open cation membr<strong>an</strong>e ch<strong>an</strong>nel. GluRδ2 receptors are<br />
preferentially expressed in Purkinje cells (PCs) <strong>an</strong>d in heterozygous Lurcher mut<strong>an</strong>ts<br />
(GluRδ2 +/Lc ), PCs become chronically depolarized (P5-P6) be<strong>for</strong>e <strong>the</strong>y begin to degenerate.<br />
Virtually all GluRδ2 +/Lc PCs eventually degenerate by mech<strong>an</strong>isms that have been hypo<strong>the</strong>sized<br />
to involve necrosis, autophagy, or apoptosis. PKC enzymes are potent effectors of apoptosis <strong>an</strong>d<br />
neurodegeneration both in vivo <strong>an</strong>d in vitro. Previous studies have shown that of <strong>the</strong> various<br />
PKC inhibitors, only <strong>the</strong> PKC inhibitor Gö6976 efficiently promotes Purkinje cell survival <strong>an</strong>d<br />
axodendritic differentiation in org<strong>an</strong>otypic cultures. In a previous study we demonstrated that<br />
application of Gö6976 in cerebellar slice cultures <strong>from</strong> <strong>the</strong> beginning of cell culture dramatically<br />
increased GluRδ2 +/+ <strong>an</strong>d GluRδ2 +/Lc PC survival. In order to determine if Gö6976 rescued PCs<br />
<strong>from</strong> developmental <strong>an</strong>d/or pathological cell death in this experiment we initiated PKC inhibitor<br />
treatments <strong>from</strong> <strong>the</strong> seventh day in vitro (DIV 7), which is shortly be<strong>for</strong>e GluRδ2 +/Lc PCs begin<br />
to degenerate in vitro. Wild type (WT) <strong>an</strong>d GluRδ2 +/Lc parasagittal cerebellar slices (350µm)<br />
<strong>from</strong> P0 mice were tr<strong>an</strong>sferred onto membr<strong>an</strong>es of Millipore culture inserts. Slices were
maintained in culture in medium supplemented with 25% horse serum at 35°C in 5% CO2/95%<br />
air. Inhibitor was added to <strong>the</strong> media at 7 DIV <strong>an</strong>d <strong>the</strong> media was ch<strong>an</strong>ged every 3 rd day until<br />
DIV14. Slice cultures were <strong>the</strong>n fixed <strong>an</strong>d PCs identified by immunolabeling <strong>for</strong> calbindin. The<br />
number of PCs per culture was determined by systematically counting all labeled PCs in <strong>the</strong><br />
slices. After 14 DIV in control cultures, GluRδ2 +/Lc Purkinje survival was reduced by over 70%<br />
compared to WT PCs (625 ± 65 GluRδ2 +/Lc PCs; n = 13 vs. 2216 ± 266 WT PCs; n = 10).<br />
Exposing cultures to Gö6976 <strong>from</strong> 1 DIV increases <strong>the</strong> number of surviving WT PCs 15 fold <strong>an</strong>d<br />
GluRδ2 +/Lc PC survival by 80 fold. However, treatment with Gö6976 starting <strong>from</strong> 7 DIV<br />
increased <strong>the</strong> number of surviving wild type PCs by only 1.5 fold (3,706 ± 495; n = 9) <strong>an</strong>d<br />
GluRδ2 +/Lc PC survival increased by about three fold (1,858 ± 170; n = 8). These results suggest<br />
activation of PKC is involved both in GRID2 +/Lc developmental <strong>an</strong>d pathological Purkinje cell<br />
death. To determine <strong>the</strong> contribution of specific PKC iso<strong>for</strong>ms to <strong>the</strong> Lurcher PC death we are in<br />
process of testing different PKC iso<strong>for</strong>ms in our cell cultures system in collaboration of KAI<br />
pharmaceuticals (S<strong>an</strong> Fr<strong>an</strong>cisco, CA).<br />
Disclosures: H.S. Z<strong>an</strong>j<strong>an</strong>i, None; V. Gau<strong>the</strong>ron, None; M.W. Vogel, None; J. Mari<strong>an</strong>i,<br />
None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.11/B32<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Title: Delta protein kinase C expression in <strong>the</strong> brain during development<br />
Authors: *I. ZEMLYAK, A. B. CUTLER, R. M. SAPOLSKY;<br />
St<strong>an</strong><strong>for</strong>d, St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Delta Protein Kinase C (δPKC) helps to mediate <strong>the</strong> apoptosis associated with a wide<br />
variety of triggers, such as activation of death receptors, DNA damage, exposure to excitotoxins,<br />
<strong>an</strong>d necrotic insults. Within neurons, <strong>the</strong> protein occurs in a full length 75kDa <strong>for</strong>m <strong>an</strong>d a<br />
catalytic fragment of 38kDa, both of which are believed to have roles in neuron survival.<br />
Following a necrotic insult, <strong>the</strong> full length protein tr<strong>an</strong>slocates <strong>from</strong> <strong>the</strong> cytoplasm to <strong>the</strong> nucleus<br />
(<strong>an</strong>d mitochondria), where it is cleaved into its catalytic <strong>for</strong>m. In this study, we aimed to<br />
determine <strong>the</strong> expression pattern of δPKC during development in both cortical <strong>an</strong>d hippocampal<br />
neurons. Brains of Sprague-Dawley rats were collected <strong>from</strong> P1 through 4 months old, as well as<br />
<strong>from</strong> adults. Hippocampus <strong>an</strong>d cortex were removed <strong>from</strong> each brain, sliced <strong>an</strong>d subjected to
Oxygen Glucose Deprivation (OGD) <strong>for</strong> 3h <strong>an</strong>d 3h of reperfusion. Protein was extracted <strong>an</strong>d<br />
using Western blot <strong>an</strong>alysis, we detected full size <strong>an</strong>d catalytic <strong>for</strong>ms (75kDa <strong>an</strong>d 38kDa,<br />
respectively) of δPKC. During <strong>the</strong> first ten days after birth, <strong>the</strong> 75kDa <strong>for</strong>m of <strong>the</strong> protein is <strong>the</strong><br />
domin<strong>an</strong>t type found in both Control <strong>an</strong>d OGD-treated samples. Starting <strong>from</strong> day 15, <strong>the</strong> 38kDa<br />
cleaved <strong>for</strong>m is <strong>the</strong> domin<strong>an</strong>t type in both Control <strong>an</strong>d OGD-treated brains. This pattern is<br />
observed in both <strong>the</strong> cortex <strong>an</strong>d hippocampus. Fur<strong>the</strong>rmore, we demonstrated differences in<br />
expression of both 75kDa <strong>an</strong>d 38kDa proteins over <strong>the</strong> course of <strong>the</strong> first month in response to<br />
OGD. However, no ch<strong>an</strong>ges were observed in <strong>the</strong> expression of <strong>the</strong> same protein in adult brains<br />
in response to OGD.<br />
We suggest that ch<strong>an</strong>ges in expression of δPKC evidence about its ch<strong>an</strong>ging role in <strong>the</strong><br />
development of <strong>the</strong> brain (<strong>an</strong>d, strikingly, a similar developmental shift in PKC expression<br />
profiles has been reported in cardiomyocytes). These marked ch<strong>an</strong>ges during brain development<br />
may prove helpful in underst<strong>an</strong>ding <strong>the</strong> dramatic developmental shifts that c<strong>an</strong> occur in<br />
vulnerability to necrotic insults.<br />
Disclosures: I. Zemlyak, None; A.B. Cutler, None; R.M. Sapolsky, None.<br />
Poster<br />
509. Molecular Mech<strong>an</strong>isms Regulating Neuronal Survival <strong>an</strong>d Death<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 509.12/B33<br />
Topic: A.06.a. Developmental cell death: Biological effects<br />
Support: CONACYT<br />
DGAPA IN-214607-3<br />
Title: Rotenona induces progressive motor deficits in rats<br />
Authors: *M. GOMEZ-CHAVARIN 1 , R. MORALES 2 , C. TORNER 4 , G. ROLDAN 3 , J.<br />
FERNANDEZ 3 ;<br />
2 3 1<br />
Med. Faculty, Microbiology Dpt, Med. Faculty, Physiol. Dpt, UNAM, Mexico D. F., Mexico;<br />
4<br />
Hlth. Care, UAM, D.F., Mexico<br />
Abstract: Epidemiologic studies suggest that <strong>the</strong> use of pesticides like rotenone (ROT) in <strong>the</strong><br />
control of plagues have <strong>the</strong> capacity to produce damage in <strong>the</strong> nigrostriatal dopaminergic system<br />
(NSDS). For this reason, it c<strong>an</strong> be considered <strong>an</strong> environmental risk factor <strong>for</strong> <strong>the</strong> development<br />
of Parkinson’s disease (PD). The presence of this xenobiotic during <strong>the</strong> embryonic <strong>an</strong>d perinatal
development, may alter <strong>the</strong> proper maturation of <strong>the</strong> NSDS <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e, to facilitate <strong>the</strong><br />
development of PD later in life. Although a single exposition could not be enough to induce<br />
parkinsonism, <strong>the</strong> produced damage could remain silent; later on, future aggressions to <strong>the</strong><br />
NSDS might produce fur<strong>the</strong>r damage <strong>an</strong>d facilitate <strong>the</strong> m<strong>an</strong>ifestation of symptoms characteristic<br />
of <strong>the</strong> PD.<br />
We study <strong>the</strong> NSDS vulnerability to ROT, through <strong>the</strong> <strong>an</strong>alysis of <strong>the</strong> motor deficit caused by <strong>the</strong><br />
exposition to ROT in prenatal <strong>an</strong>d perinatal ages of <strong>the</strong> rat, <strong>an</strong>d after repeating <strong>the</strong> exposition at<br />
adult age.<br />
Methods: Pregn<strong>an</strong>t Wistar rats received a sc dose of 250 µg ROT daily during <strong>the</strong> 21 days of<br />
gestation <strong>an</strong>d also during 30 days of nursing. Pups at 1 <strong>an</strong>d 2 months of age were trained <strong>an</strong>d<br />
tested in <strong>the</strong> Drucker´s task <strong>for</strong> motor coordination, that consisted in crossing a beam of different<br />
widths in a ceiling time (CT) of 120 sec (J. Neurosci 39(1991):153-161). Then, at 4.5 months<br />
<strong>the</strong>se adult rats received a second dose of ROT. Finally, <strong>the</strong>y were evaluated in <strong>the</strong> beam at 6, 9<br />
<strong>an</strong>d 12 months of age. A second group of intact rats were also trained <strong>an</strong>d tested in <strong>the</strong> beam at 2<br />
months age, <strong>an</strong>d <strong>the</strong>n at 4.5 months <strong>the</strong>y were injected daily sc with 250 µg of ROT by 51 days<br />
in order to be tested again at 6, 9 <strong>an</strong>d 12 months of age.<br />
Results: Of <strong>the</strong> <strong>an</strong>imals that were exposed to ROT during early development, only 40% of <strong>the</strong>m<br />
were able to execute <strong>the</strong> task (CT= 102.60 + 24.45 sec) at <strong>the</strong> 2 months test. Fur<strong>the</strong>rmore, at <strong>the</strong><br />
12 months test after receiving <strong>the</strong> second dose of ROT none of <strong>the</strong>m could execute it (CT=120 +<br />
0 sec). In contrast 100% of <strong>the</strong> intact <strong>an</strong>imals executed <strong>the</strong> test at <strong>the</strong> 2 months age (CT=95.5 +<br />
7.29 sec) <strong>an</strong>d 60% at <strong>the</strong> 12 months age, after one ROT administration (CT=72.2 + 14.34 sec).<br />
Conclusion: Early exposure in uterus <strong>an</strong>d during nursing to ROT produces motor deficits in<br />
young adult rats. This effect is potentiated when rats receive a second dose of ROT in adult age.<br />
It remains to be studied if ROT injections that do not produce <strong>an</strong>y overt deficits also make <strong>the</strong><br />
system more vulnerable to future exposures.<br />
Disclosures: M. Gomez-Chavarin, None; R. Morales, None; C. Torner, None; G. Rold<strong>an</strong>,<br />
None; J. Fern<strong>an</strong>dez, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.1/B34<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Swedish Research Council<br />
Title: The MAP- <strong>an</strong>d SAP-kinase pathways in Schw<strong>an</strong>n cells of <strong>the</strong> injured sciatic nerve
Authors: L. B. MÅRTENSSON 1 , L. B. DAHLIN 2 , *M. E. KANJE 3 ;<br />
1 Dept. of cell <strong>an</strong>d org<strong>an</strong>ism biology, Lund, Sweden; 2 Dept. of clinical sciences - h<strong>an</strong>d surgery,<br />
Lund, Sweden; 3 Cell <strong>an</strong>d Org<strong>an</strong>ism Biol., Lund, Sweden<br />
Abstract: Signal tr<strong>an</strong>sduction in Schw<strong>an</strong>n cells in response to <strong>an</strong> injury of <strong>the</strong> rat sciatic nerve<br />
was studied. Focus was directed on activation <strong>an</strong>d up-regulation of signalling molecules in <strong>the</strong><br />
MAP- <strong>an</strong>d SAP- kinase pathways, including <strong>the</strong> signalling elements Erk1/2 <strong>an</strong>d JNK as well as<br />
<strong>the</strong> tr<strong>an</strong>scription factors c-Jun <strong>an</strong>d ATF-3.<br />
We raised <strong>the</strong> question if Erk1/2 activation in <strong>the</strong> MAP-kinase pathway could be linked to <strong>the</strong><br />
SAP-kinase pathway, since we have shown that Erk 1/2 is activated in Schw<strong>an</strong>n cells at <strong>the</strong> site<br />
of injury (Mårtensson et al 2007).<br />
After axotomy in vivo, JNK immunoreactivity was increased in Schw<strong>an</strong>n cells, but not until after<br />
24 h. p-JNK immunoreactivity showed <strong>an</strong> irregular pattern. There was <strong>an</strong> early (1 h) upregulation<br />
of c-Jun in Schw<strong>an</strong>n cells both distal <strong>an</strong>d proximal to <strong>the</strong> site of <strong>the</strong> tr<strong>an</strong>section, while<br />
pc-Jun immunoreactivity increased in a biphasic m<strong>an</strong>ner. There was also <strong>an</strong> up-regulation of<br />
ATF-3 both distal <strong>an</strong>d proximal to <strong>the</strong> site of <strong>the</strong> nerve lesion; this increase in ATF-3<br />
immunoreactivity was first visible at 30 min <strong>an</strong>d continued up to 48 h.<br />
In cultured pieces of <strong>the</strong> sciatic nerve, which are amenable to pharmacological m<strong>an</strong>ipulations,<br />
alterations in JNK, c-Jun, pc-Jun <strong>an</strong>d ATF-3 in Schw<strong>an</strong>n cells were similar to that observed in<br />
vivo. However, p-JNK exhibited a tr<strong>an</strong>sient increase peaking at 6 h.<br />
In <strong>the</strong> cultured sciatic nerve pieces <strong>the</strong> inhibitor of <strong>the</strong> SAP-kinase pathway (SP600125) had no<br />
effect on <strong>the</strong> immunoreactivity of JNK, p-JNK, c-Jun, pc-Jun, Erk 1/2 or ATF-3 in <strong>the</strong> Schw<strong>an</strong>n<br />
cells. However, in DRG nerve cell nuclei SP600125 inhibited up-regulation of ATF-3. In nerve<br />
pieces cultured with <strong>the</strong> MAPK inhibitor U0126, <strong>the</strong> phosphorylation of Erk1/2 in <strong>the</strong> Schw<strong>an</strong>n<br />
cell cytoplasm was blocked, but it had no inhibitory effect on JNK, p-JNK, c-jun or pc-jun. In<br />
<strong>the</strong> nerves cultured with <strong>the</strong> MAPK inhibitor <strong>the</strong> number of proliferating Schw<strong>an</strong>n cells<br />
incorporating BrdU was reduced, but <strong>the</strong>re was no inhibitory effect on <strong>the</strong> up-regulation of ATF-<br />
3.<br />
We conclude that <strong>the</strong>re is activation of <strong>the</strong> MAP- <strong>an</strong>d SAP-kinase pathways in Schw<strong>an</strong>n cells in<br />
response to a nerve injury, but <strong>the</strong> pathways are not interconnected.<br />
Ref.<br />
Mårtensson L, Gustavsson P, Dahlin LB <strong>an</strong>d K<strong>an</strong>je M. Activation of extracellular-signal-related<br />
kinase-1/2 precedes <strong>an</strong>d is required <strong>for</strong> injury-induced Schw<strong>an</strong>n cell proliferation. Neuroreport<br />
2007; 18:957-961.<br />
Disclosures: L.B. Mårtensson, None; L.B. Dahlin, None; M.E. K<strong>an</strong>je, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 510.2/B35<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Swedish Medical Research Council<br />
Title: Injury-induced activation of ERK1/2 in sciatic nerve of healthy <strong>an</strong>d diabetic rats<br />
Authors: *L. B. DAHLIN 1 , L. STENBERG 2 , L. MÅRTENSSON 3 , M. KANJE 3 ;<br />
1 Sc<strong>an</strong>ia H<strong>an</strong>d Ctr. AB, S-200 42 Malmo, Sweden; 2 H<strong>an</strong>d Surgery, Dept Clin. Sciences, Malmo,<br />
Malmo, Sweden; 3 Dept of Cell <strong>an</strong>d Org<strong>an</strong>ism Biol., Lund, Sweden<br />
Abstract: Diabetes mellitus (DM), which affects 6 % of <strong>the</strong> world’s population, frequently<br />
induces neuropathy not only in <strong>the</strong> lower but also in <strong>the</strong> upper extremity. Neuropathy is related<br />
to morbidity <strong>an</strong>d mortality. The pathophysiology of neuropathy in diabetes is still not clarified.<br />
Extracellular-signal regulated kinase (ERK 1/2), which is governed by cell surface receptors, is<br />
activated by nerve damage <strong>an</strong>d its activation precedes proliferation of Schw<strong>an</strong>n cells. In this<br />
study, phosphorylated-ERK 1/2 (p-ERK 1/2) intensity in injured sciatic nerve ends was<br />
investigated with immunohistochemistry in healthy rats <strong>an</strong>d in two <strong>an</strong>imal models of diabetes.<br />
The rat models consist of streptozotocin (STZ)-induced DM <strong>an</strong>d Biobreeding (BB) rats, which<br />
spont<strong>an</strong>eously develops DM. p-ERK 1/2 activation was examined at three different time points<br />
(30 min, 1 h <strong>an</strong>d 48 h) after nerve tr<strong>an</strong>section. After tr<strong>an</strong>section rat sciatic nerves showed <strong>an</strong><br />
increased intensity of p-ERK 1/2 in <strong>the</strong> proximal <strong>an</strong>d in <strong>the</strong> distal nerve ends at all time points as<br />
compared to <strong>an</strong> uninjured control nerve. The staining was confined to Schw<strong>an</strong>n cells with only<br />
occasional faint staining in single axons. In both models of diabetic rats, a signific<strong>an</strong>tly lower<br />
intensity was found at 1 h in <strong>the</strong> distal nerve end <strong>an</strong>d at 48 h in <strong>the</strong> proximal nerve end th<strong>an</strong> in<br />
tr<strong>an</strong>sected non-diabetic nerves. In addition, in both models of diabetic rats <strong>an</strong> increased number<br />
of 4`,6-diamino-2-phenylindole (DAPI) stained cells (total number of cells) were observed in <strong>the</strong><br />
proximal <strong>an</strong>d distal nerve ends after injury at 48 h. ERK 1/2 is activated differentially after nerve<br />
injury in diabetic compared to healthy rats; findings that may shed fur<strong>the</strong>r light on how diabetes<br />
affects <strong>the</strong> peripheral nervous system with development of neuropathy.<br />
Disclosures: L.B. Dahlin, None; L. Stenberg, None; L. Mårtensson, None; M. K<strong>an</strong>je, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.3/B36
Topic: A.08.b. Regeneration: PNS<br />
Support: CIHR<br />
AHFMR<br />
Deutsche Forschungsgemeinschaft SFB470<br />
Title: Chemical communication between regenerating motor axons <strong>an</strong>d Schw<strong>an</strong>n cells in <strong>the</strong><br />
growth pathway<br />
Authors: T. GORDON 1 , *D. G. EVERAERT 2 , N. TYREMAN 1 , M. SCHACHNER 3 , G.<br />
VRBOVA 4 ;<br />
1 Ctr. <strong>for</strong> Neuroscience, Div. of Physical Med. <strong>an</strong>d Rehabil., 2 Univ. of Alberta, Edmonton, AB,<br />
C<strong>an</strong>ada; 3 Zentrum fur Molekulare Neurobiologie, Univ. Hamburg, Hamburg, Germ<strong>an</strong>y;<br />
4 Autonomic Neurosci. Ctr., Royal Free <strong>an</strong>d Univ. Col. Med. Sch., London, United Kingdom<br />
Abstract: It is well recognized that Schw<strong>an</strong>n cells are essential <strong>for</strong> axonal regeneration.<br />
Regeneration fails if <strong>the</strong> Schw<strong>an</strong>n cells undergo severe atrophy <strong>an</strong>d/or <strong>the</strong>y are not present. Even<br />
central axons regenerate in nerve pathways that contain Schw<strong>an</strong>n cells. Yet <strong>the</strong> mech<strong>an</strong>isms by<br />
which <strong>the</strong> Schw<strong>an</strong>n cells support <strong>the</strong> growth of regenerating axons have remained largely<br />
unknown. There are receptors on denervated Schw<strong>an</strong>n cells that may respond to <strong>the</strong><br />
neurotr<strong>an</strong>smitters acetylcholine (ACh) <strong>an</strong>d ATP that are released <strong>from</strong> growth cones of<br />
regenerating motor axons. In order to ascertain whe<strong>the</strong>r <strong>the</strong> interaction of <strong>the</strong> tr<strong>an</strong>smitters <strong>an</strong>d<br />
<strong>the</strong>ir receptors does play a role during axon regeneration, we hypo<strong>the</strong>sized that pharmacological<br />
block of <strong>the</strong> interaction would reduce <strong>the</strong> number of motoneurons that regenerate <strong>the</strong>ir axons<br />
after nerve section <strong>an</strong>d surgical repair. The tibial nerve was cut <strong>an</strong>d cross-sutured to <strong>the</strong> distal<br />
stump of <strong>the</strong> common peroneal nerve in <strong>the</strong> hindlimbs of adult Sprague Dawley rats. The sciatic<br />
nerve was cut <strong>an</strong>d resutured in C57BL/6J mice. Various drugs were applied to <strong>the</strong> peripheral<br />
nerve stump ei<strong>the</strong>r directly or via mini-osmotic pumps over a 2-4 week period to block <strong>the</strong><br />
binding of ACh to nicotinic <strong>an</strong>d muscarinic receptors (AChRs: α-bungarotoxin, tubocurarine,<br />
atropine <strong>an</strong>d gallamine) <strong>an</strong>d binding of ATP to P2Y receptors (suramin) in experimental groups<br />
of <strong>an</strong>imals. Saline was administered in <strong>the</strong> control group of <strong>an</strong>imals. The number of motoneurons<br />
that had regenerated <strong>the</strong>ir axons was counted after backlabelling of <strong>the</strong> regenerated axons with<br />
fluororuby. The nicotinic AChR <strong>an</strong>tagonistic drugs α-bungarotoxin <strong>an</strong>d tubocurarine, <strong>the</strong> nonspecific<br />
AChR <strong>an</strong>tagonist gallamine, <strong>an</strong>d <strong>the</strong> P2Y receptor <strong>an</strong>tagonist suramin, signific<strong>an</strong>tly<br />
reduced <strong>the</strong> number of tibial motoneurons that regenerated <strong>the</strong>ir axons through <strong>the</strong> common<br />
peroneal distal nerve stumps. In mice, suramin signific<strong>an</strong>tly reduced <strong>the</strong> upregulation of <strong>the</strong><br />
regeneration conducive HNK-1 (natural killer cell) carbohydrate on <strong>the</strong> Schw<strong>an</strong>n cells in <strong>the</strong><br />
sciatic distal nerve stump that normally occurs during motor axon regeneration. These data<br />
indicate that chemical communication between regenerating axons <strong>an</strong>d Schw<strong>an</strong>n cells during<br />
axon regeneration via released neurotr<strong>an</strong>smitters <strong>an</strong>d <strong>the</strong>ir receptors may play <strong>an</strong> import<strong>an</strong>t role<br />
in axon regeneration in <strong>the</strong> peripheral nervous system.<br />
Disclosures: T. Gordon, None; D.G. Everaert, None; N. Tyrem<strong>an</strong>, None; M. Schachner,<br />
None; G. Vrbova, None.
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.4/B37<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Adelson Medical Research Foundation<br />
Title: Differential gene expression in denervated motor <strong>an</strong>d sensory Schw<strong>an</strong>n cells<br />
Authors: *M. C. WRIGHT 1 , G. COPOLLA 4 , D. H. GESCHWIND 4 , T. BRUSHART 2 , A.<br />
HOKE 3 ;<br />
1 2 3 4<br />
Neurol., Orthopedics, Neurol. <strong>an</strong>d Neurosci., Johns Hopkins, Baltimore, MD; Neurol.,<br />
UCLA, Los Angeles, CA<br />
Abstract: Previous studies <strong>from</strong> our lab examined growth factor expression in subsets of<br />
Schw<strong>an</strong>n cells (SCs) <strong>an</strong>d demonstrated that SCs express distinct motor <strong>an</strong>d sensory phenotypes<br />
that influence modality specific axonal regeneration (Höke, J Neurosci. 2006; 26 :9646-55). The<br />
present experiments exp<strong>an</strong>ded on <strong>the</strong>se findings <strong>an</strong>d using microarray <strong>an</strong>alysis, examined<br />
differential gene expression in denervated dorsal roots, a pure population of sensory SCs, <strong>an</strong>d<br />
denervated ventral roots, a pure population of motor SCs. The purpose of <strong>the</strong>se studies is to<br />
identify specific regeneration associated genes that are selectively expressed in motor <strong>an</strong>d<br />
sensory SCs <strong>an</strong>d to determine <strong>the</strong> contribution of <strong>the</strong>se genes in modality specific axonal<br />
regeneration. The L3, L4, <strong>an</strong>d L5 dorsal <strong>an</strong>d ventral roots were unilaterally denervated in adult<br />
female rats. Fifteen days later, <strong>the</strong> denervated dorsal <strong>an</strong>d ventral roots <strong>an</strong>d contralateral,<br />
uninjured dorsal <strong>an</strong>d ventral roots were harvested. Ch<strong>an</strong>ges in gene expression were examined by<br />
microarray <strong>an</strong>d real-time RT-PCR <strong>an</strong>alysis. Over 75 genes considered signific<strong>an</strong>tly upregulated<br />
in ei<strong>the</strong>r denervated dorsal or ventral roots in <strong>the</strong> microarray experiments were selected based on<br />
available literature <strong>an</strong>d <strong>the</strong>ir potential to influence sensory or motor axon regeneration. These<br />
genes included a number of tr<strong>an</strong>scription factors, secreted proteins, cell-membr<strong>an</strong>e associated<br />
proteins <strong>an</strong>d receptors. The upregulation of <strong>the</strong> selected genes was subsequently confirmed using<br />
real-time RT-PCR. From <strong>the</strong> RT-PCR experiments, 10 genes have been selected <strong>for</strong> fur<strong>the</strong>r<br />
<strong>an</strong>alysis. The contribution of <strong>the</strong>se genes in sensory <strong>an</strong>d motor nerve regeneration is presently<br />
being tested using a number of in vitro <strong>an</strong>d in vivo models of axonal outgrowth <strong>an</strong>d regeneration.<br />
Fur<strong>the</strong>r investigation of <strong>the</strong> identified genes <strong>an</strong>d <strong>the</strong>ir role in axonal regeneration may offer<br />
<strong>the</strong>rapeutic avenues to enh<strong>an</strong>ce motor <strong>an</strong>d sensory specific axonal regeneration.
Disclosures: M.C. Wright, None; G. Copolla, None; D.H. Geschwind, None; T. Brushart,<br />
None; A. Hoke, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.5/B38<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: The Brody Bro<strong>the</strong>rs Foundation<br />
Title: The role of Dicer-dependent miRNA pathway in peripheral nerve regeneration<br />
Authors: *D. WU, A. RAAFAT, A. NAGLER, E. PAK, A. MURASHOV;<br />
East Carolina Univ., Greenville, NC<br />
Abstract: MicroRNAs (miRNAs) are small, non-coding RNAs that function as import<strong>an</strong>t<br />
regulators of gene expression. Dysregulation of miRNAs has been implicated in m<strong>an</strong>y diseases<br />
including neurological disorders. Work <strong>from</strong> our laboratory, as well as <strong>from</strong> o<strong>the</strong>rs, showed that<br />
peripheral nerve axons in vivo <strong>an</strong>d in vitro contain pivotal miRNA machinery proteins. While<br />
previous studies demonstrated <strong>the</strong> response of miRNA biogenesis to injury, <strong>the</strong> physiological<br />
signific<strong>an</strong>ce of it remains to be elucidated. There<strong>for</strong>e, <strong>the</strong> aim of this study was to investigate <strong>the</strong><br />
role of Dicer, a key enzyme responsible <strong>for</strong> generation of miRNAs, on peripheral nerve<br />
regeneration. We presumed that deletion of Dicer would disrupt production of Dicer-dependent<br />
small RNAs <strong>an</strong>d negatively impact regenerative axon growth. A model of sciatic nerve crush<br />
injury was used on tamoxifen-inducible knockout mice CAG-CreERt:Dicer fl/fl . Functional<br />
behavioral test showed that sciatic functional index, <strong>an</strong> indicator of functional recovery, was<br />
lower in tamoxifen treated CAG-CreERt:Dicer fl/fl mice compared to non-treated group<br />
throughout regeneration. Electrophysiological study on regenerating sciatic nerves of tamoxifen<br />
treated <strong>an</strong>d non-treated CAG-CreERt:Dicer fl/fl revealed <strong>the</strong> effect of Dicer deletion on nerve<br />
conduction velocity <strong>an</strong>d compound action potentials. Regenerative axon growth after sciatic<br />
nerve injury was also examined with immunohistochemistry <strong>an</strong>d <strong>an</strong>alysis of semi-thin sections<br />
<strong>for</strong> <strong>the</strong> total number of regenerating nerve fibers <strong>an</strong>d me<strong>an</strong> axonal area of myelinated fiber after<br />
injury in tamoxifen treated CAG-CreERt:Dicer fl/fl knockout mice. Our results suggest that<br />
deletion of Dicer may markedly decelerate peripheral nerve regeneration <strong>an</strong>d functional<br />
recovery. Our data provides <strong>an</strong> insight on <strong>the</strong> role of Dicer-dependent miRNA pathway in<br />
regulation of regenerative axon growth.
Disclosures: D. Wu , None; A. Raafat, None; A. Nagler, None; E. Pak, None; A. Murashov,<br />
None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.6/B39<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Adelson Program in Neural Repair <strong>an</strong>d Rehabilitation<br />
NIH gr<strong>an</strong>t 5R01NS041269-07<br />
Title: Loss of Merkel cells in <strong>the</strong> hind foot skin of mice with inhibition of TrkC signaling<br />
Authors: *B. PAN 1 , D. GINTY 2,3 , J. W. GRIFFIN 1,2 ;<br />
1 2 3<br />
Neurol., Neurosci., Johns Hopkins Sch. Med., Baltimore, MD; Howard Hughes Med. Inst.,<br />
Baltimore, MD<br />
Abstract: Neurotrophins (NT) <strong>an</strong>d <strong>the</strong>ir high affinity Trk receptors are not only import<strong>an</strong>t <strong>for</strong> <strong>the</strong><br />
differentiation <strong>an</strong>d survival of specific neurons in sympa<strong>the</strong>tic <strong>an</strong>d dorsal root g<strong>an</strong>glia, but also in<br />
target innervation <strong>an</strong>d nerve regeneration. NT3 <strong>an</strong>d its receptor TrkC, a member of <strong>the</strong> Trk<br />
family of neurotrophin receptors, are essential in <strong>the</strong> development of large sensory fibers that<br />
innervate Merkel cells in touch dome <strong>an</strong>d hair follicles, as well as muscle spindles <strong>an</strong>d Golgi<br />
tendon org<strong>an</strong>s. In <strong>the</strong> present study, we studied <strong>the</strong> role of TrkC signaling during adulthood in<br />
mice with Trk knock-in mutation TrkC F617A that allows <strong>for</strong> pharmacological control of Trk kinase<br />
activity (Chen et al, 2005). It has been previously characterized that n<strong>an</strong>omolar concentrations of<br />
1NMPP1, a derivative of <strong>the</strong> general kinase inhibitor PP1, inhibit NGF <strong>an</strong>d BDNF signaling in<br />
TrkA F592A <strong>an</strong>d TrkB F616A neurons, respectively, but no such Trk inhibition is observed in wildtype<br />
neurons. The present work was aimed to study <strong>the</strong> function of TrkC signaling in adult mice<br />
using Merkel cell as <strong>the</strong> index. Homozygous TrkC F617A are viable <strong>an</strong>d fertile with no observable<br />
phenotype. Footpad skins were immediately embedded <strong>an</strong>d frozen in OCT. Cryostat sections<br />
were cut at 16 µm. Merkel cells were labeled by immunohistochemistry using <strong>an</strong> <strong>an</strong>tibody<br />
against cytokeratin-20. The number of Merkel cells was qu<strong>an</strong>tified <strong>an</strong>d compared between<br />
groups using one way ANOVA <strong>an</strong>alysis. Compared to wild-type mice, <strong>the</strong>re was a 30% decrease<br />
in <strong>the</strong> number of Merkel cells in TrkC F617A mice (p< 0.05), suggesting that <strong>the</strong>se <strong>an</strong>imals are<br />
mildly hypomorphic. Ten week old wild-type <strong>an</strong>d TrkC F617A mice were treated with 1NMPP1 (40<br />
µM 1NMPP1 in drinking water) or vehicle DMSO <strong>for</strong> 20 days. Footpad skin was collected <strong>from</strong>
<strong>the</strong> hind paw under deep <strong>an</strong>es<strong>the</strong>sia. Animals were <strong>the</strong>n perfusion fixed. INMPP1 treatment, a<br />
fur<strong>the</strong>r reduction of 35% in Merkel cell numbers was observed in TrkC F617A mice, but not in<br />
wild-type mice (p< 0.001). Despite a reduction in <strong>the</strong> density of Merkel cells, cell size <strong>an</strong>d<br />
morphology remain similar to that of <strong>the</strong> wild-type. These results indicate that TrkC activity is<br />
required <strong>for</strong> <strong>the</strong> mainten<strong>an</strong>ce of Merkel cells <strong>an</strong>d/or <strong>the</strong>ir afferents in <strong>the</strong> hind paw skin.<br />
Disclosures: B. P<strong>an</strong>, None; D. Ginty, None; J.W. Griffin, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.7/B40<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH gr<strong>an</strong>t NS39098<br />
The Miami Project to Cure Paralysis<br />
Title: Acute stimulation of neurons tr<strong>an</strong>spl<strong>an</strong>ted into peripheral nerve improves motoneuron<br />
survival<br />
Authors: *R. M. GRUMBLES, C. M. THOMAS, K. MORRIS, Y. LIU, C. K. THOMAS;<br />
Miami Project Cure Paralysis, Univ. Miami Sch. Med., Miami, FL<br />
Abstract: Trauma to <strong>the</strong> spinal cord <strong>an</strong>d disease result in death of motoneurons, often across<br />
several spinal levels. Skeletal muscles are denervated, paralyzed, <strong>an</strong>d atrophy rapidly. There is<br />
no effective <strong>the</strong>rapy to restore muscle function when motoneuron death is widespread. One week<br />
after sciatic nerve section in adult Fischer rats, we tr<strong>an</strong>spl<strong>an</strong>ted dissociated embryonic day 14-15<br />
ventral spinal cord cells into <strong>the</strong> tibial nerve as <strong>the</strong> only source of neurons <strong>for</strong> muscle<br />
reinnervation. Our aim was to test whe<strong>the</strong>r depolarization of <strong>the</strong> tr<strong>an</strong>spl<strong>an</strong>t ch<strong>an</strong>ged motoneuron<br />
survival. In one group of <strong>an</strong>imals <strong>the</strong> embryonic cells were stimulated electrically at 20 Hz <strong>for</strong> 1<br />
hour immediately after tr<strong>an</strong>spl<strong>an</strong>tation. The o<strong>the</strong>r group received cells but no stimulation. Ten<br />
weeks later, physiological recordings showed that more muscles contracted in <strong>an</strong>imals that<br />
received cells <strong>an</strong>d stimulation versus only cells. Electrical stimulation of <strong>the</strong> cells at <strong>the</strong> time of<br />
tr<strong>an</strong>spl<strong>an</strong>tation also resulted in greater numbers of neurons (NeuN-positive), ChAT positive<br />
neurons, <strong>an</strong>d myelinated axons. These data suggest that acute neuron depolarization immediately<br />
after cell tr<strong>an</strong>spl<strong>an</strong>tation improves long-term neuron survival, axon regeneration <strong>an</strong>d muscle<br />
function.
Disclosures: R.M. Grumbles, None; C.M. Thomas, None; K. Morris, None; Y. Liu,<br />
None; C.K. Thomas, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.8/B41<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NS057190<br />
Title: Limited peripheral axon regeneration in conditional BDNF knockout mice<br />
Authors: *J. C. WILHELM, D. CUCORANU, J. GU, A. MULLIGAN, A. W. ENGLISH;<br />
Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: After peripheral nerve injury, <strong>the</strong> upregulation of neurotrophins has been shown to<br />
have a key role in <strong>the</strong> regeneration of axotomized axons. In this study, we evaluated <strong>the</strong><br />
requirement <strong>for</strong> brain derived neurotrophic factor (BDNF) <strong>for</strong> <strong>the</strong> regeneration of peripheral<br />
axons after injury. The common fibular or tibial nerves in thy-1-YFP-H mice, a strain in which a<br />
subset of peripheral axons express yellow fluorescent protein (YFP), were cut bilaterally <strong>an</strong>d<br />
repaired using a graft of <strong>the</strong> same nerve <strong>from</strong> adult, strain-matched, non-fluorescent wild-type<br />
(WT) or BDNF knockout (BDNF -/-) mice. BDNF -/- nerve graft donors were produced using a<br />
Cre/loxP-system <strong>for</strong> conditional gene deletion. In one strain, BDNF was constitutively knocked<br />
out in a cell-type specific m<strong>an</strong>ner by <strong>the</strong> expression of Cre recombinase in all CNTF-expressing<br />
cells (CNTF-Cre + ). In <strong>the</strong> o<strong>the</strong>r strain, conditional BDNF knock out was achieved systemically<br />
by activation of Cre recombinase through oral administration of tamoxifen (tam-Cre + ).<br />
There<strong>for</strong>e, <strong>the</strong> grafts <strong>from</strong> ei<strong>the</strong>r of <strong>the</strong>se strains lacked BDNF in Schw<strong>an</strong>n cells. Two weeks<br />
post-repair, <strong>the</strong> grafts were harvested, <strong>an</strong>d <strong>the</strong> lengths of profiles of regenerating YFP + axons<br />
were measured <strong>from</strong> optical sections using fluorescence microscopy. The lengths of profiles of<br />
axons growing into BDNF-/- grafts were less th<strong>an</strong> one third <strong>the</strong> length of those growing into WT<br />
grafts, emphasizing <strong>the</strong> import<strong>an</strong>ce of Schw<strong>an</strong>n cell BDNF <strong>for</strong> peripheral axon regeneration.<br />
There was no signific<strong>an</strong>t difference in <strong>the</strong> lengths of axon profiles in grafts <strong>from</strong> <strong>the</strong> CNTF-Cre +<br />
or tam-Cre + strains. Thus, it is unlikely that our results are due to potential effects of developing<br />
without BDNF or to tamoxifen treatment. In addition, axon profile lengths in grafts <strong>from</strong><br />
tamoxifen-treated WT or tam-Cre + mice with only one floxed BDNF allele (BDNF floxed/wt ) were<br />
not signific<strong>an</strong>tly different <strong>from</strong> those in grafts <strong>from</strong> untreated WT mice. The lengths of YFP +<br />
axons in grafts <strong>from</strong> untreated tam-Cre + mice or BDNF floxed/wt ::CNTF-Cre + mice also did not
differ signific<strong>an</strong>tly when compared to lengths measured in WT grafts. Taken toge<strong>the</strong>r, <strong>the</strong><br />
reduction in profile length in BDNF -/- grafts is consistent with <strong>the</strong> idea that BDNF is required<br />
<strong>for</strong> normal regeneration of peripheral axons. Although <strong>the</strong> proximal segment of <strong>the</strong> cut nerves<br />
was BDNF +/+, regenerating axons did not grow into <strong>the</strong> distal segment; thus, <strong>the</strong> requirement<br />
<strong>for</strong> BDNF must be <strong>from</strong> <strong>the</strong> Schw<strong>an</strong>n cells in <strong>the</strong> distal segment ra<strong>the</strong>r th<strong>an</strong> <strong>from</strong> proximal<br />
Schw<strong>an</strong>n cells or <strong>from</strong> <strong>the</strong> regenerating axons <strong>the</strong>mselves.<br />
Disclosures: J.C. Wilhelm, None; D. Cucor<strong>an</strong>u, None; J. Gu, None; A. Mullig<strong>an</strong>,<br />
None; A.W. English, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.9/B42<br />
Topic: A.08.b. Regeneration: PNS<br />
Title: Role of c-Jun <strong>an</strong>d its N-terminal phosphorylation in facial nerve regeneration<br />
Authors: C. RUFF 1 , C. DA COSTA 2 , N. STAAK 3 , P. RUMAJOGEE 1 , M. MAKWANA 1 , A.<br />
ACOSTA-SALTOS 1 , M. KASWICH 3 , S. BRECHT 3 , M. HRISTOVA 1 , A. S. NATERI 2 , L.<br />
RIERA-SANS 2 , R. MIRSKY 4 , T. HERDEGEN 3 , K. R. JESSEN 4 , A. BEHRENS 2 , *G.<br />
RAIVICH 1 ;<br />
1 Perinatal Brain Repair Group, Inst. Women's Health, Univ. Col. London, London, United<br />
Kingdom; 2 Mammali<strong>an</strong> Genet. Laboratory, C<strong>an</strong>cer Res. UK, London, United Kingdom; 3 Dept<br />
Neuropharmacology, Univ. of Kiel, Kiel, Germ<strong>an</strong>y; 4 Dept. of Cell <strong>an</strong>d Developmental Biology,<br />
Univ. Col. London, London, United Kingdom<br />
Abstract: C-Jun is <strong>an</strong> immediate early gene that is <strong>an</strong> integral component of <strong>the</strong> AP1<br />
tr<strong>an</strong>scription complex. Previous studies link prolonged upregulation of c-Jun to successful<br />
neuronal regeneration, with Jun N-Terminal Kinases (JNKs) playing <strong>an</strong> influential role in Jun<br />
signal tr<strong>an</strong>sduction. However, <strong>the</strong>re is still a question whe<strong>the</strong>r c-Jun phosphorylation is needed<br />
<strong>for</strong> regeneration.<br />
Here, we have investigated <strong>the</strong> role of c-Jun <strong>an</strong>d its phosphorylation in axonal outgrowth, motor<br />
neuron survival <strong>an</strong>d cellular response after facial motor nerve axotomy. Brain-specific deletion<br />
of c-Jun, using Nestin::Cre <strong>an</strong>d floxed jun resulted in defects in neuronal chromatolysis,<br />
perineuronal sprouting, lymphocyte recruitment <strong>an</strong>d microglial activation. Motor neurons also<br />
exhibited <strong>an</strong> atrophic phenotype, reduced target reinnervation <strong>an</strong>d were resist<strong>an</strong>t to cell death.<br />
Schw<strong>an</strong>n cell-selective P0::Cre deletion of floxed c-Jun did not affect <strong>the</strong> initial neuronal cell
ody response, but interfered with axonal regeneration as well as strongly enh<strong>an</strong>ced neuronal cell<br />
death. In contrast, <strong>the</strong> homozygous deletion of JNK1, JNK2 or JNK3, or substitution of <strong>the</strong> c-Jun<br />
N-terminal serine phosphoacceptor sites (ser63&73), with al<strong>an</strong>ines (JunAA), did not produce a<br />
difference in response to injury. In fact, <strong>the</strong>re were similar levels of perineuronal sprouting,<br />
microglial <strong>an</strong>d astrocyte activation in <strong>the</strong> JNK1-3kos <strong>an</strong>d JunAA mut<strong>an</strong>ts compared to <strong>the</strong>ir wild<br />
type controls. Motor neuron cell death, as well as functional <strong>an</strong>d <strong>an</strong>atomical reinnervation was<br />
also not affected by JNK1, 2, or 3 deletions or <strong>the</strong> JunAA mutation.<br />
The current results indicate that N-terminal phosphorylation of ser63&73 does not play <strong>an</strong><br />
essential function <strong>for</strong> axonal regeneration in vivo. They shed light on <strong>the</strong> essential, non-JNKdependent<br />
role of neuronal <strong>an</strong>d Schw<strong>an</strong>n cell c-Jun in regulating neuronal survival <strong>an</strong>d<br />
promoting axonal regeneration.<br />
Disclosures: C. Ruff, None; C. Da Costa, None; N. Staak, None; P. Rumajogee, None; M.<br />
Makw<strong>an</strong>a, None; A. Acosta-Saltos, None; M. Kaswich, None; S. Brecht, None; M. Hristova,<br />
None; A.S. Nateri, None; L. Riera-S<strong>an</strong>s, None; R. Mirsky, None; T. Herdegen, None; K.R.<br />
Jessen, None; A. Behrens, None; G. Raivich, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.10/B43<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH Gr<strong>an</strong>t 5R01AG32322-2 (MPI)<br />
Title: Monitoring nerve regeneration in aged <strong>an</strong>imal in vivo with a new video-rate 2-photon<br />
microscope<br />
Authors: *H. KANG, J. W. LICHTMAN;<br />
Ctr. <strong>for</strong> Brain Sci., Harvard Univ., Cambridge, MA<br />
Abstract: Sarcopenia, <strong>the</strong> degenerative loss of skeletal muscle mass <strong>an</strong>d strength associated with<br />
aging is a major health concern as it limits <strong>the</strong> mobility <strong>an</strong>d independence of aged people. The<br />
cause remains unknown. Denervation <strong>an</strong>d failure of reinnervation of muscle fibers may be part<br />
of <strong>the</strong> problem.<br />
Our aim is to study how regenerating axons grow <strong>an</strong>d come to reoccupy <strong>for</strong>merly denervated<br />
junctions in aged <strong>an</strong>imals. In vivo time-lapse imaging of tr<strong>an</strong>sgenic mice that express fluorescent<br />
protein in <strong>the</strong>ir axons provides valuable data of nerve growth <strong>an</strong>d synapse <strong>for</strong>mation in situ.
However, <strong>an</strong>imal movements due to pulse <strong>an</strong>d breathing do not allow <strong>the</strong> slow image acquisition<br />
of conventional confocal or multiphoton sc<strong>an</strong>ning microscopes as <strong>the</strong>se movements cause<br />
distortions in images. To overcome this problem, we constructed a video-rate 2-photon<br />
microscope using a reson<strong>an</strong>ce sc<strong>an</strong>ner which allows acquisitions more th<strong>an</strong> 60 frames per<br />
second. Combining of fast sc<strong>an</strong>ning ability <strong>an</strong>d femtoliter volume excitability of multiphoton<br />
photon microscopy has allowed us to obtain high spatiotemporal resolution images of growing<br />
axons in living <strong>an</strong>imals.<br />
To address <strong>the</strong> questions of nerve regeneration <strong>an</strong>d synapse <strong>for</strong>mation in aged <strong>an</strong>imals, we<br />
imaged regenerating motor axons in <strong>the</strong> sternomastoid following nerve damage by simple crush.<br />
The results show that axon growth in aged <strong>an</strong>imal is impaired in part by excessive nerve<br />
br<strong>an</strong>ching. Our data suggests that <strong>the</strong> fundamental process of axon regeneration is disrupted with<br />
adv<strong>an</strong>ced age.<br />
Disclosures: H. K<strong>an</strong>g, None; J.W. Lichtm<strong>an</strong>, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.11/B44<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: AxoGen Inc.<br />
Title: Sensory, motor <strong>an</strong>d mixed donor nerves are equally effective as cellular <strong>an</strong>d<br />
decellularized, chondroitinase processed grafts in rat peripheral nerve reconstruction<br />
Authors: J. B. GRAHAM 1 , D. NEUBAUER 1 , *D. F. MUIR 2 ;<br />
1 Pediatric Neurol., 2 Univ. of Florida Col. of Med., Gainesville, FL<br />
Abstract: Mixed nerves (containing both sensory <strong>an</strong>d motor axons) have been used successfully<br />
<strong>for</strong> nerve autografting in <strong>an</strong>imals. Also, m<strong>an</strong>y studies find that both sensory <strong>an</strong>d motor nerves are<br />
equivalent as grafts in <strong>the</strong> support of nerve regeneration. However, recent reports, including<br />
Nichols et al. (Exp. Neurol. 2004; 190: 347-355), conclude that sensory nerve grafts are inferior<br />
to motor <strong>an</strong>d mixed nerve grafts <strong>for</strong> repair of a mixed nerve defect. This raises concerns in<br />
clinical nerve grafting because sensory nerves are most often selected <strong>for</strong> use as autografts. The<br />
aim of <strong>the</strong> present study was to determine if this same disparity found with cellular graft<br />
subtypes exists <strong>for</strong> detergent decellularized, chondroitinase ABC processed rat nerve grafts. We<br />
hypo<strong>the</strong>size that this processing ei<strong>the</strong>r improves or neutralizes <strong>the</strong> presumptive inferior
properties of sensory nerve grafts. Grafts were prepared <strong>from</strong> <strong>the</strong> saphenous (sensory), femoral<br />
quadriceps (motor) <strong>an</strong>d tibial (mixed) nerves of Lewis rats. Size disparity between <strong>the</strong>se grafts<br />
was compensated <strong>for</strong> by construction of a three-cable sensory graft, a two-cable motor graft <strong>an</strong>d<br />
a single-cable mixed graft, which provided grafts of comparable diameter. Grafts (5 mm length)<br />
were used in tensionless nerve reconstruction of syngenic Lewis rat tibial (mixed) nerve. Nerve<br />
regeneration was evaluated 21 days after nerve grafting. Axons were scored 3 mm distal to <strong>the</strong><br />
graft by two methods, total axons by neurofilament-immunolabeling <strong>an</strong>d myelinated axons by<br />
toluidine blue staining of semi-thin sections. First, we retested <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong>re is a<br />
difference in <strong>the</strong> outcome with cellular sensory, motor <strong>an</strong>d mixed nerve grafts, duplicating <strong>the</strong><br />
experimental design of Nichols et al. Using <strong>the</strong> same scoring methods (toluidine blue staining),<br />
we found no signific<strong>an</strong>t difference in <strong>the</strong> myelinated axon counts <strong>for</strong> <strong>the</strong> three cellular graft<br />
subtypes (sensory 1672 ±188; motor 1814 ±72; mixed 1864 ±81). Fur<strong>the</strong>rmore, scoring<br />
neurofilament immunopositive axons (which also accounts <strong>for</strong> unmyelinated axons) showed that<br />
cellular sensory grafts were somewhat superior to mixed <strong>an</strong>d motor nerve grafts in <strong>the</strong> repair of a<br />
mixed nerve defect (sensory 9273 ±523; motor 7431 ±453; mixed 8133 ±387). Similar results<br />
were obtained <strong>for</strong> acellular processed nerve grafts, <strong>for</strong> which no signific<strong>an</strong>t differences in<br />
neurofilament-immunopositive axon counts were found between sensory (5687 ±211), motor<br />
(5093 ±478) <strong>an</strong>d mixed (5718 ±865) nerve grafts. These findings show that <strong>the</strong> type of nerve<br />
(sensory, motor, mixed) used as a source <strong>for</strong> ei<strong>the</strong>r cellular or acellular grafts has no signific<strong>an</strong>t<br />
impact on <strong>the</strong> short-term outcome of nerve regeneration in a mixed nerve repair model.<br />
Disclosures: J.B. Graham, None; D. Neubauer, None; D.F. Muir, D. Muir is <strong>an</strong> inventor of<br />
nerve grafting technology discussed in this study <strong>an</strong>d holds equity in AxoGen, Inc., a comp<strong>an</strong>y<br />
commercializing <strong>the</strong> processed allograft technology., E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.12/B45<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH NS041596<br />
NIH NS049041<br />
The Adelson Medical Foundation
The Christopher & D<strong>an</strong>a Reeve Foundation<br />
The Nemours Foundation<br />
Title: Co-tr<strong>an</strong>sport of axonal mRNAs by ZBP1 facilitates neurite growth<br />
Authors: *C. J. DONNELLY 1 , D. E. WILLIS 2 , M. XU 3 , S. YOO 2 , I. GOMEZ-CURET 2 , M.<br />
KENDALL 2 , M. ERENSHTEYN 2 , A. ENGLISH 3 , W. WANG 2 , G. J. BASSELL 3 , J. TWISS 2,1 ;<br />
1 Dept. of Biol. Sci., Univ. of Delaware, Newark, DE; 2 Nemours Biomed. Res., Alfred I duPont<br />
Hosp. <strong>for</strong> Children, Wilmington, DE; 3 Dept. of Cell Biol., Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Localized protein syn<strong>the</strong>sis in axons contributes to axonal pathfinding, injury<br />
responses <strong>an</strong>d regeneration. Surprisingly, a large population of mRNAs have been detected in<br />
axons of cultured neurons. Increasing evidence fur<strong>the</strong>r suggests that mRNAs are tr<strong>an</strong>sported into<br />
<strong>the</strong> axonal compartment in vivo during development <strong>an</strong>d after injury. It is clear that only some<br />
mRNAs localize into axons, pointing to specific mech<strong>an</strong>isms <strong>for</strong> targeting <strong>the</strong>se tr<strong>an</strong>scripts <strong>for</strong><br />
subcellular tr<strong>an</strong>sport. Looking at individual tr<strong>an</strong>scripts, only a fraction of <strong>the</strong> total cellular pool<br />
of <strong>an</strong> mRNA is localized suggesting that <strong>the</strong> machinery needed to tr<strong>an</strong>sport <strong>the</strong> mRNAs are in<br />
limited supply. To test this possibility, we used <strong>the</strong> well-characterized β-actin mRNA<br />
localization system. Subcellular localization of β-actin mRNA requires binding to a cis-acting<br />
element in 3’UTR by <strong>the</strong> mRNA binding protein, zipcode binding protein 1 (ZBP1).<br />
Overexpression of a dzGFPmyr reporter containing <strong>the</strong> 3'UTR of β-actin mRNA in DRG<br />
neurons cultured <strong>from</strong> adult rats decreased axonal localization of <strong>the</strong> endogenous β-actin mRNA.<br />
This was accomp<strong>an</strong>ied by attenuated axonal outgrowth in both adult DRG neurons <strong>an</strong>d<br />
embryonic primary motor neuron cultures. DRG neurons cultured <strong>from</strong> tr<strong>an</strong>sgenic mice<br />
expressing dzGFPmyr3' β-actin under control of Tα1 promoter also showed decreased axonal<br />
outgrowth. Tr<strong>an</strong>sfection of DRG neurons <strong>from</strong> dzGFPmyr3' β-actin mice with ZBP1 rescued this<br />
growth deficit, indicating that this RNA binding protein is in limited supply. However,<br />
tr<strong>an</strong>sfection with a construct expressing β-actin protein + 3'UTR, to compete with <strong>the</strong> tr<strong>an</strong>sgene<br />
<strong>for</strong> ZBP1 binding, does not rescue <strong>the</strong> growth deficit. This suggests that tr<strong>an</strong>sport of o<strong>the</strong>r<br />
mRNAs by ZBP1 in addition to β-actin mRNA contribute to neurite growth. Consistent with this<br />
idea, DRG neurons expressing <strong>the</strong> dzGFPmyr + 3'UTR of β-actin showed signific<strong>an</strong>t depletion<br />
of GAP-43 <strong>an</strong>d neuritin mRNAs <strong>from</strong> <strong>the</strong>ir axons. Conversely, tr<strong>an</strong>sfection with ZBP1 increased<br />
axonal localization of GAP-43 <strong>an</strong>d β-actin mRNAs above control. These studies indicate that<br />
RNA binding protein availability is a critical <strong>an</strong>d specific determin<strong>an</strong>t of axonal mRNA levels.<br />
Fur<strong>the</strong>rmore, locally syn<strong>the</strong>sized protein products of mRNAs cotr<strong>an</strong>sported by ZBP1 facilitate<br />
axonal growth.<br />
Disclosures: C.J. Donnelly, None; D.E. Willis, None; M. Xu, None; S. Yoo, None; I. Gomez-<br />
Curet, None; M. Kendall, None; M. Erenshteyn, None; A. English, None; W. W<strong>an</strong>g,<br />
None; G.J. Bassell, None; J. Twiss, None.<br />
Poster
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.13/B46<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: CIHR<br />
AHFMR<br />
Title: Accelerated axon outgrowth <strong>an</strong>d target reinnervation <strong>from</strong> tr<strong>an</strong>sected sciatic peripheral<br />
nerves following brief electrical stimulation<br />
Authors: B. SINGH 1 , Q.-G. XU 1 , C. DALTON 1 , T. GORDON 2 , *R. MIDHA 1 , D. W.<br />
ZOCHODNE 1 ;<br />
1 2<br />
Clin. Neurosci, Univ. Calgary, Calgary, AB, C<strong>an</strong>ada; Ctr. <strong>for</strong> Neurosci., Univ. of Alberta,<br />
Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: Despite assumptions o<strong>the</strong>rwise, injury to peripheral neurons is associated with<br />
incomplete <strong>an</strong>d delayed regeneration. Brief electrical stimulation (ES) applied to injured<br />
peripheral nerves immediately after injury has been discovered to improve axon regeneration.<br />
This study explored ES <strong>an</strong>d additional facets of regeneration: early axon <strong>an</strong>d Schw<strong>an</strong>n cell<br />
outgrowth <strong>from</strong> tr<strong>an</strong>section injuries, distal target reinnervation <strong>an</strong>d neurite outgrowth <strong>from</strong> adult<br />
neurons in vitro.<br />
Mice were divided into ES <strong>an</strong>d sham (S) stimulation groups (n=17/group). Sciatic nerves in each<br />
group were tr<strong>an</strong>sected <strong>an</strong>d repaired within a silicone tube (2 mm gap). The ES paradigm, as<br />
identified previously in <strong>the</strong> Gordon laboratory (0.1ms, 3V, 20Hz <strong>for</strong> 1 hr) was applied<br />
immediately <strong>the</strong>reafter. At 7 days <strong>the</strong> regenerating cables were harvested <strong>for</strong> neurofilament <strong>an</strong>d<br />
GFAP immunostaining <strong>an</strong>d in additional mice, cables were harvested at 21d to address early<br />
repopulation of myelinated axons. In a separate study, fluorescent YFP axons of thy-1 mice were<br />
live imaged <strong>an</strong>d followed up to 10 weeks <strong>for</strong> parallel <strong>an</strong>alysis of axon outgrowth, functional<br />
recovery <strong>an</strong>d foot skin reinnervation. A fur<strong>the</strong>r approach involved two epochs of stimulation<br />
(day 0 <strong>an</strong>d 5) <strong>an</strong>d <strong>an</strong>alysis of NF <strong>an</strong>d GFAP outgrowth on day 10. Dissociated adult sensory<br />
neurons <strong>from</strong> Sprague-Dawley rats grown on a microelectrode array underwent <strong>an</strong> identical ES<br />
paradigm <strong>for</strong> 1 hr. <strong>an</strong>d were compared with unstimulated neurons 24 hours later to <strong>an</strong>alyze<br />
neurite number <strong>an</strong>d outgrowth.<br />
In vivo, by 7d after tr<strong>an</strong>section <strong>an</strong>d ES, numbers of outgrowing axons <strong>an</strong>d activated Schw<strong>an</strong>n<br />
cells of stimulated nerves were higher in number <strong>an</strong>d grew greater dist<strong>an</strong>ces th<strong>an</strong> controls. By<br />
21d, more bridges in <strong>the</strong> ES group were repopulated with myelinated axons <strong>an</strong>d <strong>the</strong> overall<br />
number of myelinated axons was greater. Moreover in some mice of <strong>the</strong> ES group, myelinated<br />
axons were evident in <strong>the</strong> distal nerve stump. A second stimulation epoch did not increase<br />
outgrowth dist<strong>an</strong>ce but numbers of outgrowing axons were fur<strong>the</strong>r increased. The fluorescent
axon bridges in <strong>the</strong> stimulated thy-1 YFP mice were thicker <strong>an</strong>d more complete at day 21<br />
compared to control <strong>an</strong>imals <strong>an</strong>d at later time points this improvement was correlated with<br />
improved sensation <strong>an</strong>d foot skin reinnervation. In vitro, stimulated sensory neurons had higher<br />
neurite numbers <strong>an</strong>d longer neurites.<br />
These studies confirm that ES promotes outgrowth of axons <strong>an</strong>d Schw<strong>an</strong>n cells after tr<strong>an</strong>section<br />
nerve injuries. The benefits persist during repopulation of regenerative bridges by myelinated<br />
axons <strong>an</strong>d include improved reinnervation of targets.<br />
Disclosures: B. Singh, None; Q. Xu, None; C. Dalton, None; T. Gordon, None; R. Midha,<br />
None; D.W. Zochodne, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.14/B47<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Errett Fisher Foundation<br />
Title: Keratin biomaterials activate schw<strong>an</strong>n cells via integrin-mediated signaling <strong>an</strong>d promote<br />
regeneration of large peripheral nerve defects in a rabbit model<br />
Authors: *L. A. PACE 1 , P. SIERPINSKI HILL 1 , P. APEL 2 , B. TAWFIK 3 , J. BARNWELL 2 , T.<br />
SMITH 2 , L. KOMAN 2 , A. ATALA 1 , M. VAN DYKE 1 ;<br />
1 2 3<br />
Wake Forest Inst. <strong>for</strong> Regenerative Med., Dept. of Orthopaedic Surgery, Wake Forest Univ.<br />
Sch. of Med., Winston-Salem, NC<br />
Abstract: Large peripheral nerve defects c<strong>an</strong> be surgically repaired by autologous grafts or<br />
impl<strong>an</strong>tation of nerve guid<strong>an</strong>ce conduits. Nerve regeneration across a larger gap dist<strong>an</strong>ce c<strong>an</strong> be<br />
enh<strong>an</strong>ced with <strong>the</strong> introduction of biomaterial fillers into <strong>the</strong> conduit lumen. Hum<strong>an</strong> hair consists<br />
of mainly alpha <strong>an</strong>d gamma keratin proteins which c<strong>an</strong> be extracted <strong>an</strong>d processed into a<br />
hydrogel that promotes regeneration of peripheral nerves. We found that mouse tibial nerves<br />
treated with a crude gel consisting of alpha <strong>an</strong>d gamma keratins resulted in a >300%<br />
improvement in conduction delay <strong>an</strong>d ~150% increase in <strong>the</strong> compound muscle action potential<br />
(CMAP) after 6 weeks. We hypo<strong>the</strong>size that this rapid regeneration is due to keratin’s ability to<br />
stimulate Schw<strong>an</strong>n cell (SC) activity. The mech<strong>an</strong>ism of this activity is not known, but we<br />
postulate that it may be dependent on <strong>the</strong> primary amino acid (AA) structure of <strong>the</strong> keratins.<br />
Published hum<strong>an</strong> hair keratin (HHK) sequences were <strong>an</strong>alyzed <strong>for</strong> <strong>the</strong> presence of peptide
domains that are known to promote cell adhesion. AA <strong>an</strong>alysis was per<strong>for</strong>med using <strong>the</strong> Pico-<br />
Tag method <strong>an</strong>d results showed that most HHKs have at least one peptide binding domain. These<br />
cell binding domains are most prevalent in alpha keratins, 74% of which were found to have at<br />
least one binding domain. Integrins are cell surface receptors known to mediate cell signaling<br />
<strong>an</strong>d cell adhesion to extracellular matrix components or o<strong>the</strong>r cells. Total cell lysates of SC<br />
cultures were <strong>an</strong>alyzed by Western blot <strong>an</strong>d qu<strong>an</strong>tification showed upregulation of beta 1 <strong>an</strong>d<br />
alpha V integrin subunits in SCs cultured on keratin substrates versus uncoated dishes. To assess<br />
<strong>the</strong> ability of a keratin gel to promote regeneration of large peripheral nerve gaps, a critical size<br />
defect model was developed in rabbits. Following model development, 35 rabbits were<br />
r<strong>an</strong>domized into 3 groups: 1) Empty conduit, 2) Keratin-filled conduit, or 3) Autograft. At 3<br />
months, nerves were tested by electrophysiology, harvested <strong>an</strong>d assessed histologically.<br />
Assessment of regeneration in a rabbit model showed that a gel composed of alpha <strong>an</strong>d gamma<br />
keratins promotes functional recovery across a 2 cm defect. At 3 months, keratin treated nerves<br />
had signific<strong>an</strong>tly better conduction delay (4.5% vs. 21%) <strong>an</strong>d improved CMAP (51% vs. 36%)<br />
over empty conduit controls. Keratins are a family of structural proteins that c<strong>an</strong> be processed<br />
into biomaterial scaffolds <strong>for</strong> nerve regeneration. We show that a keratin gel composed of alpha<br />
<strong>an</strong>d gamma keratins has <strong>an</strong> effect on <strong>the</strong> expression of specific integrin receptors in Schw<strong>an</strong>n<br />
cells <strong>an</strong>d promotes regeneration across a critical-sized defect in a rabbit model.<br />
Disclosures: L.A. Pace, None; P. Sierpinski Hill, None; P. Apel, None; B. Tawfik, None; J.<br />
Barnwell, None; T. Smith, None; L. Kom<strong>an</strong>, None; A. Atala, None; M. V<strong>an</strong> Dyke, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.15/B48<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: AxoGen, Inc.<br />
Title: Evaluation of a decellularized, chondroitinase processed nerve allograft in a 3-cm rabbit<br />
nerve graft model<br />
Authors: *R. D. JOHNSON 1 , V. P. DUGAN 2 , J. B. GRAHAM 3 , D. NEUBAUER 3 , D. MUIR 3 ;<br />
2 Physiological Sci., 3 Pediatric Neurol., 1 Univ. Florida, Gainesville, FL<br />
Abstract: There is a pressing need <strong>for</strong> <strong>an</strong> effective <strong>an</strong>d practical alternative to nerve autografting<br />
<strong>for</strong> peripheral nerve reconstruction. Acellular allografts avoid m<strong>an</strong>y of <strong>the</strong> difficulties associated
with availability <strong>an</strong>d procurement of autografts. In this study we evaluated a nerve allograft that<br />
was decellularized with a series of detergents, treated with chondroitinase ABC to degrade<br />
inhibitory chondroitin sulfate proteoglyc<strong>an</strong>, <strong>an</strong>d irradiated <strong>for</strong> sterility (DCI-allograft). The DCIallograft<br />
(processed by AxoGen, Inc.) was compared to direct repair, a collagen conduit<br />
(NeuraGen® Nerve Guide) <strong>an</strong>d cellular autograft in a 3-cm rabbit common peroneal nerve graft<br />
model. Evaluations were per<strong>for</strong>med 17 weeks after grafting to assess recovery of function (toespread),<br />
tr<strong>an</strong>s-graft electrophysiological impulse conduction, <strong>an</strong>d axon counts in <strong>the</strong> distal nerve.<br />
While under isoflur<strong>an</strong>e <strong>an</strong>es<strong>the</strong>sia in a terminal procedure, <strong>the</strong> common peroneal nerve was<br />
isolated 1-2 cm proximal to <strong>the</strong> proximal coaptation point <strong>an</strong>d distally near its entry into <strong>the</strong><br />
cr<strong>an</strong>iolateral compartment of tarsal flexor muscles. Each of <strong>the</strong> nerve ends were placed on a pair<br />
of platinum fine wire electrodes <strong>for</strong> stimulation <strong>an</strong>d recording of a whole nerve compound action<br />
potential (CAP) <strong>an</strong>d <strong>the</strong> subsequent off-line calculation of conduction velocity (CV). Direct<br />
repair <strong>an</strong>d autografts achieved greater me<strong>an</strong> CV of <strong>the</strong> fastest fibers (≈41 m/sec, 42% of normal<br />
uninjured nerve values) compared to DCI-grafts (31 m/sec), <strong>an</strong>d were far superior to collagen<br />
conduits (7.7 m/sec). The size of <strong>the</strong> CAP (integrated wave<strong>for</strong>m) had a similar trend.<br />
Postmortem histological <strong>an</strong>alysis showed <strong>the</strong> three graft types were well tolerated, integrated<br />
with <strong>the</strong> recipient nerve <strong>an</strong>d supported abund<strong>an</strong>t axonal regrowth <strong>from</strong> <strong>the</strong> recipient proximal<br />
nerve. Axon counts in <strong>the</strong> distal host nerves did not fully explain differences in CV <strong>an</strong>d CAP in<br />
<strong>the</strong> four repair conditions. Instead, distinct patterns of axon collaterals, regeneration <strong>an</strong>d<br />
myelination associated with <strong>the</strong> disparate graft types were likely involved. Proximodistal<br />
<strong>an</strong>alysis of axon regrowth <strong>an</strong>d action potential propagation suggested that <strong>the</strong>re was a somewhat<br />
greater latency in nerve regeneration with <strong>the</strong> DCI-allografts compared to autografts <strong>an</strong>d direct<br />
repair. Signs of toe-spread function first appeared only weeks prior to <strong>the</strong> end of <strong>the</strong> study,<br />
indicating regeneration was ongoing <strong>an</strong>d 17 weeks was insufficient <strong>for</strong> signific<strong>an</strong>t functional<br />
reinnervation. In conclusion, <strong>the</strong> 3-cm DCI-allografts at 17 weeks supported action potential<br />
propagation, robust axon growth, remyelination, vascularity, <strong>an</strong>d features associated with<br />
successful, ongoing yet incomplete, nerve regeneration.<br />
Disclosures: R.D. Johnson, None; V.P. Dug<strong>an</strong>, None; J.B. Graham, None; D. Neubauer,<br />
None; D. Muir, D. Muir is <strong>an</strong> inventor of <strong>the</strong> patented nerve grafting technology discussed in<br />
this study <strong>an</strong>d holds equity in AxoGen, Inc., a comp<strong>an</strong>y commercializing <strong>the</strong> allograft<br />
technology., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.16/B49<br />
Topic: A.08.b. Regeneration: PNS
Support: CIHR<br />
AHFMR<br />
Title: Side-to-side nerve grafts sustain chronically denervated nerve pathways during axon<br />
regeneration resulting in improved reinnervation<br />
Authors: *A. LADAK 1,2 , P. SCHEMBRI 1 , N. TYREMAN 3 , E. UDINA 3 , J. OLSON 2 , T.<br />
GORDON 3 ;<br />
1 Fac. of Med. <strong>an</strong>d Dent., 2 Div. of Plastic <strong>an</strong>d Reconstructive Surgery, 3 Ctr. <strong>for</strong> Neurosci. <strong>an</strong>d<br />
Div. of Physical Med. <strong>an</strong>d Rehabil., Univ. of Alberta, Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: Peripheral nerve injuries result in signific<strong>an</strong>t morbidity <strong>an</strong>d functional disability.<br />
There is a limited time during which axonal regeneration c<strong>an</strong> occur following nerve injury such<br />
that long-term neuronal axotomy <strong>an</strong>d chronic denervation of nerve stumps progressively reduce<br />
regenerative capacity <strong>an</strong>d in turn, functional recovery. In <strong>the</strong> case of chronic denervation of <strong>the</strong><br />
distal nerve stump of injured peripheral nerves, <strong>the</strong> number of motoneurons that regenerate <strong>an</strong>d<br />
reinnervate skeletal muscle is less th<strong>an</strong> 10% within 6 months of <strong>the</strong> nerve repair. In this study,<br />
side-to-side nerve grafts were used to join a donor nerve to a denervated distal nerve stump of a<br />
tr<strong>an</strong>sected nerve as a me<strong>an</strong>s to sustain <strong>the</strong> chronically denervated stump. Thereby we asked<br />
whe<strong>the</strong>r <strong>the</strong>se nerve grafts ‘sustain’ <strong>the</strong> distal nerve stumps to promote axon regeneration<br />
through <strong>the</strong> ‘protected’ distal nerve stump. Adult Sprague Dawley rats were divided into 2<br />
groups, both of which underwent a unilateral tr<strong>an</strong>section of <strong>the</strong> common peroneal (CP) nerve. In<br />
group 1 (<strong>the</strong> unprotected group), <strong>the</strong> ends of <strong>the</strong> severed CP nerve were sutured back to muscle<br />
to prevent reinnervation. In group 2 (<strong>the</strong> protected group), 3 side-to-side nerve bridges (obtained<br />
<strong>from</strong> <strong>the</strong> contralateral CP nerve) were used to join <strong>the</strong> tibial (TIB) nerve to <strong>the</strong> distal nerve stump<br />
of <strong>the</strong> CP nerve, while <strong>the</strong> proximal CP nerve stump was sutured back to muscle. Both groups of<br />
rats were left to convalesce <strong>for</strong> 4 months, following which <strong>the</strong> proximal <strong>an</strong>d distal nerve stumps<br />
of <strong>the</strong> CP nerve in both groups were aligned <strong>an</strong>d surgically repaired primarily. The rats were left<br />
to recover <strong>for</strong> 6 months, at which time axonal regeneration was qu<strong>an</strong>tified by counting <strong>the</strong> <strong>the</strong><br />
motoneurons that were backlabelled with fluorescent retrograde dyes. Fluororuby <strong>an</strong>d<br />
Fluorogold were applied to each of <strong>the</strong> cut ends of <strong>the</strong> CP <strong>an</strong>d tibial distal nerve stumps prior to<br />
4% para<strong>for</strong>maldehyde fixation of <strong>the</strong> spinal cord 4-6 days later. The me<strong>an</strong> number (+SE) of<br />
motoneurons that regenerated axons through <strong>the</strong> CP nerve in <strong>the</strong> protected group (132.2+28) was<br />
signific<strong>an</strong>tly higher th<strong>an</strong> in <strong>the</strong> unprotected group (76.7+33). Tibialis <strong>an</strong>terior muscle weights<br />
were recorded <strong>an</strong>d used as a measure of functional reinnervation. The me<strong>an</strong> muscle weight (+SE)<br />
of <strong>the</strong> protected group (363.5+21 g) was signific<strong>an</strong>tly higher th<strong>an</strong> in <strong>the</strong> unprotected group<br />
(219.3+29 g). Results of this study demonstrate <strong>the</strong> potential of using side-to-side nerve bridges<br />
to maintain <strong>the</strong> integrity of a chronically denervated nerve, <strong>the</strong>reby promoting functional<br />
recovery. (Grateful th<strong>an</strong>ks to <strong>the</strong> CIHR <strong>an</strong>d AHFMR)<br />
Disclosures: A. Ladak, None; P. Schembri, None; N. Tyrem<strong>an</strong>, None; E. Udina, None; J.<br />
Olson, None; T. Gordon, None.
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.17/B50<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH Gr<strong>an</strong>t NS059003<br />
Title: HSV-mediated tr<strong>an</strong>sfer of Sox11 <strong>an</strong>d peripheral nerve regeneration<br />
Authors: *X. JING, T. WANG, S. HUANG, D. M. BRIAN, J. C. GLORIOSO, K. M.<br />
ALBERS;<br />
Univ. Pittsburgh, Pittsburgh, PA<br />
Abstract: The Sry box containing tr<strong>an</strong>scription factor 11, a member of <strong>the</strong> group C high<br />
mobility group (HMG) tr<strong>an</strong>scription factor family, is a critical tr<strong>an</strong>scriptional regulator during<br />
embryonic development <strong>an</strong>d org<strong>an</strong>ogenesis. In addition to its role in development, Sox11 is <strong>an</strong><br />
essential component of a regenerative tr<strong>an</strong>scriptional control repertoire. Previous studies indicate<br />
that DRG neurons treated with Sox11-targeted siRNAs inhibited neurite outgrowth <strong>an</strong>d<br />
decreased expression of genes associated with this process. Myelinated <strong>an</strong>d unmyelinated axonal<br />
regeneration are inhibited when Sox11 is knocked down by Sox11-siRNA in vivo. To fur<strong>the</strong>r<br />
explore <strong>the</strong> potential function of Sox11 during peripheral nerve regeneration, we used a herpes<br />
simplex virus (HSV)-based gene tr<strong>an</strong>sfer approach to determine if enh<strong>an</strong>ced expression of Sox11<br />
modulates properties of DRG neurons. We found that HSV-Sox11 mediated gene tr<strong>an</strong>sfer was<br />
effective both in vivo <strong>an</strong>d in vitro. In primary DRG cultures, <strong>the</strong> expression of Sox11 protein<br />
increased at 2 <strong>an</strong>d 3 days after infection. In adult mice, Sox11 mRNA expression increased<br />
115% at 5 days following a hind paw inoculation with HSV-Sox virus. To determine if preexpression<br />
of Sox11 improves recovery of DRG neurons following nerve crush, mice were<br />
injected with virus <strong>an</strong>d 3 days later, saphenous nerve injury was per<strong>for</strong>med. At 3, 5, 12 <strong>an</strong>d 30<br />
days after saphenous injury, <strong>the</strong> nerve, DRG <strong>an</strong>d skin were collected <strong>an</strong>d <strong>an</strong>alyzed using<br />
immunochemical <strong>an</strong>d molecular biological techniques. The results indicate that Sox11<br />
overexpression modulates genes associated with survival <strong>an</strong>d outgrowth of injured peripheral<br />
neurons. For inst<strong>an</strong>ce, <strong>the</strong> expression of TRAF family member-associated NFκB activator<br />
(TANK), a strong c<strong>an</strong>didate target gene of Sox11, showed a 2-fold increase 5 days after crush in<br />
DRG neurons (lumbar L2, 3) following HSV-Sox inoculation. The increase in o<strong>the</strong>r injury<br />
responsive genes, such as GAP-43, β-tubulin III, Sprr1, N-Cadherin, <strong>an</strong>d Mash1, were<br />
unch<strong>an</strong>ged by enh<strong>an</strong>ced expression of Sox11. These results suggest that <strong>the</strong> expression of Sox11<br />
upon injury selectively modulates tr<strong>an</strong>scription of genes associated with neuron survival <strong>an</strong>d<br />
regeneration.
Disclosures: X. Jing, None; T. W<strong>an</strong>g, None; S. Hu<strong>an</strong>g, None; D.M. Bri<strong>an</strong>, None; J.C.<br />
Glorioso, None; K.M. Albers, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.18/B51<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: CIHR<br />
AHFMR<br />
Title: Crush injury to motor nerves in <strong>the</strong> SOD1 G93A tr<strong>an</strong>sgenic mouse model of amyotrophic<br />
lateral sclerosis (ALS) promotes muscle reinnervation <strong>an</strong>d enh<strong>an</strong>ced survival of functionally<br />
intact nerve-muscle connections<br />
Authors: *T. GORDON 1 , N. TYREMAN 2,3 , K. J. JONES 2,4 , P. SHARP 2 ;<br />
1 Univ. Alberta Fac Med., Edmonton, AB, C<strong>an</strong>ada; 2 Ctr. <strong>for</strong> Neurosci., 3 Div. of Physical Med.<br />
<strong>an</strong>d Rehabil., 4 Fac. of Physical Educ. <strong>an</strong>d Recreation, Univ. of Alberta, Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: Whilst <strong>the</strong> first signific<strong>an</strong>t decline in motoneuron numbers occurs at 80 days of age in<br />
<strong>the</strong> SOD1 G93A tr<strong>an</strong>sgenic mouse ALS model, we reported a dramatic decline in numbers of<br />
functionally intact motor units (MUs: motoneurons with intact neuromuscular connections) presymptomatically<br />
<strong>from</strong> 40 days of age, reaching a plateau by 90 days in fast-twitch muscles<br />
(Hegedus et al, Neurobiol Dis 28: 154-164, 2007). Selective progressive loss of <strong>the</strong> largest <strong>an</strong>d<br />
fastest MUs suggested <strong>the</strong> possibility that <strong>the</strong> smaller axon caliber of surviving MUs is import<strong>an</strong>t<br />
<strong>for</strong> survival. Yet despite <strong>the</strong> supportive findings that axotomized motoneurons with smaller<br />
axons survived longer (Kong et al J Comp Neurol 412: 373-380, 1999), reduced axon caliber<br />
does not prolong survival of motoneurons at end-stage disease in SOD1 G37R SH tr<strong>an</strong>sgenic mice<br />
in which axon size was reduced by disrupting one allele of each neurofilament gene (Nguyen et<br />
al PNAS 97: 12306-12311, 2000). In this study, we crushed <strong>the</strong> sciatic nerve in SOD1 G93A <strong>an</strong>d<br />
SOD1 WT tr<strong>an</strong>sgenic mice at 40 days of age <strong>an</strong>d recorded reinnervated muscle <strong>an</strong>d MU isometric<br />
twitch <strong>an</strong>d tet<strong>an</strong>ic contractions of 4 hindlimb muscles that included tibialis <strong>an</strong>terior (TA), at 90<br />
days of age. MU number was determined by <strong>the</strong> ratio of <strong>the</strong> maximal muscle twitch <strong>for</strong>ce, <strong>the</strong><br />
<strong>for</strong>ce of all <strong>the</strong> MUs, <strong>an</strong>d <strong>the</strong> me<strong>an</strong> MU <strong>for</strong>ce. The me<strong>an</strong> (+SE) of 408+36mN <strong>for</strong> muscle tet<strong>an</strong>ic<br />
contractile <strong>for</strong>ce of <strong>the</strong> reinnervated muscles was signific<strong>an</strong>tly higher th<strong>an</strong> 73+12mN in <strong>the</strong> intact<br />
muscles of <strong>the</strong> SOD1 G93A tr<strong>an</strong>sgenic mice. There was <strong>an</strong> associated 3-fold increase in both MU
numbers <strong>an</strong>d size: 34+3.4 vs 12+2.5 functionally intact MUs in <strong>the</strong> reinnervated <strong>an</strong>d intact<br />
SOD1 G93A tr<strong>an</strong>sgenic mouse muscles as compared to 54+6.9 vs 71+9.7 in <strong>the</strong> SOD1 WT mice.<br />
Import<strong>an</strong>tly, <strong>the</strong> size of <strong>the</strong> MUs was signific<strong>an</strong>tly increased in <strong>the</strong> reinnervated vs intact<br />
SOD1 G93A mouse muscles (4.2+0. vs 2.3+0.3mN) whilst <strong>the</strong> MU twitch <strong>for</strong>ces were <strong>the</strong> same in<br />
<strong>the</strong> SOD1 WT mice (7.3+0.9 vs 6.1+0.7mN). The increased MU <strong>for</strong>ce demonstrated <strong>the</strong> survival<br />
of more intact <strong>an</strong>d enlarged MUs after nerve injury <strong>an</strong>d muscle reinnervation in <strong>the</strong> SOD1 G93A<br />
tr<strong>an</strong>sgenic mouse. Whe<strong>the</strong>r this enh<strong>an</strong>ced MU survival with indications of conversion to more<br />
fatigue resist<strong>an</strong>t type MUs, is linked to <strong>the</strong> reduced size of <strong>the</strong> regenerating axons <strong>an</strong>d/or whe<strong>the</strong>r<br />
<strong>the</strong> survival is linked to <strong>the</strong> activation of regeneration associated genes, is not known presently. It<br />
is possible that <strong>the</strong> nerve injury provokes gene expression that signific<strong>an</strong>tly delays <strong>the</strong> onset of<br />
<strong>the</strong> deleterious ch<strong>an</strong>ges in <strong>the</strong> motoneurons that include misfolding of <strong>the</strong> mut<strong>an</strong>t superoxide<br />
dismutase (SOD1) enzyme, oxidative stress <strong>an</strong>d excitatoxicity. (Grateful th<strong>an</strong>ks to <strong>the</strong> funding<br />
agencies of <strong>the</strong> AHFMR <strong>an</strong>d CIHR of C<strong>an</strong>ada).<br />
Disclosures: T. Gordon, None; N. Tyrem<strong>an</strong>, None; K.J. Jones, None; P. Sharp, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.19/B52<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: The Packard Center <strong>for</strong> ALS Research at Johns Hopkins<br />
Hospital<br />
NCI CA69129<br />
<strong>the</strong> Americ<strong>an</strong> Leb<strong>an</strong>ese Syri<strong>an</strong> Associated Charities, St. Jude Children’s Research<br />
Title: Anaplastic lymphoma kinase (ALK) <strong>an</strong>d motor nerve regeneration<br />
Authors: *R. MI 1 , M. WRIGHT 1 , R. THAKRAR 1 , W. CHEN 1 , L. XUE 2 , S. W. MORRIS 2 , M.<br />
VIGNY 3 , A. HOKE 1 ;<br />
1 2<br />
Johns Hopkins Univ. Sch. Med., Baltimore, MD; St. Jude Children’s Res. Hosp., Memphis,<br />
TN; 3 INSERM/UPMC, Pairs, Fr<strong>an</strong>ce<br />
Abstract: The receptor tyrosine kinase product of <strong>the</strong> <strong>an</strong>aplastic lymphoma kinase (ALK) gene<br />
is tr<strong>an</strong>siently expressed in specific regions of <strong>the</strong> central <strong>an</strong>d peripheral nervous systems during
development <strong>an</strong>d has been implicated in regulation of neuronal development based on its<br />
expression pattern. However, its role in <strong>the</strong> adult nervous system has remained poorly<br />
understood. Our previous study indicated that ALK might act as <strong>the</strong> receptor mediating<br />
neurotrophic properties of Pleiotrophin (PTN) in motor neurons. To fur<strong>the</strong>r explore <strong>the</strong><br />
relationship between PTN <strong>an</strong>d ALK signaling in motor neuron biology, we examined <strong>the</strong> effect<br />
of agonist <strong>an</strong>ti-ALK <strong>an</strong>tibodies mAb46 <strong>an</strong>d mAb48 in spinal cord expl<strong>an</strong>ts <strong>an</strong>d <strong>an</strong> immortalized<br />
motor neuron line, MN1 cells. We found that both of <strong>the</strong>se <strong>an</strong>tibodies induced axonal outgrowth<br />
<strong>from</strong> motor neurons in spinal cord expl<strong>an</strong>t cultures <strong>an</strong>d promoted neurite growth in <strong>the</strong> MN1 cell<br />
line. Conversely, <strong>the</strong> <strong>an</strong>tagonist <strong>an</strong>ti-ALK <strong>an</strong>tibody mAb30 inhibited axon outgrowth of motor<br />
neurons in spinal cord expl<strong>an</strong>ts induced by PTN. In order to fur<strong>the</strong>r explore <strong>the</strong> role of ALK, we<br />
per<strong>for</strong>med a st<strong>an</strong>dard sciatic nerve crush in Alk knockout mice. Our results showed that <strong>the</strong>re<br />
was a four-day delay in <strong>the</strong> emergence of compound motor action potentials in <strong>the</strong> foot muscles<br />
of <strong>the</strong> Alk knockout mice compared to wild-type littermates. The me<strong>an</strong> compound motor action<br />
potential amplitudes were signific<strong>an</strong>tly decreased in <strong>the</strong> Alk knockout mice 4 weeks after sciatic<br />
nerve crush, but <strong>the</strong> numbers of reinnervated neuromuscular junctions were similar. These<br />
findings suggest that <strong>the</strong>re is a defect in motor nerve regeneration in Alk knockout mice <strong>an</strong>d that<br />
ALK-PTN signaling may play a role in motor nerve regeneration in <strong>the</strong> adult peripheral nervous<br />
system.<br />
Disclosures: R. Mi , None; M. wright, None; R. Thakrar, None; W. Chen, None; L. Xue,<br />
None; S.W. Morris, None; M. Vigny, None; A. Hoke, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.20/B53<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Adelson Medical Research Foundation<br />
Title: Brn3a is upregulated in Schw<strong>an</strong>n cells associated with injured axons<br />
Authors: *A. HOKE 1 , A. YARON 3 , M. FAINZILBER 3 , M. C. WRIGHT 2 ;<br />
1 2 3<br />
Depts Neurol, Neurosci, Neurol., Johns Hopkins Univ., Baltimore, MD; Dept. of Biol. Chem.,<br />
Weizm<strong>an</strong>n Inst. of Sci., Rehovot, Israel<br />
Abstract: Peripheral nerves are capable of regenerating following traumatic injury <strong>an</strong>d research<br />
clearly demonstrates that Schw<strong>an</strong>n cells (SCs) are a critical component required <strong>for</strong> this
egenerative process to proceed <strong>an</strong>d <strong>for</strong> functional recovery to occur. Despite extensive ef<strong>for</strong>t,<br />
<strong>the</strong> regulatory mech<strong>an</strong>isms that are responsible <strong>for</strong> <strong>the</strong> ability of SCs to provide a supportive<br />
environment <strong>for</strong> peripheral nerve regeneration remain unclear. Recently, we have identified a<br />
novel SC tr<strong>an</strong>scription factor, Brn3a that appears to be involved in response of SCs to peripheral<br />
nerve injury. In o<strong>the</strong>r cell populations, Brn3a has been shown to regulate a number of factors that<br />
have previously been implicated in SC-axon interactions during regeneration, including certain<br />
growth factors (e.g. IGF1, VEGF), growth factor receptors (e.g. TrkA, p75, <strong>an</strong>d <strong>the</strong> GDNFR<br />
alpha), <strong>an</strong>d members of <strong>the</strong> cell adhesion molecule family known to promote axon outgrowth<br />
(e.g. NCAM <strong>an</strong>d L1-CAM). Preliminary results <strong>from</strong> our labs demonstrate that Brn3a expression<br />
in SCs follows a distinct spatial <strong>an</strong>d temporal pattern following injury <strong>an</strong>d during axonal<br />
regeneration. Using both immunocytochemistry <strong>an</strong>d qu<strong>an</strong>titative RT-PCR <strong>an</strong>alysis, Brn3a<br />
mRNA <strong>an</strong>d protein levels were found to increase in SCs associated with regenerating axons<br />
following sciatic nerve tr<strong>an</strong>section <strong>an</strong>d during regeneration following sciatic nerve crush. Current<br />
experiments examining <strong>the</strong> ability of SCs that lack Brn3a to support neurite outgrowth <strong>an</strong>d<br />
peripheral nerve regeneration will provide import<strong>an</strong>t in<strong>for</strong>mation regarding <strong>the</strong> contribution of<br />
Brn3a in axon-SC communication, as well as a greater underst<strong>an</strong>ding of <strong>the</strong> overall role of SCs<br />
in axonal regeneration.<br />
Disclosures: A. Hoke, None; A. Yaron, None; M. Fainzilber, None; M.C. Wright, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.21/B54<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Carter Ch<strong>an</strong>cellor Ursche Neurobiology Research Fund<br />
Texas Scottish Rite Hospital <strong>for</strong> Children<br />
Title: Vascular endo<strong>the</strong>lial growth factor (VEGF) <strong>an</strong>d pleiotrophin (PTN) stimulate nerve<br />
regeneration across long-gap peripheral nerve defects<br />
Authors: *A. DAWOOD 1,2 , A. VARSHNEY 2 , R. CORNELIA 3 , S. BIDIC 2 , M. ROMERO<br />
ORTEGA 1,2,3 ;<br />
1 Bioengineering, Univ. of Texas Arlington, Arlington, TX; 2 Plastic Surgery, Univ. of Texas<br />
Southwestern Med. Ctr., Dallas, TX; 3 Res. Dept., Texas Scottish Rite Hosp. For Children,<br />
Dallas, TX
Abstract: Peripheral nerve gaps that result <strong>from</strong> tissue loss due to traumatic injury or tumor<br />
resection are routinely repaired by autogenous nerve grafts despite poor recovery <strong>an</strong>d <strong>the</strong><br />
expense of healthy donor nerves. To obviate <strong>the</strong> need of harvesting donor tissue, short nerve<br />
gaps (i.e., 1-2.5 cm) have been effectively bridged by syn<strong>the</strong>tic nerve guides. However, nerve<br />
regeneration across gaps longer th<strong>an</strong> 3 cm has failed when repaired using collagen or L-polylactic<br />
acid nerve scaffolds. We hypo<strong>the</strong>size that neurotrophic support <strong>an</strong>d early vascularization<br />
of <strong>the</strong> regenerated nerve are critical <strong>for</strong> successful nerve regeneration across long-gap nerve<br />
defects. Vascular endo<strong>the</strong>lial growth factor (VEGF) <strong>an</strong>d Pleiotrophin (PTN) are potent<br />
endo<strong>the</strong>lial cell mitogens that promote <strong>an</strong>giogenesis. In addition, both PTN <strong>an</strong>d VEGF have<br />
growth promoting effects on neurons in <strong>the</strong> central <strong>an</strong>d peripheral nervous systems. The<br />
combination of neurotrophic <strong>an</strong>d <strong>an</strong>giogenic effects of <strong>the</strong>se molecules suggests <strong>the</strong>y may be<br />
c<strong>an</strong>didates to promote simult<strong>an</strong>eous vascularization <strong>an</strong>d nerve regeneration across long-gap<br />
injuries in peripheral nerves. To test this hypo<strong>the</strong>sis, <strong>the</strong> regenerative response across a 3 cm<br />
nerve gap in <strong>the</strong> injured rabbit common peroneal nerve was evaluated when repaired with: a)<br />
collagen filled tube, b) multiluminal biosyn<strong>the</strong>tic nerve impl<strong>an</strong>t (BNI) filled with collagen, or c)<br />
BNI supplemented with VEGF or PTN. Histological evaluation five weeks post impl<strong>an</strong>tation<br />
revealed that nerve defects repaired with simple tubes failed to regenerate across <strong>the</strong> long gap<br />
while those bridged with collagen-filled BNIs showed vascularized nerves within <strong>the</strong> hydrogel<br />
micro-ch<strong>an</strong>nels. Moreover, qu<strong>an</strong>tification by optical densitometry revealed that <strong>the</strong> number of<br />
neurofilament-positive axons within <strong>the</strong> BNIs supplemented with VEGF or PTN increased by<br />
approximately threefold. Functional recovery is currently being ascertained by evaluating<br />
compound action potentials across <strong>the</strong> regenerated tissue <strong>an</strong>d by sensory-motor assays. Our study<br />
suggests that adding both nerve <strong>an</strong>d vascular growth factor support to <strong>the</strong> physical bridge seems<br />
necessary to overcome <strong>the</strong> lack of nerve regeneration in long-gap peripheral nerve defects.<br />
Disclosures: A. Dawood, None; A. Varshney, None; R. Cornelia, None; S. Bidic, None; M.<br />
Romero Ortega, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.22/B55<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH Gr<strong>an</strong>t HD032571<br />
Title: Misdirection of regenerating motor axons has little effect on functional recovery following<br />
peripheral nerve injury
Authors: *A. W. ENGLISH 1 , S. K. HAMILTON 2 , M. L. HINKLE 2 , L. N. RAMBO 2 , M.<br />
KAUFMAN 3 , J. NICOLINI 3 , S. ROSE 3 , D. BACKUS 2 , M. J. SABATIER 4 ;<br />
2 3 1 4<br />
Div. of Physical Therapy, Dept. of Cell Biol., Emory Univ. Sch. Med., Atl<strong>an</strong>ta, GA; Clayton<br />
State Col. <strong>an</strong>d Univ., Morrow, GA<br />
Abstract: Poor functional recovery following peripheral nerve injury is sometimes attributed to<br />
<strong>the</strong> misdirection of regenerating motor axons to innervate functionally inappropriate target<br />
muscles. We investigated <strong>the</strong> effect of intentionally increasing misdirection on muscle<br />
reinnervation <strong>an</strong>d functional recovery. In adult rats, brief electrical stimulation (ES) (20 Hz,<br />
2XT) was applied to <strong>the</strong> common fibular (CF) nerve <strong>for</strong> one hour prior to complete sciatic nerve<br />
tr<strong>an</strong>section <strong>an</strong>d end-to-end repair. This treatment was expected to enh<strong>an</strong>ce <strong>the</strong> regeneration of<br />
axons <strong>from</strong> <strong>the</strong> proximal site of <strong>the</strong> CF but not <strong>the</strong> tibial (TIB) nerve. The timing of regeneration<br />
of axons in <strong>the</strong> two br<strong>an</strong>ches was monitored by recording compound muscle action potentials<br />
(M-responses) in <strong>the</strong> soleus (SOL) <strong>an</strong>d tibialis <strong>an</strong>terior (TA) muscles during a ten week recovery<br />
period. Functional recovery was assayed using hindlimb kinematics <strong>an</strong>d SOL <strong>an</strong>d TA EMG<br />
activity during treadmill locomotion. Terminal experiments using motor unit number estimation<br />
or sequential double retrograde labeling were used to evaluate <strong>the</strong> extent of inappropriate<br />
reinnervation of muscle targets by TIB <strong>an</strong>d CF motor axons. Data <strong>from</strong> rats exposed to selective<br />
ES were compared to results obtained <strong>from</strong> unstimulated but sciatic nerve tr<strong>an</strong>sected controls.<br />
Selective ES resulted in restored M-responses at earlier times th<strong>an</strong> unstimulated controls in<br />
BOTH TA <strong>an</strong>d SOL muscles. SOL was reinnervated by stimulated (<strong>an</strong>d misdirected) common<br />
fibular axons to a greater extent th<strong>an</strong> unstimulated rats. The extent of innervation of TA by<br />
misdirected tibial axons was not signific<strong>an</strong>tly different in selective ES <strong>an</strong>d unstimulated rats.<br />
During locomotion, TA <strong>an</strong>d SOL were co-activated in both stimulated <strong>an</strong>d unstimulated rats, but<br />
<strong>the</strong> precise timing of that activity, demonstrated using principal components <strong>an</strong>alysis, differed<br />
signific<strong>an</strong>tly between groups. During <strong>the</strong> st<strong>an</strong>ce phase of locomotion, both stimulated <strong>an</strong>d<br />
untreated rats maintained a pl<strong>an</strong>tigrade posture of <strong>the</strong> foot. Throughout <strong>the</strong> step cycle <strong>the</strong><br />
hindlimbs were maintained at a more acute <strong>an</strong>gle to <strong>the</strong> treadmill belt in both stimulated <strong>an</strong>d<br />
control rats th<strong>an</strong> found in intact rats. Enh<strong>an</strong>ced axon regeneration with degraded fidelity of<br />
muscle reinnervation resulted in ch<strong>an</strong>ges in <strong>the</strong> locomotor activation of <strong>the</strong> reinnervated muscles<br />
but has little effect on functional recovery, which seems to be determined by compensatory limb<br />
movement strategies adopted by <strong>the</strong> rats irrespective of source of muscle reinnervation.<br />
Disclosures: A.W. English, None; S.K. Hamilton, None; M.L. Hinkle, None; L.N. Rambo,<br />
None; M. Kaufm<strong>an</strong>, None; J. Nicolini, None; S. Rose, None; D. Backus, None; M.J. Sabatier,<br />
None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 510.23/B56<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: Swedish Research Council<br />
Title: Polystyrene n<strong>an</strong>owires as substrate <strong>for</strong> neurite outgrowth<br />
Authors: *F. JOHANSSON, A. BOMAN, B. OTT, M. KANJE;<br />
Lund Univ., Lund, Sweden<br />
Abstract: The surface on which neurons grow <strong>an</strong>d extend neurites affects <strong>the</strong> outgrowth rate,<br />
adhesion <strong>an</strong>d even guid<strong>an</strong>ce. We <strong>an</strong>d o<strong>the</strong>rs have demonstrated that artificial surfaces with<br />
different n<strong>an</strong>otopography affect neurite outgrowth in vitro [1]. Axonal outgrowth on porous<br />
silicon surfaces is dependent of <strong>the</strong> pore size <strong>an</strong>d <strong>the</strong> reversed surface (protrusions) i.e. a surface<br />
endowed with n<strong>an</strong>owires c<strong>an</strong> be used to guide axonal outgrowth [2, 3].<br />
Here we used polystyrene (PS) surfaces with vertical, densely packed n<strong>an</strong>owires with diameters<br />
of 100 nm <strong>an</strong>d 200 nm produced with a template-based casting technique. A PS film was<br />
prepared <strong>an</strong>d <strong>the</strong> template, commercially available Anodisc Al2O3 membr<strong>an</strong>es (Whatm<strong>an</strong>®),<br />
was placed on top of <strong>the</strong> film. The preparation was s<strong>an</strong>dwiched between two glass slides <strong>an</strong>d<br />
heated above <strong>the</strong> glass tr<strong>an</strong>sition temperature of PS. The <strong>an</strong>nealing time, determined <strong>the</strong> lengths<br />
of <strong>the</strong> wires produced. The template was <strong>the</strong>n dissolved in NaOH <strong>an</strong>d finally, after rinsing, <strong>the</strong><br />
PS surface was used as <strong>an</strong> org<strong>an</strong> culture substrate. Dorsal root g<strong>an</strong>glia (DRG) were cultured on<br />
<strong>the</strong> wires <strong>for</strong> 5 days (in serum free medium containing nerve growth factor (NGF) to promote<br />
outgrowth of axons. We also cultured primary macrophages on <strong>the</strong> n<strong>an</strong>owire substrates to<br />
estimate <strong>the</strong> macrophage reaction to <strong>the</strong> n<strong>an</strong>owire surfaces.<br />
The removal of <strong>the</strong> filter by NaOH exposed <strong>the</strong> n<strong>an</strong>owire-covered surface. The length of <strong>the</strong><br />
n<strong>an</strong>owires was dependent on <strong>the</strong> <strong>an</strong>nealing time <strong>an</strong>d <strong>the</strong> n<strong>an</strong>owire-covered surfaces was found to<br />
be more hydrophobic th<strong>an</strong> smooth PS as estimated by water droplet contact <strong>an</strong>gle measurements.<br />
The hydrophobicity also varied with <strong>the</strong> length of <strong>the</strong> wires.<br />
Surfaces with short n<strong>an</strong>owires supported axonal outgrowth. Axons extended along <strong>the</strong> tips of <strong>the</strong><br />
wires but could grow down <strong>the</strong> wells <strong>an</strong>d lines of smooth PS. The growth cones were more<br />
flattened <strong>an</strong>d thus larger on <strong>the</strong> smooth areas as compared to <strong>the</strong> wire-areas. On surfaces<br />
endowed with longer n<strong>an</strong>owires outgrowth appeared inhibited. We assumed this is related to <strong>an</strong><br />
increase in hydrophobicity. Macrophages seeded onto smooth <strong>an</strong>d n<strong>an</strong>owire surfaces had<br />
different morphologies.They were rounded on <strong>the</strong> n<strong>an</strong>owires but spread out on <strong>the</strong> smooth PS.<br />
We conclude that n<strong>an</strong>owire surfaces inhibit macrophage adhesion <strong>an</strong>d mobility.The results<br />
suggest that axonal outgrowth <strong>an</strong>d macrophage adhesion on plastic n<strong>an</strong>owires are dependent on<br />
<strong>the</strong> hydrophobicity of <strong>the</strong> surface.<br />
[1] F. Joh<strong>an</strong>sson, W. Hallstrom, P. Gustavsson, L. Wallm<strong>an</strong>, C. Prinz, L. Montelius, M. K<strong>an</strong>je, J<br />
of Vacuum Sci & Tech B 26 (2008).<br />
[2] F. Joh<strong>an</strong>sson, M. K<strong>an</strong>je, C.E. Linsmeier, L. Wallm<strong>an</strong>, IEEE Tr<strong>an</strong>s Biomed Eng 55 (2008).<br />
[3] C. Prinz, W. Hallstrom, T. Martensson, L. Samuelson, L. Montelius, M. K<strong>an</strong>je, N<strong>an</strong>otech 19<br />
(2008).<br />
Disclosures: F. Joh<strong>an</strong>sson, None; A. Bom<strong>an</strong>, None; B. Ott, None; M. K<strong>an</strong>je, None.
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.24/B57<br />
Topic: A.08.b. Regeneration: PNS<br />
Title: Schw<strong>an</strong>n cell-like differentiation of adipose-derived mesenchymal stem cells<br />
Authors: *C. RADTKE 1,2 , P. M. VOGT 2 , B. SCHMITZ 2 , M. SPIES 2 , J. D. KOCSIS 1 ;<br />
1 Yale Univ. Sch. Med., West Haven, CT, CT; 2 Dept. of Plastic, H<strong>an</strong>d <strong>an</strong>d Reconstructive<br />
Surgery, H<strong>an</strong>nover Medicla Sch., H<strong>an</strong>nover, Germ<strong>an</strong>y<br />
Abstract: Recent attention has focused on <strong>the</strong> potential use of bone marrow cells which contain<br />
mesenchymal stem cells to repair <strong>the</strong> CNS because it contains populations of precursors that are<br />
multipotent <strong>an</strong>d c<strong>an</strong> differentiate into a number of cell types including bone, cartilage <strong>an</strong>d muscle<br />
cells. Mesenchymal stem cells c<strong>an</strong> be isolated <strong>from</strong> adipose tissue of <strong>the</strong> adult rat. These cells<br />
have been shown to differentiate after appropriate induction into various cell lines, e.g. fat cells,<br />
muscle, cartilage <strong>an</strong>d bone as mesenchymal stem cells <strong>from</strong> bone marrow. In addition,<br />
mesenchymal stem cells s derived <strong>from</strong> bone marrow were able to tr<strong>an</strong>sdiffentiate into glia-like<br />
cells. The question we asked was if adipose derived stem cells c<strong>an</strong> also be differentiated <strong>an</strong>d<br />
tr<strong>an</strong>sdifferentiate into glia-like cells. Adipose-derived mesenchymal stem cells were isolated<br />
<strong>from</strong> adult rats, cultured <strong>an</strong>d differentiation was induced by basis fibroblast growth factor <strong>an</strong>d<br />
epidermal growth factor in vitro. Morphology <strong>an</strong>d fur<strong>the</strong>r immunocytochemical markers of<br />
differentiated cells <strong>an</strong>d undifferentiated cells were compared. Here, we demonstrate that adiposederived<br />
mesenchymal stem cells <strong>for</strong>m nestin-positive neurosphere-like structures after induction.<br />
Fur<strong>the</strong>r differentiation leads to signific<strong>an</strong>t morphological ch<strong>an</strong>ges <strong>an</strong>d to expression of <strong>the</strong><br />
characteristic Schw<strong>an</strong>n cell marker S100 <strong>an</strong>d p75 nerve growth factor receptor <strong>an</strong>d <strong>the</strong><br />
characteristic marker GFAP <strong>for</strong> astrocytes. The simult<strong>an</strong>eous expression of characteristic glia<br />
marker of <strong>the</strong> central nervous system <strong>an</strong>d <strong>the</strong> peripheral nervous system is <strong>an</strong> exceptional feature<br />
of olfactory ensheathing cells with unique properties regarding remyelination <strong>an</strong>d enh<strong>an</strong>cement<br />
of axonal regeneration. These results indicate that adipose-derived mesenchymal stem cells<br />
grown as neuropsheres c<strong>an</strong> differentiate morphological <strong>an</strong>d functional into <strong>an</strong> Schw<strong>an</strong>n cell-like<br />
glia population.<br />
Disclosures: C. Radtke, None; P.M. Vogt, None; B. Schmitz, None; M. Spies, None; J.D.<br />
Kocsis, None.
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.25/B58<br />
Topic: A.08.b. Regeneration: PNS<br />
Support: NIH K08EB03996<br />
Army (MURI) W911NF0610218<br />
Title: DRG neurite growth on laminin-coated electrospun fibers is independent of Schw<strong>an</strong>n cell<br />
migration<br />
Authors: T. M. REGAN, M. K. LEACH, S. S. KHANNA, A. J. CHACON-SAAVEDRA, C. C.<br />
GERTZ, *J. M. COREY;<br />
Dept Neurol, Univ. Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Aligned polymer n<strong>an</strong>ofibers direct regenerating neurites in injured peripheral nerve by<br />
providing a topographical path. It has been established that Schw<strong>an</strong>n cells (SC) are critical to<br />
successful nerve repair, <strong>an</strong>d m<strong>an</strong>y nerve guides seeded with SC promote better regeneration th<strong>an</strong><br />
those without SC. Because of robust neurite growth we have observed on n<strong>an</strong>ofibers, we<br />
hypo<strong>the</strong>sized that neurite growth could be independent of SC migration. We per<strong>for</strong>med in vitro<br />
experiments to test neurite growth in <strong>the</strong> presence of aphidicolin, a mitosis inhibitor known to<br />
impair SC growth. Aligned poly-L-lactide (PLLA) n<strong>an</strong>ofibers were electrospun on substrates <strong>an</strong>d<br />
coated with polylysine, collagen I, fibronectin, or laminin. Embryonic-day-15 DRG were<br />
cultured on protein-coated n<strong>an</strong>ofibers <strong>for</strong> 3 days, <strong>the</strong>n fixed <strong>an</strong>d stained with <strong>an</strong>tibody to<br />
neurofilament <strong>an</strong>d S-100 as well as DAPI. On each DRG, <strong>the</strong> length of <strong>the</strong> 10 most prominent<br />
neurites were measured along with <strong>the</strong> greatest dist<strong>an</strong>ce of migrating SC. Additionally, total SC<br />
<strong>for</strong> each expl<strong>an</strong>t were counted using <strong>an</strong> image <strong>an</strong>alysis program written in MATLAB.<br />
Fibronectin <strong>an</strong>d laminin produced longer neurite growth th<strong>an</strong> collagen <strong>an</strong>d polylysine. SC<br />
migrated along neurites <strong>an</strong>d were found right behind growth cones. Aphidicolin in <strong>the</strong> media<br />
decreased <strong>the</strong> number of SC on <strong>the</strong> fibers by <strong>an</strong> average of 50%. Aphidicolin also decreased <strong>the</strong><br />
dist<strong>an</strong>ce of SC migration along <strong>the</strong> fibers. Most import<strong>an</strong>tly, neurite length in <strong>the</strong> presence of<br />
aphidicolin was reduced by 50% on n<strong>an</strong>ofibers coated with fibronectin but was essentially<br />
unch<strong>an</strong>ged on those coated with laminin. These results suggest that neurite growth on<br />
fibronectin-coated n<strong>an</strong>ofibers is dependent on SC migration <strong>an</strong>d that neurite outgrowth on<br />
laminin-coated n<strong>an</strong>ofibers is independent of SC. Laminin produced <strong>the</strong> longest neurites in<br />
aphidicolin, demonstrating itself to be a superior coating without Schw<strong>an</strong>n cells. Future work<br />
centers on covalent binding of <strong>the</strong> integrin-binding domains of laminin on <strong>the</strong> surfaces of<br />
n<strong>an</strong>ofibers to maximize <strong>the</strong> speed of regeneration.
Disclosures: T.M. Reg<strong>an</strong>, None; M.K. Leach, None; S.S. Kh<strong>an</strong>na, None; A.J. Chacon-<br />
Saavedra, None; C.C. Gertz, None; J.M. Corey, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.26/B59<br />
Topic: B.07.a. Presynaptic org<strong>an</strong>ization <strong>an</strong>d structure<br />
Support: CIHR<br />
Title: A putative calcium binding protein, LCaBP regulates neurite regeneration<br />
Authors: *N. NEJATBAKHSH 1 , T. LU 1 , G. CONGHUI 1 , R. VAN KESTERN 2 , Z.-P. FENG 1 ;<br />
1 Physiol., Univ. Toronto, Toronto, ON, C<strong>an</strong>ada; 2 Inst. <strong>for</strong> Neurosciences- De Boelela<strong>an</strong> 1085,<br />
Dept of Mol. <strong>an</strong>d Cell. Neurobio. - Room C-356, 1081 HV Amsterdam, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Neuro-regeneration following neuronal injury requires a multitude of processes,<br />
including syn<strong>the</strong>sis of local dendritic proteins. Thus far, <strong>the</strong> axonal or dendritic tr<strong>an</strong>scripts that<br />
have been identified are mainly related to structural proteins in both vertebrates <strong>an</strong>d<br />
invertebrates. We recently identified <strong>the</strong> tr<strong>an</strong>scripts of a novel calcium binding protein, LCaBP,<br />
in <strong>the</strong> neurites <strong>an</strong>d growth cones of Lymnaea central neurons. In this study we investigated <strong>the</strong><br />
role of LCaBP in neurite regeneration in cultured PeA cells. We first used in situ hybridization to<br />
demonstrate that LCaBP tr<strong>an</strong>scripts are distributed in specific regions of cultured PeA cells,<br />
including <strong>the</strong> soma <strong>an</strong>d growth cones. We <strong>the</strong>n constructed a specific <strong>an</strong>tibody against LCaBP<br />
protein <strong>an</strong>d used it to demonstrate that LCaBP protein levels at <strong>the</strong> soma, neurites <strong>an</strong>d growth<br />
cones of cells is ubiquitous. The effective knockdown of both LCaBP tr<strong>an</strong>script <strong>an</strong>d protein in<br />
cells treated with LCaBP siRNA was demonstrated using western blot <strong>an</strong>d immunocytochemitry<br />
combined with confocal microscopy. Using this gene silencing approach, we showed that<br />
reduced levels of LCaBP protein result in a reduction of neurite outgrowth in both intact neurites<br />
<strong>an</strong>d neurites which had undergone axotomy. In cells treated with ei<strong>the</strong>r scrambled siRNA or<br />
LCaBP siRNA, we measured <strong>the</strong> regenerative capacity of neurites which had undergone<br />
axotomy <strong>an</strong>d compared <strong>the</strong>se to <strong>the</strong> rates of growth of intact neurite over a period of 30 hours. In<br />
cells treated with scrambled siRNA, we found that 3 hours after axotomy, intact neurites of<br />
injured cells experienced signific<strong>an</strong>tly greater elongation compared to neurites of intact cells.<br />
However, all neurites belonging to injured cells treated with LCaBP siRNA experienced a net<br />
retraction compared to intact neurites. This suggests that during early stages of regeneration,<br />
LCaBP is involved in a systemic upregulation of regenerative pathways which indirectly
promote <strong>the</strong> elongation of intact neurites of injured cells compared to neurites of intact cells.<br />
Fur<strong>the</strong>rmore, 30 hours following axotomy, <strong>the</strong> proximal lengths of injured control neurites is not<br />
signific<strong>an</strong>tly different <strong>from</strong> <strong>the</strong> length of neurites of both intact <strong>an</strong>d injured cells. This is in<br />
contrast to cells treated with siRNA, where <strong>the</strong> proximal neurites of injured cells undergo a<br />
signific<strong>an</strong>t amount of retraction. This data indicates that LCaBP plays a critical role in <strong>the</strong><br />
elongation of intact neurites <strong>an</strong>d <strong>the</strong> regeneration <strong>an</strong>d continued elongation of injured neurites.<br />
Disclosures: N. Nejatbakhsh, None; T. Lu, None; G. Conghui, None; R. v<strong>an</strong> Kestern,<br />
None; Z. Feng, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.27/B60<br />
Topic: A.08.c. Regeneration: CNS<br />
Title: Hydrogel-based nerve guid<strong>an</strong>ce ch<strong>an</strong>nels <strong>for</strong> peripheral nerve regeneration<br />
Authors: *S. LIM 1 , R. MI 3 , A. HÖKE 3 , H.-Q. MAO 2 ;<br />
1 Biomed. Engin., 2 Materials Sci. <strong>an</strong>d Engin., Johns Hopkins Univ., Baltimore, MD; 3 Neurol.,<br />
Johns Hopkins Univ. Sch. of Med., Baltimore, MD<br />
Abstract: Autografts are <strong>the</strong> current gold st<strong>an</strong>dard of treatment <strong>for</strong> nerve tr<strong>an</strong>section injuries.<br />
Shortcomings of this approach are donor site morbidity as well as a finite supply of impl<strong>an</strong>table<br />
material. Syn<strong>the</strong>tic nerve guid<strong>an</strong>ce conduits (NGCs) offer <strong>an</strong> attractive alternative because <strong>the</strong>y<br />
c<strong>an</strong> be mass-produced <strong>an</strong>d tailored to match <strong>the</strong> dimensions of <strong>the</strong> nerve defect. However,<br />
clinical success of commercially-available NGCs has been limited to short gap lengths, likely<br />
because <strong>the</strong>y only provide gross physical guid<strong>an</strong>ce to <strong>the</strong> regenerating nerve stump. We<br />
hypo<strong>the</strong>size that repair over long defects requires additional cues in <strong>the</strong> <strong>for</strong>m of chemoattractive<br />
molecules or n<strong>an</strong>oscale contact guid<strong>an</strong>ce.<br />
We designed <strong>an</strong>d tested <strong>the</strong> efficacy of a hydrogel-based NGC in a rat sciatic nerve model of<br />
peripheral nerve injury. GDNF was encapsulated within a polymeric hydrogel with pore size<br />
tailored to permit prolonged growth factor release following impl<strong>an</strong>tation. Physical guid<strong>an</strong>ce <strong>for</strong><br />
axons was provided in <strong>the</strong> <strong>for</strong>m of aligned polymer n<strong>an</strong>ofibers within <strong>the</strong> NGC lumen.<br />
Additionally, <strong>the</strong> possible synergistic impact of extracellular matrix (ECM) molecules was tested<br />
by conjugating <strong>the</strong> surface of <strong>the</strong> fibers with ei<strong>the</strong>r laminin or heparin. The success of<br />
regeneration within our NGCs over a 15-mm gap was compared with Neuragen tubes. After<br />
three months in vivo, regeneration was evaluated by histomorphometry, retrograde tr<strong>an</strong>sport of
FluoroGold dye <strong>from</strong> <strong>the</strong> distal nerve stump, as well as electrophysiological recordings of <strong>the</strong><br />
nerve.<br />
Regeneration in empty gel tubes was poor, with only 2/8 <strong>an</strong>imals showing retro-labeled neuronal<br />
cell bodies; this was associated with a lack of myelinated axons <strong>an</strong>d lack of functional recovery.<br />
Both functional recovery <strong>an</strong>d presence of myelinated axons within <strong>the</strong> regenerated nerve cables<br />
was observed in <strong>an</strong>imals impl<strong>an</strong>ted with tubes containing GDNF, demonstrating that release of<br />
growth factor plays a vital role in promoting regeneration. However, <strong>the</strong>re was no signific<strong>an</strong>t<br />
enh<strong>an</strong>cement in regeneration within tubes containing additional n<strong>an</strong>ofiber guid<strong>an</strong>ce cues. It is<br />
likely that <strong>the</strong> ECM molecules provided <strong>an</strong> overly permissive microenvironment that disfavored<br />
axons <strong>from</strong> extending beyond <strong>the</strong> distal end of <strong>the</strong> NGC.<br />
Our results demonstrate that growth factors encapsulated within our NGCs maintain bioactivity<br />
<strong>an</strong>d promote regeneration when released in a controlled m<strong>an</strong>ner into a nerve gap. Studies are<br />
currently under way to optimize ECM coatings onto n<strong>an</strong>ofiber cues to best complement <strong>the</strong><br />
effect of <strong>the</strong> chemoattractive cues. The eventual goal of <strong>the</strong> study is to develop a novel NGC<br />
containing <strong>the</strong> ideal combination of cues capable of promoting regeneration over long dist<strong>an</strong>ces.<br />
Disclosures: S. Lim, None; R. Mi, None; A. Höke, None; H. Mao, None.<br />
Poster<br />
510. Regeneration: PNS<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 510.28/B61<br />
Topic: A.04.h. Axon growth <strong>an</strong>d guid<strong>an</strong>ce: O<strong>the</strong>r<br />
Support: National Science Foundation<br />
Davidson College<br />
Howard Hughes Medical Institute<br />
Title: Zebrafish motoneuron axonal pathfinding is compromised by enh<strong>an</strong>ced cholinergic<br />
activity<br />
Authors: N. L. MEYER 1 , *B. M. LOM 2 ;<br />
1 Biol. Dept., 2 Biol. Dept. <strong>an</strong>d Program in Neurosci., Davidson Col., Davidson, NC<br />
Abstract: To determine if cholinergic activity plays a role in zebrafish motoneuron axonal<br />
pathfinding, we elevated cholinergic signaling by inhibiting acetylcholinesterase (AChE) <strong>an</strong>d
examined <strong>the</strong> consequences on axonal morphology. Malathion, <strong>an</strong> org<strong>an</strong>ophosphate pesticide<br />
that inhibits AChE, is commonly used to control mosquito populations <strong>an</strong>d consequently<br />
contaminates water, where it c<strong>an</strong> exert harmful effects on non-target aquatic org<strong>an</strong>isms such as<br />
fish <strong>an</strong>d amphibi<strong>an</strong>s. We treated islet1-GFP zebrafish embryos with 2.5 mg/L of malathion,<br />
vehicle control (0.025% acetone), or dilution control (embryo medium) beginning at 0 hours post<br />
fertilization (hpf) <strong>for</strong> 72 or 96 hours. Confocal microscopy was <strong>the</strong>n used to examine <strong>the</strong><br />
morphology of GFP+ secondary motoneuron axons within a defined segment of <strong>the</strong> tail just<br />
posterior to <strong>the</strong> gut. Our results indicated that malathion exposure decreased both total axon<br />
length <strong>an</strong>d br<strong>an</strong>ching of secondary motoneurons at 72 <strong>an</strong>d 96 hpf, without affecting <strong>the</strong> <strong>an</strong>gle of<br />
axon trajectory. These results suggest that malathion exposure influences axon extension <strong>an</strong>d<br />
br<strong>an</strong>ching but does not appear to compromise axon pathfinding. However, when cholinergic<br />
activity was enh<strong>an</strong>ced beginning at 24 hpf, axon length <strong>an</strong>d br<strong>an</strong>ching were unaffected,<br />
suggesting that alterations in cholinergic activity in <strong>the</strong> first 24 hours, prior to axon extension <strong>an</strong>d<br />
pathfinding, c<strong>an</strong> influence axon length <strong>an</strong>d br<strong>an</strong>ching at <strong>the</strong> target. Shorter exposure durations<br />
<strong>from</strong> 0-12 or 12-24 hpf did not alter axonal morphology, suggesting that both window <strong>an</strong>d<br />
duration of alterations in cholinergic activity are both critical factors. Taken toge<strong>the</strong>r, <strong>the</strong>se<br />
results indicate that ch<strong>an</strong>ges in cholinergic activity during early development c<strong>an</strong> influence<br />
motoneuron axonal development <strong>an</strong>d differentiation.<br />
Disclosures: N.L. Meyer, None; B.M. Lom , None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.1/B62<br />
Topic: B.02.i. Non-NMDA glutamate receptors: Localization <strong>an</strong>d trafficking<br />
Title: Regulation of AMPA receptor trafficking <strong>an</strong>d degradation by Nedd4-mediated<br />
ubiquitination<br />
Authors: *A. W. LIN, L. JARZYLO, Q. M. HOU, S. AMATO, H. MAN;<br />
Boston Univ., Boston, MA<br />
Abstract: AMPA receptors (AMPARs) play a critical role in mediating <strong>the</strong> majority of fast<br />
excitatory synaptic tr<strong>an</strong>smission in <strong>the</strong> brain, where alterations in receptor expression,<br />
distribution <strong>an</strong>d trafficking have been shown to underlie synaptic plasticity <strong>an</strong>d higher brain<br />
function. However, <strong>the</strong> molecular mech<strong>an</strong>isms regulating AMPAR surface expression <strong>an</strong>d<br />
turnover are still not fully understood. We report that mammali<strong>an</strong> AMPARs are subject to post-
tr<strong>an</strong>slational modification by ubiquitin in heterologous cells <strong>an</strong>d rat cortical neurons. AMPAR<br />
ubiquitination enh<strong>an</strong>ced receptor degradation <strong>an</strong>d reduced AMPAR cell-surface expression.<br />
Conversely, inhibition of proteasomal activity caused AMPAR accumulation. To fur<strong>the</strong>r<br />
examine <strong>the</strong> molecular underpinnings, we used site-directed mutagenesis to replace each of four<br />
lysine residues available as putative ubiquitination sites at <strong>the</strong> GluR1 C-terminal with <strong>an</strong> arginine<br />
<strong>an</strong>d identified critical residues <strong>for</strong> ubiquitination <strong>an</strong>d receptor degradation. Consistent with <strong>the</strong><br />
role of protein ubiquitination, lysine mutation reduced <strong>the</strong> efficiency of AMPAR endocytosis. In<br />
addition, we have also identified Nedd4 as <strong>an</strong> E3 ligase <strong>for</strong> AMPAR ubiquitination. These data<br />
summarily indicate a major role <strong>for</strong> Nedd4-mediated ubiquitination in AMPAR trafficking,<br />
sorting <strong>an</strong>d degradation.<br />
Disclosures: A.W. Lin, None; L. Jarzylo, None; Q.M. Hou, None; S. Amato, None; H. M<strong>an</strong>,<br />
None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.2/B63<br />
Topic: B.02.e. NMDA receptors: Physiology<br />
Title: Proteomic identification of Nedd4 as a specific E3 ubiquitin ligase <strong>for</strong> NMDA receptor<br />
subunit NR2D<br />
Authors: *V. GAUTAM 1 , J. C. TRINIDAD 3 , R. A. RIMERMAN 2 , B. MATHIAS-COSTA 2 , A.<br />
L. BURLINGAME 3 , D. T. MONAGHAN 2 ;<br />
2 Pharmacol. <strong>an</strong>d Exptl. Neurosci., 1 Univ. Neb Med. Ctr., Omaha, NE; 3 Dept. of Pharmaceut.<br />
Chem., Univ. of Cali<strong>for</strong>nia S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: NMDA receptors are glutamate-gated ion ch<strong>an</strong>nels <strong>an</strong>d are known to be involved in<br />
<strong>the</strong> complex process of synaptic plasticity <strong>an</strong>d neuronal survival/excitotoxicity. NMDA receptors<br />
are subjected to various post-tr<strong>an</strong>slation modifications, which play <strong>an</strong> import<strong>an</strong>t role in<br />
regulating ch<strong>an</strong>nel properties as well as downstream signal tr<strong>an</strong>sduction pathways.<br />
Ubiquitination is one such post-tr<strong>an</strong>slational modification that plays <strong>an</strong> essential role in activitydependent<br />
regulation of synaptic proteins but not much has been known about <strong>the</strong> role of this<br />
post-tr<strong>an</strong>slation modification in affecting NMDA receptor activity.<br />
Using a proteomic screen, here we have identified Nedd4, <strong>an</strong> E3 ubiquitin ligase that interacts<br />
with <strong>the</strong> cytoplasmic region of NMDA receptor subunit NR2D. The direct physical interaction<br />
between NR2D cytoplasmic tail <strong>an</strong>d Nedd4 was verified by both <strong>an</strong> in vivo <strong>an</strong>d in vitro approach
using co-immunoprecipitation <strong>an</strong>d direct yeast two-hybrid system. Immunofluorescence studies<br />
also corroborated this finding as Nedd4 co-localize with NR2D in both primary hippocampal<br />
neurons as well as in a heterologous cell line. Using deletion mutagenesis strategy we have<br />
fur<strong>the</strong>r identified that <strong>the</strong> PPXY motif present on <strong>the</strong> NR2D cytoplasmic region <strong>an</strong>d <strong>the</strong> WW<br />
domain of Nedd4 play a critical role in mediating this novel interaction as a single point mutation<br />
in ei<strong>the</strong>r of <strong>the</strong>se two regions severely abrogated <strong>the</strong>ir interaction. This association of Nedd4<br />
with NMDA receptor subunit NR2D appears to cause its ubiquitination as coexpression of<br />
Nedd4 with NR2D in mammali<strong>an</strong> cells signific<strong>an</strong>tly enh<strong>an</strong>ces its ubiquitination <strong>an</strong>d degradation<br />
through proteosomal mediated pathways. Fur<strong>the</strong>rmore, coexpressing Nedd4 along with NR2D in<br />
<strong>an</strong> oocyte system also signific<strong>an</strong>tly downregulated its activity. Taken toge<strong>the</strong>r, <strong>the</strong>se results<br />
identify Nedd4, <strong>an</strong> E3 ubiquitin ligase, as a novel binding partner <strong>for</strong> NR2D <strong>an</strong>d suggest a<br />
possible mech<strong>an</strong>ism <strong>for</strong> regulating <strong>the</strong> activity of NMDA receptors that contains NR2D subunit<br />
through its ubiquitination.<br />
Disclosures: V. Gautam, None; J.C. Trinidad, None; R.A. Rimerm<strong>an</strong>, None; B. Mathias-<br />
Costa, None; A.L. Burlingame, None; D.T. Monagh<strong>an</strong>, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.3/B64<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: CIHR Fellowship to KFS Bell<br />
Title: Synaptic activity is neuroprotective via <strong>the</strong> downregulation of pro-apoptotic machinery<br />
Authors: *K. F. BELL 1 , S. PAPADIA 1 , F. LEVEILLE 1 , C. IKONOMIDOU 2 , G. E.<br />
HARDINGHAM 1 ;<br />
1 Ctr. Integrative Physiol., Univ. Edinburgh, Edinburgh, United Kingdom; 2 Dept. of Pediatric<br />
Neurol., Tech. Univ. Dresden, Dresden, Germ<strong>an</strong>y<br />
Abstract: NMDA receptor activity is a key source of activity-dependent intra-neuronal calcium<br />
tr<strong>an</strong>sients. While too much NMDA receptor activity c<strong>an</strong> be harmful in excitotoxic trauma,<br />
normal patterns of activity have been shown to be protective both in vitro <strong>an</strong>d in vivo. Outright<br />
blockade of NMDA receptor activity promotes apoptosis <strong>an</strong>d exacerbates ongoing<br />
neurodegeneration, particularly in <strong>the</strong> developing CNS. Our current underst<strong>an</strong>ding of <strong>the</strong> m<strong>an</strong>ner<br />
in which synaptic activity is protective remains poor. Work <strong>from</strong> our lab has identified a down-
egulation of genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic machinery following synaptic NMDA receptor<br />
activity in cortical neurons, as initiated via network disinhibition with Biciculline. NMDA<br />
receptor <strong>an</strong>tagonist MK801 is known to protect cells <strong>from</strong> toxic insults such as hydrogen<br />
peroxide, glutamate <strong>an</strong>d oxygen glucose deprivation. However, on its own MK801<br />
administration causes <strong>an</strong> upregulation of specific genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic machinery.<br />
Mem<strong>an</strong>tine, a non-competitive <strong>an</strong>tagonist of <strong>the</strong> NMDA receptor is also able to protect cortical<br />
neurons following ischemia or glutamate toxicity. Import<strong>an</strong>tly however, mem<strong>an</strong>tine application<br />
does not appear to induce genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic machinery, nor does it interfere with<br />
<strong>the</strong> down-regulation of <strong>the</strong>se genes following synaptic activity. In vivo administration of MK801<br />
to mice was found to induce certain genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic machinery, while<br />
mem<strong>an</strong>tine administration did not. Acute neuronal stimulus with ei<strong>the</strong>r oxygen glucose<br />
deprivation or toxic levels of glutamate failed to induce genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic<br />
machinery, however longer time-points post insult may be required <strong>for</strong> identifiable ch<strong>an</strong>ges in<br />
gene expression. Fur<strong>the</strong>r work will investigate <strong>the</strong> induction of genes import<strong>an</strong>t <strong>for</strong> pro-apoptotic<br />
machinery following oxygen glucose deprivation, as well as mem<strong>an</strong>tine’s ability to prevent this<br />
induction. This work should help characterize <strong>the</strong> molecular events underlying endogenous pro<br />
survival signaling by synaptic NMDA receptors. It also serves to highlight a potential <strong>the</strong>rapeutic<br />
role <strong>for</strong> mem<strong>an</strong>tine in selectively blocking pathological signaling, while maintaining <strong>the</strong><br />
beneficial aspects of physiological synaptic NMDA receptor signaling.<br />
Disclosures: K.F. Bell, None; S. Papadia, None; F. Leveille, None; C. Ikonomidou,<br />
None; G.E. Hardingham, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.4/B65<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NIH Gr<strong>an</strong>t NS46742<br />
NIH Gr<strong>an</strong>t NS45693<br />
Title: Epinegetic regulation of NMDA receptor NR2 subunit switch in early postnatal develop<br />
Authors: *A. RODENAS-RUANO, R. ZUKIN, M. COSSIO;<br />
Neurosci., Albert Einstein Col. Med., Bronx, NY
Abstract: A hallmark feature of NMDARs is a switch in <strong>the</strong>ir molecular <strong>an</strong>d functional<br />
signature during brain development. At birth, NMDARs contain NR1 <strong>an</strong>d NR2B subunits.<br />
During postnatal development, NR2B expression declines <strong>an</strong>d <strong>the</strong>re is a progressive inclusion of<br />
<strong>the</strong> NR2A subunit at synapses of <strong>the</strong> hippocampus. The switch between NR2B <strong>an</strong>d NR2Acontaining<br />
receptors coincides with closure of <strong>the</strong> “critical period”, a developmental window<br />
when <strong>the</strong> brain is highly plastic, <strong>an</strong>d c<strong>an</strong> be regulated by environmental cues. However, <strong>the</strong><br />
molecular mech<strong>an</strong>ism by which <strong>the</strong> switch in NMDAR phenotype occurs is, as yet, unknown.<br />
Epigenetic regulation is a powerful me<strong>an</strong>s to regulate synapse structure <strong>an</strong>d function in response<br />
to experience. The tr<strong>an</strong>scriptional repressor REST/NRSF c<strong>an</strong> silence a network of neuronspecific<br />
genes including <strong>the</strong> NMDAR subunit NR2B. Here we show that a decline in NR2B<br />
mRNA levels during postnatal development, coincides with <strong>the</strong> recruitment of <strong>the</strong> REST<br />
repressor complex <strong>an</strong>d of histone silencing marks to <strong>the</strong> NR2B promoter.These studies af<strong>for</strong>d<br />
insight into <strong>the</strong> developmental switch in NMDAR phenotype at hippocampal synapses that<br />
accomp<strong>an</strong>ies closure of <strong>the</strong> critical period, <strong>an</strong>d document a role <strong>for</strong> epigenetic remodeling in<br />
brain development.<br />
Disclosures: A. Rodenas-Ru<strong>an</strong>o, None; R. Zukin, None; M. Cossio, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.5/B66<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Title: Epigenetic regulation of <strong>the</strong> NMDAR2B gene in P19 cells<br />
Authors: *A. ANJI, M. KUMARI;<br />
K<strong>an</strong>sas State Univ, Col. Vet Med., M<strong>an</strong>hatt<strong>an</strong>, KS<br />
Abstract: N-methyl-D-aspartate (NMDA) receptors, one of three subtypes of glutamate<br />
receptors, mediate excitatory neurotr<strong>an</strong>smission in <strong>the</strong> brain. These receptors occur as multiple<br />
subtypes that differ in <strong>the</strong>ir <strong>an</strong>atomical distribution, properties <strong>an</strong>d regulation. The NR2B subunit<br />
containing NMDA receptors play import<strong>an</strong>t roles in NMDA receptor localization, endocytosis as<br />
well as in learning <strong>an</strong>d memory <strong>for</strong>mation. They have also been suggested to contribute to <strong>the</strong><br />
pathophysiology of neurodegenerative disorders such as Alzheimer's <strong>an</strong>d Huntington's disease.<br />
In this study we have examined <strong>the</strong> role of epigenetic processes, specifically <strong>the</strong> dynamic process<br />
of methylation of cytosine residues in CpG dinucleotides in regulation of <strong>the</strong> NR2B gene in P19<br />
cells. P19 cells are derived <strong>from</strong> murine pluripotent embryonal carcinoma cells <strong>an</strong>d do not
express NMDA receptors. However in <strong>the</strong> presence of all-tr<strong>an</strong>s-retinoic acid <strong>the</strong>se cells c<strong>an</strong> be<br />
induced to differentiate into post-mitotic neurons that express NR2B receptors <strong>an</strong>d thus offer <strong>an</strong><br />
excellent in vitro model system to study regulation of NR2B subunit containing receptor gene<br />
expression.<br />
The mouse NR2B gene has a CpG isl<strong>an</strong>d located in <strong>the</strong> 5’ non-coding region <strong>an</strong>d <strong>the</strong> promoter<br />
region. Exon 2 of <strong>the</strong> mouse NR2B gene has <strong>the</strong> maximum number of CpG dinucleotides.<br />
Genomic DNA <strong>from</strong> undifferentiated as well as differentiated P19 cells was digested with <strong>the</strong><br />
methyl sensitive enzyme, Hha I. Following restriction digestion, a known amount of DNA was<br />
used as a template <strong>for</strong> PCR amplification using NR2B gene specific primers. Amplified PCR<br />
products were separated on agarose gels <strong>an</strong>d photographed using Kodak EDAS system. Data<br />
<strong>an</strong>alysis showed that certain CpG dinucleotide containing regions of <strong>the</strong> NR2B promoter <strong>an</strong>d<br />
non-coding regions are hypomethylated irrespective of NR2B gene expression.<br />
Disclosures: A. Anji, None; M. Kumari, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.6/B67<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: KAKENHI 17002016 (S.N.)<br />
Title: Mech<strong>an</strong>ism of subunit switching <strong>from</strong> NR2B to NR2C in mouse cerebellar gr<strong>an</strong>ule cell<br />
cultures<br />
Authors: *K. IIJIMA 1 , H. ABE 1,2 , M. OKAZAWA 1 , K. MORIYOSHI 2 , S. NAKANISHI 1 ;<br />
1 Osaka Biosci. Inst., Suita, Osaka, Jap<strong>an</strong>; 2 Kyoto Univ., Kyoto, Jap<strong>an</strong><br />
Abstract: In <strong>the</strong> developing cerebellum, gr<strong>an</strong>ule cells switch subunit composition of NMDA<br />
receptors <strong>from</strong> NR2B to NR2C, parallel to <strong>the</strong> synapse <strong>for</strong>mation with mossy fibers. This<br />
switching alters properties of glutamatergic tr<strong>an</strong>smission onto gr<strong>an</strong>ule cell <strong>an</strong>d is a key <strong>for</strong><br />
cerebellar network maturation. We investigated mech<strong>an</strong>isms <strong>for</strong> this switching in primary<br />
cultures of mouse gr<strong>an</strong>ule cells at <strong>the</strong> physiological KCl concentration (5 mM). Gr<strong>an</strong>ule cells<br />
down-regulated NR2B mRNA <strong>an</strong>d up-regulated NR2C mRNA, consistent with <strong>the</strong> prominent<br />
extension of neuritic processes. This dual regulation of <strong>the</strong> 2 mRNAs was dependent on neural<br />
activities that were sensitive to TTX <strong>an</strong>d <strong>an</strong>tagonists selective <strong>for</strong> AMPA receptors <strong>an</strong>d NMDA<br />
receptors. Suppression of dual regulation under TTX was restored by NMDA stimulation, but
not by AMPA stimulation. Import<strong>an</strong>tly, <strong>the</strong> NMDA receptor activation drove <strong>the</strong> NR2B/2Cswitching<br />
of NMDA receptors in <strong>the</strong> cell-surface membr<strong>an</strong>e. This investigation demonstrates<br />
that stimulation on NMDA receptors following cell excitation via AMPA receptors plays a key<br />
role in functional subunit switching of NMDA receptors in maturing processes of gr<strong>an</strong>ule cells.<br />
Disclosures: K. Iijima, None; H. Abe, None; M. Okazawa, None; K. Moriyoshi, None; S.<br />
Nak<strong>an</strong>ishi, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.7/B68<br />
Topic: B.02.k. Non-NMDA glutamate receptors: Gene regulation<br />
Support: NSF Gr<strong>an</strong>t IBN0344559<br />
Title: Emotional stress induces GluR2 gene tr<strong>an</strong>scription <strong>an</strong>d a switch in synaptic AMPA<br />
receptor phenotype<br />
Authors: *Y. LIU, I. SAVTCHOUK, S.-Q. J. LIU;<br />
Dept. of Biol., Penn State Univ., State College, PA<br />
Abstract: Emotional stress promotes <strong>the</strong> release of noradrenaline <strong>an</strong>d c<strong>an</strong> alter <strong>an</strong>imal behavior<br />
including learning <strong>an</strong>d memory. Noradrenaline (NA), a neuromodulator, plays <strong>an</strong> import<strong>an</strong>t role<br />
in modulating long-term neuronal plasticity. Adrenergic neurons in <strong>the</strong> locus ceoruleus innervate<br />
<strong>the</strong> cerebellum where NA acts via β-adrenergic receptors to increase <strong>the</strong> action potential firing<br />
rate of inhibitory stellate cells <strong>an</strong>d alters <strong>the</strong> spont<strong>an</strong>eous firing of Purkinje neurons. Here we<br />
tested <strong>the</strong> hypo<strong>the</strong>sis that emotional stress produces a long-lasting ch<strong>an</strong>ge in synaptic<br />
tr<strong>an</strong>smission in <strong>the</strong> cerebellum. Exposure of mice to fox urine, a natural fear-inducing stimulus,<br />
caused innate fear. To determine <strong>the</strong> subunit composition of synaptic AMPA receptors<br />
(AMPARs), we included spermine in <strong>the</strong> patch electrode <strong>an</strong>d measured spont<strong>an</strong>eous EPSCs at<br />
several potentials. Intracellular spermine is known to block GluR2-lacking receptors (but not<br />
GluR2-containing AMPARs) at depolarized potentials, giving rise to <strong>an</strong> inwardly rectifying I-V<br />
relationship. The amplitude of sEPSCs in stellate cells <strong>from</strong> control slices was strongly<br />
attenuated at positive potentials, indicating <strong>the</strong> presence of GluR2-lacking receptors. In contrast,<br />
synaptic currents recorded at 3 h or 15 h after fox urine exposure had a nearly linear I-V<br />
relationship. This suggests that a single exposure to a fear-inducing stimulus c<strong>an</strong> induce a longlasting<br />
increase in <strong>the</strong> expression of GluR2-containing AMPARs in stellate cells. Consistent with
this observation, IEM-1480, a subunit-selective blocker of GluR2-lacking AMPARs, reduced <strong>the</strong><br />
EPSC amplitude in control cells by ~50%. At 3 h after fox urine exposure <strong>the</strong> degree of block by<br />
IEM-1480 was diminished to 20%. Thus emotional stress c<strong>an</strong> ch<strong>an</strong>ge <strong>the</strong> AMPAR phenotype<br />
<strong>from</strong> GluR2-lacking to GluR2-containing at stellate cell synapses. β-adrenergic receptors are<br />
known to mediate fear-induced freezing behavior. We found that propr<strong>an</strong>olol, a blocker of βadrenergic<br />
receptors, also prevented <strong>the</strong> switch in AMPARa induced by fox urine exposure.<br />
Fur<strong>the</strong>rmore incubation of cerebellar slices in NA <strong>for</strong> 3 h caused a switch in EPSC I-V<br />
relationship <strong>from</strong> inwardly rectifying to nearly linear. Thus, noradrenaline <strong>an</strong>d β-adrenergic<br />
receptors likely mediate <strong>the</strong> stress-induced ch<strong>an</strong>ge in synaptic AMPAR phenotype. Using<br />
qu<strong>an</strong>titative single cell RT-PCR we found that fox urine exposure selectively increased GluR2<br />
mRNA abund<strong>an</strong>ce in individual stellate cells by 63 ± 19% at 3 h <strong>an</strong>d 103 ± 36% at 15 h.<br />
There<strong>for</strong>e, <strong>the</strong> stress-induced incorporation of functional GluR2-containing AMPARs at<br />
cerebellar stellate cell synapses was associated with <strong>an</strong> increase in <strong>the</strong> GluR2 mRNA content of<br />
individual stellate cells.<br />
Disclosures: Y. Liu, None; I. Savtchouk, None; S.J. Liu, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.8/B69<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: gr<strong>an</strong>ts-in-aid <strong>for</strong> scientific research <strong>from</strong> <strong>the</strong> MEXT<br />
Title: Involvement of NMDAR2A tyrosine phosphorylation in depression-related behavior<br />
Authors: *T. NAKAZAWA 1 , S. TANIGUCHI 1 , T. TEZUKA 1 , Y. KIYAMA 2 , T. MANABE 2,3 ,<br />
T. YAMAMOTO 1 ;<br />
1 Oncology, 2 Neuronal Network, Inst. Med. Sci, Univ. Tokyo, Tokyo, Jap<strong>an</strong>; 3 Core Res. <strong>for</strong><br />
Evolutional Sci. <strong>an</strong>d Technol. (CREST), Jap<strong>an</strong> Sci. <strong>an</strong>d Technol. Agency (JST), Kawaguchi,<br />
Jap<strong>an</strong><br />
Abstract: Growing evidence implicates glutamate signaling in depression-related behavior,<br />
though <strong>the</strong> molecular mech<strong>an</strong>ism by which glutamate signaling regulates depression-related<br />
behavior remains unclear. In this study, we provide evidence suggesting that <strong>the</strong> tyrosine<br />
phosphorylation of <strong>the</strong> NMDA receptor contributes to depression-related behavior. The NR2A<br />
subunit of <strong>the</strong> NMDA receptor is tyrosine-phosphorylated, with Tyr-1325 as its one of <strong>the</strong> major
phosphorylation site. We have generated mice expressing mut<strong>an</strong>t NR2A with a Tyr-1325-Phe<br />
(Y1325F) mutation to prevent phosphorylation of this site in vivo. The homozygous knock-in<br />
mice show signific<strong>an</strong>tly less immobility in <strong>the</strong> tail suspension test <strong>an</strong>d in <strong>the</strong> <strong>for</strong>ced swim test,<br />
suggesting that <strong>the</strong> YF mutation induces <strong>an</strong>tidepress<strong>an</strong>t-like effects in mice. In <strong>the</strong> striatum of <strong>the</strong><br />
knock-in mice, DARPP-32 (dopamine- <strong>an</strong>d cAMP-regulated phosphoprotein, 32kD)<br />
phosphorylation at Thr-34, which is import<strong>an</strong>t <strong>for</strong> <strong>the</strong> regulation of depression-related behavior,<br />
is increased. We also show that <strong>the</strong> Tyr-1325 phosphorylation site is required <strong>for</strong> Src-mediated<br />
potentiation of <strong>the</strong> recombin<strong>an</strong>t NMDA receptor ch<strong>an</strong>nel. These data argue that <strong>the</strong> Tyr-1325<br />
phosphorylation regulates NMDA receptor ch<strong>an</strong>nel properties <strong>an</strong>d <strong>the</strong> NMDA receptor-mediated<br />
downstream signaling to modulate depression-related behavior.<br />
Disclosures: T. Nakazawa, None; S. T<strong>an</strong>iguchi, None; T. Tezuka, None; Y. Kiyama,<br />
None; T. M<strong>an</strong>abe, None; T. Yamamoto, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.9/B70<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: NIAAA gr<strong>an</strong>t AA12070<br />
Title: Removal of <strong>the</strong> 3’untr<strong>an</strong>slated region of <strong>the</strong> NMDAR1 mRNA facilitates its decay<br />
Authors: *M. KUMARI, A. ANJI;<br />
K<strong>an</strong>sas State Univ., M<strong>an</strong>hatt<strong>an</strong>, KS<br />
Abstract: Messenger RNA stability is <strong>an</strong> import<strong>an</strong>t regulatory mech<strong>an</strong>ism in <strong>the</strong> cell as ch<strong>an</strong>ges<br />
in mRNA half-life c<strong>an</strong> modulate <strong>the</strong> expression of genes. Growing evidence supports <strong>the</strong> notion<br />
that <strong>the</strong> 3’ Untr<strong>an</strong>slated region (3’ UTR) of mRNAs play <strong>an</strong> import<strong>an</strong>t role in regulation of<br />
mRNA half-life. In this study we determined <strong>the</strong> import<strong>an</strong>ce of <strong>the</strong> NMDA R1 (NR1) mRNA<br />
3’UTR in regulating its decay using cell-free mRNA decay assays. These assays were per<strong>for</strong>med<br />
using polysomal fraction (= endoplasmic reticulum) <strong>an</strong>d S130 fraction (= cytosol without major<br />
cell org<strong>an</strong>elles) prepared <strong>from</strong> fetal cortical neurons (FCN) cultured in <strong>the</strong> presence or absence of<br />
eth<strong>an</strong>ol (50 mM, 5 days). The polysomal fraction was used <strong>for</strong> NR2B mRNA <strong>an</strong>d NR1<br />
constructs (full length <strong>an</strong>d mut<strong>an</strong>ts) while S130 fraction was used <strong>for</strong> c-myc decay assays. The<br />
fidelity of <strong>the</strong> cell free mRNA decay assays was examined using c-myc <strong>an</strong>d NR2B cRNAs. All<br />
plasmid constructs were linearized by restriction digestion <strong>an</strong>d sense RNAs were tr<strong>an</strong>scribed in
vitro in <strong>the</strong> presence of [32P]-α-CTP <strong>an</strong>d 7mGpppGTP. A known amount of 32P-labeled c-myc<br />
cRNA was incubated with S130 fraction <strong>an</strong>d 32P-labeled NR2B or NR1 cRNAs with polysomal<br />
fractions <strong>for</strong> various time intervals at 37 degree C. Incubation was stopped by adding 1 ml of<br />
Trizol followed by purification of <strong>the</strong> RNA. RNA <strong>from</strong> all <strong>the</strong> time points were separated on<br />
agarose gel, blotted <strong>an</strong>d exposed to PhosphorImager screen. Data <strong>an</strong>alysis showed no signific<strong>an</strong>t<br />
difference in half-life of c-myc mRNA in <strong>the</strong> presence or absence of eth<strong>an</strong>ol. <strong>When</strong> tested in<br />
vitro, using polysomal fraction, <strong>the</strong> half-life of NR2B mRNA was found to be 10 h as opposed to<br />
14 h in cultured neurons reported previously (Kumari <strong>an</strong>d Ticku, 1998). The NR1 mut<strong>an</strong>t<br />
constructs lacking 3’ UTR decayed rapidly as opposed to when <strong>the</strong> 3’ UTR was present. The in<br />
vitro data was confirmed by FCN tr<strong>an</strong>sfection experiments. Taken toge<strong>the</strong>r, our data strongly<br />
suggests that <strong>the</strong> NR1 mRNA 3’ UTR plays <strong>an</strong> import<strong>an</strong>t role in regulating decay of NR1<br />
mRNA.<br />
Disclosures: M. Kumari, None; A. Anji, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.10/B71<br />
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: FIS PI060767<br />
Title: Early modifications in NMDA receptor subunit gene expression in <strong>an</strong> oxygen <strong>an</strong>d glucose<br />
deprivation model using rat hippocampal brain slices<br />
Authors: *A. FERNANDEZ-LOPEZ 1 , S. DOS-ANJOS 1 , S. MONTORI 1 , I. LORENZO 1 , T.<br />
BURGIN 1 , C. C. PÉREZ-GARCÍA 2 , B. MARTINEZ-VILLAYANDRE 1 ;<br />
1 2<br />
Univ. de Leon, Dpt. Biol. Mol., Leon, Spain; Dpt. Medicina y Cirugía Animal, Univ. de Leon,<br />
Leon, Spain<br />
Abstract: Glutamatergic NMDA receptors (NMDARs) are considered to play a key role in<br />
ischemia-induced damage. Here we report ch<strong>an</strong>ges in <strong>the</strong> mRNA levels of <strong>the</strong> major<br />
hippocampal NMDAR subunits (NR1, NR2A <strong>an</strong>d NR2B) as well as c-fos (<strong>an</strong> immediate early<br />
response gene) in <strong>an</strong> ex vivo ischemia model using hippocampal slices. NMDAR subunit mRNA<br />
levels signific<strong>an</strong>tly decreased after 30 minutes of oxygen <strong>an</strong>d glucose deprivation (OGD)<br />
followed by partial recovery of <strong>the</strong> NMDAR subunit mRNA levels when <strong>the</strong> tissues were<br />
returned to <strong>the</strong> bal<strong>an</strong>ced salt solution (reperfusion-like period) <strong>for</strong> 3 hours. Since OGD-induced
damage has been reported to be a consequence of <strong>the</strong> increased OGD-dependent glutamate<br />
release, we <strong>an</strong>alyzed NMDAR gene expression following external glutamate addition (in <strong>the</strong><br />
same r<strong>an</strong>ge as that produced during OGD period) in hippocampal slices. Fur<strong>the</strong>rmore, we<br />
detected a signific<strong>an</strong>t decrease in NMDAR expression as a consequence of <strong>the</strong> presence of MK-<br />
801 (a selective NMDAR <strong>an</strong>tagonist) or CNQX (a selective AMPA/Kainate receptor <strong>an</strong>tagonist)<br />
in <strong>the</strong> incubation solution.<br />
This work has been gr<strong>an</strong>ted by Fondo de Investigaciones S<strong>an</strong>itarias ref. FIS PI060767<br />
Disclosures: A. Fern<strong>an</strong>dez-Lopez, None; S. Dos-Anjos, None; S. Montori, None; I. Lorenzo,<br />
None; T. Burgin, None; C.C. Pérez-García, None; B. Martinez-Villay<strong>an</strong>dre, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.11/B72<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NINDS Intramural Research Program<br />
Title: Regulation of NMDA receptors by casein kinase 2<br />
Authors: *A. SANZ-CLEMENTE, K. W. ROCHE;<br />
NIH, Be<strong>the</strong>sda, MD<br />
Abstract: NMDA receptors (NMDARs) are ion ch<strong>an</strong>nels that play a key role in synaptic<br />
function <strong>an</strong>d neuronal development. They are functional tetramers composed of two NR1<br />
subunits <strong>an</strong>d two NR2A-D <strong>an</strong>d NR3 subunits. Synaptic NMDAR number <strong>an</strong>d subunit<br />
composition are strongly regulated by several mech<strong>an</strong>isms including phosphorylation <strong>an</strong>d<br />
interactions with PDZ domain-containing proteins. It is well known that <strong>the</strong> composition of<br />
synaptic NMDARs ch<strong>an</strong>ges during development. During early stages, NR2B-containing<br />
NMDARs are predomin<strong>an</strong>t, whereas NR2A-containing NMDARs are <strong>the</strong> most abund<strong>an</strong>t at<br />
adulthood. However, <strong>the</strong> precise molecular mech<strong>an</strong>isms mediating <strong>the</strong> switch remain obscure.<br />
One study showed that NR2¬B is phosphorylated in its PDZ binding domain (S1480) by casein<br />
kinase 2 (CK2), which disrupts <strong>the</strong> binding with PSD-95 <strong>an</strong>d SAP102 <strong>an</strong>d decreases <strong>the</strong> amount<br />
of NR2B at <strong>the</strong> cell surface (Chung HJ et al, J Neurosci 24: 10248-59). We have now compared<br />
CK2 phosphorylation of NR2B vs NR2A <strong>an</strong>d found that, despite its high homology, NR2A is not<br />
phosphorylated by this kinase. This difference me<strong>an</strong>s that NR2A <strong>an</strong>d NR2B binding to PDZ<br />
domain-containing proteins is differentially regulated, despite <strong>the</strong> conserved PDZ lig<strong>an</strong>ds. CK2
is highly expressed in <strong>the</strong> brain <strong>an</strong>d increases during development. There<strong>for</strong>e, we tested <strong>the</strong><br />
hypo<strong>the</strong>sis that CK2 might differentially affect NR2A <strong>an</strong>d NR2B synaptic distribution during<br />
development. We observed <strong>an</strong> increase in <strong>the</strong> levels of NR2B-S1480 phosphorylation during <strong>the</strong><br />
period of <strong>the</strong> developmental NMDAR switch as well as <strong>an</strong> elevated association of CK2 with <strong>the</strong><br />
synaptic plasma membr<strong>an</strong>e during <strong>the</strong> same period. The NMDAR subunit switch may require<br />
sequential steps including <strong>the</strong> synaptic removal of NR2B induced by CK2-mediated endocytosis,<br />
which allows <strong>the</strong> subsequent insertion of NR2A into synapses. Consistent with this model, we<br />
found that inhibition of CK2 causes a reduction in <strong>the</strong> levels of surface NR2A in cortical primary<br />
cultures. Since a developmental increase in synaptic NR2A is highly correlated with <strong>an</strong><br />
enh<strong>an</strong>cement of its total expression, we <strong>an</strong>alyzed NR2A knockout mice. Interestingly, NR2B-<br />
S1480 phosphorylation levels <strong>an</strong>d CK2 association with synaptic plasma membr<strong>an</strong>e were<br />
reduced in <strong>the</strong>se mice in comparison with wild-type <strong>an</strong>imals, revealing a potential interplay<br />
between NR2A expression <strong>an</strong>d CK2 phosphorylation of NR2B.<br />
Disclosures: A. S<strong>an</strong>z-Clemente, None; K.W. Roche, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.12/B73<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NIH Gr<strong>an</strong>t HD18655<br />
NIH Gr<strong>an</strong>t NS38475<br />
The ELA Foundation<br />
Title: Functional NMDA receptors are present on developing <strong>an</strong>d mature rat oligodendrocytes in<br />
culture<br />
Authors: *T. M. DESILVA, A. Y. KABAKOV, A. E. ARMSTRONG, J. J. VOLPE, P. A.<br />
ROSENBERG;<br />
Children's Hosp & Harv Med., Boston, MA<br />
Abstract: Excitotoxicity to oligodendrocytes (OLs) was thought to be mediated exclusively by<br />
AMPA receptors. Recently, NMDA receptors have been localized on OLs in vivo at all stages of<br />
development <strong>an</strong>d are capable of mediating excitotoxic injury to OLs. However, NMDA receptors
have not been found previously in cultured OLs. Here, we demonstrate that functional NMDA<br />
receptors are present on cultured rat OLs. Immunoblot <strong>an</strong>alysis demonstrated <strong>the</strong> obligatory NR1<br />
subunit in preoligodendrocytes (preOLs), immature OLs, <strong>an</strong>d mature OLs in culture.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> NR2B subunit was downregulated with maturation, showing increased<br />
expression in preOLs, compared to immature <strong>an</strong>d mature OLs. Conversely, expression of <strong>the</strong><br />
NR3A subunit was upregulated with maturation <strong>an</strong>d enriched in mature OLs compared to<br />
developing OLs. Immunocytochemical labeling with <strong>the</strong> <strong>an</strong>ti-NR1, NR2A, NR2B, <strong>an</strong>d NR3A<br />
<strong>an</strong>tibodies was detected in cell bodies <strong>an</strong>d processes. Ca 45 influx was evoked by exposure to<br />
NMDA in preOLs, immature OLs, <strong>an</strong>d mature OLs, increased with maturation, <strong>an</strong>d was blocked<br />
by <strong>the</strong> NMDA <strong>an</strong>tagonists MK801 <strong>an</strong>d 5,7-dichlorokynurenic acid. Patch-clamp recording was<br />
used to fur<strong>the</strong>r characterize NMDA receptors on OLs in culture. In mature OLs, glycine (0.5<br />
µM) <strong>an</strong>d glutamate (10 µM) co-application resulted in currents of 7.1 ± 2.4 pA (n = 8); glycine<br />
increased glutamate activated current by 46 ± 14% (n = 3). This current was partially inhibited<br />
by 1 µM MK801 (35 ± 6%) <strong>an</strong>d completely by 100 µM 5,7-dichlorokynurenic acid (DCK) (n =<br />
5). Co-application of glutamate <strong>an</strong>d glycine evoked currents (20 ± 5 pA; n = 3; Vm = +30 mV)<br />
in preOLs that were more th<strong>an</strong> 50% blocked by 1 µM MK801 or 10 µM DCK. Since NR2B<br />
confers increased calcium permeability, <strong>the</strong> upregulation of <strong>the</strong> NR2B subunit in preOLs may<br />
make <strong>the</strong>m particularly vulnerable to excitotoxicity during brain development. Fur<strong>the</strong>rmore, in<br />
mature OLs, upregulation of <strong>the</strong> NR3A subunit, with its decreased magnesium sensitivity, <strong>an</strong>d<br />
<strong>the</strong> overall increase in expression of NMDA receptors in processes of mature OLs suggests that<br />
<strong>the</strong>se receptors may play a role in signaling during myelination. To gain additional evidence<br />
regarding NMDA receptor expression in myelin in vivo, immunocytochemistry of NR1 was<br />
per<strong>for</strong>med in 11 hum<strong>an</strong> cases (23 weeks to 2.5 postnatal years) in <strong>the</strong> frontal <strong>an</strong>d parietal<br />
cerebral white matter. Progressively more intense NR1 labeling correlated with labeling of<br />
myelin sheaths. These data suggest a role <strong>for</strong> glutamatergic signaling between axons <strong>an</strong>d OLs in<br />
myelination <strong>an</strong>d value <strong>for</strong> in vitro models to define <strong>the</strong>se interactions.<br />
Disclosures: T.M. DeSilva, None; A.Y. Kabakov, None; A.E. Armstrong, None; J.J. Volpe,<br />
None; P.A. Rosenberg, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.13/B74<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NIH Gr<strong>an</strong>t NS057415, RR15640
Title: Developmental switch of NR2A <strong>an</strong>d NR2B subunits in <strong>the</strong> GABAergic interneurons of<br />
barrel cortex<br />
Authors: *Z. ZHANG, Q.-Q. SUN;<br />
Univ. Wyoming, Laramie, WY<br />
Abstract: N-methyl-D-aspartate receptors (NMDARs) are excitatory ionotropic glutamate<br />
receptors that are import<strong>an</strong>t <strong>for</strong> neuronal development, synaptic tr<strong>an</strong>smission <strong>an</strong>d synaptic<br />
plasticity. The NMDARs are heteromers that contain two NR1 subunits <strong>an</strong>d two o<strong>the</strong>r mixtures<br />
of NR2A-D or NR3A-B subunits that determine receptor distinct physiological properties <strong>an</strong>d<br />
functions. There is a developmental, experience-dependent switch of NR2B- to NR2A- subunitcontaining<br />
NMDARs in <strong>the</strong> pyramidal neurons of <strong>the</strong> hippocampus <strong>an</strong>d neocortex. However, it is<br />
unclear whe<strong>the</strong>r <strong>the</strong>re is a developmental switch of NR2 subunits in GABAergic interneurons<br />
<strong>an</strong>d if so, how this switch leads to <strong>the</strong> ch<strong>an</strong>ges in NMDARs functions. Here we demonstrate that<br />
NR2A-<strong>an</strong>d NR2B-subunit-mediated EPSCs exhibited developmental switch in GAD67-GFP<br />
positive interneurons in layer II/III of barrel cortex. Western blotting experiments showed that<br />
<strong>the</strong> NR2A <strong>an</strong>d NR2B subunit protein level increased during development <strong>an</strong>d peak around<br />
P19/20, with a larger increase in <strong>the</strong> NR2A th<strong>an</strong> NR2B protein level. This is <strong>the</strong> first report to<br />
show that NMDARs exhibited developmental switch in GABAergic interneurons.<br />
Gr<strong>an</strong>t support: National Institutes of Health (NS057415, RR15640).<br />
Disclosures: Z. Zh<strong>an</strong>g, None; Q. Sun, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.14/B75<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NINDS Intramural Program<br />
Title: Experience-dependent modulation of NMDA-receptor mediated tr<strong>an</strong>smission controls<br />
functional feed-<strong>for</strong>ward inhibition during barrel cortex development<br />
Authors: *R. CHITTAJALLU, J. T. R. ISAAC;<br />
NINDS, NIH, Be<strong>the</strong>sda, MD
Abstract: In <strong>the</strong> barrel cortex, feed-<strong>for</strong>ward inhibition (FFI) onto layer IV excitatory stellate<br />
cells (SCs) is mediated by thalamocortical activation of fast spiking (FS) interneurons. FFI<br />
results in a narrow time window over which integration of excitatory inputs c<strong>an</strong> occur. This<br />
integration window (IW) is a pre-requisite <strong>for</strong> temporal precision needed <strong>for</strong> <strong>the</strong> faithful<br />
representation of sensory input. During early postnatal development, FS interneurons are<br />
effectively recruited into <strong>the</strong> circuit by a series of coordinated synaptic <strong>an</strong>d cellular events<br />
resulting in a rapid emergence of FFI. In <strong>the</strong> current study, we examine <strong>the</strong> effect this<br />
developmental increase in FFI has on IW as determined by <strong>the</strong> EPSP ½ width. The amount of<br />
FFI on average, measured as <strong>the</strong> IPSC to EPSC amplitude ratio (IE ratio), in SCs increases<br />
between P6-8 <strong>an</strong>d P9-11 as previously described. However, we observed that at <strong>the</strong>se two<br />
developmental stages, SCs with essentially <strong>the</strong> same FFI (2
Support: NIH NRSA F31 DA023785-02<br />
Title: The effect of chronic prenatal caffeine exposure on <strong>the</strong> activity <strong>an</strong>d expression of NMDA<br />
<strong>an</strong>d AMPA receptors in <strong>the</strong> neonatal hippocampus<br />
Authors: *D. E. SOELLNER 1 , X. ZHOU 2 , H. WANG 1 , J. L. NUNEZ 1 ;<br />
1 Neurosci Prgm, 2 Physiol., Michig<strong>an</strong> St Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Caffeine use during pregn<strong>an</strong>cy is common despite uncertainty about its effects on<br />
neurodevelopment. Concern has been raised because caffeine <strong>an</strong>d its primary metabolites cross<br />
<strong>the</strong> placental <strong>an</strong>d blood brain barriers to reach <strong>the</strong> developing fetal brain. Within <strong>the</strong> fetus, <strong>the</strong>se<br />
subst<strong>an</strong>ces have a prolonged half-life due to <strong>the</strong> immaturity of cytochrome P-450 metabolism<br />
<strong>an</strong>d have been shown to accumulate during chronic exposure. The effects of caffeine within <strong>the</strong><br />
fetal brain are not well studied. It is known that caffeine functions as <strong>an</strong> adenosine <strong>an</strong>tagonist at<br />
<strong>the</strong> A1 <strong>an</strong>d A2A adenosine receptors. Antagonism of adenosine receptors has been shown to<br />
increase glutamate release in <strong>the</strong> hippocampus. This study aimed to determine whe<strong>the</strong>r chronic<br />
prenatal caffeine exposure alters <strong>the</strong> expression <strong>an</strong>d activity of NMDA <strong>an</strong>d AMPA receptors in<br />
<strong>the</strong> neonatal hippocampus during a critical period of glutamate receptor development. Pregn<strong>an</strong>t<br />
dams were administered ei<strong>the</strong>r tap water or caffeinated tap water (75, 150, or 300 mg/L) <strong>from</strong><br />
gestational day 4 until birth. On postnatal days 1, 5, 10, <strong>an</strong>d 20, bilateral hippocampi were<br />
removed <strong>from</strong> male <strong>an</strong>d female offspring <strong>an</strong>d processed <strong>for</strong> Western blot <strong>an</strong>alysis. The tissue<br />
was <strong>an</strong>alyzed <strong>for</strong> both <strong>the</strong> phosphorylated <strong>an</strong>d total protein expression of <strong>the</strong> NR1, NR2A,<br />
NR2B, GluR1, <strong>an</strong>d GluR2 subunits, as well as pERK, pCaMKII, <strong>an</strong>d pCREB proteins. Our<br />
results suggest that prenatal caffeine increases glutamatergic activity within <strong>the</strong> neonatal brain<br />
with long-term effects on protein expression of as a function of age <strong>an</strong>d dose.<br />
Disclosures: D.E. Soellner, None; X. Zhou, None; H. W<strong>an</strong>g, None; J.L. Nunez, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.16/B77<br />
Topic: B.02.k. Non-NMDA glutamate receptors: Gene regulation<br />
Support: Gr<strong>an</strong>ts-in-Aid <strong>for</strong> Scientific Research 19500446<br />
Title: Modification of cerebellar AMPA receptor by bal<strong>an</strong>ce exercise in rats
Authors: *H. MAEJIMA 1 , S. SAKANO 2 , T. OTANI 1 , T. KUROSE 1 , M. DEIE 1 ;<br />
1 Hiroshima Univ., Hiroshima, Jap<strong>an</strong>; 2 Nishihiroshima Rehabil. Hosp., Hiroshima, Jap<strong>an</strong><br />
Abstract: The AMPA receptor is crucial <strong>for</strong> neuroplasticity in <strong>the</strong> central nervous system. Long<br />
term depression (LTD) at gr<strong>an</strong>ule cell - Purkinje cell synapses in <strong>the</strong> cerebellar cortex underlie<br />
various <strong>for</strong>ms of motor learning in <strong>the</strong> cerebellum. Purkinje neurons contain AMPA receptors<br />
with a high proportion of GluR2 but few GluR1 subunits. Recent studies indicate<br />
phosphorylation of GluR2 at Ser-880 site <strong>an</strong>d endocytosis of GluR2 subunits play import<strong>an</strong>t<br />
roles in cellebellar LTD. Physical exercies facilitate motor skills based on motor learning.<br />
Especially, bal<strong>an</strong>ce exercises facilitate postural control based on celebellar motor learning. In<br />
this study, we assessed <strong>the</strong> effects of bal<strong>an</strong>ce exercises on <strong>the</strong> expression <strong>an</strong>d <strong>the</strong> modification of<br />
AMPA receptors in rat cerebellar cortex.<br />
As a bal<strong>an</strong>ce exercise, rats (7-week old at <strong>the</strong> beginning of experiments) walked on rotor rod ten<br />
trials every day <strong>for</strong> 10 days or 4 weeks. Rotor rod time (endur<strong>an</strong>ce time) was monitored, <strong>an</strong>d <strong>the</strong><br />
average time of 10 trials was measured every day. Control groups <strong>for</strong> both intervention groups<br />
were also prepared. Each group was composed of 5 rats. After <strong>the</strong> interventions, <strong>the</strong> rats were<br />
killed <strong>an</strong>d <strong>the</strong> cellebellar cortex was deprived <strong>for</strong> Western blotting <strong>an</strong>d qu<strong>an</strong>titative PCR<br />
<strong>an</strong>alyses. In Western blotting, a protein-blotted membr<strong>an</strong>e was probed by <strong>an</strong>ti-GluR2 <strong>an</strong>tibody,<br />
followed by reprobing with <strong>an</strong> <strong>an</strong>tibody against phosphorylated GluR2 subunit at Ser-880 site. In<br />
qu<strong>an</strong>titative PCR, expression of GluR2 mRNA was measured.<br />
Rotor rod time reached plateau level around 7th day <strong>an</strong>d gradually decreased.<br />
After 10 days intervention, <strong>the</strong>re was no signific<strong>an</strong>t difference in <strong>the</strong> expression of GluR2<br />
mRNA between exercise group <strong>an</strong>d control group, where <strong>the</strong> phosphorylation at Ser-880 of<br />
exercise group was signific<strong>an</strong>tly higher th<strong>an</strong> that of control group.<br />
After 4 weeks intervention, GluR2 mRNA expression of exercise group was signific<strong>an</strong>tly lower<br />
th<strong>an</strong> that of control group, where <strong>the</strong>re was no signific<strong>an</strong>t difference between exercise group <strong>an</strong>d<br />
control group in phosphorylation of GluR2 at Ser-880. Our data showed that bal<strong>an</strong>ce exercises<br />
modified <strong>the</strong> expression <strong>an</strong>d <strong>the</strong> function of AMPA receptor (GluR2) depending on intervention<br />
term <strong>an</strong>d <strong>the</strong> acquisition of bal<strong>an</strong>ce skill. It was suggested that such modification might be<br />
related to LTD at gr<strong>an</strong>ule cell - Purkinje cell synapses.<br />
Disclosures: H. Maejima, None; S. Sak<strong>an</strong>o, None; T. Ot<strong>an</strong>i, None; T. Kurose, None; M.<br />
Deie, None.<br />
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.17/B78
Topic: B.02.f. NMDA receptors: Gene regulation<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes <strong>for</strong> Health Research<br />
Huntington <strong>Society</strong> of C<strong>an</strong>ada<br />
Title: Synaptic versus extrasynaptic N-methyl-D-aspartate receptor signaling in corticostriatal<br />
co-culture<br />
Authors: *A. KAUFMAN 1,2 , A. MILNERWOOD 2 , L. A. RAYMOND 2 ;<br />
1 V<strong>an</strong>couver, BC, C<strong>an</strong>ada; 2 Psychiatry, Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: The subcellular localization of <strong>the</strong> N-methyl-D-aspartate receptor (NMDAR)<br />
profoundly affects its signaling. Extrasynaptic NMDAR activation triggers death-related<br />
processes, such as DNA damage. In contrast, when synaptic NMDARs are stimulated, pathways<br />
associated with synaptic plasticity <strong>an</strong>d survival are activated. These results have been reported in<br />
cortical <strong>an</strong>d hippocampal neurons; however, it is unknown whe<strong>the</strong>r striatal medium-sized spiny<br />
projection neurons (MSNs) that bear <strong>the</strong> brunt of degeneration in Huntington disease, follow <strong>the</strong><br />
same pattern. In fact, published studies, as well as unpublished data <strong>from</strong> <strong>the</strong> Raymond lab,<br />
suggest that NMDAR-activated cell death signaling pathways are different in striatal MSNs<br />
compared with cortical <strong>an</strong>d hippocampal pyramidal neurons. Here, we test <strong>the</strong> hypo<strong>the</strong>sis that<br />
NMDAR-mediated cell death vs. survival signaling occurs via distinct pathways in striatal MSNs<br />
compared to cortical pyramidal neurons.<br />
Striatal MSNs <strong>an</strong>d cortical pyramidal cells are co-cultured <strong>an</strong>d allowed to create synaptic<br />
connections. In each coculture, ei<strong>the</strong>r striatal MSNs or cortical cells are nucleofected with YFP<br />
in order to distinguish <strong>the</strong> two neuronal types. Preliminary results indicate that MSNs grown in<br />
coculture possess <strong>an</strong> elaborate network of projections that is not seen in MSN monoculture. In<br />
addition, <strong>the</strong>y show spine-like processes which are not seen in monoculture, indicating <strong>the</strong><br />
<strong>for</strong>mation of synaptic connections. Synaptic NMDARs are selectively activated by network<br />
disinhibition, as previously described in hippocampal cells. A cocktail of bicuculline to block<br />
GABAA receptors, 4-aminopyridine (4-AP) to increase cell depolarization, <strong>an</strong>d nifedipine to<br />
ensure that <strong>the</strong> calcium influx is through NMDARs ra<strong>the</strong>r th<strong>an</strong> L-type Ca2+ ch<strong>an</strong>nels is added to<br />
<strong>the</strong> medium.<br />
Extrasynaptic NMDARs are stimulated by exposing <strong>the</strong> cells to <strong>the</strong> above drugs in <strong>the</strong> presence<br />
of MK-801, <strong>an</strong> irreversible open-ch<strong>an</strong>nel NMDAR blocker, followed by exposure to glutamate<br />
or NMDA <strong>an</strong>d <strong>the</strong> NMDAR co-agonist glycine. We determine <strong>the</strong> effects of <strong>the</strong>se two<br />
m<strong>an</strong>ipulations on downstream signaling pathways, comparing striatal <strong>an</strong>d cortical neurons.<br />
Assays include immunohistochemical <strong>an</strong>alysis of key molecules such as <strong>the</strong> Cre-response<br />
element binding protein (CREB) <strong>an</strong>d brain-derived neurotrophic factor (BDNF). This research<br />
may help provide targets <strong>for</strong> pharmacological interventions in <strong>the</strong> treatment of Huntington<br />
disease.<br />
Disclosures: A. Kaufm<strong>an</strong>, None; A. Milnerwood, None; L.A. Raymond, None.
Poster<br />
511. Glutamate Receptors: Gene Regulation <strong>an</strong>d Development<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 511.18/B79<br />
Topic: B.02.g. NMDA receptors: Development<br />
Support: NSF 0744979 (TJP, JRL, BLB)<br />
NARSAD (CPF)<br />
NIH MH-64611 (CPF)<br />
Title: Hypoxia resist<strong>an</strong>ce <strong>an</strong>d neonatal characteristics in brain tissue <strong>from</strong> adult naked mole-rats<br />
Authors: B. L. BROWN 1 , J. R. LARSON 2 , S. M. LOGAN 2 , *C. P. FALL 3 , T. J. PARK 1 ;<br />
1 Biol., 2 Psychiatry, 3 BioEngineering, Univ. of Illinois at Chicago, Chicago, IL<br />
Abstract: Naked mole-rats survive in a chronically hypoxic environment due to <strong>the</strong>ir unusual<br />
lifestyle: this species combines a subterr<strong>an</strong>e<strong>an</strong> life with a preference <strong>for</strong> large number of<br />
individuals (hundreds) living toge<strong>the</strong>r. M<strong>an</strong>y <strong>an</strong>imals respiring in a confined space leads to<br />
depleted oxygen. Previously, we have shown that brain tissue <strong>from</strong> adult naked mole-rats is<br />
extremely toler<strong>an</strong>t to hypoxia. Hypoxia toler<strong>an</strong>ce is also a characteristic of m<strong>an</strong>y neonatal<br />
mammals. This is due, in part, to <strong>an</strong> NMDA receptor profile that prevents calcium entry into<br />
neurons. Neonatal NMDA receptors have a high proportion of NR2D subunits, which respond to<br />
hypoxia by closing <strong>the</strong> NMDA receptor-gated ion ch<strong>an</strong>nel. In older <strong>an</strong>imals, which have less<br />
NR2D, hypoxia leads to massive calcium entry through NMDA ch<strong>an</strong>nels <strong>an</strong>d subsequent<br />
excitotoxic cell death. Here we tested <strong>the</strong> hypo<strong>the</strong>sis that adult naked mole-rats are hypoxiatoler<strong>an</strong>t<br />
due to retention of a neonatal-like NMDA receptor profile characterized by a high<br />
proportion of NR2D. We used a Western blot assay to compare <strong>the</strong> relative amounts of NMDA<br />
receptor subunits in neonatal <strong>an</strong>d adult naked mole-rat <strong>an</strong>d mouse brains. The results show that<br />
naked mole-rats retained signific<strong>an</strong>tly more NR2D into adulthood compared to mice. Our data<br />
support <strong>the</strong> hypo<strong>the</strong>sis that adult naked mole-rat brain tissue may use a similar protective<br />
mech<strong>an</strong>ism to that used by neonatal brains. We are currently addressing this issue fur<strong>the</strong>r using<br />
calcium imaging <strong>an</strong>d electrophysiological techniques. Underst<strong>an</strong>ding mech<strong>an</strong>isms of natural<br />
hypoxia resist<strong>an</strong>ce in <strong>an</strong> adult model system may lead to new strategies <strong>for</strong> protecting brain<br />
tissue in adult hum<strong>an</strong>s exposed to hypoxic assault.<br />
Disclosures: B.L. Brown, None; J.R. Larson, None; S.M. Log<strong>an</strong>, None; C.P. Fall, None; T.J.<br />
Park, None.
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.1/B80<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: Hitchcock Foundation<br />
Title: Activation in three dimensions: Visualizing <strong>the</strong> domain architecture of ionotropic<br />
glutamate receptors<br />
Authors: *D. R. MADDEN, C. R. MIDGETT, A. BIRDSEY-BENSON, A. GILL;<br />
Dartmouth Med. Sch., H<strong>an</strong>over, NH<br />
Abstract: Crystallographic studies of glutamate receptor lig<strong>an</strong>d-binding domains have focused<br />
attention on <strong>the</strong> functional import<strong>an</strong>ce of <strong>an</strong> agonist-associated cleft closure in triggering ch<strong>an</strong>nel<br />
activation <strong>an</strong>d in regulating relative agonist efficacy. However, recent studies suggest that<br />
additional factors are import<strong>an</strong>t as well, including <strong>the</strong> stability of <strong>the</strong> closed-cleft state <strong>an</strong>d <strong>the</strong><br />
precise orientation of <strong>the</strong> axis of rotation. In all cases, it has appeared that <strong>the</strong> mech<strong>an</strong>ism of<br />
ch<strong>an</strong>nel activation involves <strong>the</strong> extent <strong>an</strong>d stability of separation between tr<strong>an</strong>smembr<strong>an</strong>e<br />
attachment points within subunit dimer pairs. However, <strong>the</strong> domain architecture of AMPA<br />
receptors has not been clearly defined, preventing <strong>an</strong>alysis of interactions between dimer pairs<br />
<strong>an</strong>d with <strong>the</strong> tr<strong>an</strong>smembr<strong>an</strong>e domains. Here, we present <strong>the</strong> single-particle electron microscopic<br />
<strong>an</strong>alysis of recombin<strong>an</strong>t AMPA-Rs, alone <strong>an</strong>d with domain-specific fusion tags, permitting a first<br />
look at <strong>the</strong> domain architecture <strong>an</strong>d subunit packing interactions in this lig<strong>an</strong>d-gated ion ch<strong>an</strong>nel.<br />
Our model suggests <strong>the</strong> import<strong>an</strong>ce of rotation as well as separation in ch<strong>an</strong>nel activation <strong>an</strong>d has<br />
implications <strong>for</strong> <strong>the</strong> mech<strong>an</strong>ism of subunit assembly.<br />
Disclosures: D.R. Madden, None; C.R. Midgett, None; A. Birdsey-Benson, None; A. Gill,<br />
None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.2/B81<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: New Zeal<strong>an</strong>d Foundation <strong>for</strong> Research, Science <strong>an</strong>d Technology, contract #<br />
CAWX0201<br />
Title: Qu<strong>an</strong>titative structure-activity relationships <strong>for</strong> domoic acid <strong>an</strong>d its isomers<br />
Authors: *D. KERR 1 , P. M. SAWANT 1 , J. TYNDALL 2 , D. O. MOUNTFORT2 3 , P.<br />
HOLLAND 3 ;<br />
1 Univ. Otago Med. Sch., Dunedin, New Zeal<strong>an</strong>d; 2 Sch. of Pharm., Univ. of Otago, Dunedin,<br />
New Zeal<strong>an</strong>d; 3 Biotoxins Section, Cawthron Inst., Nelson, New Zeal<strong>an</strong>d<br />
Abstract: Isomers of domoic acid (DA) sometimes occur in relative abund<strong>an</strong>ce. Early<br />
radiolig<strong>an</strong>d binding studies suggested that DA isomers containing a C1’-C2’ double bond at <strong>the</strong><br />
hexadienyl side chain in a ‘Z’ configuration exhibit high-affinity at kainate <strong>an</strong>d AMPA receptors<br />
(Hampson et al., 1992). Here we have observed that DA <strong>an</strong>d isodomoic acids-A through -F<br />
produced signific<strong>an</strong>t dose <strong>an</strong>d time-dependent increases in seizure activity following<br />
intrahippocampal injection; doses producing half-maximal seizure scores (ED50) were 137, 171,<br />
13000, 3154, 3317, 4000, <strong>an</strong>d 368 pmoles respectively. Competitive radiolig<strong>an</strong>d binding studies<br />
at rat cerebral KA <strong>an</strong>d AMPA receptors, indicate that <strong>the</strong> seizurogenic potencies of DA (Z,E<br />
configuration at 2 double bonds <strong>from</strong> C1’), Iso-A (Z,E) <strong>an</strong>d Iso-F (E,Z) closely correlate with<br />
<strong>the</strong>ir affinities at kainate <strong>an</strong>d AMPA receptor sites, whereas isodomoic acids-C (no double bond-<br />
E), -D (Z,E), -E (E,E) <strong>an</strong>d -B (E,E), which exhibit distinctly lower seizurogenic potencies, are<br />
weak competitive displacers. Taken toge<strong>the</strong>r, <strong>the</strong>se findings call into question previous structureactivity<br />
rules. Signific<strong>an</strong>tly, in our h<strong>an</strong>ds, Iso-D was ten-fold less potent th<strong>an</strong> Iso-F. To fur<strong>the</strong>r<br />
assess apparent discrep<strong>an</strong>cies in activity r<strong>an</strong>kings, molecular modeling was employed <strong>an</strong>d results<br />
indicated strong correlations with <strong>the</strong> r<strong>an</strong>k order of functional potency <strong>an</strong>d binding data observed<br />
here. Here we have proposed a generalised con<strong>for</strong>mational rule where DA, Iso-A <strong>an</strong>d Iso-F,<br />
which contain opposite stereochemistry (Z,E or E,Z) at two double bonds at <strong>the</strong> side chain, have<br />
higher seizurogenic potencies <strong>an</strong>d binding affinities, as compared to <strong>the</strong> compounds with <strong>the</strong><br />
same stereochemistry (Z,Z; E,E) such as isodomoic acids-D, -E <strong>an</strong>d -B. These data shed new<br />
light on structural relationships affecting isodomoic acid functional potency <strong>an</strong>d binding at highaffinity<br />
KA receptors, <strong>an</strong>d provide a firm foundation <strong>for</strong> future work in areas of AMPA <strong>an</strong>d KA<br />
receptor modeling.<br />
Disclosures: D. Kerr, None; P.M. Saw<strong>an</strong>t, None; J. Tyndall, None; D.O. Mount<strong>for</strong>t2,<br />
None; P. Holl<strong>an</strong>d, None.<br />
Poster
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.3/B82<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: JST SICP<br />
HS KHB1010<br />
JSPS KAKENHI 20360014<br />
Title: Orientation of reconstituted AMPA receptors in artificial lipid bilayer<br />
Authors: *N. KASAI 1 , T. BALOIS 1 , C. S. RAMANUJAN 2 , Y. SHINOZAKI 1 , K.<br />
SUMITOMO 1 , J. F. RYAN 2 , K. TORIMITSU 1 ;<br />
1 NTT Corp, Atsugi, Jap<strong>an</strong>; 2 Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom<br />
Abstract: Protein reconstitution into artificial lipid bilayer is <strong>an</strong> essential technique <strong>for</strong> studying<br />
<strong>the</strong> structure of membr<strong>an</strong>e proteins. Fur<strong>the</strong>rmore, it is import<strong>an</strong>t to determine <strong>an</strong>d control <strong>the</strong><br />
orientation of <strong>the</strong> reconstituted protein <strong>for</strong> pharmacological application <strong>an</strong>d <strong>for</strong> tr<strong>an</strong>smembr<strong>an</strong>e<br />
protein-containing n<strong>an</strong>o devices. However, few studies have been done to determine or control<br />
<strong>the</strong> orientation of <strong>the</strong> reconstituted membr<strong>an</strong>e proteins.<br />
Our group has examined <strong>the</strong> structure of <strong>the</strong> reconstituted α-amino-3-hydroxy-5-methyl-4isoxazole<br />
propionic acid receptor (AMPAR) by atomic <strong>for</strong>ce microscopy (AFM). AMPARs were<br />
reconstituted into artificial lipid bilayer <strong>for</strong>ming proteoliposomes, which were <strong>the</strong>n ruptured <strong>an</strong>d<br />
fused with a substrate <strong>for</strong> structural examination by AFM. Using <strong>an</strong>tibodies, we showed that <strong>the</strong><br />
reconstituted AMPARs oriented <strong>the</strong>ir N-terminal domain away <strong>from</strong> <strong>the</strong> substrate. The<br />
orientation was determined <strong>from</strong> <strong>the</strong> height histogram of <strong>the</strong> proteins, using <strong>an</strong>ti-AMPAR N- or<br />
C-terminal <strong>an</strong>tibodies <strong>an</strong>d a 10-nm-Au particle conjugated secondary <strong>an</strong>tibody. Applying Nterminal<br />
<strong>an</strong>tibodies shifted <strong>the</strong> height histogram of <strong>the</strong> protein protrusions shifted to higher<br />
values, whereas <strong>the</strong> C-terminal <strong>an</strong>tibody-application did not result in <strong>the</strong> obvious height<br />
differences.<br />
In this study, we examined <strong>the</strong> orientation of <strong>the</strong> reconstituted AMPAR in <strong>the</strong> <strong>for</strong>m of<br />
proteoliposomes by dynamic light scattering technique (DLS). GluR2 was first purified <strong>from</strong> rat<br />
brains, mixed with detergent, n-octyl-β-glucopyr<strong>an</strong>oside, <strong>an</strong>d different phospholipids, <strong>an</strong>d <strong>the</strong>n<br />
reconstituted by dialysis. The proteoliposomes were <strong>the</strong>n treated with <strong>the</strong> same immunoreactions<br />
as in <strong>the</strong> previous study. The size <strong>an</strong>d <strong>the</strong> qu<strong>an</strong>tity of <strong>the</strong> proteoliposomes were examined by<br />
DLS using latex beads as a st<strong>an</strong>dard.<br />
<strong>When</strong> <strong>the</strong> mixture of <strong>the</strong> differently sized latex beads was measured, DLS showed <strong>the</strong> two clear<br />
peaks, indicating <strong>the</strong> presence of <strong>the</strong> two beads with <strong>the</strong>ir actual sizes <strong>an</strong>d <strong>the</strong> amounts. <strong>When</strong> <strong>the</strong><br />
mixture of liposomes <strong>an</strong>d <strong>the</strong> latex beads was measured, two clear peaks were again observed
indicating <strong>the</strong> presence of <strong>the</strong> two subst<strong>an</strong>ces. DLS c<strong>an</strong> provide size distribution of pure or mixed<br />
materials, <strong>an</strong>d <strong>the</strong>se results indicate that DLS c<strong>an</strong> also be used to <strong>an</strong>alyze <strong>the</strong> size <strong>an</strong>d <strong>the</strong> relative<br />
qu<strong>an</strong>tity of <strong>the</strong> proteoliposomes to <strong>the</strong> st<strong>an</strong>dard. Thus DLS is a useful tool <strong>for</strong> qu<strong>an</strong>titative<br />
<strong>an</strong>alysis of <strong>the</strong> orientation of <strong>the</strong> reconstituted AMPARs, <strong>an</strong>d <strong>the</strong> fur<strong>the</strong>r structural <strong>an</strong>alysis of<br />
AMPAR is <strong>an</strong>ticipated.<br />
Disclosures: N. Kasai, None; T. Balois, None; C.S. Ram<strong>an</strong>uj<strong>an</strong>, None; Y. Shinozaki,<br />
None; K. Sumitomo, None; J.F. Ry<strong>an</strong>, None; K. Torimitsu, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.4/B83<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: Royal <strong>Society</strong><br />
MRC<br />
Title: Structure <strong>an</strong>d function of <strong>the</strong> AMPA receptor N-terminal domain<br />
Authors: I. H. GREGER 1 , M. ROSSMANN 1 , M. SUKUMARAN 1 , A. C. PENN 1 , *H.<br />
YOSHIDA 2 , D. B. VEPRINTSEV 3 ;<br />
1 Neurobio. Div., MRC LMB, Cambridge, United Kingdom; 2 MRC Lab. Mol Biol, Cambridge,<br />
United Kingdom; 3 Ctr. <strong>for</strong> Protein Engin., MRC, Cambridge, United Kingdom<br />
Abstract: Ionotropic glutamate receptors (iGluRs) mediate <strong>the</strong> bulk of fast excitatory<br />
tr<strong>an</strong>smission in vertebrate central nervous systems. iGluRs are modular polypeptides, composed<br />
of three major domains - <strong>the</strong> extracellular portion comprises two periplasmic-protein like lig<strong>an</strong>d<br />
binding devices (PBPs), which are fused to a membr<strong>an</strong>e embedded ion ch<strong>an</strong>nel domain. The<br />
ch<strong>an</strong>nel proximal device is a type 2 PBP, which binds glutamate <strong>an</strong>d gates <strong>the</strong> ch<strong>an</strong>nel. The<br />
structure of this domain has been reported <strong>for</strong> all three major iGluR subfamilies, as well as <strong>for</strong><br />
<strong>the</strong> delta iGluR. The structure <strong>an</strong>d function of <strong>the</strong> distal, N-terminal domain (<strong>the</strong> NTD), which<br />
resembles a type 1 PBP <strong>an</strong>d is unique to eukaryotic iGluRs, has been elusive. The NTD has been<br />
implicated in subfamily selective subunit assembly (in nonNMDARs), <strong>an</strong>d in allosteric<br />
regulation of conduct<strong>an</strong>ce properties in NMDARs.<br />
Here we present high resolution structures of <strong>the</strong> GluR2 NTD determined by x-ray<br />
crystallography. This structure reveals 1) properties of <strong>the</strong> interface <strong>for</strong>med between subunit
dimers, 2) atomic resolution of <strong>the</strong> lig<strong>an</strong>d binding pocket located in <strong>the</strong> crevice between <strong>the</strong> two<br />
lobes of <strong>the</strong> domain, <strong>an</strong>d 3) <strong>the</strong> orientation <strong>an</strong>d potential trajectory of <strong>the</strong> two lobes. Structures<br />
obtained <strong>from</strong> two different crystal <strong>for</strong>ms reveal a likely tetrameric arr<strong>an</strong>gement of <strong>the</strong> receptor<br />
at <strong>the</strong> level of <strong>the</strong> N-terminus. We also present biophysical data, which reveal a tight association<br />
of NTD pairs in solution. Finally, using high-resolution electrophysiological recordings we<br />
describe <strong>the</strong> impact of <strong>the</strong> NTD on AMPA receptor function.<br />
Disclosures: I.H. Greger, None; M. Rossm<strong>an</strong>n, None; M. Sukumar<strong>an</strong>, None; A.C. Penn,<br />
None; H. Yoshida, None; D.B. Veprintsev, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.5/B84<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: John Merck Fund<br />
UCSD start-up<br />
Title: The structures of dimer intermediates of AMPA receptors during subunit assembly<br />
Authors: N. F. SHANKS, T. MARUO, A. N. FARINA, M. H. ELLISMAN, *T.<br />
NAKAGAWA;<br />
Chem <strong>an</strong>d Biochem, UCSD, La Jolla, CA<br />
Abstract: AMPA receptor (AMPA-R) trafficking <strong>an</strong>d subunit assembly are critical components<br />
of synaptic plasticity. These processes, however, are still poorly understood at <strong>the</strong> molecular<br />
level. We established a system in which we could study <strong>the</strong> molecular structure of AMPA-Rs<br />
during subunit assembly by using a combination of genetic engineering <strong>an</strong>d single particle<br />
electron microscopy. We doxycycline (DOX)-inducibly expressed GluR2 in HEK cells <strong>an</strong>d<br />
purified <strong>the</strong> receptors. The tetramers of GluR2 were structurally indistinguishable <strong>from</strong> those<br />
purified <strong>from</strong> rat brain. Fur<strong>the</strong>rmore, by purifying at earlier timepoints after DOX induction, we<br />
were able to enrich AMPA-R dimer biosyn<strong>the</strong>tic intermediates. Dimers emerged be<strong>for</strong>e <strong>the</strong><br />
tetramers, <strong>an</strong>d <strong>the</strong> majority of <strong>the</strong> dimer-to-tetramer tr<strong>an</strong>sition occurred between 12-24 hours<br />
post-induction. In <strong>the</strong> structure of dimeric GluR2, <strong>the</strong> N-terminal domains (NTDs) <strong>an</strong>d <strong>the</strong><br />
tr<strong>an</strong>smembr<strong>an</strong>e domains (TMDs) were fused, however, <strong>the</strong> lig<strong>an</strong>d binding domains (LBDs) were<br />
separated. The two separated LBDs connected <strong>the</strong> NTDs <strong>an</strong>d <strong>the</strong> TMDs. To study <strong>the</strong> functional
signific<strong>an</strong>ce of <strong>the</strong> structure of GluR2 wildtype dimers, we identified GluR2L504Y as a mut<strong>an</strong>t<br />
defective in dimer-to-tetramer tr<strong>an</strong>sition, determined <strong>the</strong> structure of <strong>the</strong> dimers of this mut<strong>an</strong>t,<br />
<strong>an</strong>d compared its structure with <strong>the</strong> wildtype. A signific<strong>an</strong>t difference was detected between <strong>the</strong><br />
dimeric structures of <strong>the</strong> wildtype <strong>an</strong>d <strong>the</strong> L504Y mut<strong>an</strong>t. In contrast to <strong>the</strong> wildtype whose<br />
LBDs are separated, <strong>the</strong> LBDs of <strong>the</strong> L504Y mut<strong>an</strong>t were detected as a single density. Our<br />
results provide direct structural evidence that separation of <strong>the</strong> LBDs within <strong>the</strong> intact dimeric<br />
subunits is critical <strong>for</strong> efficient tetramerization in <strong>the</strong> endoplasmic reticulum <strong>an</strong>d fur<strong>the</strong>r<br />
trafficking of AMPA-Rs. The structures of <strong>the</strong> dimer intermediates of AMPA-Rs provide<br />
insights into <strong>the</strong> process of dimer-to-tetramer tr<strong>an</strong>sition <strong>an</strong>d <strong>the</strong> domain arr<strong>an</strong>gements in <strong>the</strong><br />
mature tetramers. Finally, we introduced <strong>the</strong> AMPA-R auxiliary subunit stargazin into our<br />
recombin<strong>an</strong>t expression system <strong>an</strong>d are currently evaluating <strong>the</strong> effect of stargazin on modulating<br />
AMPA-R subunit assembly.<br />
Disclosures: N.F. Sh<strong>an</strong>ks, None; T. Maruo, None; A.N. Farina, None; M.H. Ellism<strong>an</strong>,<br />
None; T. Nakagawa, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.6/B85<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: SFB636/A4<br />
NGFNplus/SP10<br />
Title: Endogenous modulators of AMPA receptors<br />
Authors: *R. SPRENGEL 1 , J. HONEK 2 , A. VOIGT 2 , V. MACK 3 , J. VON ENGELHARDT 3 ,<br />
L. LAYER 1 , P. H. SEEBURG 1 , H. MONYER 3 ;<br />
1 Dept. of Mol. Neurobiology, Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Med. Res., Heidelberg, Germ<strong>an</strong>y; 2 Inst. <strong>for</strong><br />
Pharm. <strong>an</strong>d Mol. Biotech., 3 Dept. Clin. Neurobio., Univ. of Heidelberg, Heidelberg, Germ<strong>an</strong>y<br />
Abstract: The alpha-amino-3-hydroxy-5-methyl-4-isocazolepropionic acid (AMPA) receptors<br />
are ionotropic receptor ch<strong>an</strong>nels responsible <strong>for</strong> <strong>the</strong> fast chemical synaptic tr<strong>an</strong>smission at most<br />
glutamatergic synapses. The receptors are activated by glutamate as well as its <strong>an</strong>alog AMPA<br />
<strong>an</strong>d <strong>the</strong>y are critically involved in <strong>the</strong> <strong>for</strong>mation of memory. Recently, several endogenous novel<br />
AMPA receptor modulating membr<strong>an</strong>e sp<strong>an</strong>ning proteins were identified by us <strong>an</strong>d o<strong>the</strong>rs. One
of our AMPA receptor modulators, with a single membr<strong>an</strong>e sp<strong>an</strong>ning region, is a novel protein<br />
with no real counterpart in <strong>the</strong> current nucleotide <strong>an</strong>d protein databases.<br />
However, in <strong>an</strong> exhaustive database search a number of related proteins c<strong>an</strong> be predicted after<br />
m<strong>an</strong>ual reassembly of related genomic segments using conserved protein segments, topology<br />
predictions <strong>an</strong>d conserved exon/intron boundaries. The preliminary data show that <strong>the</strong>se novel<br />
AMPA receptor modulating proteins are specifically expressed in <strong>the</strong> brain <strong>an</strong>d moreover are<br />
conserved just in vertebrates. The evolutionarily youngest member has homologs in mammali<strong>an</strong>s<br />
only indicating that <strong>the</strong>se putative AMPA receptor modulators appeared late in evolution <strong>an</strong>d<br />
might have exclusive functions in a complex CNS. Toge<strong>the</strong>r with <strong>the</strong> phylogenetic org<strong>an</strong>ization,<br />
<strong>the</strong> predicted structure will be presented combined with a functional model of <strong>the</strong> new AMPA<br />
receptor modulators.<br />
Disclosures: R. Sprengel, None; J. Honek, None; A. Voigt, None; V. Mack, None; J. von<br />
Engelhardt, None; L. Layer, None; P.H. Seeburg, None; H. Monyer, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.7/B86<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: Academy of Finl<strong>an</strong>d Gr<strong>an</strong>t #110900<br />
Title: N-terminal domain modulates AMPA receptor desensitization <strong>an</strong>d pharmacology<br />
Authors: *K. P. KEINANEN 1 , A. SEMENOV 1 , T. MÖYKKYNEN 2 , S. K. COLEMAN 1 , E. R.<br />
KORPI 2 ;<br />
1 Dept Biol & Envrn. Sci., 2 Inst. of Biomedicine, Univ. Helsinki, Helsinki, Finl<strong>an</strong>d<br />
Abstract: The functional role of <strong>the</strong> large N-terminal domain (NTD) of AMPA receptors is<br />
poorly understood. Mutational studies indicate that NTD is not necessary <strong>for</strong> basic lig<strong>an</strong>d-gated<br />
ch<strong>an</strong>nel function, but may be involved in heteromeric receptor assembly <strong>an</strong>d in synaptic protein<br />
interactions. In <strong>the</strong> present study, we have <strong>the</strong> studied <strong>the</strong> effects of NTD deletion on <strong>the</strong><br />
functional properties of homomeric GluA4 (GluR-D, GluR4) ch<strong>an</strong>nels (of flip iso<strong>for</strong>m)<br />
expressed in cultured mammali<strong>an</strong> cell lines <strong>an</strong>d in Xenopus laevis oocytes. Voltage-clamp<br />
<strong>an</strong>alysis of agonist-triggered steady-state currents in oocytes indicated that glutamate had closely<br />
similar efficacy <strong>an</strong>d potency <strong>for</strong> both full-length GluA4 <strong>an</strong>d truncated GluA4_deltaNTD receptor<br />
ch<strong>an</strong>nels. In contrast, relative efficacy of kainate was strongly reduced: <strong>the</strong> ratio of currents
elicited by kainate <strong>an</strong>d glutamate, both at 100 microM, decreased <strong>from</strong> 2.1 in <strong>the</strong> wild-type to 0.3<br />
in <strong>the</strong> truncated receptor. In whole-cell patch clamp recordings of tr<strong>an</strong>sfected HEK293 cells,<br />
deletion of NTD had little or no effect on glutamate-induced (10 mM) peak currents, whereas <strong>the</strong><br />
steady-state current was strongly increased <strong>for</strong> GluA4_deltaNTD. Kinetic <strong>an</strong>alysis indicated that<br />
while GluA4 <strong>an</strong>d GluA4_deltaNTD had similar rates of <strong>the</strong> onset of desensitization, <strong>the</strong><br />
truncated receptors recovered <strong>from</strong> desensitization much faster: time-const<strong>an</strong>ts of recovery <strong>from</strong><br />
desensitization were 26 ms <strong>an</strong>d 10 ms, <strong>for</strong> <strong>the</strong> full-length <strong>an</strong>d <strong>the</strong> deletion mut<strong>an</strong>t, respectively.<br />
This study demonstrates that NTD has a previously unrecognized role in <strong>the</strong> modulation of<br />
AMPA receptor ch<strong>an</strong>nels. Our findings are consistent with a model in which NTD <strong>for</strong>ms direct<br />
contacts with <strong>the</strong> lig<strong>an</strong>d-binding domain in <strong>the</strong> kainate-stabilized <strong>an</strong>d in <strong>the</strong> desensitized states<br />
but not (or less) with <strong>the</strong> glutamate-induced active con<strong>for</strong>mation.<br />
Disclosures: K.P. Kein<strong>an</strong>en, None; A. Semenov, None; T. Möykkynen, None; S.K. Colem<strong>an</strong>,<br />
None; E.R. Korpi, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.8/B87<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: NS 051187<br />
P20 RR015567<br />
Title: Characterization of turtle glutamate receptor subunit 4 (GluR4) <strong>an</strong>d its alternatively<br />
spliced iso<strong>for</strong>ms<br />
Authors: *B. E. SABIRZHANOV, Z. ZHENG, J. KEIFER;<br />
Neurosci Grp, Univ. South Dakota Sch. Med., Vermillion, SD<br />
Abstract: Glutamate receptors underlie fast neurotr<strong>an</strong>smission in <strong>the</strong> brain <strong>an</strong>d play <strong>an</strong> import<strong>an</strong>t<br />
role in neural plasticity, neural development <strong>an</strong>d neurodegeneration. Previous studies using <strong>an</strong> in<br />
vitro model of eyeblink classical conditioning in pond turtles suggest that acquisition of<br />
conditioned responses (CRs) is associated with synaptic insertion of AMPARs containing GluR4<br />
subunits (e.g., J. Neurophysiol. 101: 2549, <strong>2009</strong>). In order to fur<strong>the</strong>r investigate <strong>the</strong> role of<br />
GluR4-containing AMPARs in conditioning, we characterized <strong>the</strong> sequence <strong>an</strong>d domain<br />
structure of turtle GluR4 (tGluR4) <strong>an</strong>d its splice vari<strong>an</strong>ts. We identified <strong>the</strong> sequence of tGluR4,
tGluR4c <strong>an</strong>d tGluR4s vari<strong>an</strong>ts <strong>from</strong> turtle brain stem (pons) by RT-PCR. tGluR4 <strong>an</strong>d tGluR4c<br />
were found to express both flip <strong>an</strong>d flop vari<strong>an</strong>ts. No evidence <strong>for</strong> <strong>the</strong> existence of tGluR4d in<br />
turtle brain stem was found. Using 5’- <strong>an</strong>d 3’-RACE, we characterized two groups of tGluR4<br />
truncated vari<strong>an</strong>ts (tGluR4tr). One group is truncated on <strong>the</strong> C-terminus (tGluR4trc) <strong>an</strong>d <strong>the</strong><br />
o<strong>the</strong>r is on <strong>the</strong> N-terminus (tGluR4trn). Three tr<strong>an</strong>scripts <strong>for</strong> alternatively spliced iso<strong>for</strong>ms of<br />
tGluR4trc were identified (tGluR4trc 1-3). tGluR4trc1 contains only <strong>the</strong> LIVBP-like domain,<br />
whereas tGluR4trc2 <strong>an</strong>d trc3 contain <strong>the</strong> LIVBP domain <strong>an</strong>d slightly different segments of <strong>the</strong><br />
S1 lig<strong>an</strong>d-binding domain. Two N-terminal truncated vari<strong>an</strong>ts were also sequenced <strong>an</strong>d both lack<br />
<strong>the</strong> signal peptide cleavage site. There<strong>for</strong>e, alternative splicing allows <strong>for</strong> 10 identified<br />
tr<strong>an</strong>scripts of GluR4 in turtle brain stem, although whe<strong>the</strong>r all of <strong>the</strong>se vari<strong>an</strong>ts are expressed as<br />
protein <strong>an</strong>d are functional has yet to be determined. Previous work has identified truncated<br />
vari<strong>an</strong>ts of AMPAR subunits in chicken, rat, <strong>an</strong>d hum<strong>an</strong>. To test our hypo<strong>the</strong>sis that synaptic<br />
insertion of GluR4 AMPAR subunits is critical <strong>for</strong> in vitro conditioning, <strong>the</strong>se sequence data<br />
were used to generate several siRNAs to knockdown expression of tGluR4 in brain stem<br />
preparations. The siRNAs targeted sequences in <strong>the</strong> lig<strong>an</strong>d-binding domain that are specific to<br />
GluR4 subunits <strong>an</strong>d preliminary data suggest <strong>the</strong>y selectively decrease expression of GluR4<br />
subunits ra<strong>the</strong>r th<strong>an</strong> GluR1-3. Future work will fur<strong>the</strong>r characterize <strong>the</strong> relative abund<strong>an</strong>ce of<br />
tGluR4 splice iso<strong>for</strong>ms in <strong>the</strong> brain stem, <strong>an</strong>d <strong>the</strong> effect of <strong>the</strong> <strong>an</strong>ti-tGluR4 siRNAs on<br />
conditioning.<br />
Disclosures: B.E. Sabirzh<strong>an</strong>ov, None; Z. Zheng, None; J. Keifer, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.9/B88<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Title: Structure-function study of ionotropic glutamate delta-1 receptor<br />
Authors: *R. YADAV, M. ROENFELD, M. SCOFIELD, S. DRAVID;<br />
Pharmacol., Creighton Univ., Omaha, NE<br />
Abstract: Glutamate delta-1 receptor (GluRdelta1) is a member of <strong>the</strong> ionotropic glutamate<br />
receptor family <strong>an</strong>d is expressed in <strong>the</strong> adult hippocampus <strong>an</strong>d in <strong>the</strong> inner ear <strong>an</strong>d has recently<br />
been shown to be import<strong>an</strong>t in high frequency hearing. Similar to its closest homologue, <strong>the</strong><br />
GluRdelta2 receptor, no agonist induced currents are observed <strong>from</strong> <strong>the</strong> GluRdelta1 receptor. A<br />
naturally occurring mutation present in <strong>the</strong> activation gate in <strong>the</strong> third tr<strong>an</strong>smembr<strong>an</strong>e domain of
<strong>the</strong> GluRdelta2 receptor in <strong>the</strong> lurcher strain of mice converts <strong>the</strong> receptor into a constitutively<br />
open cation permeating ch<strong>an</strong>nel. In <strong>an</strong> ef<strong>for</strong>t to underst<strong>an</strong>d <strong>the</strong> structure <strong>an</strong>d function of<br />
GluRdelta1 receptors we probed <strong>the</strong> conserved tr<strong>an</strong>smembr<strong>an</strong>e domain 3 of <strong>the</strong> receptor<br />
mutating amino-acids in <strong>the</strong> putative activation gate of <strong>the</strong> receptor. Our results suggest that<br />
GluRdelta1 receptor may assemble as homomers. Fur<strong>the</strong>rmore, experiments with chimeric<br />
Delta1-Delta2 receptors suggest that <strong>the</strong> residues <strong>for</strong>ming <strong>the</strong> agonist binding domain in<br />
GluRDelta2 may be conserved in GluRDelta1 receptor. Additionally extracellular Ca 2+ may<br />
have similar potentiation effect as in Delta2 lurcher.<br />
Disclosures: R. Yadav, None; M. Roenfeld, None; M. Scofield, None; S. Dravid, None.<br />
Poster<br />
512. Non-NMDA Glutamate Receptor Structure<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 512.10/B89<br />
Topic: B.02.h. Non-NMDA glutamate receptors: Structure<br />
Support: SNSF Gr<strong>an</strong>t 3100A0-117717<br />
Title: Functionnal <strong>an</strong>alysis of a conserved histidine residue of <strong>the</strong> extracellular loop of acidsensing<br />
ion ch<strong>an</strong>nel 1a (ASIC1a)<br />
Authors: *B. BARGETON, S. KELLENBERGER;<br />
Dept. of Pharmacol. <strong>an</strong>d Toxicology, Univ. of Laus<strong>an</strong>ne, Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d<br />
Abstract: ASICs are non-voltage-gated neuronal sodium ch<strong>an</strong>nels activated by protons.<br />
Activation of ASICs induces a membr<strong>an</strong>e depolarization <strong>an</strong>d <strong>the</strong>re is evidence that ASICs<br />
contribute to long term potentiation, <strong>the</strong> expression of fear, <strong>the</strong> termination of epileptic seizures,<br />
nociception <strong>an</strong>d damages after ischemia. These functions make ASICs potential drug targets.<br />
Functional ASICs are homo- or heterotrimeric assemblies of homologous subunits. The crystal<br />
structure of <strong>the</strong> chicken ASIC1 in its inactivated con<strong>for</strong>mation has recently been determined.<br />
Each subunit has two tr<strong>an</strong>smembr<strong>an</strong>e domains <strong>an</strong>d a large extracellular loop (ECL), <strong>the</strong> N- <strong>an</strong>d<br />
C-termini being cytoplasmic. The ECL is <strong>the</strong> sensor of <strong>the</strong> extracellular acidity <strong>an</strong>d <strong>the</strong> target of<br />
several modulators as <strong>for</strong> example toxins <strong>an</strong>d divalent cations. Zinc has been shown to potentiate<br />
<strong>the</strong> acid activation of ASIC2a-containing ch<strong>an</strong>nels. Two protonable residues (rASIC2a His162<br />
<strong>an</strong>d His339) are essential <strong>for</strong> this effect. Of <strong>the</strong>se two residues only <strong>the</strong> His162 is conserved in all<br />
ASICs (His163 in hum<strong>an</strong> ASIC1a) <strong>an</strong>d is located in a contact region between <strong>the</strong> three domains<br />
knuckle, finger <strong>an</strong>d β-ball. The conserved residues Asn96, His163 <strong>an</strong>d Gln225 belong
espectively to each of <strong>the</strong> three loops, <strong>an</strong>d His163 <strong>for</strong>ms H-Bonds with <strong>the</strong>se two residues. To<br />
test <strong>the</strong>ir functional relev<strong>an</strong>ce, we mutated <strong>the</strong>se <strong>an</strong>d neighboring residues by site-directed<br />
mutagenesis to cys. Most mut<strong>an</strong>t ch<strong>an</strong>nels showed poor expression in Xenopus oocytes<br />
suggesting that this part of <strong>the</strong> ECL is essential <strong>for</strong> <strong>the</strong> ch<strong>an</strong>nel folding. The mut<strong>an</strong>t ch<strong>an</strong>nels<br />
were however functional <strong>an</strong>d were unlike <strong>the</strong> wild type (wt) ch<strong>an</strong>nel inhibited by Cd 2+ . The Cd 2+<br />
IC50 values were 71±10µM <strong>an</strong>d 73±53µM <strong>for</strong> <strong>the</strong> N96C <strong>an</strong>d Q225C mut<strong>an</strong>ts versus 331±36µM<br />
<strong>for</strong> H163C, suggesting toge<strong>the</strong>r with <strong>the</strong> structural in<strong>for</strong>mation that H163 participates in Cd 2+<br />
chelation in <strong>the</strong> mut<strong>an</strong>t ch<strong>an</strong>nels. Covalent modification by <strong>the</strong> charged meth<strong>an</strong>ethiosulfonate<br />
reagent MTSET was used to fur<strong>the</strong>r investigate <strong>the</strong> structural <strong>an</strong>d functional role of <strong>the</strong> His163<br />
<strong>an</strong>d its environment. After MTSET exposure <strong>the</strong> ASIC1a H163C ch<strong>an</strong>nel current decreased to<br />
4±1% <strong>an</strong>d <strong>the</strong> pH dependence of activation was shifted by -0.24 pH units, while <strong>the</strong> pH<br />
dependence of inactivation was not affected. The unitary conduct<strong>an</strong>ce of <strong>the</strong> modified H163C<br />
mut<strong>an</strong>t was not different <strong>from</strong> <strong>the</strong> conduct<strong>an</strong>ce of unmodified H163C or wt ASIC1a. These<br />
observations suggest a role of this region in <strong>the</strong> gating, but likely not <strong>the</strong> pH-sensing of ASIC1a.<br />
In conclusion our study identifies a conserved region around His163 as critical determin<strong>an</strong>t of<br />
ASIC1a expression <strong>an</strong>d function.<br />
Disclosures: B. Bargeton, None; S. Kellenberger, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.1/B90<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: Wellcome Trust gr<strong>an</strong>t 71436<br />
Epilepsy Research UK gr<strong>an</strong>t 0802<br />
Title: Thalamic extrasynaptic GABAA receptor gain-of-function is necessary <strong>an</strong>d sufficient <strong>for</strong><br />
absence epilepsy<br />
Authors: G. DI GIOVANNI, G. ORBAN, S. J. FYSON, V. CRUNELLI, *D. W. COPE;<br />
Cardiff Univ., Cardiff, United Kingdom<br />
Abstract: Aberr<strong>an</strong>t GABAergic inhibition in thalamo-cortical networks has been identified as a<br />
potential mech<strong>an</strong>ism <strong>for</strong> spike-<strong>an</strong>d-wave discharge (SWD) generation. Thalamocortical (TC)<br />
neurons in <strong>the</strong> ventrobasal (VB) thalamus receive both ‘phasic’ <strong>an</strong>d ‘tonic’ GABAA receptor
mediated inhibition, generated by synaptic <strong>an</strong>d extrasynaptic delta-subunit containing receptors,<br />
respectively [Cope et al., 2005; J. Neurosci. 25: 11553]. We have shown a selective increase of<br />
tonic GABAA receptor-mediated inhibition in pharmacological as well as in polygenic <strong>an</strong>d<br />
monogenic rat <strong>an</strong>d mouse models of absence epilepsy due to <strong>an</strong> impairment of GABA<br />
tr<strong>an</strong>sporter-1 (GAT-1) activity [<strong>Society</strong> <strong>for</strong> Neuroscience 2007, 142.7, 142.8, 142.9]. There<strong>for</strong>e,<br />
we suggested that extrasynaptic GABAA receptor gain-of-function in VB TC neurons is a<br />
necessary requirement <strong>for</strong> <strong>the</strong> appear<strong>an</strong>ce of SWDs. To directly test this hypo<strong>the</strong>sis we have<br />
pharmacologically <strong>an</strong>d genetically targeted extrasynaptic GABAA receptors in VB <strong>an</strong>d<br />
monitored EEG <strong>an</strong>d behavioural correlates of absence epilepsy in normal Wistar rats, Genetic<br />
Absence Epilepsy Rats <strong>from</strong> Strasbourg (GAERS) <strong>an</strong>d GABAA receptor delta-subunit knockout<br />
mice. Reverse microdialysis of <strong>the</strong> selective extrasynaptic GABAA agonist THIP (70 <strong>an</strong>d 100<br />
µM, both n=5) <strong>an</strong>d <strong>the</strong> selective GAT-1 inhibitor NO-711 (200 µM, n=5) into <strong>the</strong> VB induced<br />
SWDs <strong>an</strong>d behavioural correlates of absence seizures in normal Wistar rats. THIP- <strong>an</strong>d NO-711induced<br />
SWDs were suppressed by systemic administration of <strong>the</strong> <strong>an</strong>ti-absence drug<br />
ethosuximide (ETX, 100-200 mg/kg, n=5). In addition, we “knocked-down” delta-subunit<br />
containing GABAA receptors in TC cells of GAERS by directly infusing <strong>an</strong> <strong>an</strong>tisense<br />
oligodeoxynucleotide (ODN, 1 <strong>an</strong>d 2 nMol/µl, n=5 <strong>an</strong>d 6, respectively) into <strong>the</strong> VB. The<br />
<strong>an</strong>tisense ODN produced a marked reduction (~70% at 2 nMol) of <strong>the</strong> total time spent in seizures<br />
<strong>an</strong>d <strong>the</strong> number of SWDs, whilst infusion of a missense ODN (1-2 nMol/µl, n=5) was<br />
ineffective. Lastly, systemic administration of 50 mg/kg of gamma-butyrolactone induced<br />
absence seizures in wild type mice, which were signific<strong>an</strong>tly reduced in <strong>the</strong> delta-subunit<br />
knockout mice (84% decrease in <strong>the</strong> total time spent in seizures <strong>an</strong>d 53% reduction in number of<br />
SWDs).<br />
Our data demonstrate that extrasynaptic GABAA receptor gain-of-function in VB TC neurons is<br />
a necessary <strong>an</strong>d sufficient requirement <strong>for</strong> <strong>the</strong> appear<strong>an</strong>ce of a pure absence epilepsy phenotype,<br />
<strong>an</strong>d that GAT-1 critically controls SWD genesis.<br />
Disclosures: G. Di Giov<strong>an</strong>ni, None; G. Orb<strong>an</strong>, None; S.J. Fyson, None; V. Crunelli,<br />
None; D.W. Cope, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.2/B91<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Title: Differentially altered contribution of δ subunit-containing GABAA receptors to loss of<br />
thalamocortical tonic currents in epileptic mice
Authors: *K. P. MANGAN, M. V. JONES;<br />
Univ. Wisconsin-Madison, Madison, WI<br />
Abstract: The R43Q mutation in <strong>the</strong> γ2 subunit of <strong>the</strong> GABAA receptor (γ2R43Q) causes<br />
Generalized Epilepsy with Febrile Seizures Plus (GEFS+) (Nat Genet, 28:49). This mutation<br />
alters receptor kinetics (PNAS 99:15170) <strong>an</strong>d expression (J Biol Chem, 279:47034; J Neurosci,<br />
26:2590; Mol Cell Neurosci, 29:120), <strong>an</strong>d confers absence epilepsy in knock-in mice, but has<br />
relatively subtle effects on synaptic inhibition (PNAS, 104:17536). Thus, <strong>the</strong> primary mech<strong>an</strong>ism<br />
underlying epilepsy remains unknown. Here we examined <strong>the</strong> involvement of δ subunitcontaining<br />
receptors in <strong>the</strong> abolition of tonic GABAergic inhibition in γ2R43Q knock-in mice.<br />
Whole-cell voltage-clamp recordings were made <strong>from</strong> pyramidal cells of layer II/III<br />
somatosensory cortex or thalamic relay cells in slices <strong>from</strong> 16-22 day old male wild type (RR) or<br />
heterozygous knock-in (RQ) C57B6 mice (-60 mV; st<strong>an</strong>dard aCSF + 5 µM DNQX + 25 µM AP5<br />
+ 100 nM TTX; KCl-based intracellular solution; room temp.). No exogenous GABA was<br />
applied. The magnitude of GABAergic tonic current was defined as <strong>the</strong> ch<strong>an</strong>ge in holding<br />
current caused by bath application of bicuculline (100 µM). To separate tonic current <strong>from</strong><br />
phasic mIPSCs, all-points amplitude histograms were constructed <strong>from</strong> 30-second stretches of<br />
data <strong>an</strong>d fit with a Gaussi<strong>an</strong> function over only <strong>the</strong> outward half of <strong>the</strong> histogram, thus omitting<br />
<strong>the</strong> inward tail produced by mIPSCs.<br />
Tonic current densities (pA/pF) were small but consistent in RR mouse cortex (0.5 ± 0.1, n = 4, n<br />
= 4) <strong>an</strong>d thalamus (0.4 ± 0.1, n = 10), yet were essentially absent <strong>from</strong> both regions in RQ mice<br />
(cortex: -0.1 ± 0.2, n = 4, p ≤ 0.05; thalamus: 0 ± 0, n = 6, p ≤ 0.05). Because receptors<br />
containing <strong>the</strong> δ subunit contribute to tonic currents in several brain regions, we examined <strong>the</strong><br />
effects of <strong>the</strong> δ-preferring activators THIP (1 µM) <strong>an</strong>d allopregn<strong>an</strong>olone (APG, 60 nM). There<br />
were no differences in <strong>the</strong> effects of <strong>the</strong>se drugs in cortex between RR <strong>an</strong>d RQ mice (THIP: RR:<br />
1.07 ± 0.16, n = 4; RQ: 1.31 ± 0.14, n = 5; APG: RR: 0.81 ± 0.04, n = 4; RQ: 0.72 ± 0.05, n = 5).<br />
In thalamus, however, both drugs activated ~50% smaller currents in RQ th<strong>an</strong> in RR (THIP: RR:<br />
5.15 ± 0.49, n = 4; RQ: 2.74 ± 0.70, n = 14, p ≤ 0.05; APG: RR: 1.33 ± 0.11, n = 5; RQ: 0.50 ±<br />
0.04, n = 3, p ≤ 0.05).<br />
These results demonstrate that <strong>the</strong> γ2R43Q mutation abolishes tonic currents in thalamocortical<br />
principal neurons <strong>an</strong>d suggest that this occurs via loss of <strong>the</strong> δ subunit in thalamus, but not in<br />
cortex. These findings are compatible with studies that show <strong>the</strong> γ2 subunit interferes with <strong>the</strong><br />
expression of multiple tonic current-related subunits in o<strong>the</strong>r systems (e.g., δ: J Neurosci<br />
27:7520; α5: J Neurosci 27:14108), <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e implicate <strong>the</strong> abolition of tonic current as a<br />
c<strong>an</strong>didate mech<strong>an</strong>ism <strong>for</strong> epileptogenesis.<br />
Disclosures: K.P. M<strong>an</strong>g<strong>an</strong>, None; M.V. Jones, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.3/B92<br />
Topic: B.02.p. GABA(A) receptors: Gene regulation<br />
Support: NS050393-010A<br />
Title: Iso<strong>for</strong>m-specific regulation of Egr3: A seizure-induced regulator of GABA-A receptor<br />
subunit expression<br />
Authors: *J. H. KIM 1,2 , A. R. BROOKS-KAYAL 3 , S. J. RUSSEK 1,2 ;<br />
1 Pharmacol. <strong>an</strong>d Exptl. Therapeut., 2 Program in Biomed. Neurosci., Boston Univ. Sch. of Med.,<br />
Boston, MA; 3 Pediatrics, Univ. of Colorado, Denver, Denver, CO<br />
Abstract: Temporal lobe epilepsy (TLE) is a disease of <strong>the</strong> brain characterized by <strong>the</strong><br />
appear<strong>an</strong>ce of spont<strong>an</strong>eous seizures arising after a latent period of days to years <strong>from</strong> <strong>an</strong> initial<br />
brain insult that often occurs during childhood. TLE accounts <strong>for</strong> 40% of all epilepsy cases in <strong>the</strong><br />
adult population. Despite high prevalence, <strong>the</strong>re is no cure <strong>for</strong> this devastating disease that leaves<br />
its mark on individuals through <strong>the</strong> comorbidity of clinical depression <strong>an</strong>d cognitive disorders.<br />
Currently, most of <strong>the</strong> <strong>an</strong>ti-convuls<strong>an</strong>t drugs in use target a variety of neurotr<strong>an</strong>smitter receptors<br />
<strong>an</strong>d ion ch<strong>an</strong>nels, including <strong>the</strong> gamma-aminobutyric acid (GABA) type A receptors<br />
(GABAARs) that mediate <strong>the</strong> majority of synaptic inhibition in <strong>the</strong> brain. GABAARs are<br />
heteropentameric comprised of different combinations of subunits. In both hum<strong>an</strong> patients <strong>an</strong>d<br />
TLE models, alteration in GABAAR subunit composition is believed to decrease inhibitory<br />
control in <strong>the</strong> dentate gyrus (DG), gatekeeper of <strong>the</strong> hippocampal <strong>for</strong>mation. In particular, <strong>an</strong><br />
increase in alpha4 subunit levels, <strong>an</strong>d decrease in alpha1, is associated with spont<strong>an</strong>eous seizure<br />
development after status epilepticus (SE). Receptors containing alpha4 <strong>an</strong>d delta are mostly<br />
extrasynaptic, mediating tonic inhibition. However, work <strong>from</strong> our laboratory has shown that<br />
after SE, alpha4 co-assembles with gamma2, a subunit that localizes it to <strong>the</strong> synapse. Because<br />
alpha4/gamma2-containing receptors desensitize rapidly, <strong>the</strong>ir appear<strong>an</strong>ce may underlie<br />
decreased GABAAR function after SE. Results of our studies also suggest that alpha4 subunit<br />
levels increase via a ch<strong>an</strong>ge in <strong>the</strong> tr<strong>an</strong>scription of <strong>the</strong> alpha4 subunit gene (Gabra4) after<br />
binding of early growth response factor 3 (Egr3). Egr3 is critical <strong>for</strong> processes of learning <strong>an</strong>d<br />
memory <strong>an</strong>d it has also been implicated in schizophrenia; however, <strong>the</strong> underlying mech<strong>an</strong>isms<br />
of Egr3 regulation in <strong>the</strong> brain have yet to be elucidated. We hypo<strong>the</strong>size that a ch<strong>an</strong>ge in<br />
expression <strong>an</strong>d/or nuclear localization of Egr3 occurs in response to SE altering DG inhibition<br />
<strong>an</strong>d contributes to spont<strong>an</strong>eous seizure development. Specifically, we investigated <strong>the</strong><br />
biochemical properties of <strong>the</strong> two major iso<strong>for</strong>ms of Egr3 (Egr3 alpha <strong>an</strong>d beta) in <strong>the</strong> brain, <strong>an</strong>d<br />
found that Egr3 is expressed <strong>an</strong>d regulated upon cellular activation in <strong>an</strong> iso<strong>for</strong>m-specific<br />
m<strong>an</strong>ner. Findings <strong>from</strong> this study allow us to move closer to developing novel <strong>an</strong>tiepileptic<br />
<strong>the</strong>rapeutics that c<strong>an</strong> selectively target Egr3 <strong>an</strong>d abrogate disease progression by reducing<br />
expression of <strong>the</strong> alpha4 subunit gene.<br />
Disclosures: J.H. Kim, None; A.R. Brooks-Kayal, None; S.J. Russek, None.
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.4/B93<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: GM058037<br />
Title: The γ2(K289M) GABAA receptor epilepsy mutation <strong>an</strong>d homologous syn<strong>the</strong>tic mutation<br />
in <strong>the</strong> α subunit affect GABA efficacy <strong>an</strong>d modulation by propofol<br />
Authors: *T. G. DEEB 1 , D. SHARP 2 , T. G. HALES 3 ;<br />
1 Neurosci., 2 Dept. of Anes<strong>the</strong>siol. <strong>an</strong>d Critical Care Med., 3 Pharmacol. <strong>an</strong>d Physiol., George<br />
Washington Univ., Washington, D.C., DC<br />
Abstract: The γ2(K289M) epilepsy mutation causes a reduction in ch<strong>an</strong>nel me<strong>an</strong> open time of<br />
α1β2γ2 GABAA receptors. We previously demonstrated that mutation of <strong>the</strong> homologous residue<br />
in <strong>the</strong> α1(K278M) causes a similar functional deficit <strong>an</strong>d reduces GABA potency. In this study<br />
we fur<strong>the</strong>r characterized <strong>the</strong>se mutations <strong>an</strong>d examined whe<strong>the</strong>r <strong>the</strong> functional deficits c<strong>an</strong> be<br />
corrected by two allosteric modulators, <strong>the</strong> general <strong>an</strong>es<strong>the</strong>tic propofol <strong>an</strong>d <strong>the</strong> hypnotic<br />
benzodiazepine flunitrazepam, which enh<strong>an</strong>ce GABAA function through different mech<strong>an</strong>isms.<br />
Propofol, but not flunitrazepam, supramaximally potentiated GABA-evoked currents mediated<br />
by α1(K278M)β2γ2 <strong>an</strong>d α1β2γ2(K289M) receptors, but not wild type receptors. There<strong>for</strong>e, <strong>the</strong>se<br />
mutations alter <strong>the</strong> ch<strong>an</strong>nel gating equilibrium const<strong>an</strong>t. Consistent with this propofol also<br />
increased <strong>the</strong> me<strong>an</strong> open time of α1(K278M)β2γ2 single ch<strong>an</strong>nels activated by saturating GABA.<br />
Nei<strong>the</strong>r <strong>the</strong> α1(K278M) nor <strong>the</strong> γ2(K289M) mutation altered <strong>the</strong> potency of bicuculline as <strong>an</strong><br />
inhibitor of GABAA receptors activated by propofol, demonstrating that <strong>the</strong> affinity <strong>an</strong>d negative<br />
efficacy of bicuculline are unaltered. However, <strong>the</strong> α1(K278M) mutation did increase <strong>the</strong><br />
potency of bicuculline as <strong>an</strong> inhibitor of GABA-evoked currents <strong>an</strong>d increased <strong>the</strong> Hill-slope of<br />
<strong>the</strong> GABA concentration-response relationship in <strong>the</strong> presence of bicuculline, indicating reduced<br />
GABA affinity. We demonstrated that bicuculline increased <strong>the</strong> rate of deactivation of GABAevoked<br />
currents mediated by α1(K278M)β2γ2 receptors, indicating a reduction in <strong>the</strong> prevalence<br />
of <strong>the</strong> higher affinity agonist trapped state. These data suggest that <strong>the</strong> Lys to Met mutation<br />
reduces efficacy through two independent mech<strong>an</strong>isms.<br />
Disclosures: T.G. Deeb, None; D. Sharp, None; T.G. Hales, None.
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.5/B94<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: NIH Gr<strong>an</strong>t<br />
Title: Comparison of effects of isoflur<strong>an</strong>e <strong>an</strong>d etomidate on GABAA,slow IPSCs in org<strong>an</strong>otypic<br />
hippocampal cultured slices<br />
Authors: *S. DAI;<br />
Dept Anes<strong>the</strong>siol, Univ. Wisconsin Madison, Madison, WI<br />
Abstract: Background: The molecular mech<strong>an</strong>ism of volatile <strong>an</strong>es<strong>the</strong>sia is currently uncertain,<br />
GABARs, NMDARs, HCN <strong>an</strong>d K + ch<strong>an</strong>nels are potential molecular targets. Etomidate<br />
selectively modulates GABARs <strong>an</strong>d produces amnesia at low doses in vivo. To evaluate <strong>the</strong><br />
contribution of GABARs to <strong>an</strong>es<strong>the</strong>tic-induced amnesia, we compare effects of isoflur<strong>an</strong>e with<br />
etomidate on GABAergic inhibition. GABAA,slow IPSCs has been proposed to control synaptic<br />
plasticity <strong>an</strong>d memory <strong>for</strong>mation. Here we have tested effects of two <strong>an</strong>es<strong>the</strong>tics on GABAA,slow<br />
IPSCs in cultured hippocampal slices.<br />
Methods: Cultured hippocampal slices, were prepared <strong>from</strong> postnatal P3-P8 mice <strong>an</strong>d cultured<br />
<strong>for</strong> 10-14 days. The slices were continuously superfused with artificial cerebrospinal fluid<br />
(ACSF) at room temperature (22+1 0 C) oxygenated with carbogen (95%O2+5%CO2).<br />
Electrophsyiological recordings were obtained <strong>from</strong> neurons within <strong>the</strong> pyramidal cell layer of<br />
<strong>the</strong> CA1 area. GABAA,slow IPSCs were isolated by placed stimulating electrode in <strong>the</strong> stratum<br />
lacunosum-moleculare (SL-M) with presence of CNQX <strong>an</strong>d APV in ACSF. Data are presented<br />
as me<strong>an</strong> ± SD.<br />
Results: Both isoflur<strong>an</strong>e <strong>an</strong>d etomidate reversibly prolonged both <strong>the</strong> decay of GABAA,slow<br />
IPSCs in a dose-dependent m<strong>an</strong>ner. Isoflur<strong>an</strong>e at a concentration that produces partial amnesia<br />
(100 µM) signific<strong>an</strong>tly (69.9+11.6 ms vs. 86.5+9.4 ms <strong>for</strong> control <strong>an</strong>d drug, respectively, ratio:<br />
1.25+0.04,p
GABAA,slow IPSCs but 100 µM isoflur<strong>an</strong>e (about 125% to EC50-amnesia) increased only 125%<br />
on decay of GABAA,slow IPSCs. Etomidate had much stronger effects on decay of GABAA,slow<br />
IPSCs th<strong>an</strong> isoflur<strong>an</strong>e did: 0.5 <strong>an</strong>d 1 µM etomidate prolonged τdecay by 253 <strong>an</strong>d 363% <strong>for</strong><br />
GABAA,slow, but 200 <strong>an</strong>d 400µM isoflur<strong>an</strong>e only by 165 <strong>an</strong>d 277%. Comparing with etomiate,<br />
effects of isoflur<strong>an</strong>e on GABAA,slow IPSCs only partially contribute to isoflur<strong>an</strong>e-induced<br />
amnesia.<br />
Disclosures: S. Dai, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.6/B95<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: NIH Gr<strong>an</strong>t R01GM66724<br />
NIH Gr<strong>an</strong>t P01GM58448<br />
Title: Relating etomidate effects on deactivation rate vs. GABA EC50 in GABAA receptors<br />
Authors: *R. DESAI, A. LIU, S. A. FORMAN;<br />
Massachusetts Genl Hosp., Boston, MA<br />
Abstract: The potent intravenous general <strong>an</strong>es<strong>the</strong>tic etomidate acts via GABAA receptors.<br />
Electrophysiological studies indicate that etomidate enh<strong>an</strong>ces apparent GABA affinity (reducing<br />
GABA EC50) <strong>an</strong>d slows current deactivation. Assuming etomidate acts by stabilizing open states<br />
<strong>an</strong>d slowing ch<strong>an</strong>nel closing, simple models of receptor activation predict that deactivation rates<br />
should slow in proportion to <strong>the</strong> inverse square of EC50. We tested whe<strong>the</strong>r this relationship was<br />
evident in α1β2γ2L GABAARs.<br />
Experiments were done on stable HEK293 cell lines expressing α1, β2, <strong>an</strong>d γ2L. GABAdependent<br />
activation was characterized using patch-clamp electrophysiology. Rapid superfusion<br />
in <strong>an</strong> "artificial synapse" was used to pre-equilibrate cells with etomidate (0 to 30 µM), activate<br />
ch<strong>an</strong>nels with high (10 mM) GABA pulses, <strong>an</strong>d observe current deactivation in <strong>the</strong> presence of<br />
etomidate. Deactivation traces were <strong>an</strong>alyzed by non-linear least-squares fitting to doubleexponential<br />
functions, <strong>an</strong>d weighted time const<strong>an</strong>ts were calculated. Average deactivation time<br />
const<strong>an</strong>ts <strong>an</strong>d τw values were calculated <strong>for</strong> each etomidate concentration tested. GABA EC50s at<br />
each etomidate concentration were estimated with concentration-response studies.
As etomidate concentrations increased, both fast <strong>an</strong>d slow deactivation rates slowed, <strong>an</strong>d <strong>the</strong><br />
portion of slow deactivation increased relative to fast deactivation. A plot of -log(GABA EC50)<br />
vs. log (τw) showed <strong>an</strong> initial slope near 1 <strong>an</strong>d a reduced slope at etomidate concentrations above<br />
1 mM. Nei<strong>the</strong>r fast nor slow time const<strong>an</strong>ts displayed <strong>the</strong> slope of 2 predicted by simple models.<br />
Simple models assuming stabilization of a single type of open state do not accurately predict <strong>the</strong><br />
relationship between GABA EC50 <strong>an</strong>d τw at different concentrations of etomidate. These data<br />
suggest alternative models with multiple open states, where etomidate selectively stabilizes longlived<br />
states more th<strong>an</strong> short-lived states.<br />
Disclosures: R. Desai, None; A. Liu, None; S.A. Form<strong>an</strong>, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.7/B96<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: BBSRC<br />
Anonymous Trust<br />
TENOVUS Tayside<br />
Epilepsy Research UK<br />
Title: Investigating <strong>the</strong> effects of THIP <strong>an</strong>d etomidate on thalamocortical neuron excitability:<br />
The role of synaptic <strong>an</strong>d extrasynaptic GABAA receptors<br />
Authors: *M. B. HERD, A. R. BROWN, J. J. LAMBERT, D. BELELLI;<br />
Ctr. <strong>for</strong> Neuroscience, Div. of Med. Sci., Univ. of Dundee, Dundee, United Kingdom<br />
Abstract: Thalamocortical (TC) networks are a crucial component of <strong>the</strong> neuronal circuitry<br />
controlling <strong>the</strong> sleep-wake cycle <strong>an</strong>d it has been proposed that <strong>the</strong> thalamus represents a key<br />
substrate <strong>for</strong> <strong>the</strong> actions of sedative/hypnotics <strong>an</strong>d general <strong>an</strong>es<strong>the</strong>tics, which pharmacologically<br />
induce a ‘sleep-like’ state. The activity of TC relay neurons are tightly regulated by GABA-ergic<br />
neurons of <strong>the</strong> nucleus reticularis thalami which act, in part, via two distinct GABAA receptor<br />
populations on TC neurons: synaptic receptors composed of α1β2γ2 subunits <strong>an</strong>d extrasynaptic<br />
receptors composed of α4β2δ subunits. Synaptic receptors mediate ‘phasic’ inhibition, whereas
extrasynaptic receptors mediate sustained ‘tonic’ inhibition, <strong>an</strong>d <strong>the</strong>se distinct inhibitory modes<br />
exhibit differential sensitivity to sedative/hypnotic agents such as THIP <strong>an</strong>d <strong>the</strong> general<br />
<strong>an</strong>es<strong>the</strong>tic etomidate. However, <strong>the</strong> mech<strong>an</strong>ism whereby enh<strong>an</strong>cement of phasic <strong>an</strong>d/or tonic<br />
inhibition tr<strong>an</strong>slates into sedation is unknown. It is <strong>the</strong>re<strong>for</strong>e import<strong>an</strong>t to underst<strong>an</strong>d how<br />
enh<strong>an</strong>ced inhibition impacts on <strong>the</strong> firing properties of neurons within relev<strong>an</strong>t regions of <strong>the</strong><br />
sleep-wake circuitry. Thus, we have per<strong>for</strong>med current clamp recordings <strong>from</strong> TC neurons<br />
within mouse brain slices containing <strong>the</strong> ventrobasal nucleus using <strong>the</strong> gramicidin per<strong>for</strong>ated<br />
patch technique. Local pressure ejection of etomidate (300 µM, 10-20ms duration,
Authors: *J. ADAMS, T. G. SMART;<br />
Neuroscience, Physiol. <strong>an</strong>d Pharmacol., Univ. Col. London|910005393|0, London, United<br />
Kingdom<br />
Abstract: GABAA receptors are responsible <strong>for</strong> inhibitory synaptic tr<strong>an</strong>smission in <strong>the</strong> brain.<br />
Endogenous agents that c<strong>an</strong> modulate <strong>the</strong>se receptors will <strong>the</strong>re<strong>for</strong>e have import<strong>an</strong>t<br />
consequences <strong>for</strong> neural activity. Neurosteroids <strong>an</strong>d protein kinases are among <strong>the</strong> most potent<br />
modulators of <strong>the</strong> GABAA receptor, which, individually, c<strong>an</strong> enh<strong>an</strong>ce or depress receptor<br />
function depending on <strong>the</strong> type of neurosteroid or kinase present <strong>an</strong>d <strong>the</strong> subunit composition of<br />
<strong>the</strong> receptor . However, in vivo, <strong>the</strong>se modulators are likely to act in concert to modulate GABAA<br />
receptors to achieve precise fine-tuning of inhibitory neurotr<strong>an</strong>smission.<br />
There<strong>for</strong>e, <strong>the</strong> functional relationship between <strong>the</strong>se two modulators, at <strong>the</strong> α1β3γ2L GABAA<br />
receptor expressed in HEK cells, was investigated using whole-cell patch clamp<br />
electrophysiology. The potentiation of GABA responses (EC20) by<br />
tetrahydrodeoxycorticosterone (THDOC) was reduced by staurosporine, a broad-spectrum kinase<br />
inhibitor, <strong>an</strong>d by bisindolylmaleimide I, a selective inhibitor of PKC. By contrast, <strong>the</strong><br />
potentiation remained unaffected by inhibiting PKA or PKG. The THDOC potentiation was also<br />
increased by phorbol ester activation of PKC, but <strong>the</strong> activation of PKA had no effect.<br />
As PKC c<strong>an</strong> phosphorylate GABAA receptor β3 subunits at S408 <strong>an</strong>d S409 , we evaluated<br />
neurosteroid potentiation in cells expressing α1β3 S408A,S409A γ2L GABAA receptors. GABA<br />
responses <strong>for</strong> <strong>the</strong> mut<strong>an</strong>t receptor showed no ch<strong>an</strong>ge in <strong>the</strong> magnitude of THDOC potentiation<br />
when treated with ei<strong>the</strong>r staurosporine, bisindolylmaleimide I or PMA. This indicates that<br />
phosphorylation of <strong>the</strong> GABAA receptor β subunit by PKC is import<strong>an</strong>t <strong>for</strong> regulating <strong>the</strong><br />
potentiation of GABAA receptor function by neurosteroids.<br />
By using a Western blot <strong>an</strong>alysis <strong>for</strong> wild-type receptors, cells treated with THDOC showed <strong>an</strong><br />
increase in <strong>the</strong> phosphorylation of β3 S408 <strong>an</strong>d S409. In contrast, β3 subunit phosphorylation<br />
remained unaffected in cells expressing α1 Q241W β3γ2L GABAA receptors, a mutation which<br />
prevents neurosteroid potentiation (Hosie et al, 2006).<br />
Our studies indicate a role <strong>for</strong> PKC in setting <strong>the</strong> limits of potentiation of GABAA receptor<br />
function by neurosteroids as well as <strong>the</strong> presence of a ‘reverse’ pathway in which neurosteroids<br />
c<strong>an</strong> modulate <strong>the</strong> phosphorylation of <strong>the</strong> GABAA receptor.<br />
Belelli D et al. (2005). Nature Rev Neurosci 6, 565-575.<br />
McDonald BJ et al. (1998). Nature Neurosci 1, 23-28.<br />
Moss SJ et al. (1996). Int Rev Neurobiol 39, 1-52.<br />
Hosie AM et al. (2006). Nature 444, 486-489.<br />
Disclosures: J. Adams, None; T.G. Smart, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.9/B98<br />
Topic: B.02.p. GABA(A) receptors: Gene regulation<br />
Support: AG026472<br />
AG029612<br />
HD029186<br />
RR000163<br />
Title: Influence of 17β-estradiol <strong>an</strong>d progesterone on GABAergic gene expression in <strong>the</strong> arcuate<br />
nucleus, amygdala <strong>an</strong>d hippocampus of <strong>the</strong> rhesus macaque<br />
Authors: *N. C. NORIEGA, D. H. EGHLIDI, V. T. GARYFALLOU, S. G. KOHAMA, S. G.<br />
KRYGER, H. F. URBANSKI;<br />
Neurosci. Div., Oregon Natl. Primate Res. Ctr., Beaverton, OR<br />
Abstract: Gamma-aminobutyric acid (GABA) is <strong>the</strong> major inhibitory neurotr<strong>an</strong>smitter in <strong>the</strong><br />
adult amniote brain, <strong>an</strong>d <strong>the</strong> responsiveness of neurons to GABA c<strong>an</strong> be modulated by sex<br />
steroids. To better underst<strong>an</strong>d how ovari<strong>an</strong> steroids influence GABAergic systems in <strong>the</strong> primate<br />
brain, we evaluated <strong>the</strong> expression of genes encoding GABA receptor subunits, glutamic acid<br />
decarboxylase (GAD) <strong>an</strong>d a GABA tr<strong>an</strong>sporter in 12 female rhesus macaque brains.<br />
Ovariectomized adults were subjected to a hormone replacement paradigm involving ei<strong>the</strong>r 17βestradiol<br />
(E; n=4), or E plus progesterone (P) (E+P; n=4). Untreated <strong>an</strong>imals (OVX; n=4) served<br />
as controls. Using Affymetrix gene microarray <strong>an</strong>alysis <strong>an</strong>d qu<strong>an</strong>titative real-time RT-PCR<br />
(qPCR), we examined gene expression differences within <strong>an</strong>d between <strong>the</strong> amygdala (AMD),<br />
hippocampus (HPC) <strong>an</strong>d arcuate nuclei of <strong>the</strong> medial basal hypothalamus (MBH). The results<br />
<strong>from</strong> qPCR corresponded with results <strong>from</strong> representative microarray probesets. Exposure to<br />
E+P attenuated expression of genes <strong>an</strong>notated as encoding proteins regulating GABA<br />
distribution (GAD1, GAD2 <strong>an</strong>d SLC32A1) in <strong>the</strong> AMD <strong>an</strong>d HPC, but not <strong>the</strong> MBH. GABA<br />
receptor subunit gene expression was generally higher in <strong>the</strong> AMD <strong>an</strong>d HPC th<strong>an</strong> in <strong>the</strong> MBH,<br />
with <strong>the</strong> exception of genes encoding receptor subunits ε <strong>an</strong>d γ2. Taken toge<strong>the</strong>r, <strong>the</strong> data<br />
demonstrate differential regulation of GABA receptor subunits <strong>an</strong>d GABAergic system<br />
components in <strong>the</strong> MBH compared to <strong>the</strong> AMD <strong>an</strong>d HPC of rhesus macaques. Elevated ε <strong>an</strong>d<br />
reduced δ subunit expression in <strong>the</strong> MBH compared to <strong>the</strong> AMD <strong>an</strong>d HPC supports <strong>the</strong><br />
hypo<strong>the</strong>sis that <strong>the</strong> hypothalamic GABAergic system is resist<strong>an</strong>t to <strong>the</strong> modulatory effects of sex<br />
steroids. Supported by NIH gr<strong>an</strong>ts: AG026472, AG029612, HD029186 <strong>an</strong>d RR000163.<br />
Disclosures: N.C. Noriega, None; D.H. Eghlidi, None; V.T. Garyfallou, None; S.G. Kohama,<br />
None; S.G. Kryger, None; H.F. Urb<strong>an</strong>ski, None.
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.10/B99<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: KOSEF gr<strong>an</strong>t (No. R13-2008-009-01001-0)<br />
Title: Allopregn<strong>an</strong>olone directly induces Cl - currents in hippocampal CA3 pyramidal neurons<br />
Authors: H.-M. PARK 1 , I.-S. CHOI 1 , J.-H. CHO 1 , J.-J. LEE 1 , *I.-S. JANG 2 ;<br />
2 Dept. of Pharmacol., 1 Kyungpook Natl. Univ., Daegu, Republic of Korea<br />
Abstract: The properties of allopregn<strong>an</strong>olone (AlloP)-induced membr<strong>an</strong>e currents (IAlloP) were<br />
studied in mech<strong>an</strong>ically isolated hippocampal CA3 pyramidal neurons by use of a whole-cell<br />
patch-clamp technique. AlloP elicited <strong>the</strong> slow membr<strong>an</strong>e currents in a dose-dependent m<strong>an</strong>ner.<br />
However, <strong>the</strong> intracellular loading of AlloP through a patch pipette didn’t elicit <strong>an</strong>y membr<strong>an</strong>e<br />
currents, <strong>an</strong>d <strong>the</strong> intracellular loading of γ-cyclodextrin, a steroid scavenger, through a patch<br />
pipette had no effect on <strong>the</strong> IAlloP. The reversal potential of IAlloP was similar to <strong>the</strong> ECl calculated<br />
by <strong>the</strong> Nernst equation, <strong>an</strong>d <strong>the</strong> IAlloP was gradually attenuated when <strong>the</strong> ATP-free pipette<br />
solution was used. While <strong>the</strong> IAlloP was hardly blocked by SR95531, a competitive GABAA<br />
receptor blocker, it was signific<strong>an</strong>tly blocked by picrotoxin, a non-competitive GABAA receptor<br />
blocker. The results suggest that a neuroactive steroid AlloP binds to GABAA receptors in <strong>the</strong><br />
outer leaflet of lipid bilayer, which is different <strong>from</strong> <strong>the</strong> GABA-binding site, <strong>an</strong>d directly opens<br />
GABAA receptor-Cl - ch<strong>an</strong>nel complex.<br />
Disclosures: H. Park, None; I. Choi, None; J. Cho, None; J. Lee, None; I. J<strong>an</strong>g, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.11/B100
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: NIH GM47969 to ASE, DFC, CFZ, <strong>an</strong>d JHS<br />
Title: A novel photoactive neurosteroid <strong>an</strong>alogue labels GABAA receptors<br />
Authors: *Z.-W. CHEN 1 , R. W. HASTINGS 1 , J. R. BRACAMONTES 1 , C. WANG 2 , J. H.<br />
STEINBACH 1 , S. J. MENNERICK 3 , D. F. COVEY 2 , A. S. EVERS 1 ;<br />
1 Anes<strong>the</strong>siol., 2 Developmental Biol., 3 Psychiatry, Washington Univ. In St Louis, St Louis, MO<br />
Abstract: Neurosteroids have been shown to modulate neuronal excitability via GABAA<br />
receptors. To investigate <strong>the</strong> binding sites of neurosteroids on GABAA receptors, a novel<br />
photoactive pregn<strong>an</strong>olone <strong>an</strong>alogue (CW14) containing a trifluromethyl phenyl diazirine group<br />
linked to <strong>the</strong> steroid C11 position was syn<strong>the</strong>sized. CW14 is <strong>an</strong> effective GABAA receptor<br />
modulator: it potentiates GABA-induced currents in Xenopus Leavis occytes tr<strong>an</strong>sfected with<br />
α1β2γ2 subunits, modulates [ 35 S] t-butylbicyclophosphorothionate (TBPS) binding in rat brain<br />
membr<strong>an</strong>es (RBM), <strong>an</strong>d produces <strong>an</strong>es<strong>the</strong>tic effects in pre-limb bud stage Xenopus tadpoles.<br />
Following irradiation with 350 nm light, CW14 produces irreversible enh<strong>an</strong>cement of [ 35 S]<br />
TBPS binding in RBM <strong>an</strong>d prevents fur<strong>the</strong>r enh<strong>an</strong>cement by pregn<strong>an</strong>olone; this indicates that<br />
CW14 photolabels neurosteroid binding sites on <strong>the</strong> GABAA receptors. SDS-PAGE of RBM<br />
photolabled with [ 3 H]CW14 shows major labeled b<strong>an</strong>ds at ~32 kDa <strong>an</strong>d ~55 kDa. Photolabeling<br />
of <strong>the</strong> 55 kDa proteins was increased by 3 µM GABA. Blue native PAGE demonstrated that<br />
[ 3 H]CW14 photolabels proteins at ~1200 kDa, ~250 kDa, <strong>an</strong>d ~100 kDa. Western blot shows<br />
that <strong>the</strong>se three b<strong>an</strong>ds correspond to tubulin, GABAA receptor, <strong>an</strong>d voltage-dependent <strong>an</strong>ion<br />
ch<strong>an</strong>nel-1, respectively. Photolabeling of <strong>the</strong> GABAA receptors is concentration-dependent <strong>an</strong>d<br />
stereo-specific, as <strong>the</strong> 3β-OH isomer of CW14 does not show <strong>an</strong>y photolabeling. In addition,<br />
blue native PAGE shows that [ 3 H]CW14 photolabels a b<strong>an</strong>d at ~250 kDa in membr<strong>an</strong>es <strong>from</strong><br />
TSA cells expressing α1β2 subunits of <strong>the</strong> GABAA receptor, but not in membr<strong>an</strong>es <strong>from</strong> nontr<strong>an</strong>sfected<br />
cells. These data indicate that CW14 is <strong>an</strong> effective photolabeling reagent to<br />
investigate <strong>the</strong> neurosteroid binding sites on <strong>the</strong> GABAA receptors. Fur<strong>the</strong>r sequencing studies<br />
will elucidate <strong>the</strong> specific labeling sites.<br />
Disclosures: Z. Chen, None; R.W. Hastings, None; J.R. Bracamontes, None; C. W<strong>an</strong>g,<br />
None; J.H. Steinbach, None; S.J. Mennerick, None; D.F. Covey, None; A.S. Evers, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.12/B101
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: MRC<br />
Title: Regulation of tonic GABAA receptor mediated inhibition by phosphorylation<br />
Authors: D. P. BRIGHT, *T. G. SMART;<br />
Univ. Col. London, London, United Kingdom<br />
Abstract: GABAA receptors are critically involved in controlling neuronal excitability through<br />
two <strong>for</strong>ms of inhibition, termed phasic <strong>an</strong>d tonic. Phasic inhibition is mediated by <strong>the</strong> fast <strong>an</strong>d<br />
tr<strong>an</strong>sient activation of synaptic GABAA receptors, whereas tonic inhibition is due to <strong>the</strong><br />
persistent activation of high affinity extrasynaptic GABAA receptors by low ambient levels of<br />
GABA. Phosphorylation by protein kinases has been shown to be <strong>an</strong> import<strong>an</strong>t method of<br />
regulation <strong>for</strong> <strong>the</strong> receptors that underlie phasic inhibition; however, little attention has been<br />
focused on <strong>the</strong> question of whe<strong>the</strong>r phosphorylation also modulates tonic inhibition. To address<br />
this issue, we have used whole-cell patch clamping to assess <strong>the</strong> effects of protein kinases on<br />
GABA currents mediated by extrasynaptic receptors, using native receptors in acute slices of <strong>the</strong><br />
mouse hippocampal dentate gyrus <strong>an</strong>d recombin<strong>an</strong>t receptors expressed in HEK293 cells.<br />
Experiments on recombin<strong>an</strong>t α4β2δ receptors demonstrate a temperature-dependent bal<strong>an</strong>ce<br />
between <strong>the</strong> actions of kinases <strong>an</strong>d phosphatases on receptor function. We have used bath<br />
application of a low GABA concentration to mimic <strong>the</strong> activation of native receptors by ambient<br />
GABA within <strong>the</strong> CNS. At room temperature, <strong>the</strong> broad spectrum protein kinase inhibitor,<br />
staurosporine, causes <strong>an</strong> enh<strong>an</strong>cement of receptor function, as demonstrated by <strong>an</strong> increase in<br />
current vari<strong>an</strong>ce. By contrast, <strong>the</strong> PKC activator, PMA, has no effect. At more physiological<br />
temperatures (33-35°C), PMA causes a large decrease in receptor activation, whereas<br />
staurosporine has no effect. Using mature (> P30) murine slices, we have recorded <strong>from</strong> dentate<br />
gyrus gr<strong>an</strong>ule cells. At physiological temperatures (35-36°C), <strong>the</strong> presence of a tonic GABAA<br />
receptor mediated conduct<strong>an</strong>ce was revealed by application of <strong>the</strong> GABAA <strong>an</strong>tagonist,<br />
bicuculline, which causes <strong>an</strong> outwards current <strong>an</strong>d a decrease in <strong>the</strong> RMS current noise. This<br />
conduct<strong>an</strong>ce is likely to be mediated by α4β2δ receptors <strong>an</strong>d as with <strong>the</strong> recombin<strong>an</strong>t receptor,<br />
blockade of protein kinases by staurosporine leads to <strong>an</strong> increase in receptor function. Toge<strong>the</strong>r,<br />
our results suggest that cell-signalling pathways that activate PKC may have <strong>an</strong> import<strong>an</strong>t role in<br />
<strong>the</strong> modulation of tonic GABAA-receptor mediated conduct<strong>an</strong>ces <strong>an</strong>d thus have a signific<strong>an</strong>t<br />
impact on neuronal excitability.<br />
Supported by <strong>the</strong> MRC<br />
Disclosures: D.P. Bright, None; T.G. Smart, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.13/B102<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: Wellcome Trust<br />
Medical Research Council<br />
Title: Just how responsive are extrasynaptic GABA-A receptors?<br />
Authors: D. P. BRIGHT 1 , J. BARTRAM 1 , T. P. MCGEE 1 , S. G. BRICKLEY 1 , *A. M.<br />
HOSIE 2,1 ;<br />
1 Imperial Col., London, United Kingdom; 2 Biophysics Section, London, United Kingdom<br />
Abstract: The expression of high-affinity extrasynaptic GABAA receptor populations is thought<br />
to underlie both spillover detection <strong>an</strong>d <strong>the</strong> generation of tonic inhibition. High-affinity<br />
extrasynaptic delta subunit-containing GABAA receptors are a defining feature of several<br />
neuronal populations. However, relatively little is currently known regarding <strong>the</strong> functional<br />
properties of delta subunit-containing GABAA receptors under physiological conditions. We<br />
have evaluated <strong>the</strong> properties of <strong>the</strong>se receptors at steady-state GABA concentrations <strong>an</strong>d<br />
following brief synaptic-like tr<strong>an</strong>sients at physiological temperatures. These experiments have<br />
confirmed <strong>the</strong> high-affinity of <strong>the</strong>se receptors <strong>for</strong> GABA but, contrary to some reports, also<br />
demonstrate unexpected levels of desensitization <strong>an</strong>d a distinct lack of constitutive activity. This<br />
data places constraints on <strong>the</strong> ability of extrasynaptic GABAARs to detect ch<strong>an</strong>ges in ambient<br />
GABA.<br />
Disclosures: D.P. Bright, None; J. Bartram, None; T.P. McGee, None; S.G. Brickley,<br />
None; A.M. Hosie, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.14/B103<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: CONACyT Mexico 37152N
Master scholarship CONACyT<br />
Title: Modulation of monosynaptic reflex by extrasynaptic GABAA receptors in turtle spinal<br />
cord<br />
Authors: *W. D. BAUTISTA GUZMAN 1,2 , J. AGUILAR 2 , R. DELGADO-LEZAMA 2 ;<br />
1 Physiol., Univ. of M<strong>an</strong>itoba, Winnipeg, MB, C<strong>an</strong>ada; 2 Dept. de Fisiologia Biofisica y<br />
Neurociencias, Ctr. de Investigacion y Estudios Av<strong>an</strong>zados del IPN (CINVESTAV), Mexico<br />
city, Mexico<br />
Abstract: There is growing evidence that activation of high affinity extrasynaptic GABAA<br />
receptors in <strong>the</strong> brain, cerebellum <strong>an</strong>d spinal cord subst<strong>an</strong>tia gelatinosa, results in a tonic<br />
inhibition controlling postsynaptic excitability without <strong>the</strong> need <strong>for</strong> continuous spike activity in<br />
inhibitory interneurons. The aim of <strong>the</strong> present study was to determine if extrasynaptic GABAA<br />
receptors participate in <strong>the</strong> monosynaptic reflex modulation in turtle spinal cord. In <strong>the</strong><br />
mammali<strong>an</strong> spinal cord presynaptic inhibition causes a reduction in synaptic tr<strong>an</strong>smission <strong>from</strong><br />
segmental afferents through by GABAergic interneurons. We now show that in <strong>the</strong> in vitro turtle<br />
lumbar spinal cord preparation, conditioning stimulation of a dorsal root reduces <strong>the</strong> size of a test<br />
monosynaptic reflex (MSR) at long condition-test intervals. This inhibition is similar to that seen<br />
in mammali<strong>an</strong> spinal cord <strong>an</strong>d was completely blocked by bath application of 20µM picrotoxin<br />
(PTX). Presumably this low concentration PTX blocks activation of <strong>the</strong> lower affinity,<br />
synaptically located GABAA receptors on afferent fiber terminals. In order to determine if<br />
motoneuron excitability might be regulated by higher affinity GABA receptors, <strong>the</strong> PTX<br />
concentration was increased to 100µM <strong>an</strong>d 200µM resulting in <strong>an</strong> 11.5% <strong>an</strong>d 20% potentiation<br />
of <strong>the</strong> monosynaptic reflex respectively (n=4). Furosemide is believed to be a selective<br />
<strong>an</strong>tagonist of extrasynaptic GABAA receptors affecting <strong>the</strong> α6βδ <strong>an</strong>d α4βδ receptor subtypes.<br />
Similarly to <strong>the</strong> higher PTX concentrations, 100µM <strong>an</strong>d 200µM furosemide potentiated <strong>the</strong> MSR<br />
by 12.5% <strong>an</strong>d 20% (n=4) but did not affect <strong>the</strong> dorsal root potential (DRP). This suggests that<br />
extrasynaptic GABAA receptors are present on turtle motoneurons <strong>an</strong>d produce a tonic inhibition<br />
of <strong>the</strong> MSR. The present results suggest 1) that <strong>the</strong> turtle has a mammali<strong>an</strong>-like presynaptic<br />
inhibitory system in <strong>the</strong> spinal cord <strong>an</strong>d 2) that extrasynaptic GABAA receptors produce a tonic<br />
inhibition of monosynaptic reflex.<br />
Disclosures: W.D. Bautista Guzm<strong>an</strong>, None; J. Aguilar, None; R. Delgado-Lezama, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.15/B104
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: NIH Gr<strong>an</strong>t NS035985<br />
Title: Alcohol- <strong>an</strong>d alcohol <strong>an</strong>tagonist-sensitive hum<strong>an</strong> GABAA receptors: Tracking δ subunit<br />
incorporation into functional receptors<br />
Authors: P. MEERA 1 , R. W. OLSEN 2 , T. S. OTIS 1 , *M. WALLNER 3 ;<br />
1 Neurobio., 2 Mol. <strong>an</strong>d Med. Pharmacol., 3 UCLA, Los Angeles, CA<br />
Abstract: GABAA receptors (GABAARs) have long been a focus as targets <strong>for</strong> alcohol actions.<br />
Recent work suggests that tonic GABAergic inhibition mediated by highly GABA-sensitive<br />
extrasynaptic δ subunit-containing GABAARs in neurons is uniquely sensitive to eth<strong>an</strong>ol <strong>an</strong>d<br />
enh<strong>an</strong>ced at concentrations relev<strong>an</strong>t <strong>for</strong> hum<strong>an</strong> alcohol consumption (3 - 30 mM). In addition, it<br />
has been shown that low dose eth<strong>an</strong>ol enh<strong>an</strong>cement of recombin<strong>an</strong>t α4β3δ receptors is blocked<br />
by <strong>the</strong> behavioral alcohol <strong>an</strong>tagonist Ro15-4513, suggesting that EtOH/Ro15-4513-sensitive<br />
receptors mediate import<strong>an</strong>t behavioral aspects of alcohol intoxication. However, <strong>the</strong> high EtOH<br />
sensitivity of recombin<strong>an</strong>tly reconstituted δ subunit-containing receptors has been somewhat<br />
controversial with some investigators reporting difficulties in reproducing low dose EtOH<br />
actions on <strong>the</strong>se unique receptors subtypes. In this study we expressed hum<strong>an</strong> α4β3δ receptor<br />
cDNAs clones in a hum<strong>an</strong> cell line <strong>an</strong>d carefully compared <strong>the</strong> pharmacology of α4β3δ receptors<br />
with receptors <strong>for</strong>med by α4 <strong>an</strong>d β3 subunits alone, to test <strong>the</strong> hypo<strong>the</strong>sis that discrep<strong>an</strong>t findings<br />
reported in <strong>the</strong> literature concerning <strong>the</strong> alcohol-sensitivity of such GABAA receptor subtypes are<br />
due to difficulties incorporating δ subunits into functional receptors. To track δ subunit<br />
incorporation into functional receptors we utilized a functional tag, a single amino acid ch<strong>an</strong>ge<br />
(H68A) in which a histidine residue in <strong>the</strong> δ subunit is replaced by <strong>an</strong> al<strong>an</strong>ine residue found at<br />
<strong>the</strong> homologous position in γ subunits. We demonstrate that <strong>the</strong> δ(H68A) substitution confers<br />
submicromolar diazepam sensitivity to o<strong>the</strong>rwise diazepam-insensitive α4β3δ receptors, without<br />
ch<strong>an</strong>ging <strong>the</strong> high EtOH sensitivity of <strong>the</strong>se receptors. We find that enh<strong>an</strong>cement of α4β3δH68A<br />
receptors by 1 µM diazepam, 30 mM EtOH <strong>an</strong>d 1 µM β-CCE (but not 1 µM Zn 2+ block) is<br />
correlated in individual recordings, suggesting that δ subunit incorporation into recombin<strong>an</strong>t<br />
GABAARs varies <strong>from</strong> cell to cell <strong>an</strong>d that this accounts <strong>for</strong> <strong>the</strong> variable pharmacological profile.<br />
We also confirm that <strong>the</strong> behavioral alcohol <strong>an</strong>tagonist Ro15-4513 blocks low dose EtOH<br />
enh<strong>an</strong>cement on hum<strong>an</strong> α4β3δ receptors. We conclude that δ subunit-incorporation is necessary<br />
to confer unique pharmacological properties to “binary” α4/6β3 receptors, including <strong>the</strong> high<br />
eth<strong>an</strong>ol sensitivity which is one of <strong>the</strong> defining features of native δ subunit-containing<br />
GABAARs.<br />
Disclosures: P. Meera, None; R.W. Olsen, None; T.S. Otis, None; M. Wallner, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.16/B105<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: GM47818<br />
NS56411<br />
Title: Determination of <strong>the</strong> amnesic concentration of midazolam <strong>an</strong>d its metabolites in brain<br />
tissue: Implications <strong>for</strong> in vitro studies<br />
Authors: *R. A. PEARCE 1 , A. A. SAAD 1 , Y. ZHANG 2,3 , V. RAU 2 , I. OH 2 , M. TANG 2 , S.<br />
MANOHAR 2 , J. M. SONNER 2 , E. I. EGER, II 2 ;<br />
1 Anes<strong>the</strong>siol., Univ. of Wisconsin, Madison, WI; 2 Anes<strong>the</strong>sia <strong>an</strong>d Perioperative Care, Univ. of<br />
Cali<strong>for</strong>nia - S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 3 Anes<strong>the</strong>siol., Fuwai Hosp. <strong>an</strong>d Cardiovasc. Inst.,<br />
Beijing, China<br />
Abstract: Introduction: The widely used benzodiazepine midazolam produces <strong>an</strong>xiolysis,<br />
hypnosis, <strong>an</strong>d amnesia. To establish conditions appropriate <strong>for</strong> studies of midazolam’s effects in<br />
vitro at concentrations that correspond to behaviorally defined end points, we determined <strong>the</strong><br />
brain tissue <strong>an</strong>d free aqueous concentrations of midazolam, <strong>the</strong> contribution of metabolites to<br />
<strong>the</strong>se effects, <strong>an</strong>d <strong>the</strong> diffusion <strong>an</strong>d concentration profile of midazolam <strong>an</strong>d its chief metabolite<br />
1-OH-midazolam in brain slices.<br />
Methods: We measured <strong>the</strong> suppression of contextual fear conditioning in mice by midazolam<br />
by administering a r<strong>an</strong>ge of doses via subcut<strong>an</strong>eous injection, followed by a series of foot shocks<br />
40 minutes post-injection, <strong>an</strong>d testing <strong>for</strong> freezing to context <strong>the</strong> next day. In separate mice given<br />
comparable injections, concentrations of midazolam <strong>an</strong>d its metabolites in brain tissue were<br />
measured using HPLC <strong>an</strong>alysis. Uptake studies in 400µm-thick brain slices were used to<br />
calculate drug diffusion <strong>an</strong>d partition coefficients. Physiological modulation of GABAA<br />
receptors was assessed using recombin<strong>an</strong>t α1β2γ2L <strong>an</strong>d α5β3γ2L receptors expressed in<br />
HEK293 cells.<br />
Results: Midazolam impaired contextual fear conditioning in a dose-dependent m<strong>an</strong>ner, with <strong>an</strong><br />
ED50 dose of approximately 1.25 mg/kg. This dose produced brain concentrations at <strong>the</strong> time of<br />
conditioning of 76 ±12 ng/g midazolam <strong>an</strong>d 89 ±22 ng/g 1-OH-midazolam. Nei<strong>the</strong>r 4-OHmidazolam<br />
nor 1,4-di-OH-midazolam was present at detectable concentrations. Taking into<br />
account <strong>the</strong>ir brain:ACSF partition coefficients (35.7±1.7 <strong>an</strong>d 15.9±0.9), this corresponds to <strong>an</strong><br />
EC50,amnesia aqueous concentration of 6.8 nM midazolam plus 16.4 nM 1-hydroxymidazolam. In<br />
physiological studies, both compounds enh<strong>an</strong>ced currents approximately three-fold at saturating<br />
concentrations. However, <strong>the</strong> potency of 1-OH-midazolam was approximately 6-8 times that of<br />
midazolam, both <strong>for</strong> α1β2γ2L receptors (EC50 = 29 ± 2.3 nM vs. 176 ± 30 nM) <strong>an</strong>d α5β3γ2L<br />
receptors (EC50 = 32 ± 9.4 nM vs. 238 ± 101 nM).<br />
Conclusions: Under <strong>the</strong> conditions used in <strong>the</strong> present study, amnesia <strong>from</strong> <strong>an</strong> ED50,amnesia dose
of midazolam derives primarily <strong>from</strong> its highly potent active metabolite 1-OH-midazolam, which<br />
is present at a concentration that enh<strong>an</strong>ces GABAA receptor-mediated responses by<br />
approximately 50-100 percent. This in<strong>for</strong>mation provides <strong>an</strong> estimate of <strong>the</strong> free aqueous<br />
concentrations of midazolam plus 1-OH-midazolam that correspond to EC50,amnesia, as well as <strong>the</strong><br />
degree of GABAA receptor modulation that impairs hippocampus-dependent memory <strong>for</strong>mation.<br />
Disclosures: R.A. Pearce, None; A.A. Saad, None; Y. Zh<strong>an</strong>g, None; V. Rau, None; I. Oh,<br />
None; M. T<strong>an</strong>g, None; S. M<strong>an</strong>ohar, None; J.M. Sonner, None; E.I. Eger, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.17/B106<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Title: Bidirectional modulation of <strong>the</strong> effects of intravenous general <strong>an</strong>es<strong>the</strong>tics by a Gabra3<br />
knockout<br />
Authors: *U. RUDOLPH 1 , C. J. STRAUB 1,2 , H. LAU 1 , R. PARLATO 3 , G. SCHUETZ 3 , J.-M.<br />
FRITSCHY 2 ;<br />
1 Lab. of Genet. Neuropharm., McLe<strong>an</strong> Hosp. / Harvard Med. Sch., Belmont, MA; 2 Inst. of<br />
Pharmacol. <strong>an</strong>d Toxicology, Univ. of Zurich, Zurich, Switzerl<strong>an</strong>d; 3 Mol. Biol. of <strong>the</strong> Cell I,<br />
Germ<strong>an</strong> C<strong>an</strong>cer Reserach Ctr., Heidelberg, Germ<strong>an</strong>y<br />
Abstract: GABAA receptors, <strong>the</strong> major inhibitory neurotr<strong>an</strong>smitter receptors in <strong>the</strong> CNS, have<br />
been shown to mediate <strong>the</strong> hypnotic, immobilizing <strong>an</strong>d amnestic actions of m<strong>an</strong>y intravenous<br />
general <strong>an</strong>es<strong>the</strong>tics. <strong>When</strong> per<strong>for</strong>ming stereotaxic surgery on mice lacking <strong>the</strong> GABAA receptor<br />
α3 subunit (α3KO mice), a remarkable, unexpected <strong>an</strong>d apparently paradoxical increase in<br />
sensitivity to ketamine/xylazine was observed. We <strong>the</strong>n set out to study whe<strong>the</strong>r this increased<br />
sensitivity would be due to increased NMDA receptor <strong>an</strong>tagonism <strong>an</strong>d/or α2-adrenergic<br />
agonism, or whe<strong>the</strong>r <strong>the</strong> α3KO mice display a general hypersensitivity to intravenous general<br />
<strong>an</strong>es<strong>the</strong>tics, including etomidate, midazolam, <strong>an</strong>d pentobarbital. Fur<strong>the</strong>rmore, we w<strong>an</strong>ted to test<br />
<strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> increased sensitivity to ketamine/xylazine might be caused by a lack of<br />
α3-containing GABAA receptors on noradrenergic neurons. To this end, we generated mice<br />
expressing <strong>the</strong> α3-containing GABAA receptor selectively in noradrenergic neurons. These mice<br />
carry a dopamine β hydroxylase cre tr<strong>an</strong>sgene, which is specifically expressed in noradrenergic<br />
neurons, <strong>an</strong>d <strong>an</strong> α3KO allele with a loxP-fl<strong>an</strong>ked duplicated exon. Cre-loxP-mediated<br />
recombination leads to excision of this exon <strong>an</strong>d rescue of α3 subunit expression selectively in
<strong>the</strong>se cells.<br />
α3KO mice showed <strong>an</strong> enh<strong>an</strong>ced response to <strong>the</strong> hypnotic (loss of righting reflex), immobilizing<br />
(loss of hindlimb withdrawal reflex), <strong>an</strong>d hypo<strong>the</strong>rmic actions of ketamine/xylazine, compared to<br />
wild-type mice. In mice expressing <strong>the</strong> α3 subunit exclusively in noradrenergic neurons, only <strong>the</strong><br />
hypo<strong>the</strong>rmic effect of ketamine/xylazine was increased compared to wild type mice.<br />
Medetomidine, <strong>an</strong> α2-adrenergic agonist, induced a comparable decrease in locomotor activity in<br />
α3KO <strong>an</strong>d wild-type mice, whereas <strong>the</strong> duration of <strong>the</strong> loss of righting reflex was increased <strong>an</strong>d<br />
<strong>the</strong> hypo<strong>the</strong>rmic effects were slightly less pronounced in α3KO mice. All <strong>an</strong>es<strong>the</strong>tic endpoints<br />
were comparable <strong>for</strong> both genotypes with ketamine, only <strong>the</strong> decrease in body temperature was<br />
more pronounced in α3KO mice. The hypnotic, immobilizing <strong>an</strong>d hypo<strong>the</strong>rmic responses to<br />
pentobarbital were enh<strong>an</strong>ced in α3KO mice. In contrast, <strong>the</strong> hypnotic <strong>an</strong>d hypo<strong>the</strong>rmic response<br />
to <strong>the</strong> GABAergic drugs midazolam <strong>an</strong>d etomidate was reduced in α3KO mice.<br />
Our results show that α3 subunit-containing GABAA receptors differentially modulate <strong>the</strong> effects<br />
of intravenous general <strong>an</strong>es<strong>the</strong>tics, depending on <strong>the</strong> mech<strong>an</strong>ism of action of <strong>the</strong> <strong>an</strong>es<strong>the</strong>tic<br />
agent. The increased sensitivity to ketamine/xylazine in α3KO mice is at least in part due to <strong>the</strong><br />
lack of α3 subunit-containing GABAA receptors in central noradrenergic neurons.<br />
Disclosures: U. Rudolph, None; C.J. Straub, None; H. Lau, None; R. Parlato, None; G.<br />
Schuetz, None; J. Fritschy, None.<br />
Poster<br />
513. GABA-A Receptors: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 513.18/B107<br />
Topic: B.02.o. GABA(A) receptors: Physiology<br />
Support: NIH gr<strong>an</strong>t NS045950<br />
South Carolina Honors College Science Undergraduate Research Fellowship<br />
Title: Effect of <strong>the</strong> <strong>an</strong>xiolytic SB-205384 on recombin<strong>an</strong>t GABAA receptors<br />
Authors: L. S. HEIDELBERG 1 , *J. L. FISHER 2 ;<br />
1 2<br />
Honors Col., Univ. So Carolina, Columbia, SC; Dept Pharmacol, Physiol & Neurosci, Univ. So<br />
Carolina Sch. Med., Columbia, SC<br />
Abstract: M<strong>an</strong>y drugs used to treat <strong>an</strong>xiety act as positive modulators of <strong>the</strong> GABAA receptors.<br />
The GABAA receptors mediate fast inhibitory tr<strong>an</strong>smission <strong>an</strong>d c<strong>an</strong> be assembled <strong>from</strong> a
combination of at least 16 different subunits. The subunit composition of <strong>the</strong> receptor determines<br />
its pharmacological <strong>an</strong>d functional properties <strong>an</strong>d subunit expression varies regionally<br />
throughout <strong>the</strong> brain. A major goal in treatment is to reduce unw<strong>an</strong>ted side-effects by producing<br />
drugs that are more subunit-selective <strong>an</strong>d that modulate a discrete population of receptors. The<br />
<strong>an</strong>xiolytic SB-205384 is widely considered to act selectively at α3-containing GABAA receptors.<br />
However, its activity at recombin<strong>an</strong>t receptors has been compared only among α1-, α2- <strong>an</strong>d α3containing<br />
receptors. The goal of this work was to examine <strong>the</strong> activity of SB-205384 on<br />
recombin<strong>an</strong>t receptors containing each of <strong>the</strong> six different α subunits to determine if it is truly<br />
α3-selective. The effect of SB-205384 on <strong>the</strong> response to GABA was measured with whole-cell<br />
<strong>an</strong>d excised-patch recordings <strong>from</strong> tr<strong>an</strong>siently tr<strong>an</strong>sfected HEK293T mammali<strong>an</strong> cells.<br />
We found that recombin<strong>an</strong>t receptors containing <strong>the</strong> α3, α5 <strong>an</strong>d α6 subunits were all potentiated<br />
by SB-205384, <strong>an</strong>d that <strong>the</strong> α6 subunit conferred <strong>the</strong> greatest sensitivity to this modulator. With<br />
rapid application recordings, we found that SB-205384 slowed <strong>the</strong> decay rate of <strong>the</strong> response to<br />
high GABA concentrations. To determine <strong>the</strong> structural basis <strong>for</strong> high sensitivity to SB-205384,<br />
we compared <strong>the</strong> response of α1/α6 chimeric subunits. The results showed that <strong>the</strong> extracellular<br />
N-terminal domain of <strong>the</strong> α6 subunit conferred its higher sensitivity. Our studies show that SB-<br />
205384 is not <strong>an</strong> α3-selective modulator, <strong>an</strong>d instead acts on a variety of GABAA receptor<br />
iso<strong>for</strong>ms. These findings have implications <strong>for</strong> <strong>the</strong> side-effect profile of this <strong>an</strong>xiolytic as well as<br />
<strong>for</strong> its use in neuronal <strong>an</strong>d <strong>an</strong>imal studies as a marker <strong>for</strong> contribution <strong>from</strong> α3-containing<br />
receptors.<br />
Disclosures: L.S. Heidelberg, None; J.L. Fisher, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.1/B108<br />
Topic: B.02.r. Glycine receptors<br />
Support: NIH Gr<strong>an</strong>t AA006399<br />
NIH Gr<strong>an</strong>t GM47818<br />
NIH Gr<strong>an</strong>t AA013378<br />
Title: Evidence <strong>for</strong> <strong>the</strong> assignment of intra-subunit-facing residues in tr<strong>an</strong>smembr<strong>an</strong>e segments<br />
three <strong>an</strong>d four of <strong>the</strong> glycine receptor
Authors: *M. L. MCCRACKEN 1 , L. M. MCCRACKEN 1 , D. GONG 1 , J. R. TRUDELL 2 , R. A.<br />
HARRIS 1 ;<br />
1 2<br />
Waggoner Ctr. Alcohol <strong>an</strong>d Addiction Resch, Univ. Texas Austin, Austin, TX; Anes<strong>the</strong>sia,<br />
St<strong>an</strong><strong>for</strong>d Univ. Sch. of Med., St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Glycine receptors (GlyRs) are pentameric lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels that mediate<br />
inhibitory neurotr<strong>an</strong>smission in <strong>the</strong> brainstem <strong>an</strong>d spinal cord. The tr<strong>an</strong>smembr<strong>an</strong>e (TM)<br />
domains of GlyR subunits are composed of four alpha-helical segments (TM1-4). Previous<br />
studies have investigated <strong>the</strong> orientation of amino acid residues in TM1-3, however, less is<br />
known about <strong>the</strong> orientation of residues in TM4 <strong>an</strong>d <strong>the</strong>ir proximity to TM3 residues. In <strong>the</strong><br />
present study we investigated <strong>the</strong> proximity of A288 in TM3 to residues in TM4 <strong>from</strong> M404 to<br />
K411. Eight double mut<strong>an</strong>t GlyRs (A288C/M404C, A288C/F405C, A288C/Y406C,<br />
A288C/W407C, A288C/I408C, A288C/I409C, A288C/Y410C, <strong>an</strong>d A288C/K411C) <strong>an</strong>d<br />
corresponding single mut<strong>an</strong>ts (A288C, M404C, F405C, Y406C, W407C, I408C, I409C, Y410C,<br />
<strong>an</strong>d K411C) were constructed using site-directed mutagenesis. Receptors were expressed in<br />
Xenopus laevis oocytes <strong>an</strong>d glycine responses were measured using two-electrode voltage clamp<br />
electrophysiology. Following <strong>an</strong> application of HgCl2, <strong>the</strong> glycine concentration-response curves<br />
of <strong>the</strong> A288C/W407C <strong>an</strong>d A288C/Y410C double mut<strong>an</strong>ts were shifted leftward, suggesting that<br />
both W407 <strong>an</strong>d Y410 in TM4 are able to <strong>for</strong>m cross-links with A288 in TM3. Reversal of this<br />
effect was observed following <strong>the</strong> application of <strong>the</strong> reducing agent dithiothreitol (DTT). There<br />
was no effect of HgCl2 or DTT on wild-type receptors or <strong>an</strong>y of <strong>the</strong> single mut<strong>an</strong>ts tested. In<br />
addition, ch<strong>an</strong>ges in glycine EC50 responses were observed in <strong>the</strong> A288C/Y406C,<br />
A288C/W407C, A288C/I409C, <strong>an</strong>d A288C/Y410C double mut<strong>an</strong>ts when HgCl2 was co-applied<br />
with maximal glycine concentrations suggesting that Y406 <strong>an</strong>d I409, in addition to W407 <strong>an</strong>d<br />
Y410, are in proximity to <strong>for</strong>m cross-links with A288 when <strong>the</strong> receptor is activated. Toge<strong>the</strong>r<br />
with published data <strong>from</strong> Lobo et al. (2006 <strong>an</strong>d 2008), our findings support <strong>the</strong> assignment of<br />
intra-subunit facing residues in TM3 <strong>an</strong>d TM4 of <strong>the</strong> glycine receptor, which is not in agreement<br />
with data published <strong>from</strong> <strong>the</strong> GABAA receptor.<br />
Disclosures: M.L. McCracken, None; L.M. McCracken, None; D. Gong, None; J.R.<br />
Trudell, None; R.A. Harris, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.2/B109<br />
Topic: B.02.r. Glycine receptors
Support: NIH R01 AA-15150-04<br />
CONICYT AT-24080125<br />
Title: Pharmacological concentrations of eth<strong>an</strong>ol positively modulate a chimeric α1 GlyR - γ2<br />
GABAA receptor<br />
Authors: *P. A. CASTRO, A. AVILA, L. G. AGUAYO;<br />
Physiol., Univ. Concepcion, Concepcion, Chile<br />
Abstract: The medical consequences of eth<strong>an</strong>ol abuse represent a signific<strong>an</strong>t health problem<br />
around <strong>the</strong> world. Concentrations of eth<strong>an</strong>ol in <strong>the</strong> r<strong>an</strong>ge 10-100 mM produce modifications in<br />
motor, sensorial <strong>an</strong>d cognitive functions. It is likely that some of <strong>the</strong>se effects depend on<br />
modifications of <strong>the</strong> function of glycine (GlyR) <strong>an</strong>d γ-aminobutyric acid (GABAAR) receptors.<br />
Recent studies in our laboratory showed that <strong>the</strong> sensitivity of α1 GlyR to pharmacological<br />
concentrations of eth<strong>an</strong>ol depends on its interaction with Gβγ dimers through two basic amino<br />
acid motifs located in <strong>the</strong> intracellular loop (IL, 316 RFKRK <strong>an</strong>d 385 KK). On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>the</strong><br />
mech<strong>an</strong>ism of eth<strong>an</strong>ol to potentiate GABAAR is much less understood <strong>an</strong>d <strong>the</strong> relev<strong>an</strong>ce of <strong>the</strong><br />
IL in <strong>the</strong>se receptors on eth<strong>an</strong>ol sensitivity has not been investigated. There<strong>for</strong>e, we decided to<br />
initiate a systematic evaluation on <strong>the</strong> role of <strong>the</strong> IL loop of GABAA subunits on Gβγ interaction<br />
<strong>an</strong>d eth<strong>an</strong>ol potentiation through two experimental approaches. Using fusion proteins GST-IL γ2<br />
toge<strong>the</strong>r with pull-down assays, we found that this GABAA subunit interacted with Gβγ to a<br />
similar extent as α1 GlyR. Interestingly, <strong>the</strong> binding of Gβγ was almost linearly reduced with α1,<br />
α4 <strong>an</strong>d α6 showing some specificity in <strong>the</strong> interaction. Based on strong binding shown by <strong>the</strong><br />
GABAAR γ2 subunit, we constructed a chimeric receptor between α1 GlyR <strong>an</strong>d ILγ2. Next,<br />
electrophysiological recordings <strong>from</strong> <strong>the</strong> α1-γ2 chimeric receptor revealed expression of a<br />
functional receptor activated by glycine (EC50 199 ± 44 µM), but insensitive to eth<strong>an</strong>ol (15±4%).<br />
Homologation of <strong>the</strong> TM4 region in <strong>the</strong> chimera to equivalent residues in α1 GlyR reduced <strong>the</strong><br />
sensitivity to glycine (56±3 µM). Additional sequence <strong>an</strong>alysis showed that <strong>the</strong> NH2 (near TM3)<br />
<strong>an</strong>d COOH (near TM4) terminal regions of <strong>the</strong> γ2 insertion (IL) have different residues compared<br />
to its α1 GlyR counterpart. Hence, <strong>the</strong>se two regions in <strong>the</strong> chimera were mutated to homologate<br />
α1 GlyR. Homologation of <strong>the</strong> sequence near TM3 provided a phenotype nearly identical to α1<br />
GlyR (EC50 = 49±1 µM vs EC50 = 40±1), but still insensitive to eth<strong>an</strong>ol (8±3%). Finally, unlike<br />
α1 GlyR, <strong>the</strong> COOH terminal region of <strong>the</strong> γ2 IL has <strong>an</strong> al<strong>an</strong>ine residue at position 385.<br />
Interestingly, when this residue (A385) was replaced by <strong>the</strong> counterpart K385 α1 GlyR, <strong>the</strong><br />
chimera showed nearly full sensitivity to eth<strong>an</strong>ol, similar to α1 GlyR (41±2% vs 53±5%).<br />
Altoge<strong>the</strong>r, <strong>the</strong>se findings show that <strong>the</strong> γ2 subunit of GABAAR binds Gβγ <strong>an</strong>d c<strong>an</strong> be modulated<br />
by eth<strong>an</strong>ol <strong>the</strong>reby suggesting that it could be <strong>an</strong> import<strong>an</strong>t target <strong>for</strong> eth<strong>an</strong>ol action on <strong>the</strong><br />
GABAAR.<br />
Disclosures: P.A. Castro, None; A. Avila, None; L.G. Aguayo, None.<br />
Poster
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.3/B110<br />
Topic: B.02.r. Glycine receptors<br />
Title: Cell-type specific effect of <strong>the</strong> KK385/386AA mutation on eth<strong>an</strong>ol potentiation of glycine<br />
α1 receptors expressed in xenopus oocytes <strong>an</strong>d HEK-293 cells<br />
Authors: *L. ZHANG 1 , W. XIONG 1 , D. M. LOVINGER 2 ;<br />
1 NIAAA, NIH, Be<strong>the</strong>sda, MD; 2 NIAAA/NIH, Be<strong>the</strong>sda, MD<br />
Abstract: The glycine receptors (GlyRs) are thought to be a primary target <strong>for</strong> eth<strong>an</strong>ol (EtOH)<br />
action in <strong>the</strong> central nervous system. Recent studies have reported that two positively charged<br />
residues at 385-386 in <strong>the</strong> large cytoplasmic domain of <strong>the</strong> GlyR α1 subunit are critical <strong>for</strong> <strong>the</strong><br />
sensitivity of GlyRs to G-protein <strong>an</strong>d EtOH-induced potentiation of glycine-activated current<br />
(IGly). However, <strong>the</strong> effects of <strong>the</strong>se mutations on G-protein modulation <strong>an</strong>d EtOH potentiation of<br />
GlyRs were only reported in HEK-293 cells. Xenopus oocytes have been consistently used as <strong>an</strong><br />
expression system to test <strong>the</strong> functionality <strong>an</strong>d sensitivity of recombin<strong>an</strong>t GlyRs to EtOHinduced<br />
potentiation. In this study, we examined <strong>the</strong> sensitivity of <strong>the</strong> wild type (WT) <strong>an</strong>d<br />
KK385/386AA mut<strong>an</strong>t receptors to EtOH expressed in Xenopus oocytes. Cytoplasmic<br />
application of GDPβS signific<strong>an</strong>tly reduced GTPγs potentiation of hum<strong>an</strong> α1 GlyR expressed in<br />
Xenopus oocytes. Replacement of <strong>the</strong> amino acid residues at 385-386 with al<strong>an</strong>ines slightly<br />
increased <strong>the</strong> apparent agonist affinity of <strong>the</strong> GlyRs. Consistent with a previous observation in<br />
HEK-293 cells, <strong>the</strong> point-mutations at 385/386 signific<strong>an</strong>tly inhibited <strong>the</strong> GTPγs-induced<br />
potentiation of IGly when expressed in Xenopus oocytes. However, <strong>the</strong> KK385/386AA mutation<br />
did not signific<strong>an</strong>tly affect EtOH (100 mM)-induced potentiation of IGly when <strong>the</strong> mut<strong>an</strong>t<br />
receptors were expressed in Xenopus oocytes. The magnitude of EtOH-induced potentiation of<br />
<strong>the</strong> WT <strong>an</strong>d mut<strong>an</strong>t receptors were 82 ± 13% <strong>an</strong>d 89 ± 9% (p = 0.4, n = 9-11). In contrast, <strong>the</strong><br />
mut<strong>an</strong>t receptors appeared to be less sensitive th<strong>an</strong> <strong>the</strong> WT receptors to EtOH-induced<br />
potentiation when expressed in HEK-293 cells (71 ± 11% <strong>an</strong>d 31 ± 7%, p < 0.02). Moreover, <strong>the</strong><br />
magnitude of EtOH potentiation of IGly was not signific<strong>an</strong>tly different in Xenopus oocytes<br />
previously injected with mRNAs isolated <strong>from</strong> HEK-293 cells expressing <strong>the</strong> WT <strong>an</strong>d<br />
KK385/386AA receptors. These observations suggest that <strong>the</strong> effect of <strong>the</strong> KK385/386 mutation<br />
on <strong>the</strong> sensitivity of GlyRs to EtOH is cell-type specific. Supported by <strong>the</strong> NIAAA Division of<br />
Intramural Clinical <strong>an</strong>d Basic Research.<br />
Disclosures: L. Zh<strong>an</strong>g, None; W. Xiong, None; D.M. Lovinger, None.<br />
Poster
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.4/B111<br />
Topic: B.02.r. Glycine receptors<br />
Support: NIH Gr<strong>an</strong>t ROIAA151-01<br />
Title: Multiples residues along <strong>the</strong> α2 glycine receptor control eth<strong>an</strong>ol sensitivity<br />
Authors: *G. MORAGA-CID 1 , G. E. YÉVENES 1 , J. GUZMAN 1 , M. FIGUEROA 1 , R. W.<br />
PEOPLES 2 , L. G. AGUAYO 1 ;<br />
1 Univ. Concepcion, Concepcion, Chile; 2 Marquette Univ., Milwaukee, WI<br />
Abstract: Glycine receptors (GlyRs) belong to <strong>the</strong> lig<strong>an</strong>d-gated ion ch<strong>an</strong>nel superfamily (LGIC)<br />
<strong>an</strong>d play a critical role in <strong>the</strong> control of neuronal excitability in <strong>the</strong> mammali<strong>an</strong> nervous system.<br />
At present, it is well recognized that <strong>the</strong> α1-containing GlyRs are potentiated by low eth<strong>an</strong>ol<br />
concentrations (
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.5/B112<br />
Topic: B.02.r. Glycine receptors<br />
Support: NH&MRC Project Gr<strong>an</strong>t 230806<br />
NH&MRC Research fellowship 157209<br />
Title: Con<strong>for</strong>mational ch<strong>an</strong>ges in extracellular loop 2 associated with signal tr<strong>an</strong>sduction in <strong>the</strong><br />
glycine receptor<br />
Authors: *J. M. CEDERHOLM 1,2 , N. L. ABSALOM 3 , S. SUGIHARTO 1 , W. KAPLAN 3 , T.<br />
M. LEWIS 1 , P. R. SCHOFIELD 2,3 ;<br />
1 Sch. of Med. Sciences, Univ. of New South Wales, Sydney, Australia; 2 Prince of Wales Med.<br />
Res. Inst., Sydney, Australia; 3 Garv<strong>an</strong> Inst. of Med. Res., Sydney, Australia<br />
Abstract: The Cys-loop family of lig<strong>an</strong>d gated ion ch<strong>an</strong>nels (LGICs) includes <strong>the</strong> nicotinic<br />
acetylcholine receptor (nAChR), serotonin type 3 receptor (5-HT3R), γ-aminobutyric acid type A<br />
receptors (GABAAR) <strong>an</strong>d <strong>the</strong> glycine receptor (GlyR). These receptors are pentamers with each<br />
subunit containing four tr<strong>an</strong>smembr<strong>an</strong>e domains (M1-M4) (Betz, 1990) <strong>an</strong>d a large extracellular<br />
domain (ECD). The lig<strong>an</strong>d binding site is located in <strong>the</strong> ECD <strong>an</strong>d is spatially dist<strong>an</strong>t <strong>from</strong> <strong>the</strong><br />
tr<strong>an</strong>smembr<strong>an</strong>e domain M2, which lines <strong>the</strong> pore (Unwin, 1993; Corringer et. al., 2000). Several<br />
studies have proposed that loops 2 <strong>an</strong>d 7 of <strong>the</strong> ECD might be involved in coupling lig<strong>an</strong>d<br />
binding to receptor activation. In <strong>the</strong> present study, six residues of loop 2 in <strong>the</strong> ECD of <strong>the</strong><br />
hum<strong>an</strong> GlyR believed to be critical to <strong>the</strong> signal tr<strong>an</strong>sduction process were investigated by<br />
assessing <strong>the</strong> availability of substituted cysteines <strong>for</strong> covalent modification. Mut<strong>an</strong>ts were<br />
produced using site-directed mutagenesis <strong>an</strong>d expressed in HEK293 cells. Concentrationresponse<br />
curves <strong>for</strong> three of <strong>the</strong> mut<strong>an</strong>ts showed small increases in EC50, compared to WT, while<br />
<strong>the</strong> remaining three mut<strong>an</strong>t receptors exhibited a 29-fold increase in <strong>the</strong> EC50 <strong>for</strong> glycine. Thus,<br />
introducing mutations in <strong>the</strong> ECD loop 2 disrupts <strong>the</strong> tr<strong>an</strong>sduction mech<strong>an</strong>ism that couples<br />
lig<strong>an</strong>d binding <strong>an</strong>d ch<strong>an</strong>nel opening. By using two derivates of meth<strong>an</strong>e thiosulfonate (MTSET<br />
<strong>an</strong>d MTSES) we also demonstrate that loop 2 undergoes a con<strong>for</strong>mational ch<strong>an</strong>ge upon lig<strong>an</strong>d<br />
binding. This demonstrates <strong>the</strong> import<strong>an</strong>t role played by <strong>the</strong> ECD loop 2 of <strong>the</strong> GlyR in signal<br />
tr<strong>an</strong>sduction, which is likely to be applicable to o<strong>the</strong>r members of <strong>the</strong> Cys-loop family.
Disclosures: J.M. Cederholm, None; N.L. Absalom, None; S. Sugiharto, None; W. Kapl<strong>an</strong>,<br />
None; T.M. Lewis, None; P.R. Schofield, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.6/B113<br />
Topic: B.02.r. Glycine receptors<br />
Support: Deutsche Forschungsgemeinschaft<br />
Europe<strong>an</strong> Union (Neuro Cypress, HEALTH-F4-2008-202088)<br />
Elitenetzwerk Bayern (K-BM-2003-85)<br />
Title: The TM3-4 loop of <strong>the</strong> glycine receptor subunits regulates ion ch<strong>an</strong>nel rescue upon<br />
expression of independently folding subunit fragments<br />
Authors: *C. VILLMANN 1 , H. MEISELBACH 1 , J. OERTEL 2 , C.-M. BECKER 1 ;<br />
1 2<br />
Univ. Erl<strong>an</strong>gen-Nurnberg, D-91054 Erl<strong>an</strong>gen, Germ<strong>an</strong>y; Inserm, u975, Univ. Pierre et Marie<br />
Curie, 75634 Paris, Fr<strong>an</strong>ce<br />
Abstract: Glycine receptors (GlyRs) are <strong>the</strong> major inhibitory neurotr<strong>an</strong>smitter receptors in<br />
brainstem <strong>an</strong>d spinal cord in <strong>the</strong> CNS. The adult glycine receptor is a pentameric complex of 2<br />
alpha <strong>an</strong>d 3 beta subunits comprising ei<strong>the</strong>r <strong>an</strong> alpha1/beta or <strong>an</strong> alpha3/beta subunit<br />
combination.<br />
Recently, we have shown that <strong>the</strong> ion ch<strong>an</strong>nel function of <strong>the</strong> truncated α1 vari<strong>an</strong>t <strong>from</strong> <strong>the</strong><br />
oscillator mouse was rescued by coexpression with <strong>an</strong> independent tail domain. The efficiency of<br />
rescue depends on a basic motif in <strong>the</strong> TM3-4 loop (RRKRRH), which is absent <strong>from</strong> <strong>the</strong><br />
oscillator subunit mut<strong>an</strong>t. Truncation of <strong>the</strong> tail domain (TM3-4 loop, TM4 <strong>an</strong>d <strong>the</strong> C-terminus),<br />
starting <strong>from</strong> <strong>the</strong> 5’ end of <strong>the</strong> TM3-4 loop, resulted in loss of rescue efficiency. Since <strong>the</strong><br />
sequence homology among GlyR subunits is high, except <strong>for</strong> <strong>the</strong> TM3-4 loop, we tested <strong>the</strong><br />
hypo<strong>the</strong>sis that ion ch<strong>an</strong>nel function c<strong>an</strong> be rescued by independently folding domains of<br />
different alpha-subunits. Independently folding domains of <strong>the</strong> GlyR subunits alpha1 <strong>an</strong>d alpha3<br />
that included <strong>the</strong> N-terminus, <strong>the</strong> TM1-3 <strong>an</strong>d <strong>the</strong> first amino acids of <strong>the</strong> TM3-4 loop, as well as<br />
tail domains harboring most of <strong>the</strong> TM3-4 loop, TM4 <strong>an</strong>d <strong>the</strong> short C-terminus were generated.<br />
Upon recombin<strong>an</strong>t expression in HEK293 cells, <strong>the</strong> subunit vari<strong>an</strong>ts alpha3-trc <strong>an</strong>d alpha1-trc<br />
integrated into <strong>the</strong> plasma membr<strong>an</strong>e. None of <strong>the</strong>se truncated constructs was able to mediate
glycine induced chloride currents. <strong>When</strong> alpha3 tail or alpha1 tail constructs were introduced by<br />
coexpression, functional ch<strong>an</strong>nels were observed. These results demonstrate that not only <strong>the</strong><br />
GlyR alpha1 subunit is composed of independently domains, but o<strong>the</strong>r members of this receptor<br />
family as well. Next, we attempted <strong>an</strong> inter-subunit rescue of ion ch<strong>an</strong>nel function: The truncated<br />
alpha3 subunit vari<strong>an</strong>t was paired with <strong>an</strong> alpha1 tail <strong>an</strong>d vice versa. While <strong>the</strong> truncated alpha3<br />
was functionally rescued by <strong>an</strong> alpha1 tail, <strong>the</strong> alpha1 rescue with <strong>an</strong> alpha3 tail failed.<br />
Bioin<strong>for</strong>matic prediction suggests that <strong>the</strong> reason <strong>for</strong> rescue failure was not due to differences in<br />
TM4 of alpha3 compared to alpha1. Here, we demonstrate that differences in <strong>the</strong> TM3-4 loops<br />
sequences e.g. alternative splicing cassettes, phosphorylation sites of various GlyR alpha<br />
subunits are import<strong>an</strong>t regulatory domains <strong>for</strong> ion ch<strong>an</strong>nel function.<br />
Disclosures: C. Villm<strong>an</strong>n, None; H. Meiselbach, None; J. Oertel, None; C. Becker, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.7/B114<br />
Topic: B.02.r. Glycine receptors<br />
Support: MPG<br />
DFG (PA1623/2-1; Qu116/5-1)<br />
Schram-Stiftung (BQ)<br />
Fonds der Chemische Industrie<br />
Title: Interaction between syndapins <strong>an</strong>d <strong>the</strong> beta subunit of <strong>the</strong> glycine receptor<br />
Authors: I. DEL PINO-PARIENTE 1 , D. KOCH 2 , B. QUALMANN 2 , H. BETZ 1 , *I. F.<br />
PAARMANN 1 ;<br />
1 2<br />
Max-Pl<strong>an</strong>ck Inst. Brain Res., Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y; Inst. of Biochem. I, Friedrich-<br />
Schiller-University Jena, Jena, Germ<strong>an</strong>y<br />
Abstract: The Glycine Receptor (GlyR) mediates inhibitory neurotr<strong>an</strong>smission in spinal cord<br />
<strong>an</strong>d brainstem. GlyRs are composed of alpha (1-4) <strong>an</strong>d beta subunits which assemble into<br />
alpha(5) homomeric or alpha(2)beta(3) heteromeric receptors. The beta subunit (GlyRbeta)<br />
<strong>an</strong>chors GlyRs to <strong>the</strong> subsynaptic cytoskeleton via <strong>the</strong> cytoplasmic scaffolding protein gephyrin.
With <strong>the</strong> exception of gephyrin, no proteins have yet been found to interact directly with<br />
GlyRbeta.<br />
To get fur<strong>the</strong>r insight into <strong>the</strong> molecular org<strong>an</strong>ization of inhibitory glycinergic post-synapses, a<br />
proteomic screen using a GST-fusion protein containing amino acids 400-477 of <strong>the</strong> large<br />
intracellular loop of <strong>the</strong> GlyRbeta was per<strong>for</strong>med. 50 kDa protein b<strong>an</strong>ds isolated <strong>from</strong> rat brain<br />
tissue extracts were identified by mass spectrometric <strong>an</strong>alysis as Syndapin I (SdpI). Syndapin I to<br />
III are Src-homology 3 (SH3) <strong>an</strong>d F-BAR domain-containing proteins known to be involved in<br />
<strong>the</strong> intracellular tr<strong>an</strong>sport of m<strong>an</strong>y proteins additionally to <strong>the</strong>ir function in presynaptic vesicle<br />
<strong>for</strong>mation.<br />
A direct interaction of SdpI <strong>an</strong>d both splice vari<strong>an</strong>ts of SdpII with <strong>the</strong> GlyRbeta-loop was<br />
demonstrated by GST pull-down using SdpI <strong>an</strong>d SdpII constructs expressed in E. coli. Moreover,<br />
upon coexpression in <strong>an</strong> Afric<strong>an</strong> Green Monkey SV40-tr<strong>an</strong>s<strong>for</strong>med kidney fibroblast cell line,<br />
GlyRbeta <strong>an</strong>d Sdps colocalized. Mapping experiments of <strong>the</strong> binding region <strong>for</strong> SdpI in <strong>the</strong><br />
GlyRbeta-loop showed that 22 amino acids containing <strong>an</strong> SH3 lig<strong>an</strong>d binding motif are sufficient<br />
<strong>for</strong> <strong>the</strong> interaction of <strong>the</strong> receptor with SdpI. Moreover, <strong>the</strong> SH3 domain of SdpI was found to be<br />
essential <strong>for</strong> a strong interaction with <strong>the</strong> recombin<strong>an</strong>t GST tagged GlyRbeta-loop.<br />
Double immunofluorescent labeling of SdpI <strong>an</strong>d several inhibitory pre- <strong>an</strong>d postsynaptic markers<br />
in rat spinal cord neurons revealed a colocalization of gephyrin <strong>an</strong>d vesicular inhibitory amino<br />
acid tr<strong>an</strong>sporter positive punctae with SdpI. This supports a relev<strong>an</strong>ce of <strong>the</strong> syndapin-GlyR<br />
interaction. Whe<strong>the</strong>r syndapins are crucial <strong>for</strong> a correct synaptic targeting of GlyR was examined<br />
in SdpI-deficient mice. Analyses of brainstem slices of SdpI-knock out mice, however, showed<br />
that localization <strong>an</strong>d appear<strong>an</strong>ce of GlyR <strong>an</strong>d Gephyrin clusters were unaltered. Thus, <strong>the</strong> in vivo<br />
role of <strong>the</strong> SdpI-GlyR interaction does not relate to targeting of GlyR <strong>an</strong>d <strong>the</strong> GlyR-associated<br />
scaffold protein Gephyrin to inhibitory postsynaptic sites.<br />
Disclosures: I. del Pino-Pariente, None; D. Koch, None; B. Qualm<strong>an</strong>n, None; H. Betz,<br />
None; I.F. Paarm<strong>an</strong>n, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.8/B115<br />
Topic: B.02.r. Glycine receptors<br />
Support: Thyssen-Foundation<br />
Title: Production of a monoclonal <strong>an</strong>tibody against <strong>the</strong> glycine receptor β subunit
Authors: F. WELTZIEN 1 , I. PAARMANN 1 , *G. A. O'SULLIVAN 2,1 , H. BETZ 1 ;<br />
1 2<br />
Neurochemistry, MPI <strong>for</strong> Brain Res., Fr<strong>an</strong>kfurt, Germ<strong>an</strong>y; Zerial Lab., MPI-CBG, Dresden,<br />
Germ<strong>an</strong>y<br />
Abstract: Glycine receptors (GlyRs) belong to <strong>the</strong> cis-loop lig<strong>an</strong>d-gated ion ch<strong>an</strong>nel family of<br />
neurotr<strong>an</strong>smitter receptors <strong>an</strong>d mediate fast inhibitory neurotr<strong>an</strong>smission in <strong>the</strong> central nervous<br />
system (CNS), especially in spinal cord <strong>an</strong>d brainstem. Synaptic GlyRs are composed of two α<br />
(1-4) <strong>an</strong>d three β subunits. The β subunits (GlyRβ) mediate postsynaptic <strong>an</strong>choring of GlyRs by<br />
interacting with <strong>the</strong> scaffolding protein gephyrin via <strong>the</strong> large cytosolic loop. In addition GlyRβ<br />
is involved in lig<strong>an</strong>d binding. In situ hybridization studies suggest a widespread expression<br />
throughout <strong>the</strong> mammali<strong>an</strong> CNS. However, at <strong>the</strong> protein level respective receptors have not<br />
been detected, due to <strong>the</strong> lack of a GlyRβ specific <strong>an</strong>tibody.<br />
Here, we describe <strong>the</strong> production of a monoclonal <strong>an</strong>tibody specific <strong>for</strong> <strong>the</strong> GlyRβ. For<br />
immunization of mice, we used a 120 amino acid peptide, which corresponded to <strong>the</strong> complete<br />
cytosolic loop between tr<strong>an</strong>smembr<strong>an</strong>e domains 3 <strong>an</strong>d 4.<br />
The monoclonal <strong>an</strong>tibody produced showed good specificity when used <strong>for</strong> Western blot <strong>an</strong>alysis<br />
of GlyR purified <strong>from</strong> spinal cord, where it recognized solely a b<strong>an</strong>d of 58 kDa, corresponding to<br />
<strong>the</strong> size of <strong>the</strong> GlyRβ protein. Immunocytochemical experiments with tr<strong>an</strong>sfected HEK-T 293<br />
cells confirmed specific recognition by <strong>the</strong> <strong>an</strong>tibody of GlyRβ constructs only, but not of GlyRα<br />
subunits 1-4. Staining of mouse spinal cord sections produced a punctate pattern similar to that<br />
seen with mAb4, <strong>an</strong> <strong>an</strong>tibody directed against GlyRα. Fur<strong>the</strong>rmore, GlyRβ staining prominently<br />
colocalized with <strong>the</strong> inhibitory presynaptic marker protein VIAAT <strong>an</strong>d gephyrin. Staining by <strong>the</strong><br />
<strong>an</strong>tibody could be completely blocked by pre-incubation with a peptide containing <strong>the</strong> first 20<br />
amino acids of <strong>the</strong> immunization sequence.<br />
In conclusion, data obtained demonstrate a specific interaction of <strong>the</strong> new <strong>an</strong>tibody with GlyRβ;<br />
interactions with GlyRα 1-4 were not observed. This monoclonal <strong>an</strong>tibody might be useful to<br />
unravel <strong>the</strong> nature <strong>an</strong>d distribution of GlyRβ containing receptors in <strong>the</strong> mammali<strong>an</strong> CNS.<br />
Disclosures: F. Weltzien, None; I. Paarm<strong>an</strong>n, None; G.A. O'Sulliv<strong>an</strong>, monoclonal <strong>an</strong>tibody<br />
may be licensed to as yet unspecified comp<strong>an</strong>y, C. O<strong>the</strong>r Research Support (receipt of drugs,<br />
supplies, equipment or o<strong>the</strong>r in-kind support); H. Betz, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.9/B116<br />
Topic: B.02.r. Glycine receptors
Support: BBSRC strategic studentship<br />
Title: A characterisation of inhibitory neurotr<strong>an</strong>smission in <strong>the</strong> dorsal raphe (DR)<br />
Authors: *E. P. MAGUIRE 1 , D. BELELLI 1 , J. J. LAMBERT 1 , D. J. K. BALFOUR 1 , V. J.<br />
BROWN 2 ;<br />
1 Ctr. <strong>for</strong> Neuroscience, Div. of Med. Sci., Univ. of Dundee, Dundee, United Kingdom; 2 Sch. of<br />
Psychology, Univ. of St. Andrews, St. Andrews, United Kingdom<br />
Abstract: The DR is a primary source of serotonergic projections to <strong>the</strong> <strong>for</strong>ebrain <strong>an</strong>d is<br />
implicated in <strong>the</strong> induction of depressive <strong>an</strong>d <strong>an</strong>xiety related behaviours in <strong>an</strong>imal models of<br />
stress. The DR receives both GABAergic <strong>an</strong>d glycinergic inputs. However, <strong>the</strong> role played by<br />
glycine in modulating DR activity is relatively unknown.<br />
To investigate inhibitory neurotr<strong>an</strong>smission in <strong>the</strong> DR, <strong>the</strong> whole cell patch-clamp technique was<br />
applied to <strong>an</strong> in vitro brain slice preparation. Miniature inhibitory post-synaptic currents<br />
(mIPSCs) were recorded <strong>from</strong> DR neurones of 18-30 day old mice at a holding potential of -<br />
60mV, in <strong>the</strong> presence of 2mM kynurenic acid.<br />
The GABAA receptor <strong>an</strong>tagonist bicuculline greatly reduced <strong>the</strong> frequency of mIPSCs, <strong>the</strong> few<br />
remaining events being abolished by <strong>the</strong> glycine receptor <strong>an</strong>tagonist strychnine. Application of<br />
bicuculline did not induce <strong>an</strong> outward current, fur<strong>the</strong>rmore <strong>the</strong> δ-GABAA receptor selective<br />
agonist THIP (1µM) had little or no effect on <strong>the</strong> holding curent. These observations suggest that<br />
DR neurons express few or no extrasynaptic GABAA receptors. In contrast, strychnine<br />
application revealed <strong>the</strong> presence of a relatively large tonic conduct<strong>an</strong>ce mediated by<br />
extrasynaptic ionotropic glycine receptors. Experiments utilising selective inhibitors of glycine,<br />
or taurine/β-al<strong>an</strong>ine tr<strong>an</strong>sporters revealed <strong>the</strong>se agents to greatly increase this strychninesensitive<br />
tonic conduct<strong>an</strong>ce.<br />
Collectively, <strong>the</strong>se findings indicate that <strong>the</strong> inhibitory neurotr<strong>an</strong>smitters GABA <strong>an</strong>d glycine<br />
mediate different <strong>for</strong>ms of inhibition in <strong>the</strong> DR: GABAA receptors primarily mediate phasic<br />
inhibition whereas glycine receptors are mainly located extrasynaptically <strong>an</strong>d mediate tonic<br />
inhibition. Thus, strychnine sensitive glycine receptors are likely to play a previously<br />
underestimated role in controlling <strong>the</strong> excitability of DR neurones <strong>an</strong>d may contribute to <strong>the</strong><br />
behavioural effects of amino acid tr<strong>an</strong>sporter inhibitors.<br />
Disclosures: E.P. Maguire, None; D. Belelli, None; J.J. Lambert, None; D.J.K. Balfour,<br />
None; V.J. Brown, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 514.10/B117<br />
Topic: B.09.e. O<strong>the</strong>r<br />
Support: Wellcome Project gr<strong>an</strong>t 084651<br />
OTKA 75676<br />
Title: A GABAergic-glycinergic ascending system <strong>from</strong> <strong>the</strong> brainstem reticular core to <strong>the</strong><br />
thalamus<br />
Authors: *L. ACSADY 1 , K. GIBER 1 , V. M. PLATTNER 1 , S. DIEUDONNE 2 , M. A. DIANA 2 ,<br />
H. BOKOR 3 ;<br />
1 Inst. Exp. Med. Hung Acad Sci., Budapest, Hungary; 2 Ecole Normale Supérieure, Paris, Fr<strong>an</strong>ce;<br />
3 Inst. Exp. Med. Hung. Acad. Sci., Budapest, Hungary<br />
Abstract: The ascending activating systems arising <strong>from</strong> <strong>the</strong> brainstem typically exert excitatory<br />
actions on <strong>the</strong> thalamus, which induce a switch in <strong>the</strong> firing mode of thalamocortical cells to<br />
tonic activity, a common feature of <strong>the</strong> awake state. Challenging this classical view of <strong>the</strong><br />
reticulo-thalamic system, here we describe a novel GABAergic-glycinergic pathway <strong>from</strong> <strong>the</strong><br />
midbrain reticular core that selectively innervates <strong>the</strong> intralaminar <strong>an</strong>d midline nuclei of <strong>the</strong><br />
thalamus.<br />
A dense GlyT2-positive axonal plexus was found in <strong>the</strong> intralaminar <strong>an</strong>d midline thalamic nuclei<br />
of EGFP-glycine tr<strong>an</strong>sporter type 2 (GlyT2) tr<strong>an</strong>sgenic mice. Anterograde <strong>an</strong>d retrograde tract<br />
tracing experiments demonstrated that most fibers originate in <strong>the</strong> ipsilateral pontine reticular<br />
nucleus (PnO). The glycinergic component constituted 50% of this reticulo-thalamic pathway.<br />
EGFP-GlyT2 boutons were also GABA immunopositive, <strong>an</strong>d established multiple synapses on<br />
<strong>the</strong> proximal dendrites of relay cells. Extracellular stimulation in vitro in <strong>the</strong> intralaminar nucleus<br />
of <strong>the</strong> EGFP-GlyT2 mice could evoke powerful, monosynaptic glycinergic, GABAergic, or<br />
mixed currents in thalamic relay cells.<br />
EGFP-GlyT2 positive neurons in PnO in vitro demonstrated fast, intrinsic, subthreshold<br />
oscillation of <strong>the</strong> membr<strong>an</strong>e potential. Paired recordings showed that EGFP-GlyT2 neurons c<strong>an</strong><br />
be electrically coupled. EGFP-GlyT2 positive PnO cells in vivo in ketamine-xylazine<br />
<strong>an</strong>es<strong>the</strong>tized <strong>an</strong>imals could be <strong>an</strong>tidromically activated <strong>from</strong> <strong>the</strong> thalamus with short latency<br />
(~2ms). The firing pattern of PnO GlyT2 cells was characterized by rhythmic clusters of spikes<br />
at 50-90 Hz interspersed with high frequency doublets (up to 400 Hz). Clusters occurred in tight<br />
synchrony with cortical Up states recorded in <strong>the</strong> ipsilateral prefrontal cortex. Tr<strong>an</strong>sient<br />
inactivation of prefrontal cortical activity led to <strong>an</strong> overall decrease in firing frequency <strong>an</strong>d a<br />
ch<strong>an</strong>ge in firing pattern. Rhythmic firing was tr<strong>an</strong>s<strong>for</strong>med into <strong>an</strong> irregular activity with spike<br />
clusters of variable length <strong>an</strong>d spike number, but neurons still displayed high frequency doublets.<br />
The GABAergic <strong>an</strong>d glycinergic PnO-thalamic pathway described here likely exerts a strong<br />
inhibitory action on relay cells via high presynaptic firing rates, <strong>an</strong>d <strong>the</strong> multisite innervation of<br />
proximal dendrites. Our data indicate that ra<strong>the</strong>r th<strong>an</strong> purely excitatory in nature, local<br />
interactions, cortical inputs <strong>an</strong>d behavioral state dependent ch<strong>an</strong>ges will determine <strong>the</strong> bal<strong>an</strong>ce of<br />
excitation <strong>an</strong>d inhibition in <strong>the</strong> reticulo-thalamic pathway, which is essential to maintain<br />
conscious state.
Disclosures: L. Acsady, None; K. Giber, None; V.M. Plattner, None; S. Dieudonne,<br />
None; M.A. Di<strong>an</strong>a, None; H. Bokor, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.11/B118<br />
Topic: B.02.r. Glycine receptors<br />
Support: BMBF / DIP G3.2<br />
Title: The LINE1 associated splicing defect of <strong>the</strong> glycine receptor mut<strong>an</strong>t mouse spastic:<br />
Qu<strong>an</strong>tification of aberr<strong>an</strong>t mRNA species by MALDI-TOF mass spectrometry<br />
Authors: *K. BECKER, N. BENDERSKA, M. EBERHARDT, C.-M. BECKER;<br />
Inst. of Biochemistry, Univ. of Erl<strong>an</strong>gen-Nurnberg, Erl<strong>an</strong>gen, Germ<strong>an</strong>y<br />
Abstract: Table of Contents<br />
o The spont<strong>an</strong>eous mouse mut<strong>an</strong>t spastic carries a mut<strong>an</strong>t allele of <strong>the</strong> glycine<br />
receptor beta subunit gene, Glrb, where a LINE1 element has inserted into <strong>an</strong><br />
intronic position. LINE1 elements are <strong>the</strong> most frequent autonomous<br />
retrotr<strong>an</strong>sposons in <strong>the</strong> genomes of hum<strong>an</strong>s <strong>an</strong>d mice, accounting <strong>for</strong> 20 % of <strong>the</strong><br />
total DNA. LINE1 insertions c<strong>an</strong> cause disease in hum<strong>an</strong>s <strong>an</strong>d mice when inserted<br />
into exons, leading to frameshift mutations <strong>an</strong>d premature stop-codons. The more<br />
frequent insertions within gene regulatory sequences or introns c<strong>an</strong> alter <strong>the</strong> level<br />
of mRNA by induction of exon skipping, posttr<strong>an</strong>scriptional repression,<br />
premature polyadenylation, or defective tr<strong>an</strong>scriptional elongation. We used <strong>the</strong><br />
Glrbspa allele present in <strong>the</strong> mut<strong>an</strong>t mouse spastic as a model system to study <strong>the</strong><br />
effects of <strong>an</strong> intronic LINE1 insertion on pre-mRNA splicing. Minigenes were<br />
<strong>an</strong>alyzed that contain defined LINE1 sequence elements, using in vivo splicing<br />
assays, to determine regions sufficient <strong>for</strong> exon skipping. We show that<br />
overexpression or knock-down of specific splicing regulator proteins modulate<br />
skipping in a LINE1 sequence specific m<strong>an</strong>ner. For large scale screeing of<br />
samples, a procedure utilizing mass spectrometric <strong>an</strong>alysis <strong>an</strong>d qu<strong>an</strong>tification of<br />
splice products was established <strong>an</strong>d validated by comparison to both TaqM<strong>an</strong> <strong>an</strong>d<br />
Real-time PCR approaches. We show that <strong>an</strong> intronic LINE element is able to<br />
both, regulate pre-mRNA splicing <strong>an</strong>d modulate mRNA expression levels. This
occurs by interaction with splicing regulatory proteins <strong>an</strong>d by interacting with<br />
defined exonic <strong>an</strong>d/or intronic regulatory sequences.<br />
Disclosures: K. Becker, None; N. Benderska, None; M. Eberhardt, None; C. Becker, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.12/B119<br />
Topic: B.02.r. Glycine receptors<br />
Support: "Heinrich und Alma Vogels<strong>an</strong>g Stiftung"<br />
Title: Histamine is <strong>an</strong> <strong>an</strong>tagonist of recombin<strong>an</strong>t strychnine-sensitive glycine receptors<br />
Authors: *O. KLETKE, H. HATT, G. GISSELMANN;<br />
Ruhr Univ. Bochum, Bochum, Germ<strong>an</strong>y<br />
Abstract: Histamine is a crucial cytokine in <strong>the</strong> peripheral nervous system <strong>an</strong>d <strong>an</strong> import<strong>an</strong>t<br />
neurotr<strong>an</strong>smitter <strong>an</strong>d neuromodulator in <strong>the</strong> brain. Histamine is produced <strong>an</strong>d released by<br />
basophils, mast cells, neurons <strong>an</strong>d enterochromaffin-like cells of <strong>the</strong> stomach. It is involved in<br />
several import<strong>an</strong>t physiological functions like inflammation, sleep control through<br />
tuberomammilary nucleus neurons <strong>an</strong>d as a target <strong>for</strong> metabotropic histamine receptors (H1-H4).<br />
Histamine causes vasodilation, bronchoconstriction <strong>an</strong>d stimulates gastric acid secretion. Glycine<br />
is <strong>the</strong> major inhibitory neurotr<strong>an</strong>smitter in spinal cord <strong>an</strong>d brain stem. There, glycinergic<br />
synapses control motor <strong>an</strong>d sensory pathways by activating strychnine-sensitive glycine<br />
receptors (GlyRs) which belong to <strong>the</strong> ‘Cys-loop’ family of lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels (LGICs).<br />
Several allosteric agents <strong>an</strong>d modulators are known that share <strong>the</strong>ir pharmacological profile with<br />
GABAA receptors (GABAARs).<br />
Previously, we showed that histamine is able to potentiate GABAARs mediated through <strong>the</strong> β<br />
subunit.<br />
Now we were able to show, that histamine is <strong>an</strong> <strong>an</strong>tagonist of GlyRs expressed in Xenopus<br />
oocytes <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e acts in <strong>an</strong> opposite way at <strong>the</strong> main inhibitory LGICs of <strong>the</strong> periphery <strong>an</strong>d<br />
CNS of vertebrates. We investigated homo- <strong>an</strong>d heteropentameric GlyRs with different α<br />
subunits. Histamine decreases glycine induced currents in a dose dependent m<strong>an</strong>ner. The IC50 of<br />
histamine modulation r<strong>an</strong>ges <strong>from</strong> high micromolar to low millimolar concentrations <strong>an</strong>d leads<br />
to a left shift of glycine EC50 in <strong>the</strong> dose response curve. In addition, <strong>the</strong> IC50 of histamine<br />
depends on <strong>the</strong> glycine concentration suggesting a putative competitive mech<strong>an</strong>ism. Herewith we
present histamine as a new endogenous modulator <strong>an</strong>d <strong>an</strong>tagonist of GlyRs which are <strong>an</strong><br />
import<strong>an</strong>t target <strong>for</strong> peripheral inhibition.<br />
Disclosures: O. Kletke, None; H. Hatt, None; G. Gisselm<strong>an</strong>n, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.13/B120<br />
Topic: B.02.r. Glycine receptors<br />
Support: NIH DC004450<br />
NIH NS028901<br />
Title: Control of glycine reversal potential by a Ca2+/H+-dependent mech<strong>an</strong>ism<br />
Authors: *Y. KIM, L. O. TRUSSELL;<br />
Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: The major roles of glycine <strong>an</strong>d GABA are in neural inhibition. Never<strong>the</strong>less, it is well<br />
known that glycinergic or GABAergic inputs c<strong>an</strong> be depolarizing, exerting shunting inhibition or<br />
even excitation in developing or mature neurons. Cartwheel cells are auditory interneurons<br />
whose reversal potential <strong>for</strong> glycine (Egly) c<strong>an</strong> be hyperpolarizing or depolarizing; indeed, m<strong>an</strong>y<br />
cartwheel cells are excited by glycine (Golding <strong>an</strong>d Oertel, 1996). These neurons are also<br />
characterized by <strong>the</strong> generation of Na + <strong>an</strong>d Ca 2+ dependent spike bursts, termed complex spikes.<br />
Using gramicidin per<strong>for</strong>ated-patch recording, we determined <strong>the</strong> effect of Na + <strong>an</strong>d Ca 2+ spike<br />
activity on Egly in mouse cartwheel cells in a brain slice preparation. After a period of complex<br />
spiking induced by depolarizing current injection, Egly was shifted toward more negative<br />
potentials, thus enh<strong>an</strong>cing glycine’s inhibitory effect <strong>for</strong> about 30 s following cessation of<br />
spiking. A similar effect on Egly was seen after a period of Ca 2+ spiking (in TTX); in Ca 2+ -free<br />
bath solutions this effect was abolished, revealing a positive shift triggered by passive Cl -<br />
redistribution during depolarization. Combined per<strong>for</strong>ated-patch electrophysiology <strong>an</strong>d imaging<br />
studies showed that <strong>the</strong> negative Egly shift was preceded by a rise in intracellular Ca 2+ <strong>an</strong>d a drop<br />
in intracellular pH, <strong>an</strong>d was associated with reduction in intracellular Cl - concentration. The<br />
negative Egly shift was inhibited by H2DIDS <strong>an</strong>d removal of HCO3 - /CO2 - , suggesting a role <strong>for</strong> a<br />
bicarbonate-Cl - exch<strong>an</strong>ger. We suggest that a Ca 2+ dependent mech<strong>an</strong>ism c<strong>an</strong> maintain or even<br />
enh<strong>an</strong>ce <strong>the</strong> strength of inhibition in response to increases in excitatory activity.
Disclosures: Y. Kim, None; L.O. Trussell, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.14/B121<br />
Topic: B.02.r. Glycine receptors<br />
Support: Sp<strong>an</strong>ish MCyT Gr<strong>an</strong>t BFU2006-06598<br />
Title: Localization of glycine receptors in <strong>the</strong> cat central nervous system: Immunohistochemical<br />
<strong>an</strong>d autoradiographic studies<br />
Authors: *F. J. MARTIN-CORA, R. LEIRAS, P. VELO, A. CANEDO;<br />
Dept. Physiol., Sch. Med, Univ. S<strong>an</strong>tiago Compostela, S<strong>an</strong>tiago de Compostela, Spain<br />
Abstract: Glycine receptors (GlyRs) are chloride ion ch<strong>an</strong>nels that facilitate inhibitory<br />
neurotr<strong>an</strong>smission in <strong>the</strong> mammali<strong>an</strong> spinal cord <strong>an</strong>d brain. The present knowledge on <strong>the</strong><br />
location of central GlyRs comes mainly <strong>from</strong> studies in rodents <strong>an</strong>d hum<strong>an</strong>s. Despite <strong>the</strong>se,<br />
distribution studies in o<strong>the</strong>r mammals are largely uncharacterized. The present work provides a<br />
detailed <strong>an</strong>alysis of <strong>the</strong> <strong>an</strong>atomical distribution of GlyRs immunoreactivity <strong>an</strong>d binding sites in<br />
<strong>the</strong> cat CNS. In autoradiographic studies, binding sites were labeled with [ 3 H]strychnine (10 nM)<br />
<strong>an</strong>d nonspecific binding was determined in <strong>the</strong> presence of 10 mM of glycine. In<br />
immunohistochemical studies, monoclonal <strong>an</strong>tibody Mab4a (1:1000) <strong>an</strong>d a biotinylated <strong>an</strong>timouse<br />
<strong>an</strong>tibody (1:400) were used to detect GlyRs. Immunohistochemical staining was<br />
visualized using <strong>the</strong> avidin-biotin-peroxidase complex with a nickel-intensified<br />
diaminobenzidine procedure. Analysis of <strong>the</strong> autoradiographic data revealed a discrete<br />
localization of glycine binding sites in <strong>the</strong> cat CNS. Examination of <strong>the</strong> gross regional<br />
distribution shows that <strong>the</strong> density of GlyRs decreases progressively as one ascends in <strong>the</strong><br />
neuraxis. High density of sites was detected in <strong>the</strong> gray matter of <strong>the</strong> spinal cord, medullary<br />
nuclei (i.e., cuneatus <strong>an</strong>d gracilis nuclei) <strong>an</strong>d in <strong>the</strong> pons. Lower levels of binding were seen in<br />
<strong>the</strong> diencephalon (i.e., nucleus centralis thalami) <strong>an</strong>d still lower levels in <strong>the</strong> telencephalon (i.e.,<br />
amygdala). Likewise, immunohistochemical data disclosed a discrete regional distribution of<br />
GlyRs throughout <strong>the</strong> neuraxis. GlyRs were present in high concentration in <strong>the</strong> spinal cord<br />
(immunoreactivity was intense on <strong>the</strong> surface of <strong>the</strong> motor neurons while cells in <strong>the</strong> dorsal horn<br />
were less intensely labeled) <strong>an</strong>d brainstem (cell bodies <strong>an</strong>d proximal dendrites of <strong>the</strong> cells in <strong>the</strong><br />
superior olivary complex were intensely labeled). In diencephalic <strong>an</strong>d telencephalic areas,<br />
GlyRs-immunopositive neurons were less abund<strong>an</strong>t (i.e. hippocampus <strong>an</strong>d basal g<strong>an</strong>glia). <strong>When</strong>
autoradiographic <strong>an</strong>d immunohistochemical data were compared some discrep<strong>an</strong>cies were<br />
evident. For inst<strong>an</strong>ce, autoradiography did not detect GlyRs binding sites in <strong>the</strong> olfactory bulb,<br />
but GlyRs immunoreactivity was observed in <strong>the</strong> main olfactory bulb (periglomerular cells were<br />
heavily labeled <strong>an</strong>d a few mitral cells were moderately stained) <strong>an</strong>d <strong>the</strong> accessory olfactory bulb<br />
(<strong>the</strong> large soma <strong>an</strong>d multiple apical dendrites of <strong>the</strong> accessory mitral cells were heavily stained).<br />
Despite <strong>the</strong>se differences, <strong>the</strong> overall distribution of GlyRs labeled in <strong>the</strong> cat CNS ei<strong>the</strong>r with<br />
[ 3 H]strychnine or <strong>the</strong> <strong>an</strong>tibody Mab4a closely resemble that previously described <strong>for</strong> GlyRs in<br />
o<strong>the</strong>r mammals.<br />
Disclosures: F.J. Martin-Cora, None; R. Leiras, None; P. Velo, None; A. C<strong>an</strong>edo, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.15/B122<br />
Topic: B.02.r. Glycine receptors<br />
Title: Glycine receptor expression in <strong>the</strong> <strong>for</strong>ebrain of male AA/ANA rats<br />
Authors: *S. JONSSON 1 , N. KEREKES 1 , P. HYYTIÄ 2 , M. ERICSON 1 , B. SÖDERPALM 1 ;<br />
1 Neurosci & Physiol, Go<strong>the</strong>nburg, Sweden; 2 Natl. Inst. of Hlth. <strong>an</strong>d Welfare, Helsinki, Finl<strong>an</strong>d<br />
Abstract: Eth<strong>an</strong>ol is known to directly interact with <strong>the</strong> glycine receptor (GlyR) which is a<br />
member of <strong>the</strong> cystein-loop lig<strong>an</strong>d-gated ion ch<strong>an</strong>nel family. GlyRs are pentameric membr<strong>an</strong>e<br />
proteins <strong>an</strong>d, to <strong>the</strong>ir constitution, ei<strong>the</strong>r α-homomers or α- β heteromers with a subunit<br />
stoichiometry of 2α3β. Studies previously presented by our group have suggested a role <strong>for</strong><br />
GlyRs <strong>an</strong>d its endogenous lig<strong>an</strong>ds glycine <strong>an</strong>d taurine in <strong>the</strong> mesolimbic dopamine activating<br />
<strong>an</strong>d rein<strong>for</strong>cing effects of eth<strong>an</strong>ol.<br />
In <strong>the</strong> present study we use qu<strong>an</strong>titative reverse tr<strong>an</strong>scriptase PCR (qPCR) to compare <strong>the</strong><br />
relative expression of GlyR as well as glycine- (GlyT1) <strong>an</strong>d taurine tr<strong>an</strong>sporters (TauT) in Alko<br />
Alcohol/NonAlcohol (AA/ANA) rats.<br />
These <strong>an</strong>imals have been selectively bred to create two distinct populations regarding alcohol<br />
consumption <strong>an</strong>d preference, presumably mainly due to genetic differences. The aim of this<br />
study was to examine <strong>the</strong> relative gene expression of GlyR subunits (α1-3 <strong>an</strong>d β) <strong>an</strong>d tr<strong>an</strong>sporters<br />
in different brain areas of AA <strong>an</strong>d ANA rats <strong>an</strong>d to relate it to alcohol consumption (in AA rats).<br />
The hypo<strong>the</strong>sis was that <strong>the</strong>se two rat lines are differently disposed to eth<strong>an</strong>ol consumption due<br />
to <strong>the</strong>ir tr<strong>an</strong>sporter or GlyR set-ups <strong>an</strong>d/or compositions.<br />
Results <strong>from</strong> <strong>the</strong> present study indicates that α2 is <strong>the</strong> most widely expressed α-subunit in <strong>the</strong>
<strong>for</strong>ebrain regions <strong>an</strong>d that <strong>the</strong> α2β-heteromer seems to be <strong>the</strong> most common subunit composition<br />
in this part of <strong>the</strong> CNS. Also, of <strong>the</strong> two tr<strong>an</strong>sporters TauT was <strong>the</strong> one more widely expressed.<br />
In spite of <strong>the</strong> different drinking behaviours observed in <strong>the</strong> AA <strong>an</strong>d ANA strains <strong>the</strong> <strong>an</strong>ticipated<br />
differences in mRNA expression were few. However, correlations found between alcohol<br />
consumption <strong>an</strong>d/or preference <strong>an</strong>d GlyR expression support a role <strong>for</strong> GlyRs in alcohol<br />
consumption. Tentative differences between AA <strong>an</strong>d ANA <strong>an</strong>imals related to GlyR tr<strong>an</strong>smission<br />
could <strong>the</strong>re<strong>for</strong>e lie in e.g. regulation of <strong>the</strong> levels of <strong>the</strong> endogenous lig<strong>an</strong>d(s) <strong>for</strong> <strong>the</strong> receptor or<br />
in mech<strong>an</strong>isms downstream to GlyR activation.<br />
Disclosures: S. Jonsson , None; N. Kerekes, None; P. Hyytiä, None; M. Ericson, None; B.<br />
Söderpalm, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.16/B123<br />
Topic: B.02.r. Glycine receptors<br />
Support: FCT Gr<strong>an</strong>t SFRH/BPD/34299/2006<br />
Title: Expression pattern of glycine receptor in rat hippocampus: Ch<strong>an</strong>ges <strong>from</strong> embryonic to<br />
postnatal stages<br />
Authors: *C. A. VALENTE, R. I. AROEIRA, A. M. SEBASTIAO;<br />
Inst. de Medicina Mol., Lisbon, Portugal<br />
Abstract: The inhibition in <strong>the</strong> central nervous system is mediated by both GABA <strong>an</strong>d glycine.<br />
GABA is <strong>the</strong> main inhibitory neurotr<strong>an</strong>smitter in <strong>the</strong> brain, while glycine plays its major role in<br />
<strong>the</strong> spinal cord <strong>an</strong>d brainstem. Be<strong>for</strong>e release into <strong>the</strong> synaptic cleft, presynaptic glycine <strong>an</strong>d/or<br />
GABA are packaged into vesicles via a common tr<strong>an</strong>sporter named VIAAT (Vesicular Inhibitory<br />
Amino-Acid Tr<strong>an</strong>sporter). Glycine receptors (GlyR) belong to <strong>the</strong> superfamily of lig<strong>an</strong>d-gated<br />
ion ch<strong>an</strong>nels <strong>an</strong>d <strong>for</strong>m pentameric ch<strong>an</strong>nels composed of two different subunits (α <strong>an</strong>d β)<br />
permeable to Cl - ions. The glycinergic neurotr<strong>an</strong>smission is well established in spinal cord. In <strong>the</strong><br />
hippocampus, most inhibitory synapses are known to be GABAergic, <strong>an</strong>d glycinergic ones have<br />
been less studied. However, recent studies identified GlyR in <strong>the</strong> hippocampus, as well as in<br />
cultured hippocampal neurons, increasing <strong>the</strong> interest of <strong>the</strong> scientific community towards<br />
glycine function <strong>an</strong>d <strong>the</strong> role of GlyR-mediated neurotr<strong>an</strong>smission in <strong>the</strong> brain.<br />
In <strong>the</strong> present study we per<strong>for</strong>med a spatial-temporal characterization of GlyR in rat
hippocampus in different developmental stages. The total protein expression in hippocampus was<br />
evaluated by Western Blot. RT-PCR was used to assess mRNA expression of <strong>the</strong> different GlyR<br />
subunits, namely, α1, α2 <strong>an</strong>d β. The specific localization of <strong>the</strong> receptor was examined in several<br />
areas of <strong>the</strong> hippocampus, namely Dentate Gyrus (DG) <strong>an</strong>d Cornus Ammonis (CA), through<br />
immunohistochemistry in coronal brain slices (5 µm). The postsynaptic occurrence of GlyR was<br />
confirmed by double staining of GlyR <strong>an</strong>d VIAAT.<br />
Western blot <strong>an</strong>alysis showed that <strong>the</strong>re was a 9-10 fold-increase in GlyR total expression <strong>from</strong><br />
birth (P0) to seven days postnatal (P7) (n=3, P
Abstract: The postnatal development of spinal inhibitory systems is essential <strong>for</strong> <strong>the</strong> maturation<br />
of appropriate behavioural <strong>an</strong>d neurophysiological responses to noxious stimulation, yet <strong>the</strong> roles<br />
of gamma-aminobutyric acid (GABA) <strong>an</strong>d glycine through postnatal development are not yet<br />
fully understood. Here we have studied <strong>the</strong> functional effects of cellular <strong>an</strong>d molecular ch<strong>an</strong>ges<br />
in glycine-mediated activity upon intact nociceptive spinal circuits through <strong>the</strong> first three<br />
postnatal weeks. Intra<strong>the</strong>cal (i.t.) administration of <strong>the</strong> glycine-specific <strong>an</strong>tagonist strychnine<br />
(165ng/g) resulted in a decrease in mech<strong>an</strong>ical withdrawal threshold in <strong>the</strong> adult, as expected<br />
<strong>from</strong> blockade of tonic glycinergic inhibition. In contrast, a paradoxical increase in threshold was<br />
observed following i.t. strychnine in neonatal rats. Fur<strong>the</strong>rmore in vivo electrophysiological<br />
recordings of single spinal dorsal horn neurones in isoflur<strong>an</strong>e-<strong>an</strong>aes<strong>the</strong>tised rats show a lack of<br />
tonic glycinergic inhibition in neonates compared to adults (increase over baseline: 0.003<br />
spikes.sec-1 at postnatal day 3 versus 0.16 spikes.sec-1, 20mins post strychnine). The effect of<br />
strychnine upon evoked dorsal horn neuronal activity was calculated by plotting responses to<br />
subthreshold, threshold <strong>an</strong>d suprathreshold von Frey hairs using area under <strong>the</strong> curve (AUC) as<br />
<strong>an</strong> integrated measurement. A glycinergic facilitation of dorsal horn cell cut<strong>an</strong>eous evoked<br />
activity was seen in early development (AUC post strychnine in neonates: -1.58 ±0.75 compared<br />
to adult: +0.08 ±0.04). This is consistent with <strong>the</strong> results of previous patch-clamp experiments in<br />
neonatal spinal cord slices revealing little to no glycinergic IPSCs in <strong>the</strong> newborn, despite <strong>the</strong><br />
presence of functional glycine receptors (GlyRs) <strong>from</strong> birth. We have per<strong>for</strong>med immunostaining<br />
of <strong>the</strong> glycine tr<strong>an</strong>sporter GlyT2, a marker <strong>for</strong> glycinergic neurons, <strong>an</strong>d revealed a diffuse<br />
staining throughout laminae III to V in newborn spinal cord which gradually becomes restricted<br />
to lamina III by <strong>the</strong> third week of life, coinciding with <strong>the</strong> onset of functional inhibition of<br />
nociceptive responses. In addition we have used c-fos expression following i.t. strychnine<br />
administration in young <strong>an</strong>d adult rats to map <strong>the</strong> disinhibition caused by blocking GlyRs in <strong>the</strong><br />
dorsal horn of <strong>the</strong> spinal cord. In this way neurons under both tonic <strong>an</strong>d evoked inhibitory<br />
control are compared in newborn <strong>an</strong>d older rats. Taken toge<strong>the</strong>r, our results suggest that<br />
glycinergic inhibitory activity in dorsal horn undergoes subst<strong>an</strong>tial developmental tuning which<br />
has import<strong>an</strong>t physiological effects upon nociceptive processing in <strong>the</strong> immature spinal cord.<br />
Disclosures: S.C. Koch, None; G. Brent, None; G. Hathway, None; M. Fitzgerald, None.<br />
Poster<br />
514. Glycine Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 514.18/B125<br />
Topic: B.02.r. Glycine receptors<br />
Support: Swedish <strong>Society</strong> <strong>for</strong> Medical Research
Swedish Medical Research Council<br />
Swedish <strong>Society</strong> of Medicine<br />
Tore Nilsson foundation<br />
Vilhelm och Martina Lundgren<br />
Swedish Brain Foundation<br />
Title: Subregion-specific eth<strong>an</strong>ol modulation in <strong>the</strong> striatum via GABAA <strong>an</strong>d glycine receptors<br />
Authors: *L. ADERMARK, R. CLARKE, M. ERICSON, B. SÖDERPALM;<br />
Addiction Biol. Unit, Go<strong>the</strong>nburg, Sweden<br />
Abstract: The striatum is <strong>the</strong> largest input nucleus to <strong>the</strong> basal g<strong>an</strong>glia, a brain structure<br />
implicated in motor pl<strong>an</strong>ning, action selection, <strong>an</strong>d execution. The striatum is also associated<br />
with reward-based behavior, <strong>an</strong>d <strong>the</strong> motivational aspects of rewarding stimuli. Fur<strong>the</strong>rmore,<br />
medium spiny projection neurons located in <strong>the</strong> ventral striatum regulate dopamine release <strong>from</strong><br />
<strong>the</strong> ventral tegmental area (VTA), making <strong>the</strong> striatum <strong>an</strong> import<strong>an</strong>t brain region to study with<br />
respect to <strong>the</strong> rein<strong>for</strong>cing properties of drugs of abuse. The aim of this study was to evaluate if<br />
striatal output in brain slices <strong>from</strong> <strong>the</strong> rat are modulated by acute eth<strong>an</strong>ol (EtOH) exposure. We<br />
show that EtOH (20, 50, or 80 mM) induces a biphasic GABAA receptor-dependent <strong>an</strong>d<br />
strychnine-sensitive depression of population spike (PS) amplitude in <strong>the</strong> dorsolateral striatum,<br />
while <strong>the</strong> synaptic output <strong>from</strong> <strong>the</strong> nucleus accumbens (nAc) core remains unaffected. We also<br />
show that GABAergic neurotr<strong>an</strong>smission has a stronger influence on synaptic output <strong>from</strong> <strong>the</strong><br />
dorsolateral striatum as compared to PSs evoked in nAc core, which might underlie <strong>the</strong><br />
difference in sensitivity to EtOH exposure. Fur<strong>the</strong>rmore, strychnine (1 µM) enh<strong>an</strong>ces PS<br />
amplitude in both subregions, suggesting that glycine receptors are tonically activated <strong>an</strong>d inhibit<br />
striatal output. The strychnine-mediated increase in PS amplitude is inhibited by <strong>the</strong> GABAA<br />
receptor <strong>an</strong>tagonist picrotoxin (50 µM), suggesting that glycine receptors adjust striatal output<br />
through modulation of GABAergic neurotr<strong>an</strong>smission. In conclusion, EtOH, picrotoxin, <strong>an</strong>d<br />
strychnine regulate striatal output in a subregion-specific m<strong>an</strong>ner, which is import<strong>an</strong>t <strong>for</strong><br />
underst<strong>an</strong>ding striatal microcircuits <strong>an</strong>d <strong>the</strong> rein<strong>for</strong>cing properties of drugs of abuse.<br />
Disclosures: L. Adermark, None; R. Clarke, None; M. Ericson, None; B. Söderpalm, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 515.1/C1<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: VRAID internal PUC gr<strong>an</strong>t<br />
Title: Muscarinic acetylcholine receptor activation inhibits task-like k + current in rat carotid<br />
body chemoreceptor cells<br />
Authors: *F. A. ORTIZ 1,2 , R. VARAS 2 ;<br />
1 S<strong>an</strong>tiago, Chile; 2 Physiological Sci., P. Univ. Catolica de Chile, S<strong>an</strong>tiago, Chile<br />
Abstract: The chemorreceptor carotid body (CB) cells respond to hypoxia with membr<strong>an</strong>e<br />
depolarization, voltage-gated calcium entry <strong>an</strong>d neurosecretion. A key step in <strong>the</strong> initiation of<br />
membr<strong>an</strong>e depolarization is <strong>the</strong> reversible inhibition of TASK-like K + background current by<br />
hypoxia. It has been previously reported that TASK-like K + ch<strong>an</strong>nels could be modulated by Gprotein<br />
coupled receptors, such as <strong>the</strong> muscarinic acetylcholine receptor (mAChRs). Since <strong>the</strong>re<br />
is a proposed role <strong>for</strong> ACh as <strong>an</strong> autocrine/paracrine modulator of CB chemoreceptor cells, we<br />
have investigated <strong>the</strong> effects of muscarinic-responsive pathway upon K + background current.<br />
CBs were obtained <strong>from</strong> <strong>an</strong>aes<strong>the</strong>tized neonatal rats <strong>an</strong>d enzymatic <strong>an</strong>d mech<strong>an</strong>ically<br />
disassociated. Freshly isolated cells were maintained in HAMs F-12 culture media under 5 %<br />
CO2 in air at 37 °C <strong>an</strong>d used within 2-6 hrs.<br />
Using Ca 2+ fluorometric measurements we evaluated <strong>the</strong> functional presence of mAChRs in<br />
hypoxia-sensitive cells. In 12 out of 29 cells, fast superfusion with <strong>the</strong> selective muscarinic<br />
agonist methacholine (Meth 100µM) <strong>an</strong>d/or muscarine (50 µM) evokes intracellular Ca 2+ rises<br />
even in <strong>the</strong> absence of added external Ca 2+ .<br />
TASK-like K + ch<strong>an</strong>nels activity was recorded with cell-attached voltage-clamp technique in<br />
hypoxia sensitive cells. Qu<strong>an</strong>tification of ch<strong>an</strong>nel activity was estimated by <strong>the</strong> open probability<br />
of active ch<strong>an</strong>nels in a patch (NPo). Under <strong>the</strong>se conditions TASK-like activity was reduced by<br />
~50% in <strong>the</strong> presence of Meth (100µM), effect that is fully blocked by atropine (1µM, n=4).<br />
Then, we investigated possible intracellular pathways that links mAChRs activation with TASKlike<br />
activity: Oleoylacetylglycerol (OAG, 20 µM), compound that mimics diacylglycerol,<br />
decreases ch<strong>an</strong>nel NPo by ~50% (n=4). The coapplication of OAG + Meth do not fur<strong>the</strong>r inhibits<br />
ch<strong>an</strong>nel NPo. Following our line of thought, <strong>the</strong> PKC selective inhibitors Calphostin C (100nM)<br />
or chelerythrine (50 µM) evoked <strong>an</strong> increase in NPo >50% (n=4). In addition, <strong>the</strong> PLC inhibitor<br />
U732122 (10µM) increases NPo ~ 50% compared with control.<br />
Our results suggest that <strong>the</strong> activation of mAChRs inhibits TASK-like current in rat CB<br />
chemoreceptor cells throughout a PLC/PKC-dependent pathway. These data suggest that<br />
mAChRs contribute to define membr<strong>an</strong>e potential through direct action on K + background<br />
current <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e <strong>the</strong> autocrine/paracrine cholinergic mech<strong>an</strong>ism involves a direct action<br />
upon <strong>the</strong> hypoxic responsive pathway.<br />
Disclosures: F.A. Ortiz, None; R. Varas, None.
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.2/C2<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: Heart <strong>an</strong>d Stroke Foundation of C<strong>an</strong>ada<br />
Savoy Foundation<br />
C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research<br />
Title: Cholinergic modulation of NMDA current in hippocampal CA1 pyramidal neurons is agedependent<br />
Authors: *C. TAI, B. A. MACVICAR;<br />
Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: The cholinergic system is one of <strong>the</strong> most import<strong>an</strong>t neuromodulatory<br />
neurotr<strong>an</strong>smitter systems in <strong>the</strong> CNS. Both <strong>an</strong>imal <strong>an</strong>d hum<strong>an</strong> studies indicate cholinergic<br />
system plays a key role in modulating neuronal excitability, synaptic plasticity <strong>an</strong>d neuronal<br />
intrinsic properties. One of <strong>the</strong> predomin<strong>an</strong>t effects of cholinergic agonists on hippocampal CA1<br />
neurons is potentiation of currents through <strong>the</strong> ionotropic glutamatergic N-methyl-d-aspartate<br />
(NMDA) receptors (NMDARs), which play a central role in synaptic tr<strong>an</strong>smission <strong>an</strong>d in<br />
mediating synaptic plasticity, learning <strong>an</strong>d memory. However, <strong>the</strong> mech<strong>an</strong>ism underlying <strong>the</strong><br />
cholinergic modulation of NMDARs still remains unknown. In <strong>the</strong> present study, we report that<br />
muscarinic modulation of NMDA-evoked current (INMDA) in CA1 pyramidal neurons is<br />
profoundly different at two different developmental stages. We found that muscarinic agonist<br />
carbachol potentiated INMDA in both young (5-10 postnatal days) <strong>an</strong>d old (6-8 postnatal weeks)<br />
<strong>an</strong>imals, but strikingly, via distinct downstream pathways. In young <strong>an</strong>imals, muscarinic<br />
stimulation potentiated INMDA through a [Ca2+]i-independent but PKC- <strong>an</strong>d Src-dependent<br />
pathway. In contrast, in old <strong>an</strong>imals, muscarinic stimulation potentiated INMDA through a<br />
[Ca2+]i-dependent but PKC-independent pathway. Interestingly, <strong>the</strong> activity of <strong>the</strong> Gαq-coupled<br />
M1-like muscarinic receptors was required <strong>for</strong> <strong>the</strong> potentiation of INMDA in both young <strong>an</strong>d old<br />
<strong>an</strong>imals. Our results indicate a novel developmental pattern of cholinergic modulation of<br />
INMDA in CA1 pyramidal neurons involving different downstream mech<strong>an</strong>isms. These findings<br />
may have import<strong>an</strong>t implications in <strong>the</strong> induction <strong>an</strong>d regulation of NMDA-dependent synaptic<br />
plasticity at different development stages, <strong>an</strong>d may provide a crucial mech<strong>an</strong>ism by which<br />
cholinergic input modulates learning <strong>an</strong>d memory.
Disclosures: C. Tai, None; B.A. MacVicar, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.3/C3<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NSERC<br />
FRSQ<br />
Title: Mech<strong>an</strong>isms underlying muscarinic depolarization of layer II cells of <strong>the</strong> parasubiculum<br />
Authors: *S. D. GLASGOW, C. A. CHAPMAN;<br />
Dept Psychol, Concordia Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Cholinergic receptor activation has strong neuromodulatory effects on neurons of <strong>the</strong><br />
hippocampal <strong>for</strong>mation, <strong>an</strong>d has been implicated in learning <strong>an</strong>d memory processes by<br />
promoting network-level <strong>the</strong>ta frequency synchronization. The parasubiculum sends its major<br />
output to layer II of <strong>the</strong> entorhinal cortex, <strong>an</strong>d receives strong cholinergic innervation <strong>from</strong> <strong>the</strong><br />
basal <strong>for</strong>ebrain. We have recently shown that bath application of <strong>the</strong> muscarinic agonist<br />
carbachol results in <strong>the</strong> slow depolarization of membr<strong>an</strong>e potential in morphologically-identified<br />
layer II parasubicular cells, suggesting that cholinergic innervation of <strong>the</strong> parasubiculum may<br />
serve to promote temporal coordination of network interactions with <strong>the</strong> entorhinal cortex via<br />
depolarization to near-threshold voltages. However, <strong>the</strong> conduct<strong>an</strong>ces underlying cholinergic<br />
depolarization of parasubicular neurons have yet to be fully elucidated. The present study used<br />
whole cell current- <strong>an</strong>d voltage-clamp recordings to determine how <strong>the</strong> activity of layer II<br />
parasubicular neurons may be modulated by activation of muscarinic cholinergic receptors.<br />
Carbachol-induced depolarization was completely blocked by muscarinic receptor <strong>an</strong>tagonist<br />
atropine (1 µM), indicating that <strong>the</strong> depolarization is dependent on activation of muscarinic<br />
receptors. Bath application of <strong>the</strong> selective M1 receptor <strong>an</strong>tagonist pirenzepine (1 µM), but not<br />
<strong>the</strong> M2-preferring <strong>an</strong>tagonist methoctramine (1 µM), prevented depolarization by carbachol,<br />
suggesting that <strong>the</strong> depolarization is dependent on activation of M1 receptors. Application of <strong>the</strong><br />
selective Kv7.2/3 ch<strong>an</strong>nel <strong>an</strong>tagonist XE-991 (10 µM), which blocks <strong>the</strong> muscarinicallyactivated<br />
K + -current IM, depolarized parasubicular neurons in <strong>the</strong> presence of synaptic blockers.<br />
Concurrent bath application of carbachol resulted in additional depolarization in parasubicular<br />
neurons, suggesting that o<strong>the</strong>r conduct<strong>an</strong>ces may help support <strong>the</strong> muscarinic receptor-dependent
depolarization. Voltage-clamp experiments per<strong>for</strong>med at a holding potential of -60 mV verified<br />
that muscarinic receptor stimulation in parasubicular neurons results in <strong>the</strong> development of a<br />
slow inward current deflection that is not completely occluded by coapplication of XE-991. The<br />
muscarinic depolarization of parasubicular neurons which is thought to contribute to<br />
synchronous network activity is <strong>the</strong>re<strong>for</strong>e likely dependent on multiple conduct<strong>an</strong>ces including<br />
IM.<br />
Disclosures: S.D. Glasgow, None; C.A. Chapm<strong>an</strong>, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.4/C4<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NIH Gr<strong>an</strong>t AG05214<br />
Title: Amiodarone exerts complex allosteric effects at muscarinic receptors<br />
Authors: E. STAHL, *J. ELLIS;<br />
Penn State Univ., Hershey, PA<br />
Abstract: Muscarinic acetylcholine receptors are a family of five G-protein coupled receptors<br />
(M1-M5) that are considered to be import<strong>an</strong>t pharmacological targets <strong>for</strong> treating CNS disorders,<br />
including Alzheimer’s disease <strong>an</strong>d schizophrenia. However, <strong>the</strong> known orthosteric lig<strong>an</strong>ds are<br />
not highly subtype selective, leading to dose-limiting side effects mainly involving muscarinic<br />
autonomic signaling. Fur<strong>the</strong>rmore, administration of orthosteric agonists is likely to disrupt<br />
specific spatiotemporal patterns of synaptic tr<strong>an</strong>smission, which may severely limit <strong>the</strong>ir<br />
<strong>the</strong>rapeutic utility. The potential to provide enh<strong>an</strong>ced subtype selectivity <strong>an</strong>d to maintain<br />
synaptic patterning makes allosteric modulation a more attractive option.<br />
We have previously reported that amiodarone interacts with a novel site on muscarinic receptors<br />
<strong>an</strong>d allosterically modulates binding <strong>an</strong>d response. Subsequently, we have found that although<br />
amiodarone inhibits <strong>the</strong> binding of <strong>the</strong> muscarinic <strong>an</strong>tagonist [3H]N-methylscopolamine (NMS),<br />
it is unable to completely inhibit specific binding at <strong>an</strong>y of <strong>the</strong> subtypes; this indicates strictly<br />
non-competitive interactions. Functionally, amiodarone acts cooperatively with muscarinic<br />
agonists to enh<strong>an</strong>ce [3H]arachidonic acid release <strong>from</strong> chinese hamster ovary cells expressing<br />
M5. Somewhat surprisingly, agonist potency is not enh<strong>an</strong>ced concomit<strong>an</strong>tly with <strong>the</strong> increase in<br />
efficacy of <strong>the</strong> response. Simulations generated <strong>from</strong> <strong>an</strong> allosteric functional model showed that
this is indicative of ch<strong>an</strong>ges in two separate parameters of <strong>the</strong> model. These simulated response<br />
curves suggest that amiodarone enh<strong>an</strong>ces agonist efficacy, but simult<strong>an</strong>eously inhibits agonist<br />
binding to <strong>an</strong> extent that leaves response potency essentially unaffected. Additionally, a charged<br />
amiodarone <strong>an</strong>alog that lacks <strong>the</strong> ability to enh<strong>an</strong>ce efficacy is able to reverse <strong>the</strong> enh<strong>an</strong>cing<br />
effect of amiodarone, suggesting that it is competitive at <strong>the</strong> amiodarone allosteric site. By<br />
contrast, application of <strong>the</strong> orthosteric <strong>an</strong>tagonist NMS with amiodarone produces a rightward<br />
shift in agonist dose response curves, but does not alter amiodarone’s potentiation of maximal<br />
response.<br />
In summary, we have demonstrated that amiodarone interacts allosterically with all muscarinic<br />
receptor subtypes; amiodarone also affects receptor response in a complex <strong>an</strong>d reversible m<strong>an</strong>ner<br />
at <strong>the</strong> M5 subtype, due to <strong>an</strong> interaction at a specific extracellular site.<br />
[Supported by PHS R01 05214]<br />
Disclosures: E. Stahl, None; J. Ellis, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.5/C5<br />
Topic: B.03.a. Muscarinic receptors<br />
Title: Enh<strong>an</strong>cement by G protein-coupled receptor kinase 2 of internalization of muscarinic<br />
acetylcholine receptor M4 subtype with deletion of most of <strong>the</strong> third intracellular loop<br />
Authors: R. KAKEGAI 1 , M. SASAKI 1 , K. SAITO 1 , Y. HASHIMOTO 1 , *T. HAGA 2 ;<br />
1 2<br />
Inst. <strong>for</strong> Biomolecular Science, Fac. of Sci., Insr Biomed Sci, Fac Sci., Gakushuin Univ.,<br />
Tokyo, Jap<strong>an</strong><br />
Abstract: We have examined <strong>the</strong> agonist-dependent internalization of FLAG-tagged muscarinic<br />
acetylcholine receptor M4 subtype (M4 receptor), which was tr<strong>an</strong>siently expressed in<br />
HEK293tsA201 cells. We had presumed that <strong>the</strong> internalization of M4 receptors would be<br />
enh<strong>an</strong>ced by coexpression of G protein-coupled receptor kinase 2 (GRK2) as was <strong>the</strong> case <strong>for</strong><br />
muscarinic acetylcholine receptor M2 subtype (M2 receptor), because M4 receptors as well as<br />
M2 receptors have two Ser/Thr clusters in <strong>the</strong> third intracellular loop (i3) which was supposed to<br />
be phosphorylated by GRK2. The proportion of internalized M4 receptors was increased by<br />
coexpression of GRK2 as was expected. Unexpectedly, however, <strong>the</strong> stimulatory effect of GRK2<br />
was also detected <strong>for</strong> internalization of <strong>an</strong> M4 receptor mut<strong>an</strong>t, which has al<strong>an</strong>ine residues<br />
substituted <strong>for</strong> <strong>the</strong> two Ser/Thr clusters, <strong>an</strong>d of <strong>an</strong>o<strong>the</strong>r mut<strong>an</strong>t (M4del-i3) which lacks <strong>the</strong> central
part of i3 including <strong>the</strong> two Ser/Thr clusters. Fur<strong>the</strong>rmore, <strong>the</strong> stimulatory effect of GRK2 on<br />
internalization of M4del-i3 was inhibited by coexpression of GRK2-K220R mut<strong>an</strong>t in a<br />
domin<strong>an</strong>t-negative m<strong>an</strong>ner. These results suggest that GRK2 may promote <strong>the</strong> internalization of<br />
M4 receptors by phosphorylating Ser/Thr residue(s) outside <strong>the</strong> central part of i3. We have <strong>the</strong>n<br />
examined internalization of fourteen M4 mut<strong>an</strong>ts, each of which has <strong>an</strong> al<strong>an</strong>ine residue<br />
substituted <strong>for</strong> one of fourteen Ser/Thr residues within <strong>the</strong> intracellular domain of M4del-i3, <strong>an</strong>d<br />
found that internalization of all <strong>the</strong>se mut<strong>an</strong>ts was enh<strong>an</strong>ced by coexpression of GRK2. These<br />
results suggest that M4del-i3 has more th<strong>an</strong> two phosphorylation sites by GRK2 <strong>an</strong>d that<br />
phosphorylation of one of <strong>the</strong>se sites is enough to enh<strong>an</strong>ce <strong>the</strong> internalization of M4del-i3. It is<br />
also possible to assume that internalization of M4del-i3 is enh<strong>an</strong>ced by GRK2 but not by GRK2-<br />
K220R in a m<strong>an</strong>ner independent of phosphorylation of Ser/Thr residues in <strong>the</strong> intracellular loops.<br />
Disclosures: R. Kakegai, None; M. Sasaki, None; K. Saito, None; Y. Hashimoto, None; T.<br />
Haga, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.6/C6<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NIH Gr<strong>an</strong>t 3U54MH074427 to C.D.W.<br />
NIH Gr<strong>an</strong>t 3U54MH074427-02S1 to C.D.W.<br />
NIH Gr<strong>an</strong>t 1XO1MH077606-01 to C.D.W.<br />
NIH Gr<strong>an</strong>t 1U54MH084659 to C.W.L.<br />
Dystonia Medical Research Foundation Gr<strong>an</strong>t to Z.X.<br />
PhRMA Foundation Award to D.J.S.<br />
Title: A novel selective muscarinic acetylcholine receptor subtype 1 (M1 mAChR) <strong>an</strong>tagonist<br />
reduces seizures without impairing hippocampal-dependent learning<br />
Authors: *D. J. SHEFFLER 1 , R. WILLIAMS 1 , T. M. BRIDGES 1 , Z. XIANG 1 , A. S. KANE 1 ,<br />
N. E. BYUN 2 , S. JADHAV 1 , M. M. MOCK 5 , F. ZHENG 5 , L. M. LEWIS 3 , C. K. JONES 1 , C. M.
NISWENDER 1 , C. D. WEAVER 3 , C. W. LINDSLEY 4 , P. J. CONN 1 ;<br />
1 Pharmacol., 2 Radiology <strong>an</strong>d Radiological Sci., 3 V<strong>an</strong>derbilt Inst. of Chem. Biol., 4 Chem.,<br />
V<strong>an</strong>derbilt Univ. Med. Ctr., Nashville, TN; 5 Dept. of Pharmacol. <strong>an</strong>d Toxicology, Univ. of<br />
Ark<strong>an</strong>sas Med. Ctr., Little Rock, AR<br />
Abstract: Muscarinic acetylcholine (ACh) receptors (mAChRs) are G protein-coupled receptors<br />
(GPCRs) which mediate <strong>the</strong> metabotropic actions of ACh. Five mAChR subtypes have been<br />
cloned (M1-M5) with M1, M3, <strong>an</strong>d M5 coupling via Gq to calcium mobilization <strong>an</strong>d M2 <strong>an</strong>d M4<br />
coupling to Gi <strong>an</strong>d <strong>the</strong> inhibition of adenylate cyclase. Previous studies suggest that mAChR<br />
subtype selective <strong>an</strong>tagonists may provide a novel approach <strong>for</strong> <strong>the</strong> treatment of certain CNS<br />
disorders, including epileptic disorders, Parkinson’s disease, <strong>an</strong>d dystonia. Un<strong>for</strong>tunately,<br />
previously reported <strong>an</strong>tagonists are not highly selective <strong>for</strong> specific mAChR subtypes, likely due<br />
to <strong>the</strong> conservation of <strong>the</strong> orthosteric (ACh) binding site, making it difficult to definitively<br />
establish <strong>the</strong> functional roles <strong>an</strong>d <strong>the</strong>rapeutic potential <strong>for</strong> individual subtypes of this receptor<br />
subfamily. The M1 mAChR is of particular interest as a potential target <strong>for</strong> treatment of CNS<br />
disorders. A high-throughput screen (HTS) resulted in a lead compound that displayed selectivity<br />
<strong>for</strong> M1 mAChRs, but which required fur<strong>the</strong>r chemical optimization. Fur<strong>the</strong>r structure activity<br />
relationship (SAR) studies resulted in <strong>the</strong> discovery of a novel selective <strong>an</strong>tagonist of M1<br />
mAChRs, termed VU0255035. Equilibrium radiolig<strong>an</strong>d binding <strong>an</strong>d functional studies<br />
demonstrate a greater th<strong>an</strong> 75-fold selectivity of VU0255035 <strong>for</strong> M1 mAChRs relative to M2-M5.<br />
Molecular pharmacology <strong>an</strong>d mutagenesis studies indicate that VU0255035 is a competitive<br />
orthosteric <strong>an</strong>tagonist of M1 mAChRs, a surprising finding given <strong>the</strong> high level of M1 mAChR<br />
selectivity relative to o<strong>the</strong>r orthosteric <strong>an</strong>tagonists. Whole cell patch clamp recordings<br />
demonstrate that VU0255035 inhibits potentiation of NMDA receptor currents by <strong>the</strong> muscarinic<br />
agonist carbachol in hippocampal pyramidal cells. Pharmacokinetic studies indicate that<br />
VU0255035 has excellent brain penetration in vivo <strong>an</strong>d is efficacious in reducing pilocarpineinduced<br />
seizures in mice. Surprisingly, doses of VU0255035 that reduce pilocarpine-induced<br />
seizures do not induce deficits in contextual freezing, a measure of hippocampal-dependent<br />
learning that is disrupted by nonselective mAChR <strong>an</strong>tagonists. Taken toge<strong>the</strong>r, <strong>the</strong>se data suggest<br />
that selective <strong>an</strong>tagonists of M1 mAChRs do not induce <strong>the</strong> severe cognitive deficits seen with<br />
non-selective mAChR <strong>an</strong>tagonists <strong>an</strong>d could provide a novel approach <strong>for</strong> <strong>the</strong> treatment certain<br />
of CNS disorders.<br />
Disclosures: D.J. Sheffler, None; R. Williams, None; T.M. Bridges, None; Z. Xi<strong>an</strong>g,<br />
None; A.S. K<strong>an</strong>e, None; N.E. Byun, None; S. Jadhav, None; M.M. Mock, None; F. Zheng,<br />
None; L.M. Lewis, None; C.K. Jones, None; C.M. Niswender, None; C.D. Weaver,<br />
None; C.W. Lindsley, None; P.J. Conn, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.7/C7<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NS057742<br />
Title: Mutation of amino acid residues in <strong>the</strong> C-terminal tail <strong>an</strong>d near <strong>the</strong> base of tr<strong>an</strong>smembr<strong>an</strong>e<br />
sp<strong>an</strong>ning domain 1 affect M1 receptor plasma membr<strong>an</strong>e expression<br />
Authors: *G. W. SAWYER, C. A. SHULTS;<br />
OSU Ctr. Hlth. Sci., Tulsa, OK<br />
Abstract: In this investigation, we characterized <strong>the</strong> functional role of a conserved C-terminal<br />
tail motif, 423-F(X)6LL-431, in muscarinic M1 receptors. Point mutations were introduced into<br />
this motif, <strong>an</strong>d at <strong>the</strong> base of tr<strong>an</strong>smembr<strong>an</strong>e sp<strong>an</strong>ning domain 1 (TM1), using site-directed<br />
mutagenesis. Wild-type <strong>an</strong>d mut<strong>an</strong>t M1 receptors were tr<strong>an</strong>siently expressed in CHO cells <strong>an</strong>d<br />
<strong>the</strong> amount of receptor expressed on <strong>the</strong> plasma membr<strong>an</strong>e was determined using intact, whole<br />
cell [ 3 H]-N-methylscopolamine ([ 3 H]NMS) binding assays. The cellular localization of GFPtagged<br />
wild-type <strong>an</strong>d mut<strong>an</strong>t receptors was also determined using epi-fluorescence microscopy.<br />
Of <strong>the</strong> mutations introduced into <strong>the</strong> F(X)6LL motif, mutation of Leu pair 430-LL-431<br />
(hM1AA430-431) caused <strong>the</strong> greatest impairment in <strong>the</strong> plasma membr<strong>an</strong>e expression of M1<br />
receptors, causing a 92% decrease in expression. Of <strong>the</strong> mutations introduced into TM1,<br />
mutation of 46-VL-47 (hM1AA46-47) caused <strong>the</strong> greatest impairment in M1 receptor plasma<br />
membr<strong>an</strong>e expression, resulting in a 98% decrease in expression. The plasma membr<strong>an</strong>e<br />
expression of hM1AA430-431 <strong>an</strong>d hM1AA46-47 receptors increased 7.6- <strong>an</strong>d 27.3-fold, respectively,<br />
when incubated with atropine (0.1 µM) <strong>for</strong> 18 h. Unlike GFP-tagged M1 receptors, GFP-tagged<br />
M1AA430-431 receptors colocalized extensively with <strong>the</strong> ER marker DsRed-ER. <strong>When</strong> incubated<br />
with atropine (0.1 µM) <strong>for</strong> 18 h, GFP-tagged M1AA430-431 receptors acquired a cellular<br />
localization consistent with that of wild-type receptors (i.e., expressed on <strong>the</strong> plasma membr<strong>an</strong>e).<br />
Collectively, <strong>the</strong>se data suggest that 46-VL-47 <strong>an</strong>d 430-LL-431 may play a role in folding M1<br />
receptors, which is necessary <strong>for</strong> <strong>the</strong> receptors to traffic <strong>from</strong> <strong>the</strong> ER to <strong>the</strong> plasma membr<strong>an</strong>e.<br />
Disclosures: G.W. Sawyer, None; C.A. Shults, None.<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 515.8/C8<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NIH gr<strong>an</strong>ts RO1 NS08174, RO1 GM83913, T32 GM07108<br />
HFSP<br />
Title: A kinetic model <strong>for</strong> signaling by <strong>the</strong> M1 muscarinc receptor, PIP2 depletion, <strong>an</strong>d<br />
inhibition of neuronal M (KCNQ2/3) current<br />
Authors: *B. H. FALKENBURGER, J. B. JENSEN, B. HILLE;<br />
Univ. Washington, Seattle, WA<br />
Abstract: M1 muscarinic receptors (M1R) mediate import<strong>an</strong>t effects of acetylcholine in <strong>the</strong><br />
CNS <strong>an</strong>d <strong>the</strong> autonomic nervous system. In particular, we are interested in <strong>the</strong> signaling pathway<br />
<strong>from</strong> M1R activation that results in depletion of <strong>the</strong> trace membr<strong>an</strong>e lipid phosphatidylinostitol<br />
(4,5) bisphosphate (PIP2). M<strong>an</strong>y membr<strong>an</strong>e proteins require PIP2 to function. A classical<br />
example is <strong>the</strong> KCNQ2/3 potassium ch<strong>an</strong>nel, which mediates neuronal M current.<br />
Using Forster Reson<strong>an</strong>ce Energy Tr<strong>an</strong>ser (FRET), we have previously measured time const<strong>an</strong>ts<br />
of individual steps in <strong>the</strong> M1R signaling cascade (Jensen et al., JGP <strong>2009</strong>). To construct a kinetic<br />
model of M1R signaling, we have now estimated <strong>the</strong> density of <strong>the</strong>se signaling molecules at <strong>the</strong><br />
plasma membr<strong>an</strong>e by calibrating <strong>the</strong>ir fluorescence intensity. The kinetic model encompasses<br />
cooperative binding of agonist <strong>an</strong>d G-proteins to <strong>the</strong> M1R, G-protein activation by nucleotide<br />
exch<strong>an</strong>ge, G-protein deactivation by <strong>the</strong>ir GTPase activity, activation of PLC, PIP2 hydrolysis<br />
<strong>an</strong>d phosphoinositide metabolism - <strong>the</strong> basis <strong>for</strong> PIP2 recovery, PIP2-dependent M current. To<br />
learn more about <strong>the</strong> responses to fast PIP2 depletion, we used a voltage-sensitive phosphatase<br />
(VSP), that is activated by depolarization of <strong>the</strong> membr<strong>an</strong>e potential.<br />
Our calibrations suggest that cells we select <strong>for</strong> experiments contain 2000 overexpressed<br />
receptors, G-proteins, or PLC molecules per µm 2 , <strong>an</strong>d endogenous levels of 40 G-proteins <strong>an</strong>d 4<br />
PLC per µm 2 . The kinetic model predicts that
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.9/C9<br />
Topic: B.03.a. Muscarinic receptors<br />
Title: Allosteric activators differentially activate M1 Ach receptors<br />
Authors: *G. J. DIGBY, L. E. DIAZ GIMENEZ, A. E. BRADY, E. P. LEBOIS, C. W.<br />
LINDSLEY, C. D. WEAVER, V. V. GUREVICH, P. J. CONN;<br />
Pharmacol, V<strong>an</strong>derbilt Univ., Nashville, TN<br />
Abstract: M1 allosteric agonists <strong>an</strong>d potentiators, also termed M1 allosteric activators, are<br />
potentially useful treatments <strong>for</strong> several CNS disorders. These compounds bind receptors at<br />
ectopic sites <strong>an</strong>d show greater M1 selectivity th<strong>an</strong> previously developed orthosteric M1 lig<strong>an</strong>ds.<br />
Given <strong>the</strong>se novel <strong>for</strong>ms of receptor binding, it is possible that M1 allosteric activators may also<br />
induce preferential activation of a desired M1 pathway. If so, this finding would be import<strong>an</strong>t <strong>for</strong><br />
drug discovery at M1 because undesired M1 signaling pathways could be better defined <strong>an</strong>d<br />
eliminated. Here we examine whe<strong>the</strong>r M1 allosteric activators show preference <strong>for</strong> arrestin<br />
recruitment or G protein activation, two well defined M1 pathways. By measuring intracellular<br />
calcium release, we show that allosteric agonists TBPB <strong>an</strong>d VU0184670 both activate PLC<br />
suggesting <strong>the</strong>y are efficacious at M1. Fur<strong>the</strong>rmore, <strong>the</strong> allosteric potentiator BQCA increases<br />
potency of Carbachol (CCh) in calcium assays. However, in contrast to CCh which has been<br />
found to induce recruitment of arrestin3 to active M1 receptors, TBPB <strong>an</strong>d VU0184670 failed to<br />
induce recruitment. In contrast, BQCA facilitates arrestin3 recruitment (i.e. induces recruitment<br />
at submaximal concentrations of CCh). These results suggest that allosteric lig<strong>an</strong>ds TBPB <strong>an</strong>d<br />
VU0184670 preferentially activate <strong>the</strong> PLC pathway while BQCA shows no preference <strong>for</strong><br />
ei<strong>the</strong>r pathway. Fur<strong>the</strong>rmore, given that arrestins induce receptor desensitization <strong>an</strong>d scaffold<br />
effectors <strong>for</strong> <strong>the</strong>ir activation, differential arrestin recruitment may result in different signaling<br />
outcomes such as differential M1 desensitization or absence of arrestin dependent ERK<br />
phosphorylation. Toge<strong>the</strong>r, <strong>the</strong>se data indicate that allosteric activators induce novel <strong>for</strong>ms of M1<br />
activation <strong>an</strong>d provide insight into rational drug design <strong>for</strong> <strong>the</strong> generation of pathway-selective<br />
M1 compounds.<br />
Disclosures: G.J. Digby, None; L.E. Diaz Gimenez, None; A.E. Brady, None; E.P. Lebois,<br />
None; C.W. Lindsley, None; C.D. Weaver, None; V.V. Gurevich, None; P.J. Conn, None.
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.10/C10<br />
Topic: B.03.a. Muscarinic receptors<br />
Title: Receptor domains involving in agonist-dependent muscarinic M1 receptor recycling<br />
Authors: *A. THANGARAJU, G. W. SAWYER;<br />
Oklahoma State Univ. CHS, Tulsa, OK<br />
Abstract: Muscarinic receptors are G protein-coupled receptors (GPCRs), which respond to <strong>the</strong><br />
endogenous neurotr<strong>an</strong>smitter acetylcholine. Like o<strong>the</strong>r GPCRs, muscarinic receptors are known<br />
to undergo agonist-dependent internalization, recycling <strong>an</strong>d downregulation. The goal of our<br />
study was to find signal sequences that regulate M1 receptor recycling. To do so, we made M1<br />
del 276-282 <strong>an</strong>d M1 del 447-460 mut<strong>an</strong>t receptors using site-directed mutagenesis. Wild-type<br />
<strong>an</strong>d mut<strong>an</strong>t receptors were stably expressed in CHO cells <strong>an</strong>d agonist-dependent internalization,<br />
recycling <strong>an</strong>d downregulation were characterized. In internalization assays, cells stably<br />
expressing wild-type or mut<strong>an</strong>t M1 receptors were incubated with muscarinic agonist carbachol<br />
<strong>for</strong> various time intervals <strong>for</strong> up to 240 min prior to per<strong>for</strong>ming intact whole cell [ 3 H]NMS<br />
binding assays. We found that <strong>the</strong> internalization of wild-type <strong>an</strong>d M1 del 276-282 mut<strong>an</strong>t<br />
receptors were similar. In downregulation assays, cells expressing wild-type or mut<strong>an</strong>t M1<br />
receptors were incubated with carbachol <strong>for</strong> 24 hours prior to per<strong>for</strong>ming intact whole cell<br />
binding assays. Using this approach, we determined that carbachol-induced downregulation of<br />
M1 del 276-282 receptors was signific<strong>an</strong>tly impaired when compared to wild-type. For receptor<br />
recycling, cells are incubated with carbachol to achieve <strong>an</strong> equivalent amount of receptor<br />
internalization <strong>an</strong>d <strong>the</strong>n treated with benzilyl choline mustard to alkylate <strong>the</strong> receptors remaining<br />
on <strong>the</strong> cell surface. The amount of receptors returning to <strong>the</strong> cell surface is <strong>the</strong>n determined at<br />
various periods of time up to 90 min using intact whole cell binding assays. Recycling following<br />
carbachol exposure was severely impaired <strong>for</strong> M1 del 276-282 receptors when compared to wildtype.<br />
In cursory experiments, we found that <strong>the</strong> M1 del 447-460 receptors have a similar<br />
internalization to wild-type but had impaired recycling. In summary, one or more sequences in<br />
M1 receptors may be necessary <strong>for</strong> agonist-dependent recycling.<br />
Disclosures: A. Th<strong>an</strong>garaju, None; G.W. Sawyer, None.<br />
Poster
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.11/C11<br />
Topic: B.03.a. Muscarinic receptors<br />
Title: In vivo qu<strong>an</strong>tification of phosphoinositide hydrolysis in hippocampus <strong>an</strong>d submaxillary<br />
gl<strong>an</strong>d induced by selective <strong>an</strong>d non-selective muscarinic agonists<br />
Authors: *M. L. HENDRICKSON, M. L. VERDONE, S. A. HANSON, J. D. BECK, T. M.<br />
TWOSE;<br />
Neurosci., Mithridion, Inc., Madison, WI<br />
Abstract: Measurement of phosphoinositide hydrolysis <strong>an</strong>d subsequent accumulation of myoinositol-1-phosphate<br />
(IP1) in <strong>the</strong> presence of lithium has become <strong>the</strong> st<strong>an</strong>dard functional assay<br />
<strong>for</strong> activation of M1, M3, <strong>an</strong>d M5 receptors <strong>for</strong> in vitro cell <strong>an</strong>d ex vivo tissue systems. However,<br />
<strong>an</strong>alogous in vivo assays are rarely per<strong>for</strong>med due to <strong>the</strong>ir labor-intensive nature (Bymaster et al.,<br />
1998, Brain Research, 795:179-190). Here we describe a relatively simple, robust procedure <strong>for</strong><br />
<strong>the</strong> in vivo measurement of IP1 in brain <strong>an</strong>d o<strong>the</strong>r tissues of rats <strong>an</strong>d mice. After predosing with<br />
lithium, <strong>an</strong>imals are given muscarinic agonists of interest by various administration routes.<br />
Fifteen minutes to two hours later, hippocampus <strong>an</strong>d submaxillary gl<strong>an</strong>d are collected, <strong>an</strong>d IP1<br />
levels are qu<strong>an</strong>tified using a non-radioactive, time-resolved fluorescence reson<strong>an</strong>ce energy<br />
tr<strong>an</strong>sfer assay. A series of non-selective agonists (pilocarpine, oxotremorine, <strong>an</strong>d arecoline),<br />
described selective agonists (x<strong>an</strong>omeline, talsaclidine, <strong>an</strong>d cevimeline), <strong>an</strong>d novel selective<br />
agonists were examined <strong>for</strong> <strong>the</strong>ir ability to induce IP1 accumulation. In addition, agonist<br />
concentrations in <strong>the</strong> plasma <strong>an</strong>d brain c<strong>an</strong> be correlated to muscarinic receptor activation to<br />
provide estimates of intrinsic efficacy <strong>an</strong>d <strong>the</strong>rapeutic ratio.<br />
Disclosures: M.L. Hendrickson, Mithridion, Inc., A. Employment (full or part-time);<br />
Mithridion, Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); M.L. Verdone, Mithridion, Inc., A. Employment (full or part-time); Mithridion, Inc.,<br />
E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); S.A. H<strong>an</strong>son,<br />
Mithridion, Inc., A. Employment (full or part-time); Mithridion, Inc., E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); J.D. Beck, Mithridion, Inc., A.<br />
Employment (full or part-time); Mithridion, Inc., E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); T.M. Twose, Mithridion, Inc., A. Employment (full or<br />
part-time); Mithridion, Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property).<br />
Poster
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.12/C12<br />
Topic: B.03.a. Muscarinic receptors<br />
Support: NIH Gr<strong>an</strong>t NS31173<br />
Title: Phosphorylation of HSP27 in SH-SY5Y neuroblastoma cells is <strong>an</strong> integration point <strong>for</strong><br />
muscarinic receptor-mediated signal tr<strong>an</strong>sduction<br />
Authors: *L. A. DOKAS 1 , S. GE 1 , W. S. MESSER, Jr. 1,2 ;<br />
1 Pharmacol., 2 Medicinal <strong>an</strong>d Biol. Chem., The Univ. of Toledo, Toledo, OH<br />
Abstract: Activation of muscarinic receptors on hum<strong>an</strong> SH-SY5Y neuroblastoma cells causes a<br />
pleiotropic response that includes reorg<strong>an</strong>ization of <strong>the</strong> cytoskeleton <strong>an</strong>d inhibition of apoptosis.<br />
Since <strong>the</strong> small heat shock protein, HSP27, is involved in both processes, its phosphorylation at<br />
Ser-82, a modification required <strong>for</strong> its function, was characterized by immunoblotting in<br />
response to <strong>the</strong> cholinergic agonist, carbachol (CCh). Muscarinic receptor activation by 1 mM<br />
CCh rapidly induces HSP27 phosphorylation by multiple protein kinases as evidenced by<br />
sensitivity to selective inhibitors. In contrast, phosphorylation elicited ei<strong>the</strong>r by a phorbol ester or<br />
hyperosmotic stress is more completely reduced by inhibition of protein kinase C (PKC) or p38<br />
mitogen-activated protein kinase (MAPK), respectively. Paradoxically, inhibition of<br />
phosphoinositide 3-kinase (PI3-K) by LY 294002 or Akt by Akti-1/2 increases basal HSP27<br />
phosphorylation. Akti-1/2 is more effective th<strong>an</strong> LY 294002 while rapamycin, which inhibits<br />
mTORC1, a downstream target of Akt, is without effect; thus, <strong>the</strong> stimulation occurs at <strong>the</strong> level<br />
of Akt. Conversely, when Akt phosphorylation at Ser-473, a marker <strong>for</strong> its activation, is<br />
stimulated by insulin, no phosphorylation of HSP27 is detected. In vitro, Akti-1/2 c<strong>an</strong> also<br />
inhibit MAPK kinase (MKK), <strong>the</strong> upstream activator of extracellular signal-regulated kinase1/2<br />
(ERK1/2). However, basal phosphorylation of ERK1/2 is less sensitive to Akti-1/2 th<strong>an</strong> that of<br />
HSP27 <strong>an</strong>d CCh-stimulated ERK1/2 phosphorylation is inhibited only with high concentrations<br />
of Akti-1/2 which do not reduce HSP27 phosphorylation. Moreover, direct inhibition of MKK<br />
with PD 98059 does not increase basal phosphorylation of HSP27. The stimulatory effect of<br />
Akti-1/2 on HSP27 phosphorylation is reversed by a p38 MAPK inhibitor (SB 203580)<br />
indicating <strong>an</strong> inverse relationship between Akt <strong>an</strong>d p38 MAPK activities. As in o<strong>the</strong>r cell types,<br />
<strong>the</strong>se results suggest that a protein complex may exist in SH-SY5Y cells through which <strong>the</strong><br />
activities of Akt <strong>an</strong>d p38 MAPK are regulated. Such a complex would provide a point of<br />
integration <strong>for</strong> HSP27 phosphorylation through <strong>the</strong> multiple signal tr<strong>an</strong>sduction pathways that<br />
are responsive to CCh-mediated muscarinic receptor activation.<br />
Disclosures: L.A. Dokas, None; S. Ge, None; W.S. Messer, Dr. William Messer holds several<br />
patents on muscarinic agonists. He is <strong>the</strong> Chief Scientific Officer <strong>for</strong> <strong>an</strong>d has fin<strong>an</strong>cial interest in
Mithridion, Inc., Madison WI/Toledo OH, E. Ownership Interest (stock, stock options, patent or<br />
o<strong>the</strong>r intellectual property).<br />
Poster<br />
515. Muscarinic Receptors<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 515.13/C13<br />
Topic: B.03.a. Muscarinic receptors<br />
Title: G-protein-coupled receptor modulation of N-type Ca2+ ch<strong>an</strong>nels is differentially affected<br />
in sympa<strong>the</strong>tic neurons <strong>from</strong> RGS7 mut<strong>an</strong>t mice, Rgs7tm1Lex<br />
Authors: *V. B. LU 1 , J. ZHANG 2 , W. F. SIMONDS 2 , S. R. IKEDA 1 ;<br />
1 2<br />
Lab. of Mol. Physiol., NIH/NIAAA, Rockville, MD; Metabolic Dis. Br., NIH/NIDDK,<br />
Be<strong>the</strong>sda, MD<br />
Abstract: Proteins in <strong>the</strong> R7 regulator of G-protein signaling (RGS) subfamily (including RGS6,<br />
7, 9 <strong>an</strong>d 11) bind with Gβ5 <strong>an</strong>d c<strong>an</strong> act as GTPase-activating proteins (GAPs) <strong>for</strong> several major<br />
classes of G-proteins (Gαi, Gαo, Gαq). These negative regulators attenuate or terminate activation<br />
through G-protein coupled receptor (GPCR) pathways during <strong>an</strong>d after <strong>the</strong> presence of agonist,<br />
respectively. However, <strong>the</strong> degree of selectivity of <strong>the</strong> various RGS proteins <strong>for</strong> specific Gprotein<br />
signaling pathways remains to be determined. Moreover, <strong>the</strong> role played by several<br />
similar RGS proteins in neuronal tissues is unclear.<br />
To study <strong>the</strong> specific function of RGS7, a mut<strong>an</strong>t mouse with a germline deletion of Rgs7 exon<br />
11 causing <strong>an</strong> in-frame 32 amino acid deletion in <strong>the</strong> coding sequence, Rgs7 tm1Lex , was utilized.<br />
Expression of <strong>the</strong> truncated mut<strong>an</strong>t RGS7 protein in neurons of homozygous mut<strong>an</strong>t mice was<br />
confirmed by Western blotting. Potential effects of <strong>the</strong> RGS7 mutation were assayed by<br />
recording whole-cell Ca 2+ currents, which are modulated by various GPCRs. The inhibition of<br />
voltage-gated Ca 2+ ch<strong>an</strong>nels was measured in superior cervical g<strong>an</strong>glion (SCG) neurons <strong>an</strong>d<br />
compared between Rgs7 tm1Lex homozygous mut<strong>an</strong>t mice <strong>an</strong>d wild-type littermates.<br />
The kinetics of Ca 2+ ch<strong>an</strong>nel inhibition following norepinephrine exposure (10 µM, 30 s) were<br />
similar between both groups of mice. The onset of inhibition (15 ± 2 s <strong>for</strong> controls, 20 ± 5 s <strong>for</strong><br />
mut<strong>an</strong>ts) <strong>an</strong>d time-course <strong>for</strong> G-protein deactivation (67 ± 5 s <strong>for</strong> controls, 58 ± 5 s <strong>for</strong> mut<strong>an</strong>ts)<br />
were comparable. Also, no signific<strong>an</strong>t difference was found between dose-response curves <strong>for</strong><br />
wild-type <strong>an</strong>d Rgs7 tm1Lex mut<strong>an</strong>t mice (0.51 ± 0.1 µM versus 0.56 ± 0.2 µM <strong>for</strong> EC50, p > 0.5; 55<br />
± 3% versus 52 ± 5% Ca 2+ ch<strong>an</strong>nel inhibition <strong>for</strong> Emax, p > 0.7). Thus, study of <strong>the</strong> RGS7 mut<strong>an</strong>t<br />
mouse did not implicate RGS7 in Ca 2+ ch<strong>an</strong>nel inhibition mediated by α2-adrenergic receptors.<br />
In contrast, <strong>the</strong> sensitivity of muscarinic receptors to <strong>the</strong> cholinergic agonist oxotremorine-M
(OxoM) was signific<strong>an</strong>tly greater in RGS7 mut<strong>an</strong>t mice compared with wild-type (EC50: 43 ± 8<br />
nM versus 444 ± 20 nM, p < 0.05). No ch<strong>an</strong>ge in efficacy was observed (Emax: 58 ± 8% current<br />
inhibition <strong>for</strong> controls, 65 ± 5% <strong>for</strong> mut<strong>an</strong>ts) <strong>an</strong>d pertussis toxin treatment (PTX, 0.5 µg/ml,<br />
overnight) abolished <strong>the</strong> difference in OxoM sensitivity. Also, <strong>the</strong>re was no difference in <strong>the</strong><br />
onset of Ca 2+ ch<strong>an</strong>nel inhibition (15 ± 3 s <strong>for</strong> controls, 16 ± 2 s <strong>for</strong> mut<strong>an</strong>ts) or response<br />
deactivation (65 ± 5 s <strong>for</strong> controls, 64 ± 6 s <strong>for</strong> mut<strong>an</strong>ts) following OxoM exposure (10 µM, 30<br />
s). There<strong>for</strong>e, RGS7 appears to have a selective role in altering <strong>the</strong> sensitivity of muscarinic<br />
receptor responses through PTX-sensitive G-proteins but not those coupled to adrenergic<br />
receptors.<br />
Disclosures: V.B. Lu, None; J. Zh<strong>an</strong>g, None; W.F. Simonds, None; S.R. Ikeda, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.1/C14<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NIH Gr<strong>an</strong>t MH58448<br />
Title: Is estrogen-induced desensitization of 5-HT1A receptors mediated through GPR30?<br />
Authors: *C. E. MCALLISTER, D. ROSSI, Y. DAI, J. FRANKLIN, N. MUMA, Q. LI;<br />
Dept. of Pharmacol. <strong>an</strong>d Toxicology, Univ. of K<strong>an</strong>sas, Lawrence, KS<br />
Abstract: Selective serotonin reuptake inhibitors (SSRIs) are commonly used to treat mood<br />
disorders. SSRIs are clinically effective after 2-3 weeks of treatment, <strong>the</strong> time it takes to induce<br />
desensitization of 5-HT1A receptor signaling. Estrogen treatment <strong>for</strong> 2 days causes a partial<br />
desensitization of 5-HT1A receptor signaling in <strong>the</strong> hypothalamic paraventricular nucleus<br />
(PVN), as measured by a decrease in <strong>the</strong> release of oxytocin <strong>an</strong>d adrenocorticotropin hormone<br />
(ACTH) in response to 5-HT1A receptor stimulation. The mech<strong>an</strong>ism by which estrogen induces<br />
5-HT1A receptor desensitization is unknown; however, our studies suggest that <strong>the</strong> recently<br />
identified membr<strong>an</strong>e estrogen receptor GPR30 may be involved. Female Sprague-Dawley rats<br />
were ovariectomized <strong>an</strong>d <strong>the</strong>n double barrel c<strong>an</strong>nulae were placed directly above <strong>the</strong> PVN to<br />
allow direct injections of G-1, a selective agonist <strong>for</strong> GPR30. Rats received injections of G-1<br />
(10nmol/ul/day or 100nmol/ul/day) <strong>for</strong> 2 consecutive days; 24 hours after <strong>the</strong> last injection, rats<br />
received a challenge injection of 5-HT1A receptor agonist (+)-8-hydroxy-2dipropylaminotetralin<br />
(DPAT; 200ug/kg) 15 minutes prior to decapitation. Pretreatment with
high dose of G-1 signific<strong>an</strong>tly decreased <strong>the</strong> oxytocin response to DPAT <strong>an</strong>d both doses of<br />
G-1 signific<strong>an</strong>tly decreased <strong>the</strong> ACTH response (Xu et at. <strong>2009</strong>). Western blots of PVN tissue<br />
showed a signific<strong>an</strong>t increase in GPR30 protein levels with high <strong>an</strong>d low dose G-1 pretreatment,<br />
however, <strong>the</strong>re was no signific<strong>an</strong>t ch<strong>an</strong>ge in 5-HT1A receptor, Gαz, or estrogen receptor β at<br />
ei<strong>the</strong>r dose. Two b<strong>an</strong>ds (~60kD <strong>an</strong>d ~45kD) were measured <strong>for</strong> RGSz1 protein: both are<br />
signific<strong>an</strong>tly increased in <strong>the</strong> low dose G-1 treated rats. RGSz1 is a GTPase that is highly<br />
selective <strong>for</strong> <strong>the</strong> Gαz subunit, which couples to 5-HT1A receptors in <strong>the</strong> PVN to stimulate <strong>the</strong><br />
release of hormones. RGSz1 undergoes posttr<strong>an</strong>slational modification, <strong>an</strong>d <strong>the</strong> two b<strong>an</strong>ds may<br />
represent differentially modified RGSz1 protein. To fur<strong>the</strong>r investigate whe<strong>the</strong>r GPR30 induces<br />
5-HT1A receptor desensitization, <strong>an</strong> siRNA-expressing adenoviral vector was used to silence<br />
expression of GPR30 in <strong>the</strong> PVN. Ovariectomized rats were injected with adenovirus expressing<br />
ei<strong>the</strong>r siRNA or mismatch RNA <strong>for</strong> GPR30; 5 days later rats received 2 days of pretreatment<br />
with ei<strong>the</strong>r β-estradiol-3-benzoate (2ug/kg) or vehicle followed by challenge with DPAT<br />
(200ug/kg) or saline (1ml/kg). Hormone responses will be measured to determine if reductions in<br />
GPR30 impact <strong>the</strong> desensitization of 5-HT1A receptors in <strong>the</strong> PVN. These data will confirm<br />
whe<strong>the</strong>r estrogen induces desensitization of 5-HT1A receptors through GPR30 signaling, <strong>an</strong>d<br />
fur<strong>the</strong>r investigation of <strong>the</strong> precise mech<strong>an</strong>ism is needed.<br />
Disclosures: C.E. McAllister, None; D. Rossi, None; Y. Dai, None; J. Fr<strong>an</strong>klin, None; N.<br />
Muma, None; Q. Li, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.2/C15<br />
Topic: B.03.d. Serotonin receptors<br />
Title: F15599, a selective 5-HT1A agonist: Frontal cortex <strong>an</strong>d dorsal Raphe microinjections<br />
reveal low- <strong>an</strong>d high-dose <strong>for</strong>ced swim test effects<br />
Authors: *A. NEWMAN-TANCREDI, A. AUCLAIR, C. BARRET-GRÉVOZ, A.<br />
GALINIER, M. BARRETO, R. DEPOORTÈRE;<br />
Ctr. Recherche Pierre Fabre, Castres, Fr<strong>an</strong>ce<br />
Abstract: Activation of post-synaptic 5-HT1A receptors contributes to <strong>the</strong> activity of<br />
<strong>an</strong>tidepress<strong>an</strong>ts, whereas activation of 5-HT1A autoreceptors may delay <strong>the</strong>ir <strong>the</strong>rapeutic effects<br />
by feed-back inhibition of serotonergic tr<strong>an</strong>smission. F15599 is a highly selective <strong>an</strong>d efficacious<br />
5-HT1A receptor agonist. In tests of G-protein activation, c-fos expression, ERK1/2
phosphorylation, microdialysis <strong>an</strong>d electrophysiology, F15599 preferentially activates postsynaptic<br />
medial pre-frontal cortex (mPFC) 5-HT1A receptors versus Raphe-localised 5-HT1A<br />
autoreceptors (Newm<strong>an</strong>-T<strong>an</strong>credi et al. Br J Pharmacol 156:338, <strong>2009</strong>; Celada et al. Soc.<br />
Neurosci. Abstr 33:170.27, 2007). F15599 is also remarkably potent in <strong>the</strong> rat <strong>for</strong>ced swim test<br />
(FST), completely abolishing immobility upon acute systemic administration (ED50 = 0.1 mg/kg<br />
p.o.). Here, we examined <strong>the</strong> brain regions underlying this action by local microinjection of<br />
F15599 into <strong>the</strong> mPFC, <strong>the</strong> dorsal (DRN) or medi<strong>an</strong> Raphe nucleus (MRN). The effects of<br />
F15599 were compared with those of a close chemical congener, F13714, <strong>an</strong>d of <strong>the</strong> prototypical<br />
5-HT1A receptor agonist, (+)8-OH-DPAT.<br />
Microinjection of F13714 or (+)8-OH-DPAT (0.016 to 8 µg) into <strong>the</strong> mPFC dose-dependently<br />
<strong>an</strong>d monophasically reduced immobility in <strong>the</strong> FST (maximal inhibition >60% at 8 µg). These<br />
effects were inhibited by pre-administration of <strong>the</strong> selective 5-HT1A receptor <strong>an</strong>tagonist,<br />
WAY100635 (0.63 mg/kg s.c.). In contrast, microinjection of low doses of F15599 (0.016 to 1<br />
µg) into <strong>the</strong> mPFC resulted in a V-shaped dose-response curve of immobility in <strong>the</strong> FST (~50%<br />
inhibition of immobility at 0.25 µg). The effect of 0.25 µg F15599 was <strong>an</strong>tagonised by<br />
WAY100635. Higher doses of intra-mPFC F15599 (1 to 32 µg) resulted in a progressive<br />
decrease in immobility in <strong>the</strong> FST (>70% at 32µg) which was also reversed by WAY100635.<br />
<strong>When</strong> <strong>the</strong> agonists were microinjected into <strong>the</strong> DRN, effects on FST closely resembled those<br />
observed in <strong>the</strong> mPFC. F13714 <strong>an</strong>d (+)8-OH-DPAT yielded monophasic dose-response curves<br />
whereas F15599 evoked a V-shaped dose-response curve at low doses (0.001 to 1 µg; ~70%<br />
effect at 0.016 µg) followed by a decrease of immobility at high doses (1 to 32 µg; ~60% effect<br />
at 32 µg). The effects of F15599 at both 0.016 <strong>an</strong>d at 32 µg were reversed by WAY100635.<br />
Unlike <strong>the</strong> mPFC <strong>an</strong>d DRN, microinjections into <strong>the</strong> MRN resulted in monophasic FST<br />
responses <strong>for</strong> all three agonists. These data indicate that, in contrast to F13714 or (+)8-OH-<br />
DPAT, F15599 evokes dual low-dose <strong>an</strong>d high-dose 5-HT1A receptor-mediated responses in <strong>the</strong><br />
mPFC <strong>an</strong>d in <strong>the</strong> DRN, suggesting that independent subpopulations of 5-HT1A receptors may be<br />
involved in <strong>the</strong> <strong>an</strong>tidepress<strong>an</strong>t-like properties of F15599.<br />
Disclosures: A. Newm<strong>an</strong>-T<strong>an</strong>credi, Pierre Fabre Laboratories, A. Employment (full or parttime);<br />
A. Auclair, Pierre Fabre Laboratories, A. Employment (full or part-time); C. Barret-<br />
Grévoz, Piarre Fabre Laboratories, A. Employment (full or part-time); A. Galinier, Pierre Fabre<br />
Laboratories, A. Employment (full or part-time); M. Barreto, Pierre Fabre Laboratories, A.<br />
Employment (full or part-time); R. Depoortère, Pierre Fabre Laboratories, A. Employment (full<br />
or part-time).<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.3/C16
Topic: B.03.d. Serotonin receptors<br />
Title: Aripiprazole modulates citalopram-induced 5-HT release <strong>an</strong>d MDMA-induced<br />
hyperactivity: Potential role of 5-HT2B receptor blockade<br />
Authors: *D. CUSSAC 1 , M.-B. ASSIÉ 2 , A. AUCLAIR 2 , R. DEPOORTERE 2 , I. RAULY-<br />
LESTIENNE 1 , I. RAULY-LESTIENNE 1 , J.-C. MARTEL 2 , A. NEWMAN-TANCREDI 2 ;<br />
1 Dept Cell. & Mol Bio, 2 Div. Neurobio. 2, Inst. Recherches Pierre Fabre, Castres, Fr<strong>an</strong>ce<br />
Abstract: The atypical <strong>an</strong>tipsychotic aripiprazole has shown clinical efficacy as adjunct<br />
treatment with <strong>the</strong> selective serotonin reuptake inhibitor (SSRI), citalopram, <strong>for</strong> refractory<br />
depression, but <strong>the</strong> mech<strong>an</strong>istic basis of this activity is unclear. Interestingly, whereas m<strong>an</strong>y<br />
atypical <strong>an</strong>tipsychotics potently <strong>an</strong>tagonise 5-HT2A receptors, aripiprazole preferentially<br />
<strong>an</strong>tagonises 5-HT2B receptors (Shapiro et al., Neuropsychopharmacol 28:1400, 2003), a subtype<br />
that has been shown to influence 5-HT re-uptake in serotonergic neuronal cells (Launay et al.,<br />
FASEB 20:1846, 2006). Fur<strong>the</strong>r, 5-HT2B receptors are implicated in <strong>the</strong> control of<br />
methylenedioxymethamphetamine (MDMA)-induced hyperactivity in mice (Doly et al., J<br />
Neurosci 28:2933, 2008). We <strong>the</strong>re<strong>for</strong>e compared aripiprazole <strong>an</strong>d <strong>the</strong> selective 5-HT2B receptor<br />
<strong>an</strong>tagonist LY266097 on (i) in vitro binding <strong>an</strong>d activation of cloned h5-HT2B receptors; (ii)<br />
medial prefrontal cortex 5-HT release induced by <strong>the</strong> SSRI, citalopram, in freely moving rats<br />
using in vivo microdialysis, <strong>an</strong>d (iii) MDMA-induced hyperactivity in mice.<br />
Aripiprazole exhibited marked affinity at 5-HT2B receptors (pKi = 8.8) that was in <strong>the</strong> same<br />
r<strong>an</strong>ge as that at D2 (9.2), 5-HT1A (8.4) <strong>an</strong>d 5-HT2A receptors (8.2). In sensitive tests of 5-HT2B<br />
receptor activation, aripiprazole abolished 5-HT-induced [ 35 S]GTPγS binding to Gq proteins<br />
(pKB = 7.6). LY266097 exhibited potent <strong>an</strong>tagonism (pKB = 8.2) in <strong>the</strong> same assay <strong>an</strong>d was<br />
highly selective <strong>for</strong> 5-HT2B receptors (pKi = 9.9) versus a r<strong>an</strong>ge of o<strong>the</strong>r receptors (at least 100fold<br />
affinity ratio). In microdialysis experiments, citalopram (0.16-10 mg/kg, i.p.) dosedependently<br />
increased extracellular 5-HT (up to 320% of baseline values at 10 mg/kg) <strong>an</strong>d <strong>the</strong><br />
increase in 5-HT levels induced by citalopram (0.63 mg/kg; approx 200% of baseline) was<br />
signific<strong>an</strong>tly decreased (to 122% of baseline) by pre-administration of aripiprazole (0.63 mg/kg,<br />
i.p.), although no effect of aripiprazole alone was seen at this dose. LY266097 (2.5 <strong>an</strong>d 10<br />
mg/kg, i.p.) also decreased citalopram-induced 5-HT release (to 127 <strong>an</strong>d 138% of baseline,<br />
respectively) without itself modifying 5-HT levels. MDMA (10 mg/kg i.p.) induced marked<br />
hyperactivity in mice : 4-fold increase over that of vehicle-treated controls. Aripiprazole (0.04 to<br />
0.63 mg/kg i.p.) diminished this hyperactivity (complete inhibition at 0.63), whereas LY266097<br />
(0.01 to 0.16 mg/kg, i.p) partially reduced hyperactivity.These data suggest that 5-HT2B receptor<br />
<strong>an</strong>tagonism contributes to <strong>the</strong> neurochemical <strong>an</strong>d behavioural effects of aripiprazole. Fur<strong>the</strong>r, 5-<br />
HT2B <strong>an</strong>tagonism may be of utility in <strong>the</strong> control of some aspects of neuropsychiatric disorders.<br />
Disclosures: D. Cussac , None; M. Assié, None; A. Auclair, None; R. Depoortere, None; I.<br />
Rauly-Lestienne, None; I. Rauly-Lestienne, None; J. Martel, None; A. Newm<strong>an</strong>-T<strong>an</strong>credi,<br />
None.<br />
Poster
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.4/C17<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NIH Gr<strong>an</strong>t T32M07108<br />
NIMH Gr<strong>an</strong>t 63303<br />
Title: Reduced expression of conditioned fear in rats by 5-HT(1B) agonist CP 94,253<br />
Authors: *R. A. MCDEVITT, J. F. NEUMAIER;<br />
Neurobiol & Behavior, Univ. Washington, Seattle, WA<br />
Abstract: The neurotr<strong>an</strong>smitter 5-hydroxytryptamine (5-HT; serotonin) is widely implicated in<br />
psychiatric disease states such as depression <strong>an</strong>d posttraumatic stress disorder. Some of <strong>the</strong><br />
emotional <strong>an</strong>d cognitive effects of serotonin are mediated via <strong>the</strong> 5-HT(1B) receptor, a Gi/ocoupled<br />
receptor localized primarily on axon terminals which responds to 5-HT by inhibiting<br />
neurotr<strong>an</strong>smitter release. Genetic <strong>an</strong>d pharmacological studies have shown that 5-HT(1B)<br />
receptors regulate <strong>an</strong>xiety <strong>an</strong>d <strong>the</strong> acquisition of aversive memories. Here we investigate <strong>the</strong> role<br />
of 5-HT(1B) receptors in regulating <strong>the</strong> expression, ra<strong>the</strong>r th<strong>an</strong> acquisition, of aversive memory<br />
by conditioning <strong>an</strong>imals in a drug-naive state <strong>an</strong>d injecting drug prior to <strong>the</strong> a session. We<br />
trained rats to fear a novel chamber by administering foot shocks, <strong>an</strong>d 24 hours later injected<br />
ei<strong>the</strong>r saline or <strong>the</strong> selective 5-HT(1B) agonist CP 94,253 30 minutes prior to context reexposure.<br />
Fear was measured by scoring rats <strong>for</strong> freezing behavior during <strong>the</strong> context reexposure.<br />
In experiment 1, rats were injected (i.p.) with ei<strong>the</strong>r sterile saline (n=4) or CP 94,253<br />
(n=4) at 1 mg/kg. In experiment 2, rats were injected with ei<strong>the</strong>r sterile saline (n=4) or CP<br />
94,253 (n=4) at 5 mg/kg. We found that at 1 mg/kg, injections of CP 94,253 signific<strong>an</strong>tly<br />
reduced freezing (p0.6). These<br />
results suggest that activation of 5-HT(1B) receptors reduces <strong>the</strong> expression of conditioned fear<br />
in <strong>an</strong> inverted-U shaped m<strong>an</strong>ner. To confirm this, we are currently per<strong>for</strong>ming a dose-response<br />
curve with doses of CP 94,253 r<strong>an</strong>ging <strong>from</strong> 0.3 to 10 mg/kg. Future experiments will measure<br />
<strong>the</strong> relative contributions of 5-HT(1B) receptors located on serotonergic neurons (autoreceptors)<br />
vs. on non-serotonergic neurons (hetereoreceptors). O<strong>the</strong>r experiments will explore <strong>the</strong> extent to<br />
which reduced freezing is caused by reduced emotionality vs. impaired spatial awareness <strong>an</strong>d/or<br />
recall.<br />
Disclosures: R.A. McDevitt, None; J.F. Neumaier, None.
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.5/C18<br />
Topic: B.03.d. Serotonin receptors<br />
Support: USPHS Gr<strong>an</strong>t NS 034153<br />
Loyola University Chicago Intramural Gr<strong>an</strong>t LU201328<br />
Title: In vivo 5-HT1A receptor activation of MAP kinase in <strong>the</strong> peripubertal rat hypothalamic<br />
PVN: Temporal differences in agonist-dependent activation<br />
Authors: *G. BATTAGLIA, M. L. PETRUNICH-RUTHERFORD, F. GARCIA;<br />
Loyola Univ, Stritch Sch. Med., Maywood, IL<br />
Abstract: In <strong>the</strong> adult rat hypothalamic paraventricular nucleus (PVN): (1) in vivo<br />
administration of (+)8-OH-DPAT or t<strong>an</strong>dospirone activates extracellular signal-regulated kinase<br />
(ERK) via 5-HT1A receptors <strong>an</strong>d (2) ERK activation is required <strong>for</strong> selective serotonin reuptake<br />
inhibitors (SSRIs) to desensitize 5-HT1A receptor-mediated hormone responses. (+)8-OH-DPAT<br />
<strong>an</strong>d t<strong>an</strong>dospirone activate ERK to a comparable extent (~100% over basal) <strong>an</strong>d this activation by<br />
(+)8-OH-DPAT is rapid <strong>an</strong>d tr<strong>an</strong>sient, as increases in phosphorylated (activated) ERK (pERK)<br />
occur at 5 minutes post-injection <strong>an</strong>d return to baseline by 15 minutes. As we have previously<br />
observed a number of unique aspects of 5-HT1A signaling prior to maturation, <strong>the</strong> present study<br />
investigated 5-HT1A receptor activation of ERK in <strong>the</strong> peripubertal PVN with respect to: (1) <strong>the</strong><br />
onset <strong>an</strong>d longevity of 5-HT1A receptor activation of mitogen activated protein kinase (MAP<br />
kinase) <strong>an</strong>d (2) potential agonist-dependent differences in 5-HT1A receptor activation of MAP<br />
kinase. Peripubertal male Sprague-Dawley rats (42 days old) were injected with saline (1 ml/kg,<br />
s.c.), (+)8-OH-DPAT (0.2 mg/kg, s.c.), or t<strong>an</strong>dospirone (10 mg/kg, s.c.). Rats were sacrificed 5,<br />
10, 15, 20 or 30 minutes post-injection <strong>an</strong>d brains were collected to determine levels of ERK <strong>an</strong>d<br />
phosphorylated ERK in <strong>the</strong> PVN. (+)8-OH-DPAT signific<strong>an</strong>tly increased pERK in <strong>the</strong> PVN at 5<br />
<strong>an</strong>d 10 minutes post-injection, with a gradual return to baseline by 20 minutes. In contrast,<br />
t<strong>an</strong>dospirone produced a signific<strong>an</strong>t <strong>an</strong>d tr<strong>an</strong>sient elevation of pERK in <strong>the</strong> PVN only at 10<br />
minutes <strong>an</strong>d <strong>the</strong> response was approximately 2-fold higher th<strong>an</strong> that elicited by (+)8-OH-DPAT.<br />
These data suggest that in addition to agonist-induced differences in <strong>the</strong> onset <strong>an</strong>d longevity of 5-<br />
HT1A activation of ERK in <strong>the</strong> peripubertal PVN, different classes of agonists may exhibit<br />
differences in efficacy with respect to <strong>the</strong> 5-HT1A receptor activation of ERK. Subsequent<br />
studies are required to investigate this possibility <strong>an</strong>d to determine if differences in efficacy are<br />
due to <strong>the</strong> ability of <strong>an</strong> agonist to “traffick” 5-HT1A receptors to one or more G-protein subtypes<br />
associated with ERK activation in <strong>the</strong> peripubertal PVN. These studies should identify aspects of
5-HT1A signaling pathways <strong>an</strong>d mech<strong>an</strong>isms of drug-induced desensitization that are unique to<br />
<strong>the</strong> immature brain <strong>an</strong>d may contribute to <strong>the</strong> <strong>the</strong>rapeutic <strong>an</strong>d/or side effects of serotonergic<br />
drugs used to treat children <strong>an</strong>d adolescents.<br />
Disclosures: G. Battaglia , None; M.L. Petrunich-Ru<strong>the</strong>r<strong>for</strong>d, None; F. Garcia, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.6/C19<br />
Topic: B.03.d. Serotonin receptors<br />
Support: USPHS Gr<strong>an</strong>t NS 034153<br />
Loyola University Chicago Intramural Gr<strong>an</strong>t LU201328<br />
Title: Initial identification of in vivo 5-HT1A receptor activation of protein kinase B (Akt) in<br />
peripubertal rat hypothalamic PVN: Evidence <strong>for</strong> agonist-dependent functional selectivity<br />
Authors: *M. L. PETRUNICH-RUTHERFORD, F. GARCIA, G. BATTAGLIA;<br />
Loyola Univ. Chicago, Maywood, IL<br />
Abstract: In vitro studies in cell lines or primary culture have indicated that serotonin 1A (5-<br />
HT1A) receptors increase levels of activated protein kinase B (Akt). However, 5-HT1A receptor<br />
activation of Akt has not been demonstrated in <strong>an</strong> in vivo model. We have previously shown that<br />
5-HT1A receptors in <strong>the</strong> peripubertal hypothalamic paraventricular nucleus (PVN) activate<br />
extracellular signal-regulated kinase (ERK) <strong>an</strong>d that activated ERK is required <strong>for</strong> selective<br />
serotonin reuptake inhibitors (SSRIs) to desensitize 5-HT1A receptor-mediated neuroendocrine<br />
responses. Given recent findings implicating Akt in stress <strong>an</strong>d depression, <strong>the</strong> present studies<br />
investigated 5-HT1A receptor signaling of Akt in <strong>the</strong> peripubertal PVN with respect to: (1) 5-<br />
HT1A receptor selectivity of (+)8-OH-DPAT activation of Akt, (2) <strong>the</strong> onset <strong>an</strong>d longevity of 5-<br />
HT1A receptor activation of Akt, <strong>an</strong>d (3) potential agonist-dependent differences in 5-HT1A<br />
receptor-mediated activation of Akt. In one experiment, peripubertal male Sprague-Dawley rats<br />
(42 days old) were pretreated with saline or <strong>the</strong> 5-HT1A receptor <strong>an</strong>tagonist WAY100635 (0.1<br />
mg/kg, s.c.) 30 min be<strong>for</strong>e injection of saline or (+)8-OH-DPAT (0.2 mg/kg, s.c.). Rats were<br />
sacrificed <strong>an</strong>d brains were collected to determine Akt <strong>an</strong>d phosphorylated Akt (pAkt) levels in<br />
<strong>the</strong> PVN. (+)8-OH-DPAT signific<strong>an</strong>tly elevated levels of pAkt in <strong>the</strong> PVN at 15 min postinjection,<br />
which was completely blocked by pretreatment with WAY100635. In <strong>an</strong>o<strong>the</strong>r
experiment, peripubertal male Sprague-Dawley rats (42 days old) were injected with saline, (+)8-<br />
OH-DPAT (0.2 mg/kg, s.c.), or t<strong>an</strong>dospirone (10 mg/kg, s.c.). Rats were sacrificed 5 to 30 min<br />
post-injection <strong>an</strong>d PVN levels of Akt <strong>an</strong>d pAkt were determined. (+)8-OH-DPAT signific<strong>an</strong>tly<br />
increased pAkt responses in <strong>the</strong> PVN within 5 min post-injection, <strong>an</strong>d this response was<br />
prolonged with a gradual tapering to baseline by 30 min. T<strong>an</strong>dospirone did not elicit a signific<strong>an</strong>t<br />
elevation of pAkt in <strong>the</strong> PVN until 10 min, <strong>an</strong>d this response rapidly declined to basal levels by<br />
15 min post-injection. The maximal elevation induced by t<strong>an</strong>dospirone was signific<strong>an</strong>tly (~32%)<br />
higher th<strong>an</strong> <strong>the</strong> maximal response elicited by (+)8-OH-DPAT. These data provide <strong>the</strong> first in<br />
vivo demonstration that 5-HT1A receptors c<strong>an</strong> activate Akt in <strong>the</strong> peripubertal PVN. Given our<br />
previous studies demonstrating 5-HT1A receptors c<strong>an</strong> stimulate hormone responses <strong>an</strong>d activate<br />
ERK activation in <strong>the</strong> peripubertal PVN, <strong>the</strong>se findings provide <strong>the</strong> first in vivo demonstration<br />
that 5-HT1A receptors c<strong>an</strong> mediate <strong>the</strong> concomit<strong>an</strong>t activation of three signaling pathways in<br />
peripubertal hypothalamic PVN. The clinical signific<strong>an</strong>ce of <strong>the</strong>se findings remains to be<br />
determined.<br />
Disclosures: M.L. Petrunich-Ru<strong>the</strong>r<strong>for</strong>d, None; F. Garcia, None; G. Battaglia, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.7/C20<br />
Topic: B.03.d. Serotonin receptors<br />
Support: Picower foundation<br />
Title: Serotonin receptor 1b signaling in nucleus accumbens neurons<br />
Authors: *M. ARANGO 1 , S. MUSATOV 1,2 , M. G. KAPLITT 1 ;<br />
1 Neurosurg., Weill Cornell Med. Col., New York, NY; 2 Neurologix, Inc, Fort Lee, NJ<br />
Abstract: One major hypo<strong>the</strong>sis explaining <strong>the</strong> pathophysiology of depression is <strong>the</strong><br />
deregulation of <strong>the</strong> serotonin system. P11 (S100A10) has been identified as a binding partner of<br />
serotonin receptors 5-HT1B <strong>an</strong>d 5-HT4 <strong>an</strong>d has been shown to modulate depressive phenotypes.<br />
Brain tissue <strong>from</strong> depressed patients demonstrates decreased levels of p11 <strong>an</strong>d tr<strong>an</strong>sgenic studies<br />
indicate that overexpression of p11 recapitulates rodent behavior seen after pharmacological<br />
<strong>an</strong>ti-depress<strong>an</strong>t treatment while p11 knock-out mice exhibit depression-like phenotypes. Using<br />
adeno-associated viruses type 2 (AAV2) expressing small interfering RNA against p11, our<br />
group previously demonstrated that focal p11 knockdown in <strong>the</strong> nucleus accumbens (NAcc)
produced a similar phenotype to tr<strong>an</strong>sgenic p11 KO <strong>an</strong>imals, as measured by <strong>the</strong> tail suspension<br />
test (TST) <strong>an</strong>d <strong>for</strong>ced swim test (FST). We also demonstrated that viral-mediated gene tr<strong>an</strong>sfer<br />
of p11 cDNA into <strong>the</strong> NAcc c<strong>an</strong> reverse <strong>the</strong> depressed phenotype of p11 KO mice.<br />
Previous data shows decreased 5HT1b agonist binding in <strong>the</strong> striatum of p11 knock-out mice<br />
suggesting P11 as a modulator of 5-HT1B signaling. To fur<strong>the</strong>r investigate 5HT1B signaling in<br />
<strong>the</strong> NAcc <strong>an</strong>d its effect in neuronal activity, we used calcium imaging in neuronal cultures <strong>from</strong><br />
<strong>the</strong> NAcc. Using 5HT1B agonist Anpirtoline, we found that activation of <strong>the</strong> receptor induces<br />
specific dose dependent increase in intracellular calcium, blocked by pretreatment with <strong>the</strong> 5-<br />
HT1B <strong>an</strong>tagonist GR-127935. This response appeared to be independent of extracellular<br />
calcium, indicating a release of intracellular calcium by 5-HT1B activation. 5-HT1B is a G<br />
protein coupled receptor <strong>an</strong>d we are now studying which small protein G is implicated in this<br />
response. We will ultimately address if p11 modulation has <strong>an</strong> influence in 5-HT1b signaling.<br />
A better underst<strong>an</strong>ding of serotoninergic signaling in <strong>the</strong> NAcc will eventually give some insight<br />
in <strong>the</strong> intracellular mech<strong>an</strong>isms of depression which is necessary <strong>for</strong> new <strong>an</strong>d better targeted<br />
depression treatments.<br />
Disclosures: M. Ar<strong>an</strong>go, None; S. Musatov, None; M.G. Kaplitt, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.8/C21<br />
Topic: B.03.d. Serotonin receptors<br />
Support: ANZCA Gr<strong>an</strong>t 08-010<br />
Title: Desensitization <strong>an</strong>d recovery of recombin<strong>an</strong>t hum<strong>an</strong> 5-HT1B receptor signalling in AtT-<br />
20 cells<br />
Authors: *M. CONNOR 1 , M. HEBLINSKI 2 ;<br />
1 2<br />
Univ. of Sydney, Camperdown, Australia; Nor<strong>the</strong>rn Clin. Sch., Univ. of Sydney, Sydney,<br />
Australia<br />
Abstract: Objectives: Migraine is a recurring <strong>an</strong>d debilitating disorder affecting about 15% of<br />
people. 5-HT1B/1D receptor agonists (Tript<strong>an</strong>s) are effective <strong>for</strong> m<strong>an</strong>y but not all migraineurs,<br />
<strong>an</strong>d regular or extended use of tript<strong>an</strong>s c<strong>an</strong> lead to recurring migraine. Little is known about how<br />
agonists regulate 5-HT1B receptors <strong>an</strong>d so we created a model system to study <strong>the</strong> effects of 5-<br />
HT <strong>an</strong>d <strong>an</strong>ti-migraine drugs on 5-HT1B receptors in a neuronal environment.
Methods: AtT-20 cells were stably tr<strong>an</strong>sfected with HA-tagged hum<strong>an</strong> 5-HT1B receptor cDNA.<br />
5-HT receptor signalling was assessed using whole cell patch clamp recordings of G proteingated<br />
inwardly rectifying potassium ch<strong>an</strong>nels (GIRKs).<br />
Results: Wild type AtT-20 cells did not express signific<strong>an</strong>t amounts of 5-HT1B or 5-HT1D<br />
receptor mRNA <strong>an</strong>d 5-HT (10µM) did not activate GIRK. In <strong>an</strong> AtT-20 clone expressing 5-<br />
HT1B receptors, 5-HT strongly activated GIRK, with <strong>an</strong> EC50 of 65 nM. Sumatript<strong>an</strong> activated<br />
GIRK to similar extent, with <strong>an</strong> EC50 of 165 nM. Both 5-HT <strong>an</strong>d sumatript<strong>an</strong> produced profound<br />
desensitization of 5-HT1B receptor signalling within a few minutes of continuous application,<br />
with EC50s of 120 nM <strong>an</strong>d 280 nM respectively. Both agonists produced a lesser but signific<strong>an</strong>t<br />
heterologous inhibition of GIRK activation by <strong>the</strong> native somatostatin receptors of AtT-20 cells.<br />
Receptor signalling recovered within 10 minutes of agonist washout. Inclusion of staurosporine<br />
or PP2 in <strong>the</strong> recording pipette did not affect homologous 5-HT1B receptor desensitization.<br />
However, staurosporine selectively abrogated 5HT but not sumatript<strong>an</strong>-mediated desensitization<br />
of somatostatin signalling. Inhibition of GRK2 or ERK1/2 activation did not inhibit homologous<br />
desensitization produced by ei<strong>the</strong>r 5-HT or sumatript<strong>an</strong>.<br />
Conclusions: Our data suggest that <strong>the</strong> AtT-20 cell line is appropriate <strong>for</strong> studying <strong>the</strong> regulation<br />
of 5-HT1B receptor in a neuronal-like environment. 5-HT1B receptor desensitization was rapid,<br />
profound, <strong>an</strong>d <strong>the</strong> potency of 5-HT <strong>an</strong>d sumatript<strong>an</strong> to produce desensitization of GIRK<br />
signalling was only twofold lower th<strong>an</strong> <strong>the</strong>ir potency to activate GIRK. This suggests that<br />
desensitization may occur at physiologically or <strong>the</strong>rapeutically relev<strong>an</strong>t concentrations of<br />
agonist.<br />
Disclosures: M. Connor, None; M. Heblinski, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.9/C22<br />
Topic: B.03.d. Serotonin receptors<br />
Support: Swedish Research Counsil<br />
Familjen Erling Persson Stiftelse<br />
Title: Omega-3 regulates serotonin 1B receptor distribution<br />
Authors: T. LIEBMANN 1 , N. SOURIAL BASSILLIOUS 1 , H. BRISMAR 1,2 , *L. S. SCOTT 1 ,<br />
A. APERIA 1 ;
1 2<br />
Dept Wom<strong>an</strong> Child Hlth., Karolinska Institutet, Stockholm, Sweden; KTH Royal Inst. of<br />
Technol., Stockholm, Sweden<br />
Abstract: Alterations in serotonin tr<strong>an</strong>smission in <strong>the</strong> brain are major contributors to highly<br />
prevalent mood disorders such as major <strong>an</strong>d bipolar depression. Current <strong>the</strong>rapy consists<br />
principally of neurotr<strong>an</strong>smitter reuptake inhibitors. However, <strong>the</strong>se approaches generally require<br />
long term administration <strong>an</strong>d often result with limited efficacy <strong>an</strong>d even severe side-effects in a<br />
sizeable fraction of patients. There is <strong>an</strong> obvious need to shift <strong>from</strong> <strong>the</strong> indiscriminate uptake<br />
inhibition approach to a more specific target.<br />
The serotonin 1B receptor (5-HT1B) is a good c<strong>an</strong>didate as it has been consistently implicated in<br />
depression. We previously demonstrated that 5-HT1B is expressed <strong>an</strong>d laterally mobile in<br />
dendritic membr<strong>an</strong>e of cultured hippocampal neurons. We also demonstrated predomin<strong>an</strong>ce in<br />
vesicle-like structures. This unique characteristic of 5-HT1B distinguishes it <strong>from</strong> o<strong>the</strong>r serotonin<br />
receptors <strong>an</strong>d most GPCRs. None of our previous studies of dopamine, metabotropic glutamate<br />
or serotonin receptors have revealed this distinct bimodal expression. M<strong>an</strong>ipulation of <strong>the</strong><br />
vesicular/membr<strong>an</strong>e distribution offers a targeted possibility <strong>for</strong> improving serotonin<br />
tr<strong>an</strong>smission potentially leading to mood stabilization. Here we address factors that c<strong>an</strong><br />
potentially regulate <strong>the</strong> relative receptor distribution.<br />
A promising effect was seen with treatment of omega-3 (W3) polyunsaturated fatty acids. W3<br />
has been linked to depression, but with lack of mech<strong>an</strong>istic underst<strong>an</strong>ding. We have found that<br />
exposure to W3 (100µM linolenic acid) signific<strong>an</strong>tly alters <strong>the</strong> distribution of 5-HT1B in cultured<br />
hippocampal neurons by increasing abund<strong>an</strong>ce in dendritic membr<strong>an</strong>e. This was verified by<br />
confocal imaging of fluorescent fusion proteins <strong>an</strong>d biotinylation of neuronal cultures after 2 to<br />
12 hours of W3 exposure. We also used total internal reflection fluorescence (TIRF) microscopy<br />
to monitor vesicle interaction with <strong>the</strong> membr<strong>an</strong>e <strong>an</strong>d addressed <strong>the</strong> lateral mobility of receptors<br />
in <strong>the</strong> membr<strong>an</strong>e by fluorescence recovery after photobleaching (FRAP).<br />
This study provides a novel regulation of 5-HT1B receptors in <strong>the</strong> dendritic membr<strong>an</strong>e. In<br />
addition to <strong>the</strong> suggested general health <strong>an</strong>d well-being effects of W3, <strong>the</strong>se polyunsaturated<br />
fatty acids promote a shift in 5-HT1B preference <strong>from</strong> vesicles to plasma membr<strong>an</strong>e. As 5-HT1B<br />
receptor signaling is involved in mood disorders, possibilities to alter membr<strong>an</strong>e levels provide a<br />
promising targeted approach to mediating signaling malfunctions that contribute to mood<br />
disorders such as <strong>an</strong>xiety, aggression <strong>an</strong>d depression.<br />
Disclosures: T. Liebm<strong>an</strong>n, None; N. Sourial Bassillious, None; H. Brismar, None; L.S.<br />
Scott, None; A. Aperia, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 516.10/C23<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NIH Gr<strong>an</strong>t R01NS12969<br />
Title: Expression <strong>an</strong>d distribution of 5-HT1D receptors in <strong>the</strong> murine enteric nervous system:<br />
Influence of mucosal inflammation<br />
Authors: *M.-T. LIU, M. GERSHON;<br />
Dept Pathol & Cell Bio, Columbia Univ., New York, NY<br />
Abstract: 5-HT receptors have been identified in <strong>the</strong> murine enteric nervous system (ENS).<br />
Subtypes expressed include 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT3A, 5-HT3B,<br />
5-HT4, 5-HT5A, <strong>an</strong>d 5-HT7. Qu<strong>an</strong>titative RT-PCR <strong>an</strong>d immunocytochemistry (<strong>an</strong>tibodies<br />
provided by Dr. A. Ahn [UCSF]) were used to study <strong>the</strong> expression <strong>an</strong>d distribution of 5-HT1D<br />
receptors was investigated in <strong>the</strong> ENS of adult mice. 5-HT1D immunoreactivity was found to be<br />
expressed in cell bodies of both myenteric <strong>an</strong>d submucosal neurons in all regions of <strong>the</strong> small<br />
<strong>an</strong>d large intestines. 5-HT1D-immunoreactive nerve fibers were found to be located in mucosal<br />
villi, <strong>the</strong> lamina propria, around enteric blood vessels, <strong>an</strong>d, surprisingly, between epi<strong>the</strong>lial cells,<br />
where <strong>the</strong>ir distribution was coincident with tight junction markers. 5-HT1D-containing nerve<br />
fibers were rare in <strong>the</strong> circular muscle layer. Dense varicose pericellular baskets of 5-HT1Dimmunoreactive<br />
fibers were observed around non-5-HT1D-immunoreactive neurons in <strong>the</strong><br />
myenteric plexus, but only thin varicose 5-HT1D-immunoreactive nerve fibers were observed in<br />
<strong>the</strong> submucosal plexus. About 45% of submucosal neurons <strong>an</strong>d ~11% of myenteric neurons were<br />
5-HT1D-immunoreactive. The total number of neurons was determined by immunostaining<br />
HuC/D, which is a universal neuronal marker. The majority of 5-HT1D-immunoreactive neurons<br />
were classified according to <strong>the</strong>ir shape as Dogiel type II, which suggests that <strong>the</strong>y may be<br />
sensory in function. Expression of 5-HT1D immunoreactivity in nei<strong>the</strong>r plexus was coincident<br />
with those of vasoactive intestinal peptide, NOS, TH, somatostatin, or subst<strong>an</strong>ce P; however,<br />
partial coincidence was seen with calretinin <strong>an</strong>d choline acetyltr<strong>an</strong>sferase. All CGRPimmunoreactive<br />
nerve fibers <strong>an</strong>d neurons expressed 5-HT1D immunoreactivity but <strong>the</strong>re were<br />
more 5-HT1D-immunoreactive neurons th<strong>an</strong> CGRP-immunoreactive neurons in both plexuses.<br />
CGRP marks a subset of intrinsic primary afferent neurons <strong>an</strong>d is also found in extrinsic sensory<br />
axons; thus, at least some 5-HT1D-expressing neurons <strong>an</strong>d fibers are probably sensory. To<br />
determine <strong>the</strong> effect of mucosal inflammation of 5-HT1D expressions, colitis was induced by<br />
adding dextr<strong>an</strong> sulfate sodium (DSS; 5%) to <strong>the</strong> drinking water <strong>for</strong> 6 days. The severity of <strong>the</strong><br />
resulting DSS-induced mucosal inflammation was confirmed by measuring neutrophil <strong>an</strong>d<br />
macrophage infiltration. DSS-induced colitis increased <strong>the</strong> abund<strong>an</strong>ce of tr<strong>an</strong>scripts encoding 5-<br />
HT1D receptors ~10% (p < 0.05, n = 3). These studies are compatible with <strong>the</strong> idea that primary<br />
afferent neurons <strong>an</strong>d fibers express 5-HT1D receptors <strong>an</strong>d that inflammation of <strong>the</strong> colonic<br />
mucosa increases 5-HT1D expressions.<br />
Disclosures: M. Liu, None; M. Gershon, None.
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.11/C24<br />
Topic: B.03.d. Serotonin receptors<br />
Support: The Lundbeck Foundation<br />
Faculty of Health Sciences, University of Copenhagen<br />
Title: Ch<strong>an</strong>ges in 5-HT2A-mediated behavior <strong>an</strong>d serotonin receptor binding in conditional<br />
BDNF knock-out mice<br />
Authors: *A. B. MARCUSSEN 1 , M. A. SANTINI 1 , S. AZNAR 1 , G. M. KNUDSEN 1 , M.<br />
RIOS 2 ;<br />
1 2<br />
Copenhagen Univ. Hosp., Copenhagen, Denmark; Dept. of Neuroscience, Tufts Univ. Sch. of<br />
Med., Boston, MA<br />
Abstract: Ch<strong>an</strong>ges in Brain-Derived Neurotrophic Factor (BDNF) expression in <strong>the</strong> mature<br />
brain have been implicated in <strong>the</strong> stress response <strong>an</strong>d <strong>the</strong> etiology of psychiatric disorders. To<br />
investigate <strong>the</strong> mech<strong>an</strong>isms behind this, conditional BDNF KO mice were examined. The floxed<br />
BDNF/CamKII-cre recombinase conditional BDNF KO mice (cBDNF KO mice) have normal<br />
BDNF levels during prenatal development, but are gradually depleted of this neurotrophin in <strong>the</strong><br />
brain within <strong>the</strong> first three weeks of life. Neurobiologically, a severe serotonergic impairment<br />
specific <strong>for</strong> <strong>the</strong> serotonin 2A receptor (5-HT2A) in prefrontal cortex has been described in <strong>the</strong>se<br />
mice. This is of much interest, as <strong>the</strong> 5-HT2A has been widely implicated in several<br />
neuropsychiatric disorders <strong>an</strong>d in <strong>an</strong>xiety-mediated behavior. The aim of this study was to<br />
fur<strong>the</strong>r characterize <strong>the</strong>se serotonin receptor differences <strong>an</strong>d possible functional consequences<br />
hereof. We looked <strong>for</strong> ch<strong>an</strong>ges in 5-HT1A (3H-WAY100635) <strong>an</strong>d 5-HT2A (3H-MDL100907)<br />
receptor autoradiography in frontal cortex <strong>an</strong>d hippocampus. Fur<strong>the</strong>rmore, we investigated<br />
whe<strong>the</strong>r <strong>the</strong> observed 5-HT2A ch<strong>an</strong>ges were reflected in behavioral outcome on two specific 5-<br />
HT2A-mediated tests, <strong>the</strong> head-twitch response (HTR) <strong>an</strong>d <strong>the</strong> ear-scratch response (ESR). Here,<br />
we report lower 5-HT2A receptor binding in frontal cortex, but increased binding in<br />
hippocampus. Additionally, 5-HT1A receptor binding was decreased in hippocampus in cBDNF<br />
KO mice, but unch<strong>an</strong>ged in frontal cortex. Moreover, <strong>the</strong>se receptor ch<strong>an</strong>ges were m<strong>an</strong>ifested in<br />
differences in behavioral outcome, as <strong>the</strong> cBDNF KO mice showed a clearly diminished DOIinduced<br />
ESR although curiously no difference in HTR between wildtype <strong>an</strong>d cBDNF KO was<br />
observed. The cumulative data illustrate <strong>the</strong> marked effects of perturbed BDNF signaling on <strong>the</strong><br />
serotonergic receptor system.
Disclosures: A.B. Marcussen, None; M.A. S<strong>an</strong>tini, None; S. Aznar, None; G.M. Knudsen,<br />
None; M. Rios, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.12/C25<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NIH<br />
NARSAD<br />
Title: Glutamate <strong>an</strong>tipsychotics acting at <strong>the</strong> 5-HT2A/mGluR2 complex activate both Gi/o- <strong>an</strong>d<br />
Gq/11-dependent signaling<br />
Authors: M. FRIBOURG 1 , J. L. MORENO 2 , L. ALBIZU 3 , R. MAHAJAN 4 , A. BAKI 4 , S. C.<br />
SEALFON 3 , D. E. LOGOTHETIS 4 , *J. GONZALEZ-MAESO 2 ;<br />
1 Structural <strong>an</strong>d Chem. Biol., 2 Dept Psychiatry, 3 Neurol., Mount Sinai Sch. Med., New York, NY;<br />
4 Physiol. <strong>an</strong>d Biophysics, Virginia Commonwealth Univ., Richmond, VA<br />
Abstract: Schizophrenia is a brain disease that affects perception, emotion <strong>an</strong>d cognition. In a<br />
recent clinical trial, a metabotropic glutamate 2/3 receptor (mGluR2/3) agonist has shown<br />
promise as a new <strong>the</strong>rapeutic drug <strong>for</strong> schizophrenia. A high affinity <strong>for</strong> <strong>the</strong> serotonin 5-HT2A<br />
receptor (2AR) is a common characteristic of all atypical <strong>an</strong>tipsychotics. We have recently found<br />
that 2AR <strong>an</strong>d mGluR2 interact to <strong>for</strong>m a functional receptor heterocomplex in brain cortex. The<br />
role of <strong>the</strong> 2AR/mGluR2 complex in <strong>the</strong> signaling effects of metabotropic glutamate<br />
<strong>an</strong>tipsychotics has not been studied. The 2AR <strong>an</strong>d <strong>the</strong> mGluR2 are coupled to Gq/11 <strong>an</strong>d Gi/o<br />
proteins, respectively. In order to fur<strong>the</strong>r investigate <strong>the</strong> signaling crosstalk between <strong>the</strong><br />
components of <strong>the</strong> 2AR/mGluR2 complex, we studied whe<strong>the</strong>r Gq/11 signaling could also take<br />
place when stimulating <strong>the</strong> mGluR2. We used two-electrode voltage clamp in Xenopus oocytes<br />
expressing <strong>the</strong> receptors <strong>an</strong>d used endogenous Ca2+-activated Cl- or heterologously expressed<br />
Kir ch<strong>an</strong>nels to monitor <strong>the</strong> effects of GPCR signaling. Upon mGluR2 activation, oocytes<br />
expressing mGluR2 <strong>an</strong>d 2AR, but not mGluR3 <strong>an</strong>d 2AR, elicited Ca2+-activated chloride<br />
currents, not observed in oocytes injected with <strong>the</strong> glutamate receptor alone. Fur<strong>the</strong>rmore, <strong>the</strong>se<br />
currents could be blocked by a specific regulator of Gq/11-protein signaling (RGS2) <strong>an</strong>d were<br />
accomp<strong>an</strong>ied by PIP2 hydrolysis in <strong>the</strong> membr<strong>an</strong>e, results consistent with Gq/11-signaling.<br />
Interestingly, receptor chimeric studies fur<strong>the</strong>r confirmed that <strong>the</strong> 2AR/mGluR2 complex needs
to be <strong>for</strong>med in order to allow Gi/o-Gq/11 cross activation. These data support <strong>the</strong> presence of a<br />
functional 2AR/mGluR2 complex in Xenopus oocytes cells that influences <strong>the</strong> specific pattern of<br />
G protein regulation<br />
Disclosures: M. Fribourg, None; J.L. Moreno, None; L. Albizu, None; R. Mahaj<strong>an</strong>,<br />
None; A. Baki, None; S.C. Sealfon, None; D.E. Logo<strong>the</strong>tis, None; J. Gonzalez-Maeso, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.13/C26<br />
Topic: B.03.d. Serotonin receptors<br />
Title: Long-term exposure to brain-derived neurotrophic factor (BDNF) reduces 5-HT2A<br />
receptor protein levels in hippocampal cultures<br />
Authors: *M. A. SANTINI 1 , V. TRAJKOVSKA 1 , A. B. MARCUSSEN 1 , M. S. THOMSEN 2 ,<br />
H. HANSEN 2 , J. D. MIKKELSEN 1 , L. ARNEBERG 1 , M. KOKAIA 3 , G. M. KNUDSEN 1 , S.<br />
AZNAR 1 ;<br />
1 Neurobio. Res. Unit, Rigshospitalet, Copenhagen, Denmark; 2 Neurosearch A/S, Ballerup,<br />
Denmark; 3 Lund Univ. Hosp., Lund, Sweden<br />
Abstract: Both brain-derived neurotrophic factor (BDNF) <strong>an</strong>d serotonin receptor 2A (5-HT2A)<br />
are associated with depression. Fur<strong>the</strong>r, specific 5-HT2A receptor ch<strong>an</strong>ges have been reported in<br />
BDNF conditional mut<strong>an</strong>t mice. Whe<strong>the</strong>r <strong>the</strong>re is a direct interaction between BDNF <strong>an</strong>d 5-<br />
HT2A receptor levels is, however, unknown. In this study, primary hippocampal neuronal <strong>an</strong>d<br />
mature hippocampal org<strong>an</strong>otypic cultures were exposed to BDNF at increasing concentrations<br />
<strong>for</strong> 1, 3, 5 or 7 days <strong>an</strong>d 5-HT2A <strong>an</strong>d 5-HT1A receptor protein levels in extracts were <strong>an</strong>alysed<br />
by Western blotting. To establish whe<strong>the</strong>r this interaction also applied in vivo, hippocampal 5-<br />
HT2A receptor protein levels were assessed in BDNF deficient mice (BDNF +/-). In primary<br />
neuronal cultures seven days exposure to 25 <strong>an</strong>d 50 ng/mL BDNF resulted in downregulation of<br />
5-HT2A receptor protein levels, but not of 5-HT1A. We fur<strong>the</strong>r confirmed this specific<br />
downregulation of 5-HT2A receptor protein levels in mature hippocampal org<strong>an</strong>otypic cultures.<br />
BDNF+/- mice showed a signific<strong>an</strong>t increased 5-HT2A receptor protein levels in hippocampus<br />
confirming <strong>the</strong> association between 5-HT2A <strong>an</strong>d BDNF levels in vivo. In conclusion, continuous<br />
<strong>an</strong>d long term exposure to BDNF directly induces ch<strong>an</strong>ges in 5-HT2A receptor protein levels<br />
both in primary neuronal <strong>an</strong>d org<strong>an</strong>otypic hippocampal cultures. The cellular mech<strong>an</strong>ism behind<br />
this regulation needs to be fur<strong>the</strong>r studied.
Disclosures: M.A. S<strong>an</strong>tini, None; V. Trajkovska, None; A.B. Marcussen, None; M.S.<br />
Thomsen, None; H. H<strong>an</strong>sen, None; J.D. Mikkelsen, None; L. Arneberg, None; M. Kokaia,<br />
None; G.M. Knudsen, None; S. Aznar, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.14/C27<br />
Topic: B.03.d. Serotonin receptors<br />
Support: National Centre <strong>for</strong> Biological Sciences, TIFR<br />
Department of Biotechnology, India<br />
Title: Possible roles <strong>for</strong> trace amines in <strong>the</strong> serotonergic system - Lessons <strong>from</strong> <strong>the</strong> serotonin 2A<br />
(5-HT2A) receptor<br />
Authors: *M. M. PANICKER, I. RAOTE, A. BHATTACHARYA;<br />
Natl. Ctr. For Biol. Sci. (TIFR), B<strong>an</strong>galore,, India<br />
Abstract: Serotonin (5-HT) has been written about as ‘”[being] involved in everything, but<br />
responsible <strong>for</strong> nothing,” a kind of mediator-without-portfolio’ (Lesurtel, M. et al, 2007).<br />
Interestingly, a recent report (Savelieva, K.V. et al., 2008) described a double knockout mouse<br />
with both iso<strong>for</strong>ms of <strong>the</strong> rate-limiting enzymes in serotonin biosyn<strong>the</strong>sis (TPH1 <strong>an</strong>d TPH2)<br />
eliminated, resulting in a near-complete reduction in 5-HT levels. Contrary to expectations, <strong>the</strong>se<br />
<strong>an</strong>imals showed no ‘overt phenotype.’ We hypo<strong>the</strong>sized that <strong>the</strong>re must be o<strong>the</strong>r serotoninreceptor<br />
lig<strong>an</strong>ds that are able to sufficiently compensate <strong>for</strong> <strong>the</strong> loss of 5-HT.<br />
One such lig<strong>an</strong>d we have previously described is dopamine, which activates <strong>an</strong>d induces<br />
signaling at <strong>an</strong>d trafficking of <strong>the</strong> 5-HT2A receptor at physiologically relev<strong>an</strong>t concentrations<br />
(Bhattacharyya, S. et al., 2006). This is corroborated by o<strong>the</strong>r studies which showed that<br />
dopamine also activates 5-HT1A, 5-HT2C <strong>an</strong>d 5-HT3 (Woodward, R.M. et al., 1992; Oz, M. et al.,<br />
2003). We have fur<strong>the</strong>r examined <strong>the</strong> interaction of trace amines tryptamine <strong>an</strong>d tyramine with<br />
<strong>the</strong> 5-HT2A receptor. Tryptamine <strong>an</strong>d its derivatives have been known to activate <strong>the</strong> 5-HT2A<br />
receptor (Kurrasch-Orbaugh, D.M.. et al. 2003). We now show that tyramine <strong>an</strong>d tryptamine<br />
activate 5-HT2A-EGFP receptors heterologously expressed in HEK293 cells <strong>an</strong>d induce <strong>the</strong>ir<br />
intracellular trafficking. This clearly demonstrates <strong>the</strong> possibility of o<strong>the</strong>r lig<strong>an</strong>ds taking over<br />
part of <strong>the</strong> functional role of 5-HT.<br />
This raises several interesting possibilities, including alterations in levels of tryptamine in <strong>the</strong>
TPH1 & 2 knockout mice. Ano<strong>the</strong>r import<strong>an</strong>t aspect of such interactions is <strong>the</strong> lig<strong>an</strong>d-dependent<br />
variation in intracellular tr<strong>an</strong>sduction cascades (functional selectivity) modulated by <strong>the</strong> 5-HT<br />
receptors. As we have shown earlier, functional selectivity is apparent in dopamine- vs. 5-HTmediated<br />
5-HT2A trafficking. It is probable that tryptamine <strong>an</strong>d tyramine will also display<br />
functional selectivity at this receptor. The combination of <strong>the</strong> effects modulated by <strong>the</strong>se lig<strong>an</strong>ds<br />
might compensate <strong>for</strong> <strong>the</strong> near-total lack of 5-HT. It is also likely that <strong>the</strong>se interactions have a<br />
normal physiological role but become evident in <strong>the</strong> absence of <strong>the</strong> cognate lig<strong>an</strong>d 5-HT.<br />
We will present fur<strong>the</strong>r results describing <strong>the</strong> complex interactions of trace amines at <strong>the</strong> 5-HT2A<br />
receptor.<br />
Disclosures: M.M. P<strong>an</strong>icker, None; I. Raote, None; A. Bhattacharya, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.15/C28<br />
Topic: B.03.d. Serotonin receptors<br />
Support: Supported by a CIHR gr<strong>an</strong>t to T.D.P.<br />
Supported by FER studentship (Laval University, Fac Pharmacy) to M.G.S.<br />
Title: Regulation of 5-HT1A, 5-HT2A receptors <strong>an</strong>d <strong>the</strong> activation of G protein in <strong>the</strong> brains of<br />
hemiparkinsoni<strong>an</strong> monkeys after a chronic treatment with estradiol<br />
Authors: *M. G. SANCHEZ, E. ESTRADA-CAMARENA, M. MORISSETTE, T. DI<br />
PAOLO;<br />
Lab. of Mol. Endrocrinol. <strong>an</strong>d Genomic, Laval Univ. Hosp. Res., Québec, QC, C<strong>an</strong>ada<br />
Abstract: Parkinson's disease is often associated with depression. A higher prevalence <strong>an</strong>d<br />
incidence of Parkinson's disease is observed in men. In rodents <strong>an</strong> effect of estradiol on <strong>the</strong><br />
serotonin system is well documented, whereas this is less well known in monkeys. Seven female<br />
monkeys (macacca fascicularis) were ovariectomized <strong>an</strong>d received a month later a unilateral<br />
lesion with <strong>the</strong> toxin 1-methyl 4-phenyl 1,2,3,6, tetrahydropyridine (MPTP). Four years after<br />
lesion <strong>an</strong>d ovariectomy, half received a month of treatment with 17β-estradiol <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r<br />
vehicle (saline/gelatine). The <strong>an</strong>imals were euth<strong>an</strong>ized, blood <strong>an</strong>d brain were collected <strong>an</strong>d<br />
frozen. Plasma estradiol concentrations increased in monkeys treated with estradiol. The<br />
autoradiography of specific binding of [ 3 H]8-OH-DPAT to 5-HT1A receptors showed a decrease
in <strong>the</strong> frontal cortex of monkeys treated with estradiol in both hemispheres of <strong>the</strong> brain. The<br />
autoradiography of specific binding of [ 3 H]ket<strong>an</strong>serin to 5-HT2A receptors showed <strong>an</strong> increase in<br />
all regions (dorsal, ventral, medial, lateral) of <strong>the</strong> caudate <strong>an</strong>d putamen of monkeys treated with<br />
estradiol. This increase was observed in lesioned <strong>an</strong>d intact caudate <strong>an</strong>d putamen. Specific<br />
binding of [ 3 H]ket<strong>an</strong>serin was also increased in <strong>the</strong> frontal cortex of monkeys treated with<br />
estradiol in both hemispheres of <strong>the</strong> brain. These results in monkeys are consistent with previous<br />
data in rodents, treatment with estradiol, modulates serotonin activity with <strong>an</strong> increase of 5-HT2A<br />
<strong>an</strong>d decreased 5-HT1A receptors. The autoradiography of specific binding of [ 35 S]GTPγS to 5-<br />
HT1A receptors stimulated by R-(+) 8-OH-DPAT showed a decrease in <strong>the</strong> percentage of<br />
stimulation in <strong>the</strong> frontal cortex of monkeys treated with estradiol in both hemispheres of <strong>the</strong><br />
brain. The percentage of stimulation was also decreased in <strong>the</strong> raphe nucleus in monkeys treated<br />
with estradiol. These data indicate that estradiol may be a useful adjunct to <strong>an</strong>tidepress<strong>an</strong>t<br />
treatment in postmenopausal women with mood disorders by reducing <strong>the</strong> 5-HT1A autoreceptorspecific<br />
second messenger pathway. Our results show that ch<strong>an</strong>ges in serotonin receptors in <strong>the</strong><br />
striatum <strong>an</strong>d cortex are independent of <strong>the</strong> extensive damage to <strong>the</strong> nigrostriatal dopaminergic<br />
pathway. Fur<strong>the</strong>rmore treatment with estradiol was initiated long after ovariectomy in monkeys<br />
<strong>an</strong>d serotonin receptors were still responsive. These results support a role <strong>for</strong> estradiol on<br />
serotonin activity in Parkinson's disease <strong>an</strong>d could be useful <strong>for</strong> treatment of depression<br />
associated with Parkinson's disease.<br />
Disclosures: M.G. S<strong>an</strong>chez, None; E. Estrada-Camarena, None; M. Morissette, None; T. Di<br />
Paolo, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.16/C29<br />
Topic: B.03.d. Serotonin receptors<br />
Title: Behavioral effects of <strong>the</strong> high-affinity serotonin 5-HT2A receptor agonist TCB-2<br />
Authors: *J. L. LAPORTE, A. R. BLACKLER, H. T. FRENCH, D. L. MURPHY, M. A.<br />
FOX;<br />
Lab. of Clin. Sci., NIMH, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: Typically, <strong>the</strong> syn<strong>the</strong>tic hallucinogen (+/-)-2,5-dimethoxy-4-iodophenyl-2aminoprop<strong>an</strong>e<br />
(DOI) is used to assess serotonin 5-HT2A receptor function, although this<br />
compound is also <strong>an</strong> agonist at 5-HT2C <strong>an</strong>d 5-HT2B receptors. DOI has been shown to induce
several behaviors in rodents reported to be mediated by 5-HT2A receptors. For example, DOI<br />
induces <strong>the</strong> head twitch response, suppresses feeding, <strong>an</strong>d has been shown to alter <strong>an</strong>xiety-like<br />
behaviors. Recently, a high-affinity 5-HT2A agonist (4-Bromo-3,6-dimethoxybenzocyclobuten-1yl)methylamine<br />
hydrobromide (TCB-2), a con<strong>for</strong>mationally restricted phenethylamine <strong>an</strong>alog<br />
compound, was described. Here, we provide one of <strong>the</strong> first reports of <strong>the</strong> behavioral effects of<br />
TCB-2 in C57BL/6J mice. We also assessed <strong>the</strong> effects of TCB-2 in serotonin tr<strong>an</strong>sporter<br />
(SERT)-deficient mice, as alterations in SERT expression <strong>an</strong>d function have been associated<br />
with alterations in 5-HT2A receptor function. TCB-2 induced head twitches in a dose-dependent<br />
m<strong>an</strong>ner, <strong>an</strong>d TCB-2 <strong>an</strong>d DOI induced a similar number of head twitches. Consistent with<br />
previous reports that DOI-induced head twitches are decreased ~86% in SERT knockout (-/-)<br />
mice, TCB-2-induced head twitches were decreased ~62% in SERT -/- versus SERT wildtype<br />
(+/+) mice. All head twitches were abolished by pretreatment with <strong>the</strong> selective 5-HT2A<br />
<strong>an</strong>tagonist MDL 11,939, confirming 5-HT2A mediation. Both TCB-2 <strong>an</strong>d DOI decreased feeding<br />
in food-deprived mice in a dose-dependent m<strong>an</strong>ner. Finally, TCB-2 had <strong>an</strong> <strong>an</strong>xiolytic effect in<br />
<strong>the</strong> open field, increasing <strong>the</strong> duration of time in <strong>the</strong> center, with no effects on locomotor activity<br />
or rears compared to vehicle. Toge<strong>the</strong>r, <strong>the</strong> current studies show that TCB-2 induces several<br />
behaviors previously shown to be mediated by 5-HT2A receptors. Future studies will employ<br />
selective <strong>an</strong>tagonists <strong>an</strong>d knockout mice to confirm 5-HT2A mediation of <strong>the</strong>se TCB-2-induced<br />
behaviors. Future research also needs to determine <strong>the</strong> selectivity of TCB-2 <strong>for</strong> 5-HT2A receptors<br />
compared to o<strong>the</strong>r serotonin receptor subtypes, in particular 5-HT2C receptors, in order to parse<br />
out <strong>the</strong> receptor mediation of <strong>the</strong> current behavioral responses.<br />
Disclosures: J.L. LaPorte, None; A.R. Blackler, None; H.T. French, None; D.L. Murphy,<br />
None; M.A. Fox, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.17/C30<br />
Topic: B.03.d. Serotonin receptors<br />
Title: A physiological characterization of <strong>the</strong> high-affinity 5-HT2A agonist TCB-2 in C57BL/6J<br />
mice<br />
Authors: *A. R. BLACKLER, M. A. FOX, V. Y. SCHMIDT, H. T. FRENCH, D. L.<br />
MURPHY;<br />
Lab. of Clin. Sci., Natl. Inst. of Mental Health|910004258|0, Be<strong>the</strong>sda, MD
Abstract: Recently, a high-affinity 5-HT2A agonist, 4-Bromo-3,6-dimethoxybenzocyclobuten-1yl)methylamine<br />
hydrobromide (TCB-2) was described. TCB-2 is a phenethylamine similar in<br />
structure to a commonly used syn<strong>the</strong>tic 5-HT2A/C agonist, (+/-)-2,5-dimethoxy-4-iodophenyl-2aminoprop<strong>an</strong>e<br />
(DOI). However, where DOI has a flexible side chain, TCB-2 has a<br />
con<strong>for</strong>mationally constrained side chain that lies in a pl<strong>an</strong>e perpendicular to its aromatic ring. In<br />
cell lines, this con<strong>for</strong>mational constrainment appears to cause preferential activation of <strong>the</strong><br />
PLC -mediated pathway over PLA2; resulting in 65-fold greater inositol phosphate (IP3) release<br />
over arachidonic acid (AA) release. This current report is one of <strong>the</strong> first characterizations of<br />
TCB-2 in terms of mouse physiology in a series of 5-HT2A/C mediated responses that DOI has<br />
previously been shown to elicit: hypothalamic-pituitary-adrenal (HPA) axis activation,<br />
hypo<strong>the</strong>rmia, <strong>an</strong>d activation of <strong>the</strong> MAP kinase pathway. It has been previously shown in both<br />
rats <strong>an</strong>d mice that DOI activates <strong>the</strong> HPA axis, resulting in increased plasma concentrations of<br />
oxytocin, adrenocorticotropic hormone (ACTH) <strong>an</strong>d corticosterone. In rats, this effect has been<br />
demonstrated as being 5-HT2A mediated (versus 5-HT2C), however in mice it is unclear which 5-<br />
HT2 subtype is primarily involved. Initial results demonstrate that TCB-2 administration results<br />
in a dose-dependent release of both corticosterone <strong>an</strong>d ACTH. DOI has previously been shown<br />
to induce hypo<strong>the</strong>rmia in mice; likewise TCB-2 also decreased temperature in a dose-dependent<br />
m<strong>an</strong>ner. Previous reports in mice have shown that DOI administration results in MAP kinase<br />
pathway activation in <strong>the</strong> frontal cortex, resulting in phosphorylation of ERK1/2. Our preliminary<br />
results suggest that TCB-2 treatment results in a greater increase in phosphorylated ERK1/2 th<strong>an</strong><br />
DOI. However, this difference in degree of ERK1/2 phosphorylation might be due to <strong>the</strong><br />
difference in time points used <strong>for</strong> each study (15 minutes versus 30 minutes, respectively). These<br />
studies suggest that, despite structural differences, TCB-2 elicits m<strong>an</strong>y of <strong>the</strong> same responses as<br />
DOI in mice. Future experiments utilizing pretreatment with selective 5-HT2A or 5-HT2C<br />
<strong>an</strong>tagonists will be needed to identify <strong>the</strong> relative contributions of each 5-HT2 subtype <strong>for</strong> <strong>the</strong>se<br />
effects.<br />
Disclosures: A.R. Blackler, None; M.A. Fox, None; V.Y. Schmidt, None; H.T. French,<br />
None; D.L. Murphy, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.18/C31<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NIH Gr<strong>an</strong>t DA02925
NIH Gr<strong>an</strong>t DA025412<br />
ACADIA Pharmaceuticals Inc.<br />
Veter<strong>an</strong>’s Administration VISN 22 MIRECC<br />
Title: In vitro <strong>an</strong>d in vivo characterization of a constitutively active mut<strong>an</strong>t <strong>for</strong>m of <strong>the</strong> hum<strong>an</strong> 5-<br />
HT2A receptor<br />
Authors: *A. L. HALBERSTADT 1 , J. LAMEH 2 , D. M. WEINER 2 , S. B. POWELL 1 , M. A.<br />
GEYER 1 ;<br />
1 UCSD, S<strong>an</strong> Diego, CA; 2 ACADIA Pharmaceuticals Inc., S<strong>an</strong> Diego, CA<br />
Abstract: The 5-HT2A receptor displays low levels of basal activity, but single amino acid<br />
mutations have been reported to produce 5-HT2A receptors that are constitutively active (Eg<strong>an</strong><br />
et al., J Pharmacol Exp Ther 286:85, 1998). The goal of <strong>the</strong> present investigation was to<br />
characterize <strong>the</strong> effect of a mutation in valine 175 to aspartate (V175D) on <strong>the</strong> activity of <strong>the</strong><br />
hum<strong>an</strong> 5-HT2A receptor. NIH-3T3 cells were tr<strong>an</strong>sfected with plasmid DNA <strong>for</strong> ei<strong>the</strong>r <strong>the</strong> wildtype<br />
(WT) hum<strong>an</strong> 5-HT2A receptor or <strong>the</strong> 5-HT2A-V175D mut<strong>an</strong>t. 5-HT2A receptor activity<br />
was measured in vitro using <strong>the</strong> functional assay Receptor Selection <strong>an</strong>d Amplification (RSAT;<br />
Weiner et al., J Pharmacol Exp Ther 299:268, 2001). Depending upon DNA content, <strong>the</strong> V175D<br />
mut<strong>an</strong>t receptor demonstrated up to a 4.4-fold increase in basal activity compared to <strong>the</strong> native<br />
WT receptor. Rit<strong>an</strong>serin acted as <strong>an</strong> inverse agonist at both WT <strong>an</strong>d mut<strong>an</strong>t 5-HT2A receptors,<br />
inhibiting basal receptor activity. Fur<strong>the</strong>r, <strong>the</strong> mut<strong>an</strong>t 5-HT2A receptor exhibited a leftward-shift<br />
in <strong>the</strong> dose-response curve <strong>for</strong> serotonin, whereas <strong>the</strong> dose-response <strong>for</strong> rit<strong>an</strong>serin was unaltered.<br />
All of <strong>the</strong>se findings are consistent with <strong>the</strong> V175D mutation rendering <strong>the</strong> 5-HT2A receptor<br />
constitutively active. We also examined <strong>the</strong> effect of expression of <strong>the</strong> 5-HT2A-V175D mut<strong>an</strong>t<br />
receptor on <strong>the</strong> behavior of mice on a C57 background. To generate <strong>the</strong>se mice, <strong>the</strong> hum<strong>an</strong> 5-<br />
HT2A-V175D was knocked into <strong>the</strong> mouse endogenous 5-HT2A gene locus under <strong>the</strong> control of<br />
<strong>the</strong> mouse 5-HT2A promoter by homologous recombination. Male mice heterozygous <strong>for</strong> <strong>the</strong> 5-<br />
HT2A-V175D mut<strong>an</strong>t receptor (expressing both <strong>the</strong> native mouse 5-HT2A receptor <strong>an</strong>d <strong>the</strong><br />
hum<strong>an</strong> mut<strong>an</strong>t receptor) displayed a markedly augmented startle response to loud acoustic<br />
stimuli (gene effect: F(1,36)=18.59, p=0.0001). For example, male WT mice responded to<br />
presentation of a 40-ms 120-dB pulse of broad-b<strong>an</strong>d white noise with a startle response of<br />
77.6±8.1 (me<strong>an</strong>±S.E.M.), whereas male mice heterozygous <strong>for</strong> <strong>the</strong> 5-HT2A-V175D mut<strong>an</strong>t<br />
receptor exhibited a startle response of 145.6±17.2. These findings demonstrate that amino acid<br />
mutations c<strong>an</strong> alter <strong>the</strong> activity of <strong>the</strong> 5-HT2A receptor both in vitro <strong>an</strong>d in vivo.<br />
Disclosures: A.L. Halberstadt, None; J. Lameh, None; D.M. Weiner, None; S.B. Powell,<br />
None; M.A. Geyer, S<strong>an</strong> Diego Instruments, E. Ownership Interest (stock, stock options, patent<br />
or o<strong>the</strong>r intellectual property).<br />
Poster
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.19/C32<br />
Topic: B.03.d. Serotonin receptors<br />
Title: The selectivity of [3H]Methylspiperon to D2 <strong>an</strong>d 5HT2 receptor subtypes in rat brain-- In<br />
vivo receptor occup<strong>an</strong>cy study<br />
Authors: *M. DING, J. ZYSK, J. WERKHEISER, T. PENG, P. DORFF, A. HEHMAN, L.<br />
NEVERAS, D. HEHMAN, D. MAIER;<br />
AstraZeneca, Wilmington, DE<br />
Abstract: In vivo receptor occup<strong>an</strong>cy study in rodents is a useful tool in assessing <strong>the</strong> in vivo<br />
pharmacological properties of c<strong>an</strong>didate drugs, optimizing <strong>the</strong> criteria <strong>an</strong>d condition <strong>for</strong> PET<br />
lig<strong>an</strong>ds, obtaining <strong>the</strong> tr<strong>an</strong>slation in<strong>for</strong>mation <strong>from</strong> rodent to predict <strong>an</strong>d interpret <strong>the</strong> drug<br />
efficacy in monkey <strong>an</strong>d hum<strong>an</strong>. The 5HT2 serotonin receptor class is comprised of 5HT2A,<br />
5HT2B <strong>an</strong>d 5HT2C <strong>an</strong>d <strong>the</strong> D2 dopamine receptor class includes D2, D3 <strong>an</strong>d D4 subtypes. Each<br />
subtype receptor has a unique distribution in <strong>the</strong> CNS suggesting that <strong>the</strong>y sub-serve different<br />
functions. [ 3 H]NMSP (methylspiperon) is a widely used radiolig<strong>an</strong>d <strong>for</strong> <strong>the</strong> study of D2 <strong>an</strong>d<br />
5HT2 receptors. Underst<strong>an</strong>ding <strong>the</strong> subtype selectivity of [ 3 H]NMSP <strong>for</strong> D2 <strong>an</strong>d 5HT2 receptors<br />
c<strong>an</strong> help to better evaluate a drugs effect at <strong>the</strong> subtype receptor level.<br />
Using in vivo receptor occup<strong>an</strong>cy approach, we investigated <strong>the</strong> subtype selectivity of<br />
[ 3 H]NMSP <strong>for</strong> D2 <strong>an</strong>d 5HT2 receptors in rat brain with six subtype specific lig<strong>an</strong>ds: Ket<strong>an</strong>serin<br />
<strong>for</strong> 5HT2A (10mg/kg), SB204740 <strong>for</strong> 5HT2B(3mg/kg), SB242084 <strong>for</strong> 5HT2C(5mg/kg), L741626<br />
<strong>for</strong> D2 (10mg/kg), Nafadotride <strong>for</strong> D3 (10mg/kg) <strong>an</strong>d L745870 <strong>for</strong> D4 (10mg/kg). The subtype<br />
binding profile of Quetiapine (50mg/kg) <strong>an</strong>d Norquetiapine (10mg/kg) was also examined in <strong>the</strong><br />
studies. Following 30 minutes pretreatment (sc) of <strong>the</strong> subtype specific lig<strong>an</strong>ds, [ 3 H]NMSP was<br />
administered iv at 30µCi/ml/kg <strong>for</strong> 30 minutes prior to <strong>the</strong> count of radioactivity (cpm) in <strong>the</strong><br />
dissected brain regions. Ket<strong>an</strong>cerin (5HT2A) signific<strong>an</strong>tly blocked approximately 70% of <strong>the</strong><br />
binding of [ 3 H]NMSP in frontal cortex; 33% <strong>an</strong>d 47% blocking effects were generated by<br />
L741626 (D2) <strong>an</strong>d Nafadotride (D3/D2) in striatum respectively. In <strong>the</strong> hippocampus, <strong>the</strong> D4<br />
target region, no signific<strong>an</strong>t blocking was observed. The Nucleus Accumbens, a region rich <strong>for</strong><br />
D3, moderate <strong>for</strong> D2 <strong>an</strong>d weak <strong>for</strong> D4, exhibited only 25% blocking in with L741626 (D2) versus<br />
7% with Nafadotride (D3 <strong>an</strong>tagonist). 5HT2B located mainly in cerebellum, no inhibition effect<br />
generated by SB-204741 (5HT2B).<br />
We conclude that In vivo [ 3 H]NMSP only hits D2 but not D3 <strong>an</strong>d D4, 5HT2A but not 5HT2B <strong>an</strong>d<br />
5HT2C in rat brain.<br />
Disclosures: M. Ding, None; J. Zysk, None; J. Werkheiser, None; T. Peng, None; P. Dorff,<br />
None; A. Hehm<strong>an</strong>, None; L. Neveras, None; D. Hehm<strong>an</strong>, None; D. Maier, None.
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.20/C33<br />
Topic: B.03.d. Serotonin receptors<br />
Support: Gr<strong>an</strong>t-in-Aid (No.20390417 ) <strong>from</strong> <strong>the</strong> Ministry of Education<br />
Title: The role of snoRNA RBII-52 to <strong>the</strong> serotonin 2C receptor in <strong>the</strong> rat oro-facial neuropathic<br />
pain model<br />
Authors: *K. NAKAI 1 , A. NAKAE 2 , T. TANAKA 2 , S. OBA 1 , M. SHIBATA 2 , T. MASHIMO 2 ,<br />
K. UEDA 1 ;<br />
1 Osaka Med. Col., Takatsuki, Jap<strong>an</strong>; 2 Osaka Univ. Grad. Sch. of Med., Suita, Jap<strong>an</strong><br />
Abstract: The serotonin 2C receptor is a G-protein -coupled receptor whose pre mRNA is a<br />
substrate <strong>for</strong> base modification that, via hydrolytic deamination of adenosines, yields inosines.<br />
We already studied <strong>the</strong> serotonin 2C receptor RNA editing efficiency in a rat oro-facial<br />
neuropathic pain model (Nakae et al. EJN 2008). The next, we were interested in what <strong>the</strong> factor<br />
which contributed to RNA editing of serotonin 2C receptor. In hum<strong>an</strong> study, HBII-52(one kind<br />
of small nuclear RNA; <strong>the</strong> equal function to RBII-52) is contributed to regulate <strong>the</strong> serotonin 2C<br />
receptor RNA editing (Kishore et al. Science, 2006). Our purpose of this study was to clarify <strong>the</strong><br />
relationship between RNA editing of <strong>the</strong> serotonin 2C receptor <strong>an</strong>d RBII-52.<br />
(Material <strong>an</strong>d method) We used infra-orbital loose ligation (ION-LL) model as a oro-facial<br />
neuropathic pain. Behavioral tests were carried out 3 days be<strong>for</strong>e <strong>an</strong>d 7,14,21,28 days after<br />
surgery using von Frey Filament. From post-injury day 14, fluvoxamine was administered<br />
intraperitoneally to some <strong>an</strong>imals at a dose of 30mg/kg/day <strong>for</strong> 14days.The <strong>an</strong>imals were killed<br />
by decapitation on post injury 28 <strong>an</strong>d cervical spinal cord samples were taken <strong>from</strong> each <strong>an</strong>imals<br />
<strong>an</strong>d total RNA was extracted. Then, total RNA was used <strong>for</strong> first str<strong>an</strong>d cDNA syn<strong>the</strong>sis by High<br />
Capacity RNA-to-cDNA Kit (ABI, Tokyo,Jap<strong>an</strong>). Qu<strong>an</strong>titative RT-PCR was per<strong>for</strong>med using<br />
TaqM<strong>an</strong> probes supplied by ABI. Results were <strong>the</strong>n normalized to those obtained <strong>for</strong><br />
amplifications of <strong>the</strong> same cDNA samples using 18sRNA, which acts as <strong>an</strong> internal control.Data<br />
were compared by one-way ANOVA <strong>an</strong>d statistical differences were resolved post-hoc using<br />
Tukey-Kramer multiple-comparison test (P
(Conclusion) The relationship between RNA editing of <strong>the</strong> serotonin 2C receptor <strong>an</strong>d RB-II was<br />
unclear. The current finding might suggest that RB-II was <strong>the</strong> factor which ch<strong>an</strong>ged with nerve<br />
injury<br />
Disclosures: K. Nakai, None; A. Nakae, None; T. T<strong>an</strong>aka, None; S. Oba, None; M. Shibata,<br />
None; T. Mashimo, None; K. Ueda, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.21/C34<br />
Topic: B.03.d. Serotonin receptors<br />
Support: Gr<strong>an</strong>d in aid Ministry of Jap<strong>an</strong> 20791070<br />
Title: The snoRNA RBII-52 regulates alternative splicing of <strong>the</strong> serotonin 2C receptor in <strong>the</strong> rat<br />
oro-facial neuropathic pain model<br />
Authors: *A. NAKAE 1 , K. NAKAI 2 , T. TANAKA 1 , M. YOSHIDA 1 , A. MIKAMI 1 , M.<br />
TAKASHINA 1 , S. HAGIHIRA 1 , M. SHIBATA 1 , K. UEDA 2 , T. MASHIMO 1 ;<br />
1 Osaka Univ. Grad. Sch. of Med., Suita, Jap<strong>an</strong>; 2 Osaka Med. Col., Takatsuki, Jap<strong>an</strong><br />
Abstract: [Background] Small nucleolar RNAs(snoRNAs) are non-protein-coding RNAs that<br />
are 60 to 300 nucleotides(nt) long <strong>an</strong>d that function in guiding 2’-O-methylation <strong>an</strong>d pseudouridylation<br />
in ribosomal RNAs(rRNAs), small nuclear RNAs(snRNAs), <strong>an</strong>d tRNAs.<br />
RBII-52 is a brain-specific C/D box snoRNA. RBII-52 lacks <strong>an</strong>y complementarity to <strong>an</strong> rRNA,<br />
snRNA, or tRNA. However, its <strong>an</strong>tisense element exhibits complementarity to <strong>the</strong> alternatively<br />
spliced exon Vb of <strong>the</strong> serotonin2C receptor(5-HT2CR), which is a seven-tr<strong>an</strong>smembr<strong>an</strong>e<br />
receptor located on <strong>the</strong> X chromosome. Exon V of <strong>the</strong> 5HT2CR contains at least two alternative<br />
5’ splice sites, giving rise to exon Va- <strong>an</strong>d Vb-containing iso<strong>for</strong>ms. Exon Vb encodes <strong>the</strong> second<br />
intracellular loop of <strong>the</strong> receptor, which is crucial <strong>for</strong> G protein binding. Skipping of exon Vb<br />
causes a frame shift, resulting in a receptor that is truncated after <strong>the</strong> third tr<strong>an</strong>smembr<strong>an</strong>e<br />
domain. Our purpose of this study was to clarify <strong>the</strong> relationship between alternative splicing of<br />
<strong>the</strong> 5HT2CR <strong>an</strong>d RBII-52.<br />
[Material <strong>an</strong>d Method] We used infra-orbital loose ligation (ION-LL) model as a oro-facial<br />
neuropathic pain. The <strong>an</strong>imals were decapitated <strong>an</strong>d cervical spinal cord samples were taken <strong>an</strong>d<br />
RNA was extracted 28 days after injury. Qu<strong>an</strong>titative RT-PCR was per<strong>for</strong>med <strong>for</strong> each pattern<br />
(only exon Va containing, only exon Vb containing, both exon Va <strong>an</strong>d Vb containing, <strong>an</strong>d all
pattern congaing). Results were <strong>the</strong>n normalized using 18sRNA as <strong>an</strong> internal control. Data were<br />
compared by one-way ANOVA <strong>an</strong>d statistical differences were resolved post-hoc using Tukey-<br />
Kramer multiple comparison test (P
action potential frequency. A small number of neurons (~7%) exhibited a biphasic response to 5-<br />
HT in which action potential generation was tr<strong>an</strong>siently inhibited be<strong>for</strong>e resuming at enh<strong>an</strong>ced<br />
frequencies. About 3% of neurons were not responsive to 5-HT. To rule out possible<br />
perturbations of serotonergic responsiveness due to whole-cell recording, we made extracellular<br />
recordings of spont<strong>an</strong>eous activity in neurons stimulated pharmacologically with bath-applied<br />
carbachol (50 µM) <strong>an</strong>d increased extracellular potassium (6 mM). Extracellular recordings<br />
revealed inhibitory <strong>an</strong>d excitatory responses to focal 5-HT application similar to those seen<br />
during whole-cell recording. Focal 5-HT application induced a decrease in input resist<strong>an</strong>ce in<br />
both inhibited <strong>an</strong>d excited neurons. All <strong>for</strong>ms of serotonergic modulation persisted in <strong>the</strong><br />
presence of <strong>an</strong>tagonists to fast GABAergic <strong>an</strong>d glutamatergic synaptic tr<strong>an</strong>smission, suggesting<br />
5-HT has direct effects on pyramidal neuron excitability. Currently, we are using<br />
pharmacological methods to identify <strong>the</strong> receptors <strong>an</strong>d ionic mech<strong>an</strong>isms responsible <strong>for</strong><br />
inhibitory <strong>an</strong>d excitatory serotonergic modulation of neocortical pyramidal neurons. These data<br />
demonstrate that tr<strong>an</strong>sient exposure to 5-HT c<strong>an</strong> induce ch<strong>an</strong>ges in pyramidal neuron excitability<br />
that c<strong>an</strong> persist <strong>for</strong> m<strong>an</strong>y hundreds of seconds.<br />
Disclosures: D. Avesar, None; A.T. Gulledge, None.<br />
Poster<br />
516. Serotonin Receptors II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 516.23/C36<br />
Topic: B.03.d. Serotonin receptors<br />
Support: NSERC Discovery Gr<strong>an</strong>t (EKL)<br />
Margaret S<strong>an</strong>talo fellowship (NMG)<br />
CIHR B<strong>an</strong>ting <strong>an</strong>d Best Doctoral Award (NMG)<br />
Title: Early life stress ch<strong>an</strong>ges <strong>the</strong> net effect of serotonin on layer V pyramidal neurons of <strong>the</strong><br />
prefrontal cortex<br />
Authors: *N. M. GOODFELLOW 1 , M. BENEKAREDDY 2 , V. VAIDYA 2 , E. K. LAMBE 1 ;<br />
1 2<br />
Univ. Toronto, Toronto, ON, C<strong>an</strong>ada; Dept. of Biol. Sci., Tata Inst. of Fundamental Res.,<br />
Mumbai, India
Abstract: Serotonin receptors in prefrontal cortex play a key role in mood <strong>an</strong>d <strong>an</strong>xiety. Previous<br />
work in vivo <strong>an</strong>d in vitro suggests that serotonin has a net inhibitory effect on <strong>the</strong> main output<br />
neurons of prefrontal cortex (Puig et al., 2005; Zh<strong>an</strong>g et al., 2003; Beique et al., 2004). However,<br />
<strong>the</strong>se layer V neurons are known to express a number of different 5-HT receptors in adulthood.<br />
Here, we examine <strong>the</strong> effects of different inhibitory <strong>an</strong>d excitatory 5-HT receptors on <strong>the</strong><br />
response to serotonin in layer V pyramidal neurons of <strong>the</strong> prefrontal cortex.<br />
Serotonin (10 µM, 30 s) evoked a net outward current in most layer V pyramidal neurons<br />
(27/31), measured by whole cell patch clamp recording. Blockade of <strong>the</strong> 5-HT1A receptor with<br />
WAY 100635 (30 nM, 10 min) unmasked o<strong>the</strong>r serotonin-elicited currents in <strong>the</strong> majority of<br />
layer V neurons: 17/30 neurons showed <strong>an</strong> excitatory inward current <strong>an</strong>d 5/30 neurons showed a<br />
prolonged outward current. We found that <strong>the</strong> inward currents were sensitive to <strong>the</strong> 5-HT2A<br />
<strong>an</strong>tagonist ket<strong>an</strong>serin (2 µM, 10 min). The WAY100635-insensitive outward currents were<br />
sensitive to <strong>the</strong> 5-HT5A <strong>an</strong>tagonist SB 699551 (10 µM, 20 min).<br />
Next, we examined <strong>the</strong> effects of early life stress on <strong>the</strong> serotonin currents in layer V of <strong>the</strong><br />
prefrontal cortex. Animals exposed to stress during development display a signific<strong>an</strong>tly different<br />
pattern of serotonin responses in adulthood (chi-squared test, p
a gr<strong>an</strong>t by MEXT<br />
Title: In vitro <strong>an</strong>alysis of roles of serotonin receptors in <strong>the</strong> dendrite <strong>for</strong>mation of <strong>the</strong> rat cerebral<br />
cortical neurons<br />
Authors: A. OHTANI 1 , H. NISHIYAMA 2 , M. SUGA 2 , C. SATO 3 , K. SENZAKI 1 , *T.<br />
SHIGA 1 ;<br />
1 2 3<br />
Univ. of Tsukuba, Tsukuba, Jap<strong>an</strong>; JEOL Ltd., Tokyo, Jap<strong>an</strong>; AIST, Neurosci. Res. Inst.,<br />
Tsukuba, Jap<strong>an</strong><br />
Abstract: Serotonin (5-HT) is shown to be a regulatory factor in <strong>the</strong> development of central<br />
nervous system. Serotonin receptors consist of 7 classes-14 subtypes, <strong>an</strong>d <strong>the</strong>se receptors belong<br />
to G-protein-coupled receptors (GPCRs) except <strong>for</strong> 5-HT3 receptor, which is a lig<strong>an</strong>d-gated ion<br />
ch<strong>an</strong>nel. Among <strong>the</strong>se receptors, 5-HT1A <strong>an</strong>d 5-HT2A receptors appear early in <strong>the</strong> developing<br />
cerebral cortex, but <strong>the</strong> roles of <strong>the</strong>se receptors in <strong>the</strong> cortical development are not well<br />
understood. Here, we examined roles of 5-HT1A <strong>an</strong>d 5-HT2A receptors in <strong>the</strong> dendrite <strong>for</strong>mation<br />
of rat cerebral cortex using a dissociation culture. Cerebral cortex was dissected <strong>from</strong> rat<br />
embryos (Wistar/ST) at embryonic day 16 <strong>an</strong>d dissociated cortical neurons were cultured up to 5<br />
days. During <strong>the</strong> culture, neurons were treated chronically or acutely with selective receptor<br />
agonists. After <strong>the</strong> culture, <strong>the</strong> neurons were double-immunostained by <strong>an</strong>tibodies against<br />
microtubule-associated protein 2 (MAP2) <strong>an</strong>d glutamate decarboxylase 65/67 (GAD65/67). In<br />
some cases, neurons were observed using a newly developed Atmospheric Sc<strong>an</strong>ning Electron<br />
Microscope (ASEM) at 8 nm resolution, in combination with immunohistochemistry. In this<br />
system, a vacuum-resist<strong>an</strong>t electron-permeable windowed dish is atop <strong>the</strong> inverted SEM column.<br />
The electron beam <strong>from</strong> <strong>the</strong> ASEM gun is projected upward to <strong>the</strong> cells. We found that both<br />
chronic <strong>an</strong>d acute treatment with 5-HT1A agonist (8-OH DPAT) decreased <strong>the</strong> number of primary<br />
dendrites <strong>an</strong>d <strong>the</strong> total dendiritic length of GAD65/67 negative neurons. We also investigated <strong>the</strong><br />
expression of 5-HT1A <strong>an</strong>d 5-HT2A receptors in cortical neurons cultured <strong>for</strong> 4 days. Both<br />
receptors were expressed in cell bodies <strong>an</strong>d dendrites. In conclusion, it was suggested that 5-<br />
HT1A receptor may inhibit <strong>the</strong> dendrite <strong>for</strong>mation of non-GABAergic cortical neurons.<br />
Disclosures: A. Oht<strong>an</strong>i, None; H. Nishiyama, None; M. Suga, None; C. Sato, None; K.<br />
Senzaki, None; T. Shiga, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.1/C38
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: BMBF<br />
GIF<br />
UConn<br />
Title: Temperature dependence of action potential generation in neurons <strong>an</strong>d models<br />
Authors: E. NIKITIN 1 , A. MALYSHEV 1,2 , P. BALABAN 1 , G. BARANAUSKAS 3 , M.<br />
CHISTIAKOVA 4,2 , *M. A. VOLGUSHEV 4,1 ;<br />
1 Inst. Higher Nervous Activity & Neurophysiology, Russi<strong>an</strong> Acad. of Sci., Moscow, Russi<strong>an</strong><br />
Federation; 2 Neurophysiol., Ruhr-University Bochum, Bochum, Germ<strong>an</strong>y; 3 Robotics, Brain <strong>an</strong>d<br />
Cognitive Sci., Itali<strong>an</strong> Inst. of Technol., Genova, Italy; 4 Dept Psychol, Univ. Connecticut, Storrs,<br />
CT<br />
Abstract: Action potentials (APs) in <strong>the</strong> soma of neocortical neurons exhibit a sharp, step-like<br />
onset dynamics. The rapid initiation of cortical APs may reflect genuine properties of cortical AP<br />
generation [1], in which case it may facilitate encoding of rapid signals by neuronal populations<br />
[2]. An alternative hypo<strong>the</strong>sis suggests that in neocortical neurons <strong>the</strong> APs have a smooth onset<br />
at <strong>the</strong> initiation site in <strong>the</strong> axon initial segment, while <strong>the</strong> rapid onset of somatic APs is <strong>an</strong><br />
epiphenomenon, caused by invasion of lateral current <strong>from</strong> <strong>the</strong> initiation site into <strong>the</strong> soma [3].<br />
To reproduce in <strong>the</strong> Hodgkin-Huxley type multicompartment models <strong>the</strong> onset dynamics of<br />
somatic APs, comparable to that of recorded neurons, a careful selection of parameters, which<br />
represent marginal cases of cell geometry <strong>an</strong>d electrophysiological properties was necessary.<br />
Moreover, <strong>the</strong>se simulations were per<strong>for</strong>med at virtual temperature of 37°C [3]. Since operation<br />
of ch<strong>an</strong>nels strongly depends on temperature, <strong>an</strong>d high temperature coefficients are implemented<br />
in <strong>the</strong> models (<strong>for</strong> sodium ch<strong>an</strong>nels, Q10=2.3), fastest possible ch<strong>an</strong>nel kinetics was exploited in<br />
<strong>the</strong>se simulations. Here we studied, how critical is <strong>the</strong> recording or virtual temperature as a<br />
parameter <strong>for</strong> <strong>the</strong> onset dynamics of APs in neurons <strong>an</strong>d in <strong>the</strong> models. To this end, we have<br />
compared onset dynamics of somatic APs recorded in neocortical neurons to those simulated<br />
with Hodgkin-Huxley type multicompartment models at physiological temperatures, room<br />
temperature <strong>an</strong>d 10-15°C. APs recorded in neocortical neurons exhibited sharp onset dynamics at<br />
physiological as well as at room temperatures. In some neurons, <strong>the</strong> AP onset remained fast,<br />
step-like even at 15 or 10°C. In contrast to <strong>the</strong> experimental results, <strong>the</strong> onset of somatic APs<br />
simulated with <strong>the</strong> models became invariably smooth when virtual temperature was set to 20°C<br />
or below. This discrep<strong>an</strong>cy between temperature dependence of <strong>the</strong> AP onset dynamics in <strong>the</strong><br />
neurons <strong>an</strong>d <strong>the</strong> models show that <strong>the</strong> invasion-based scenario alone c<strong>an</strong>not reconcile <strong>the</strong><br />
smooth, Hodgkin-Huxley type onset of APs at <strong>the</strong> initiation site with a sharp, step-like onset of<br />
somatic APs.<br />
[1] Naundorf et al., Nature 2006 440:1060<br />
[2] Fourcaud-Trocme et al., J Neurosci 2003 23:11628; Naundorf et al., J Comput Neurosci 2005<br />
18: 297<br />
[3] Yu et al., J Neurosci 2008 28:7260<br />
Supported by <strong>the</strong> gr<strong>an</strong>ts <strong>from</strong> <strong>the</strong> BMBF, GIF <strong>an</strong>d UConn to MV.
Disclosures: E. Nikitin, None; A. Malyshev, None; P. Balab<strong>an</strong>, None; G. Bar<strong>an</strong>auskas,<br />
None; M. Chistiakova, None; M.A. Volgushev, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.2/C39<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t F31 NS064630<br />
NIH Gr<strong>an</strong>t R37 NS36855<br />
Title: Tr<strong>an</strong>sient sodium current at subthreshold voltages: Activation by EPSP wave<strong>for</strong>ms<br />
Authors: *B. C. CARTER 1 , B. P. BEAN 2 ;<br />
2 Neurobio., 1 Harvard Med. Sch., Boston, MA<br />
Abstract: Excitatory post-synaptic potentials (EPSPs) are known to be amplified by TTXsensitive<br />
sodium current present at subthreshold voltages. This amplifying current has generally<br />
been considered to be "persistent" sodium current, a voltage-dependent but non-inactivating<br />
TTX-sensitive current that typically activates steeply between about -65 mV <strong>an</strong>d -40 mV.<br />
Persistent current is usually studied using slow ramps of voltage, <strong>an</strong>d little is known about <strong>the</strong><br />
activation <strong>an</strong>d deactivation kinetics of <strong>the</strong> current. We took adv<strong>an</strong>tage of <strong>the</strong> ability to per<strong>for</strong>m<br />
high-resolution voltage clamp experiments in acutely dissociated neurons to measure <strong>the</strong><br />
activation <strong>an</strong>d deactivation kinetics of persistent sodium currents. Experiments were done with<br />
acutely dissociated cerebellar Purkinje neurons <strong>an</strong>d CA1 hippocampal pyramidal neurons using<br />
physiological solutions <strong>an</strong>d temperature (37 ºC). In both cell types, persistent sodium current<br />
measured with slow (10 mV/s) ramps was activated detectably (> 5 pA) at voltages as negative<br />
as -75 mV. The voltage-dependence of persistent current was similar in Purkinje neurons <strong>an</strong>d<br />
CA1 pyramidal neurons, with a midpoint near -64 mV <strong>an</strong>d slope factor of ~4 mV. Activation <strong>an</strong>d<br />
deactivation kinetics were assayed using small voltage steps <strong>an</strong>d were very rapid (time const<strong>an</strong>ts<br />
< 100 microseconds). Unexpectedly, we found that small voltage steps in <strong>the</strong> subthreshold<br />
voltage r<strong>an</strong>ge activated a subst<strong>an</strong>tial component of tr<strong>an</strong>sient sodium current as well as persistent<br />
current. A tr<strong>an</strong>sient component of TTX-sensitive sodium current was evoked by voltage steps of<br />
0.5-5 mV in <strong>the</strong> voltage r<strong>an</strong>ge between -65 <strong>an</strong>d -45 mV, in both Purkinje neurons <strong>an</strong>d CA1<br />
pyramidal neurons. By comparing <strong>the</strong> TTX-sensitive current evoked by EPSP wave<strong>for</strong>ms<br />
presented at realtime speed or slowed by a factor of 100, we found that tr<strong>an</strong>sient as well as
persistent sodium current is engaged by EPSP wave<strong>for</strong>ms. In fact, >50% of <strong>the</strong> peak TTXsensitive<br />
current elicited by 5 mV EPSP wave<strong>for</strong>ms applied <strong>from</strong> holding potentials of -60 to -55<br />
mV could be ascribed to <strong>the</strong> tr<strong>an</strong>sient component of sodium current. These results show that<br />
tr<strong>an</strong>sient sodium current flows <strong>an</strong>d helps amplify EPSPs at voltages as negative as -65 mV.<br />
Disclosures: B.C. Carter, None; B.P. Be<strong>an</strong>, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.3/C40<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: Intramural Research Program of <strong>the</strong> National Institute of Neurological Disease <strong>an</strong>d<br />
Stroke, NIH<br />
NIH gr<strong>an</strong>t R37 NS36855<br />
Title: Fast real-time computation of Na ch<strong>an</strong>nel kinetic models <strong>for</strong> dynamic clamp<br />
Authors: *L. S. MILESCU 1 , J. C. SMITH 2 , B. P. BEAN 1 ;<br />
1 Neurobio., Harvard Med. Sch., Boston, MA; 2 NINDS, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: Voltage-gated Na ch<strong>an</strong>nels play a critical role in action potential generation <strong>an</strong>d<br />
control of <strong>the</strong> spiking pattern in mammali<strong>an</strong> neurons. Underst<strong>an</strong>ding how Na ch<strong>an</strong>nels regulate<br />
<strong>the</strong> firing pattern of a particular type of neuron requires not only a good underst<strong>an</strong>ding of <strong>the</strong><br />
sodium ch<strong>an</strong>nel gating kinetics, but also of <strong>the</strong> complex interactions between Na currents <strong>an</strong>d all<br />
o<strong>the</strong>r currents, some of which may be unknown or incompletely characterized.<br />
A powerful tool <strong>for</strong> studying <strong>the</strong> function of voltage gated ion ch<strong>an</strong>nels in <strong>the</strong>ir cellular context is<br />
dynamic clamp, by which individual conduct<strong>an</strong>ces c<strong>an</strong> be functionally replaced with<br />
computational models on a background of o<strong>the</strong>rwise native conduct<strong>an</strong>ces. A dynamic clamp<br />
application is a hybrid biological-computational simulator of cellular dynamics, requiring<br />
knowledge of only one ion ch<strong>an</strong>nel type, <strong>an</strong>d thus allows much more direct <strong>an</strong>d precise modeling<br />
th<strong>an</strong> a purely computational approach.<br />
However, a major challenge <strong>for</strong> exploring <strong>the</strong> role of Na ch<strong>an</strong>nels in controlling firing is that<br />
<strong>the</strong>ir kinetic properties are complex, <strong>an</strong>d realistic description requires Markov state models with<br />
a large number of states. For example, we have found that accurately describing <strong>the</strong> kinetics of<br />
Na ch<strong>an</strong>nels in medial raphe serotonergic neurons may require models with 26 states in order to
include a process of slow inactivation. Solving <strong>the</strong> differential equations <strong>for</strong> even small Markov<br />
models by traditional integration methods is computationally very dem<strong>an</strong>ding.<br />
Because of this, dynamic clamp involving Na ch<strong>an</strong>nels has mainly used Hodgkin-Huxley<br />
models, despite a growing recognition that such simplified models do not accurately capture<br />
some aspects of Na ch<strong>an</strong>nel gating (including <strong>the</strong> coupling of inactivation to activation);<br />
deterministic models also do not account <strong>for</strong> <strong>the</strong> stochastic nature of ch<strong>an</strong>nel gating <strong>an</strong>d <strong>the</strong><br />
qu<strong>an</strong>tized current levels associated with single ch<strong>an</strong>nel gating.<br />
We present new computational techniques <strong>for</strong> both deterministic <strong>an</strong>d stochastic integration by<br />
which Markov models with as m<strong>an</strong>y as 20-30 states c<strong>an</strong> be solved at rates as high as 150 kHz,<br />
while allowing complex data visualization, recording <strong>an</strong>d stimulation in a st<strong>an</strong>dard Windows<br />
environment. Fur<strong>the</strong>rmore, ensembles of as m<strong>an</strong>y as 5000 ch<strong>an</strong>nels c<strong>an</strong> be integrated<br />
stochastically at <strong>the</strong> same rates. The speed relies on efficient use of all available processors,<br />
deterministic integration using pre-computed tr<strong>an</strong>sition matrices, <strong>an</strong>d parallelized, optimized<br />
stochastic integration using Gillespie’s algorithm. We illustrate <strong>the</strong> power of <strong>the</strong> technique with<br />
several applications of realistic Markov models <strong>for</strong> sodium ch<strong>an</strong>nels in several types of central<br />
neurons.<br />
Disclosures: L.S. Milescu, None; J.C. Smith, None; B.P. Be<strong>an</strong>, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.4/C41<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: National Award <strong>for</strong> Outst<strong>an</strong>ding Young Scientist (30325021)<br />
National Basic Research Program (2006CB500804)<br />
Natural Science Foundation China (NSFC 30621130077 )<br />
Natural Science Foundation China (NSFC 30870517)<br />
CAS Program <strong>for</strong> Knowledge Innovation (KSCX2-YWR-39)<br />
Title: Axonal VGSC <strong>an</strong>d membr<strong>an</strong>e properties favor <strong>the</strong> initiation of sequential spikes
Authors: J.-H. WANG 1 , *N. CHEN 2 , H. QIAN 1 , R. GE 1 ;<br />
1 2<br />
Inst. of Biophysics, Chinese Acad. of Sci., Beijing, China; Inst. Biophys, Chin Acad Sci.,<br />
Beijing, China<br />
Abstract: An action potential is presumably initiated at axonal hillock where voltage-gated<br />
sodium ch<strong>an</strong>nels (VGSC) appear high density or low threshold. In view of sequential action<br />
potentials as essential neural codes in <strong>the</strong> brain, we investigated <strong>the</strong> locations <strong>an</strong>d mech<strong>an</strong>isms of<br />
initiating sequential spikes by recording membr<strong>an</strong>e property <strong>an</strong>d VGSC dynamics at <strong>the</strong> axon<br />
versus soma of identical neurons simult<strong>an</strong>eously in mice cortical slices. Action potentials initiate<br />
preferentially at <strong>the</strong> axon, which is associated with lower threshold <strong>an</strong>d shorter refractory period<br />
<strong>for</strong> spiking. Threshold potential <strong>for</strong> VGSC activation <strong>an</strong>d refractory period <strong>for</strong> its reactivation are<br />
lower at <strong>the</strong> axon th<strong>an</strong> soma. Immunocytochemical <strong>an</strong>d electrophysiological data show that<br />
axonal VGSCs are distributed domin<strong>an</strong>tly in a hot-spot m<strong>an</strong>ner. Computational simulation<br />
indicates that <strong>the</strong>se properties of <strong>the</strong> axons facilitate <strong>the</strong> initiation of sequential action potentials.<br />
There<strong>for</strong>e, <strong>the</strong> axon is a favorable site to initiate sequential action potentials <strong>an</strong>d write neuronal<br />
codes under physiological conditions.<br />
Disclosures: J. W<strong>an</strong>g, None; N. Chen, None; H. Qi<strong>an</strong>, None; R. Ge, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.5/C42<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Title: Role of <strong>an</strong>kyrin G in <strong>the</strong> restriction of sodium ch<strong>an</strong>nel diffusion in neurons<br />
Authors: *B. DARGENT 1 , A. BRACHET 1 , D. CHOQUET 2 , C. LETERRIER 1 ;<br />
1 2<br />
INSERM Umr641 - Univ. Mediterr<strong>an</strong>ee, Marseille Cedex 15, Fr<strong>an</strong>ce; CNRS UMR 5091 -Univ<br />
Bordeaux2, Bordeaux, Fr<strong>an</strong>ce<br />
Abstract: In neurons, generation <strong>an</strong>d propagation of action potentials require <strong>the</strong> precise<br />
accumulation of sodium ch<strong>an</strong>nels at <strong>the</strong> axonal initial segment (AIS) <strong>an</strong>d in <strong>the</strong> nodes of R<strong>an</strong>vier<br />
through <strong>an</strong>kyrin G scaffolding. It has also been shown that <strong>the</strong> AIS <strong>for</strong>ms a non specific<br />
diffusion barrier that restricts <strong>the</strong> surface mobility of membr<strong>an</strong>e proteins <strong>an</strong>d phospholipids.<br />
Genetic, biochemical <strong>an</strong>d cell biology studies converge to a crucial role of <strong>an</strong>kyrin G, as <strong>the</strong> key<br />
player in org<strong>an</strong>izing <strong>the</strong> AIS. Moreover, <strong>an</strong>kyrin G <strong>an</strong>d sodium ch<strong>an</strong>nel accumulation at <strong>the</strong> AIS<br />
is linked to <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> diffusion barrier.
In <strong>the</strong> present study, we used a single particle tracking strategy to unravel <strong>the</strong> role of specific<br />
protein-protein interactions in <strong>the</strong> <strong>for</strong>mation of a diffusion barrier. Difficulties in expressing full<br />
Nav1.2 led us to generate chimeric ion ch<strong>an</strong>nel Kv2.1-Nav1.2 as a suitable tool. The Nav1.2<br />
fragment bears <strong>the</strong> <strong>an</strong>kyrin binding motif of sodium ch<strong>an</strong>nels. We observed that <strong>the</strong> lateral<br />
diffusion of Kv2.1-Nav1.2 is highly restricted at <strong>the</strong> AIS of mature hippocampal neurons. We<br />
determined <strong>the</strong> critical residues involved in sodium ch<strong>an</strong>nel-<strong>an</strong>kyrin G interaction <strong>an</strong>d <strong>the</strong>ir<br />
effect on Kv1.2-Nav1.2 diffusion. We fur<strong>the</strong>r studied Kv2.1-Nav1.2 diffusion in young neurons,<br />
where no barrier is yet <strong>for</strong>m. In <strong>the</strong>se conditions Kv2.1-Nav1.2 diffusion is also severely<br />
restricted at <strong>the</strong> AIS, pointing to <strong>the</strong> crucial role of <strong>an</strong>kyrin G in restricting sodium ch<strong>an</strong>nel<br />
diffusion. Pharmacological inhibition of casein kinase 2 impacted Kv2.1-Nav1.2 immobilization.<br />
Our findings revealed that <strong>the</strong> <strong>an</strong>kyrin depend<strong>an</strong>t barrier <strong>for</strong>ms be<strong>for</strong>e <strong>the</strong> previously described<br />
non specific diffusion barrier.<br />
Disclosures: B. Dargent, None; A. Brachet, None; D. Choquet, None; C. Leterrier, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.6/C43<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: AFM Gr<strong>an</strong>t MNM1 2007<br />
Title: The Axon Initial Segments of mouse spinal motoneurons: Heterogeneous morphologies<br />
<strong>an</strong>d ion ch<strong>an</strong>nel compositions<br />
Authors: *M. DAVENNE, A. DUFLOCQ, B. LE BRAS, E. BULLIER, F. COURAUD;<br />
Univ. Pierre <strong>an</strong>d Marie Curie - CNRS UMR 7224 - INSERM UMRS 952, Paris, Fr<strong>an</strong>ce<br />
Abstract: The Axon Initial Segment (AIS) plays a crucial role in <strong>the</strong> physiology of neurons.<br />
Indeed, <strong>the</strong> specific aggregation of voltage-gated sodium (Nav) <strong>an</strong>d potassium (Kv) ch<strong>an</strong>nels at a<br />
high density at <strong>the</strong> AIS membr<strong>an</strong>e allows action potentials to be generated in this region.<br />
The size of <strong>the</strong> AIS, its location along <strong>the</strong> axon relative to <strong>the</strong> somato-dendritic compartment, its<br />
molecular composition in terms of Nav <strong>an</strong>d Kv ch<strong>an</strong>nel subunits <strong>an</strong>d <strong>the</strong> fine distribution of<br />
<strong>the</strong>se ch<strong>an</strong>nels within <strong>the</strong> AIS are key features that will determine <strong>the</strong> properties of action<br />
potentials generated <strong>an</strong>d thus <strong>the</strong> excitability properties along <strong>the</strong> axonal <strong>an</strong>d somato-dendritic<br />
membr<strong>an</strong>es. And variations in <strong>the</strong>se AIS parameters may underlie <strong>the</strong> existence of functionally<br />
diverse neuronal types.
Given <strong>the</strong> fact that <strong>the</strong> AIS of motoneurons has so far hardly been studied <strong>an</strong>d that motoneurons<br />
are a functionally heterogeneous population of neurons, we decided to <strong>an</strong>alyze <strong>the</strong> AIS of mouse<br />
spinal motoneurons in terms of <strong>the</strong> above-mentioned features. We focused our<br />
immunohistochemical study on <strong>the</strong> lumbar region of <strong>the</strong> mouse spinal cord.<br />
First we found that <strong>the</strong> AIS em<strong>an</strong>ates directly <strong>from</strong> <strong>the</strong> soma in 85% of motoneurons, whereas in<br />
<strong>the</strong> remaining 15% <strong>the</strong> AIS em<strong>an</strong>ates as a collateral of a MAP2-positive dendritic shaft. The<br />
population of AISs was ra<strong>the</strong>r homogeneous in terms of length <strong>an</strong>d diameter. However quite<br />
some variability could be observed as to <strong>the</strong> dist<strong>an</strong>ce between <strong>the</strong> soma <strong>an</strong>d <strong>the</strong> AIS.<br />
We <strong>the</strong>n found that Nav1.1 is expressed at <strong>the</strong> AIS in 80% of motoneurons, concentrated in its<br />
proximal portion, complementary to Nav1.6 expression more concentrated in <strong>the</strong> distal AIS. In<br />
<strong>the</strong> remaining 20% of motoneurons, Nav1.1 is absent <strong>an</strong>d Nav1.6 is expressed uni<strong>for</strong>mly<br />
throughout <strong>the</strong> AIS.<br />
We next characterized <strong>the</strong> Kv subunits expressed at <strong>the</strong> AISs of motoneurons. We found that in<br />
100% of motoneurons Kv7.2 (KCNQ2) is expressed throughout <strong>the</strong> AIS, whereas Kv1.1 <strong>an</strong>d<br />
Kv1.2 are only found in <strong>the</strong> distal AIS. Segregation of Kv1.1 <strong>an</strong>d Kv1.2 in <strong>the</strong> distal AIS thus<br />
does not correlate with <strong>the</strong> segregation of Nav1.1 <strong>an</strong>d/or Nav1.6. There<strong>for</strong>e <strong>the</strong> mech<strong>an</strong>isms<br />
responsible <strong>for</strong> <strong>the</strong> segregation of <strong>the</strong>se Kv <strong>an</strong>d Nav subunits are likely independent. Me<strong>an</strong>while<br />
Nav1.1 <strong>an</strong>d Nav1.6 are mutually exclusive within <strong>the</strong> AIS, suggesting that <strong>the</strong> mech<strong>an</strong>isms<br />
responsible <strong>for</strong> <strong>the</strong>ir respective segregation are interdependent.<br />
Spinal motoneurons thus display AISs with different morphological characteristics <strong>an</strong>d are<br />
subdivided into sub-compartments expressing different combinations of Nav <strong>an</strong>d Kv ch<strong>an</strong>nels,<br />
compositions that fur<strong>the</strong>r differ among motoneurons. These heterogenous AIS features may<br />
underlie heterogeneous excitability properties of spinal motoneurons.<br />
Disclosures: M. Davenne, None; A. Duflocq, None; B. Le Bras, None; E. Bullier, None; F.<br />
Couraud, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.7/C44<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: FUNCAP<br />
CNPQ
Title: The effects of estragole on electrophysiological parameters of <strong>the</strong> dorsal root g<strong>an</strong>glion <strong>an</strong>d<br />
sciatic nerve of rats<br />
Authors: *R. BARBOSA 1,2 , K. S. S. ALVES 2 , F. W. F. D. SILVA 2 , L. M. JUNIOR 2 , E. D. L.<br />
SIQUEIRA 2 , M. L. D. SANTOS 2 , T. D. S. NASCIMENTO 2 , A. N. C. D. SOUZA 2 , F. S. Á.<br />
CAVALCANTE 2 , V. M. CECCATTO 2 , V. M. CECCATTO 2 , P. J. C. MAGALHÃES 2 , S.<br />
LAHLOU 2 , J. L. C. D. SOUZA 2 , A. C. CASSOLA 2 , J. H. L. CARDOSO 2 ;<br />
1 Lab. Eletrofisiologia, Inst. Ciências Biomédicas, Fortaleza, Brazil; 2 Ciências Fisiológicas, Inst.<br />
Superior de Ciências Biomédicas, Fortaleza, Brazil<br />
Abstract: Estragole (EST), a low molecular weight phenylpropene, is widely utilized in food<br />
industry <strong>an</strong>d is found in essential oil of m<strong>an</strong>y pl<strong>an</strong>ts. This is a study of <strong>the</strong> effects of EST on <strong>the</strong><br />
action potentials of peripheral nerves <strong>an</strong>d neurons. We have recorded extracellularly compound<br />
action potentials (CAP) in rat sciatic nerve (CN) <strong>an</strong>d intracellular action potentials on rat dorsal<br />
root g<strong>an</strong>glion (DRG) neurons. Methods <strong>an</strong>d results: Wistar rats (200-250g) were killed by<br />
cervical dislocation.CN <strong>an</strong>d DRG were removed <strong>an</strong>d kept in ice-cold Locke solution, pH ≈ 7.40.<br />
Action potentials were elicited in CAP by electrical stimulation, <strong>an</strong>d <strong>the</strong> nerve action potential<br />
was recorde by extracellular electrodes. The waves had two positive peaks, called here as 1st <strong>an</strong>d<br />
2nd components. Averaged values of <strong>the</strong>se components in control conditions were 4.5±0.4 mV<br />
(N=24) <strong>an</strong>d 1.2±0.2 mV (N=24). EST at 4 mM reduced <strong>the</strong> amplitude of <strong>the</strong> 1st <strong>an</strong>d 2nd<br />
component to 46,1±4,9 <strong>an</strong>d 61,4±10,1 % (N=8) of control, respectively. At 6mM, EST reduced<br />
<strong>the</strong> amplitude of <strong>the</strong> 1st <strong>an</strong>d 2nd component to 13.3±2.3 <strong>an</strong>d 13.5±4.9% (N=8) of control,<br />
respectively. In DRG <strong>the</strong> action potentials were recorded <strong>from</strong> neurons through intracellular<br />
microelectrodes. Resting membr<strong>an</strong>e potential (Em), input resist<strong>an</strong>ces (Ri) <strong>an</strong>d o<strong>the</strong>r action<br />
potential parameters, like amplitude, duration <strong>an</strong>d <strong>the</strong> maximum rate of potential rising<br />
((dV/dt)max) were calculated. In 24 DRG neurons with nociceptor characteristics EST blocked<br />
<strong>the</strong> action potential in a dose related m<strong>an</strong>ner: 1, 2, 4 <strong>an</strong>d 6mM blocked <strong>the</strong> AP wave<strong>for</strong>m in<br />
0.0±0.0%, 28.6±18.4%, 50.0±16.7% <strong>an</strong>d, 75.0±25.0% of cells. In 83 DRG cells which did not<br />
show nociceptor characteristics, <strong>the</strong> blockades were 0.0±0.0%, 19.2±7.9%, 52.9±8.7%,<br />
78.6±11.4% with <strong>the</strong> same EST concentrations above. In <strong>the</strong> r<strong>an</strong>ge of EST concentration used,<br />
<strong>the</strong>re were no ch<strong>an</strong>ges in Ri <strong>an</strong>d Em in both neuronal population. Conclusion: estragole inhibits<br />
AP in CAP in neurones of DRG, in a dose-dependent . Nocicepetors <strong>an</strong>d o<strong>the</strong>r cell phenotypes<br />
are equally affected by <strong>the</strong> terpenoid. The absence of effects on Em <strong>an</strong>d Ri points to <strong>an</strong> inhibitory<br />
effect of estragole on voltage-gated Na ch<strong>an</strong>nels.<br />
Disclosures: R. Barbosa, CNPq, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); FUNCAP, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); K.S.S. Alves, None; F.W.F.D. Silva, None; L.M. Junior, None; E.D.L. Siqueira,<br />
None; M.L.D. S<strong>an</strong>tos, None; T.D.S. Nascimento, None; A.N.C.D. Souza, None; F.S.Á.<br />
Cavalc<strong>an</strong>te, None; V.M. Ceccatto, None; V.M. Ceccatto, None; P.J.C. Magalhães, None; S.<br />
Lahlou, None; J.L.C.D. Souza, None; A.C. Cassola, None; J.H.L. Cardoso, None.<br />
Poster
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.8/C45<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: National Multiple Sclerosis <strong>Society</strong><br />
Department of Veter<strong>an</strong>s Affairs<br />
Title: Identification <strong>an</strong>d characterization of novel Nav1.6 interaction partners<br />
Authors: *A. GASSER 1,2,3 , C. HAN 1,2,3 , S. STAMBOULIAN 1,2,3 , E. K. DONNELLY 1,2,3 , S. G.<br />
WAXMAN 1,2,3 , S. D. DIB-HAJJ 1,2,3 ;<br />
1 Neurol., 2 Ctr. <strong>for</strong> Neurosci. <strong>an</strong>d Regeneration Reserach, Yale Univ. Sch. of Med., New Haven,<br />
CT; 3 Rehabil. Res. Ctr., Veter<strong>an</strong>s Affairs Connecticut Healthcare Syst., West Haven, CT<br />
Abstract: Nav1.6 is <strong>the</strong> predomin<strong>an</strong>t voltage-gated sodium ch<strong>an</strong>nel at axon initial segments <strong>an</strong>d<br />
mature nodes of R<strong>an</strong>vier in myelinated axons, <strong>an</strong>d thus critically regulates excitability <strong>an</strong>d<br />
conduction in central neurons. Nav1.6 is also present in small-diameter DRG neuron cell bodies<br />
<strong>an</strong>d <strong>the</strong>ir unmyelinated axons, <strong>an</strong>d thus contributes to conduction in unmyelinated C fibers of<br />
peripheral affer<strong>an</strong>ts. Nav1.6 null mice are juvenile lethal whereas missense mutations cause<br />
motor disorders. Cytosolic proteins which interact with Nav1.6 c<strong>an</strong> modulate current amplitude<br />
<strong>an</strong>d gating properties of <strong>the</strong> ch<strong>an</strong>nel. Examples <strong>for</strong> regulatory proteins include Calmodulin <strong>an</strong>d<br />
fibroblast growth factor homologous factor (FHF) 2B, which both bind to <strong>the</strong> C-terminus of<br />
Nav1.6, <strong>an</strong>d Ankyrin-G, which interacts with <strong>the</strong> linker connecting domains II <strong>an</strong>d III.<br />
The ch<strong>an</strong>nel complex at <strong>the</strong> cell membr<strong>an</strong>e is predicted to consist of a large number of proteins.<br />
To better underst<strong>an</strong>d <strong>the</strong> complex regulation of Nav1.6 in neurons, we used genetic <strong>an</strong>d<br />
biochemical screens to identify ch<strong>an</strong>nel partners. Here we report two novel cytosolic partners of<br />
Nav1.6, which were identified by yeast-two-hybrid screening using <strong>the</strong> C-terminus of <strong>the</strong><br />
ch<strong>an</strong>nel as bait. Using a peptide <strong>from</strong> L1, <strong>the</strong> loop connecting domains I <strong>an</strong>d II of Nav1.6,<br />
<strong>an</strong>o<strong>the</strong>r ch<strong>an</strong>nel partner was identified. The full-length cDNA of each of <strong>the</strong>se targets was cloned<br />
into <strong>the</strong> Invitrogen gateway system allowing <strong>the</strong>ir expression as ei<strong>the</strong>r native proteins, fused to<br />
GFP, or tagged with a V5 <strong>an</strong>tigen. Using imaging studies, we have found that one target was<br />
exclusively located at <strong>the</strong> plasma membr<strong>an</strong>e upon expression in <strong>the</strong> neuronal cell line ND7/23,<br />
whereas <strong>an</strong>o<strong>the</strong>r protein appeared to be clustered in org<strong>an</strong>elles, which are currently identified.<br />
The interaction of <strong>the</strong>se partners with full-length Nav1.6 will be verified biochemically, <strong>an</strong>d<br />
whole-cell patch-clamp recording of Nav1.6 expressed in ND7/23 cell lines will be used to<br />
investigate <strong>the</strong> effect of this interaction on current density <strong>an</strong>d gating properties of <strong>the</strong> ch<strong>an</strong>nel.<br />
Identification <strong>an</strong>d characterization of Nav1.6 partners will enh<strong>an</strong>ce our underst<strong>an</strong>ding of <strong>the</strong><br />
cellular distribution <strong>an</strong>d modulation of biophysical properties of this ch<strong>an</strong>nel.
Disclosures: A. Gasser, None; C. H<strong>an</strong>, None; S. Stambouli<strong>an</strong>, None; E.K. Donnelly,<br />
None; S.G. Waxm<strong>an</strong>, None; S.D. Dib-Hajj, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.9/C46<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: Rehabilitation Research Service <strong>an</strong>d Medical Research, Department of Veter<strong>an</strong>s<br />
Affairs<br />
Gifts <strong>from</strong> <strong>the</strong> Erythromelagia Association<br />
Title: Contribution of sodium ch<strong>an</strong>nel Nav1.7 to action potential in DRG neurons<br />
Authors: *C. HAN 1,2 , S. D. DIB-HAJJ 1,2 , M. A. NASSAR 3 , J. N. WOOD 3 , S. G. WAXMAN 1,2 ;<br />
1 Dept. of Neurol. <strong>an</strong>d Ctr. <strong>for</strong> Neurosci. <strong>an</strong>d Regeneration Res., Yale Univ., New Haven, CT;<br />
2 Rehabil. Res. Ctr., Veter<strong>an</strong>s Affairs Connecticut Hlth. Care Syst., West Haven, CT; 3 Mol.<br />
Nociception Group, Dept. of Biol., Univ. Col. London, London, United Kingdom<br />
Abstract: Dorsal root g<strong>an</strong>glion (DRG) neurons express multiple sodium ch<strong>an</strong>nel subtypes,<br />
including TTX-Sensitive (TTX-S) sodium ch<strong>an</strong>nels Nav1.1, Nav1.6, Nav1.7 <strong>an</strong>d TTX-Resist<strong>an</strong>t<br />
(TTX-R) sodium ch<strong>an</strong>nels Nav1.8, Nav1.9. Voltage-clamp studies have suggested that Nav1.7<br />
acts as a threshold ch<strong>an</strong>nel that boosts sub-threshold stimuli. Recently, gain-of-function Nav1.7<br />
mutations have been reported to cause two hum<strong>an</strong> familial pain disorders: inherited<br />
erythromelagia (IEM) <strong>an</strong>d paroxysmal extreme pain disorder (PEPD). In contrast, loss-offunction<br />
mutations have been reported to cause ch<strong>an</strong>nelopathy-associated insensitivity to pain<br />
(CIP), consistent with <strong>an</strong> abrogated pain response to inflammation in Nav1.7 -/- mice. Voltageclamp<br />
studies have shown that IEM <strong>an</strong>d PEPD mutations of Nav1.7 ch<strong>an</strong>nels reduce <strong>the</strong> stimulus<br />
strength that is needed to activate <strong>the</strong> ch<strong>an</strong>nel or impair ch<strong>an</strong>nel inactivation, or both. Currentclamp<br />
studies have shown that mut<strong>an</strong>t Nav1.7 ch<strong>an</strong>nels render DRG neurons hyperexcitable,<br />
consistent with <strong>the</strong> role of this ch<strong>an</strong>nel in boosting normally weak stimuli. However, <strong>the</strong><br />
contribution of Nav1.7 to action potential (AP) generation in naive DRG neurons <strong>an</strong>d <strong>the</strong><br />
excitability of DRG neurons <strong>from</strong> Nav1.7 -/- mice have not been well-established.<br />
We used current-clamp recordings in small DRG neurons isolated <strong>from</strong> Nav1.7 +/+ <strong>an</strong>d Nav1.7 -/-<br />
mice to investigate <strong>the</strong> contribution of this ch<strong>an</strong>nel to electrogenesis. Our data show no<br />
signific<strong>an</strong>t difference in input resist<strong>an</strong>ce. DRG neurons <strong>from</strong> Nav1.7 -/- mice have a slightly
hyperpolarized resting membr<strong>an</strong>e potential compared with that of DRG neurons <strong>from</strong> Nav1.7 +/+<br />
mice. Additionally, AP in DRG neurons <strong>from</strong> Nav1.7 -/- mice display <strong>an</strong> increased current<br />
threshold <strong>an</strong>d a depolarized voltage threshold to both 0.5-ms <strong>an</strong>d 200-ms long step current<br />
inputs, compared with those of DRG neurons <strong>from</strong> Nav1.7 +/+ mice. These findings provide<br />
fur<strong>the</strong>r evidence that sodium ch<strong>an</strong>nel Nav1.7 regulates small DRG neuron excitability by<br />
contributing to threshold <strong>for</strong> AP firing. Additional <strong>an</strong>alysis of firing properties of DRG neurons<br />
<strong>from</strong> WT <strong>an</strong>d Nav1.7 -/- mice may shed more light on <strong>the</strong> contribution of this ch<strong>an</strong>nel to pain<br />
signaling in this <strong>an</strong>imal model.<br />
Disclosures: C. H<strong>an</strong>, None; S.D. Dib-Hajj, None; M.A. Nassar, None; J.N. Wood,<br />
None; S.G. Waxm<strong>an</strong>, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.10/C47<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: NIEHS Gr<strong>an</strong>t ES013686<br />
NIEHS Gr<strong>an</strong>t ES014591.<br />
Title: Endogenous sodium currents <strong>an</strong>d Nav1.7 sodium ch<strong>an</strong>nels in hum<strong>an</strong> embryonic kidney<br />
(HEK) cells<br />
Authors: B. HE, *D. M. SODERLUND;<br />
Dept Entomology, Cornell Univ., Geneva, NY<br />
Abstract: Hum<strong>an</strong> embryonic kidney (HEK) cells are widely used <strong>for</strong> <strong>the</strong> heterologous<br />
expression of voltage- <strong>an</strong>d lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels. Patch clamp <strong>an</strong>alysis of HEK cells in <strong>the</strong><br />
whole-cell current configuration identified voltage-gated, rapidly inactivating inward currents.<br />
Peak current amplitudes r<strong>an</strong>ged <strong>from</strong> less th<strong>an</strong> 800 pA, with <strong>the</strong> majority (60 of 90<br />
cells) in <strong>the</strong> 100-400 pA r<strong>an</strong>ge. Me<strong>an</strong> current amplitude increased <strong>from</strong> 173 pA at passage<br />
number 3 to 422 pA at passage 5 <strong>an</strong>d <strong>the</strong>n stabilized at ~280 pA at passages 10-15. Halfmaximal<br />
values <strong>for</strong> activation <strong>an</strong>d steady-state inactivation were -30.7 mV <strong>an</strong>d -65.9 mV<br />
respectively. Tr<strong>an</strong>sient inward currents were separated into two components on <strong>the</strong> basis of<br />
sensitivity to Cd++ <strong>an</strong>d tetrodotoxin (TTX). Application of Cd++ (300 µΜ) reduced current<br />
amplitude to 66% of control, consistent with <strong>the</strong> existence of current carried by <strong>the</strong> Cd++-
sensitive nonspecific cation ch<strong>an</strong>nel previously identified in HEK cells. Application of TTX (500<br />
nM) reduced current amplitude by 50%, consistent with <strong>the</strong> existence of current carried by a<br />
TTX-sensitive voltage-gated sodium ch<strong>an</strong>nel. Joint application of Cd++ <strong>an</strong>d TTX was<br />
incompletely additive, reducing current amplitude to 29% of control. The use of sodium ch<strong>an</strong>nel<br />
iso<strong>for</strong>m-specific primers in polymerase chain reaction amplifications on HEK cell first-str<strong>an</strong>d<br />
cDNA detected <strong>the</strong> expression of <strong>the</strong> hum<strong>an</strong> Nav1.7 sodium ch<strong>an</strong>nel iso<strong>for</strong>m in all cell stages<br />
that expressed <strong>the</strong> TTX-sensitive component of current. These results provide evidence <strong>for</strong> <strong>an</strong><br />
endogenous TTX-sensitive sodium current in HEK cells that is associated with <strong>the</strong> expression of<br />
<strong>the</strong> Nav1.7 sodium ch<strong>an</strong>nel iso<strong>for</strong>m.<br />
Disclosures: B. He, None; D.M. Soderlund, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.11/C48<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: Bickart Fellowship,University of Melbourne<br />
Title: Kinetic properties of heterologously-expressed Nav1.2 voltage-gated sodium ch<strong>an</strong>nels<br />
Authors: *C. FRENCH 1 , P. KWAN 2 , T. O'BRIEN 1 ;<br />
1 Univ. of Melbourne, Melbourne, Australia; 2 Chinese Univ. of Hong Kong, Hong Kong, China<br />
Abstract: Voltage gated ion ch<strong>an</strong>nels composed of hum<strong>an</strong> Nav 1.2 alpha subunits were<br />
tr<strong>an</strong>siently expressed in <strong>an</strong> HEK cell line <strong>an</strong>d examined under voltage clamp conditions using <strong>the</strong><br />
whole-cell patch-clamp method. High temporal resolution was achieved per<strong>for</strong>ming signalaveraged<br />
recordings at up to 100 kHz at reduced temperatures (<strong>from</strong> 22 to 6C). Series-resist<strong>an</strong>ce<br />
artifact was minimized by recording <strong>from</strong> cells with relatively small amplitude currents<br />
(typically
with longer pulses. Macroscopic inactivation was comparatively slow <strong>an</strong>d monoexponential at<br />
low depolarisations, but developed a bi-exponential time-course with larger depolarisations. The<br />
amplitude of <strong>the</strong> faster time-const<strong>an</strong>t was domin<strong>an</strong>t, approaching a fixed ratio of ~0.8 at maximal<br />
activation. A signific<strong>an</strong>t persistent component (“INa(p)”) was always observed with <strong>an</strong><br />
amplitude approximately 1% of peak amplitude. Steady-state activation curves were reasonably<br />
well fitted with a single Boltzm<strong>an</strong> function with slope <strong>an</strong>d half-activation potential of 6.4±0.39<br />
<strong>an</strong>d -27.4±2.2mV (n=6, me<strong>an</strong>±SEM) respectively. Steady state inactivation studies with<br />
conventional 150 ms voltage comm<strong>an</strong>ds were again reasonably well described by single<br />
Boltzm<strong>an</strong> functions with slope <strong>an</strong>d half-inactivation values of 8.9±1.4 <strong>an</strong>d -68±2.2mV (n=6).<br />
Macroscopic inactivation could be eliminated with intracellular trypsin (0.2 mg/ml). “Slow<br />
inactivation” with time const<strong>an</strong>ts of <strong>the</strong> order of 102ms could also be observed, <strong>an</strong>d was<br />
preserved with intracellular trypsin.<br />
This study provides very high resolution measurements of <strong>the</strong> kinetic properties of one of <strong>the</strong><br />
predomin<strong>an</strong>t Nav subtypes in <strong>the</strong> mammali<strong>an</strong> CNS which are inconsistent with <strong>the</strong> Hodgkin-<br />
Huxley <strong>for</strong>malism, <strong>an</strong>d demonstrates that complex features of ch<strong>an</strong>nel behaviour are preserved<br />
with solely alpha subunit composition.<br />
Disclosures: C. French, None; P. Kw<strong>an</strong>, None; T. O'Brien, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.12/C49<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: NIH R01 MH059980 (LLI)<br />
(GAP)<br />
Gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Partnership <strong>for</strong> Pediatric Epilepsy (LLI)<br />
University of Michig<strong>an</strong> Cardiovascular Center Inaugural Predoctoral Fellowship<br />
Title: The β1B splice vari<strong>an</strong>t of <strong>the</strong> voltage-gated sodium ch<strong>an</strong>nel SCN1B is a soluble protein<br />
with a possible role in axon pathfinding<br />
Authors: *G. A. PATINO 1 , W. J. BRACKENBURY 2 , L. L. ISOM 2 ;<br />
1 Neurosci. Program, 2 Dept. of Pharmacol., Univ. Michig<strong>an</strong>, Ann Arbor, MI
Abstract: Voltage-gated sodium ch<strong>an</strong>nels (VGSCs) play a key role in <strong>the</strong> depolarization of<br />
excitable cells. VGSCs are multimeric proteins composed of a pore-<strong>for</strong>ming α subunit <strong>an</strong>d two<br />
regulatory β subunits. This complex interacts with o<strong>the</strong>r membr<strong>an</strong>e, cytoskeletal, <strong>an</strong>d<br />
extracellular matrix proteins. Mutations in a number of VGSC α subunits are associated with<br />
epilepsy or cardiac arrhythmia. Mutations in SCN1B, encoding β1, are associated with both<br />
phenotypes, consistent with <strong>the</strong> expression of β1 in multiple tissues. SCN1B c<strong>an</strong> be expressed as<br />
two splice vari<strong>an</strong>ts, β1 <strong>an</strong>d β1B, which share <strong>the</strong> N-terminal signal peptide <strong>an</strong>d extracellular Ig<br />
loop domains. The C-terminus of β1B is encoded by a retained intron that shares little to no<br />
homology with <strong>the</strong> C-terminal domain of β1. In brain, β1 <strong>an</strong>d β1B display complementary<br />
developmental expression patterns. β1 is robustly expressed in postnatal brain. In contrast, β1B<br />
is abund<strong>an</strong>tly expressed during embryonic brain development. While much is known about β1,<br />
little is known about β1B. The goal of this study was to underst<strong>an</strong>d <strong>the</strong> membr<strong>an</strong>e topology <strong>an</strong>d<br />
function of β1B. In contrast to β1, bioin<strong>for</strong>matic <strong>an</strong>alysis of <strong>the</strong> amino acid sequence of β1B<br />
predicted no tr<strong>an</strong>smembr<strong>an</strong>e domain. To experimentally confirm this, we probed <strong>for</strong> <strong>the</strong> presence<br />
of β1B at <strong>the</strong> cell surface of stably tr<strong>an</strong>sfected mammali<strong>an</strong> cells using surface biotinylation<br />
assays. Despite <strong>the</strong> expression of β1B in whole-cell lysates, we were unable to detect this protein<br />
at <strong>the</strong> cell surface. In contrast, β1 was detected at <strong>the</strong> cell surface. We next asked whe<strong>the</strong>r β1B<br />
might be a secreted protein by assaying <strong>the</strong> cell medium <strong>for</strong> its presence. Western blot <strong>an</strong>alysis<br />
of <strong>the</strong> cell medium demonstrated <strong>the</strong> presence of β1B but not β1. We evaluated <strong>the</strong> effects of<br />
secreted β1B in a neurite-outgrowth assay using cerebellar gr<strong>an</strong>ule neurons (CGNs). We showed<br />
previously that β1 subunits promote neurite outgrowth in this assay via tr<strong>an</strong>s homophilic cell<br />
adhesion through <strong>the</strong> Ig loop. We found that CGNs plated on a fibroblast monolayer stably<br />
expressing β1B extended neurites to <strong>an</strong> extent similar to neurons plated on a monolayer of β1expressing<br />
cells, demonstrating that β1B promotes neurite outgrowth. Similar to β1, we propose<br />
that β1B stimulates neurite outgrowth through cell adhesive interactions. Coimmunoprecipitation<br />
experiments showed that, while β1 associates with VGSC Nav1.1 α subunits, β1B does not. In<br />
conclusion, we show <strong>for</strong> <strong>the</strong> first time that <strong>the</strong> SCN1B β1B splice vari<strong>an</strong>t is a soluble protein.<br />
Consistent with its developmental expression profile in brain, β1B may function as a soluble cell<br />
adhesion molecule that modulates neurite extension <strong>an</strong>d axon guid<strong>an</strong>ce, in addition to<br />
modulating neuronal sodium currents.<br />
Disclosures: G.A. Patino, None; W.J. Brackenbury, None; L.L. Isom, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.13/C50<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology
Support: Brain Research Trust<br />
Medical Research Council<br />
Worshipful comp<strong>an</strong>y of Pewterers<br />
Title: The functional effect of alternative splicing on Nav1.1 ch<strong>an</strong>nel kinetics<br />
Authors: *E. FLETCHER 1 , S. SCHORGE 1 , D. KULLMANN 2 ;<br />
1 Mol. Neurosci., 2 Dept. of Clin. <strong>an</strong>d Exptl. Epilepsy, Inst. of Neurol., London, United Kingdom<br />
Abstract: SCN1A, <strong>the</strong> gene that encodes <strong>the</strong> alpha subunit of <strong>the</strong> voltage-gated sodium ch<strong>an</strong>nel<br />
designated Nav1.1 is alternatively spliced at exon 5. This gene contains two copies of exon 5,<br />
denoted 5N <strong>an</strong>d 5A (<strong>for</strong> ‘Neonatal’ <strong>an</strong>d ‘Adult’ according to <strong>the</strong>ir expression during<br />
development). Both splice vari<strong>an</strong>ts code <strong>for</strong> a region within <strong>the</strong> first ch<strong>an</strong>nel domain that<br />
correspond to <strong>the</strong> end of <strong>the</strong> third tr<strong>an</strong>smembr<strong>an</strong>e segment, <strong>the</strong> extracellular linker <strong>an</strong>d <strong>the</strong> fourth<br />
voltage-sensing tr<strong>an</strong>smembr<strong>an</strong>e segment. There are 24 nucleotide differences between 5N <strong>an</strong>d<br />
5A, resulting in 3 amino acid substitutions in <strong>the</strong> extracellular linker. This includes a highly<br />
conserved switch in which 5N encodes a neutral residue, asparagine <strong>an</strong>d 5A encodes a negatively<br />
charged residue, aspartate. Although <strong>the</strong> likely functional impact has been speculated, <strong>the</strong>re is<br />
little electrophysiological data showing how <strong>the</strong> choice of exon 5 alters <strong>the</strong> ch<strong>an</strong>nel kinetics of<br />
Nav1.1.<br />
We have cloned <strong>an</strong>d expressed <strong>the</strong> alpha subunits of both splice vari<strong>an</strong>ts of hum<strong>an</strong> Nav1.1 <strong>an</strong>d<br />
used whole cell patch clamp recordings to assess ch<strong>an</strong>nel kinetics. <strong>When</strong> expressed in HEK 293<br />
cells <strong>the</strong> ‘neonatal’ ch<strong>an</strong>nel demonstrated <strong>an</strong> approximate -5mV leftward shift in both activation<br />
<strong>an</strong>d inactivation. To assess <strong>the</strong> signific<strong>an</strong>ce of <strong>the</strong> conserved amino acid substitution <strong>the</strong> ‘adult’<br />
ch<strong>an</strong>nel was mutated so that its aspartate was switched to <strong>an</strong> asparagine. Our recordings<br />
demonstrate that this mut<strong>an</strong>t ch<strong>an</strong>nel exhibits comparable activation <strong>an</strong>d inactivation kinetics to<br />
<strong>the</strong> ‘adult’ ch<strong>an</strong>nel. Suggesting this conserved single ch<strong>an</strong>ge in charge does not solely contribute<br />
to <strong>the</strong> functional differences between Nav1.1 splice vari<strong>an</strong>ts. Subtle ch<strong>an</strong>ges in this ch<strong>an</strong>nel are<br />
associated with Mendeli<strong>an</strong> epilepsies; <strong>the</strong>re<strong>for</strong>e, we hypo<strong>the</strong>size that splicing may affect <strong>the</strong><br />
development of disease.<br />
Disclosures: E. Fletcher, None; S. Schorge, None; D. Kullm<strong>an</strong>n, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.14/C51
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: MRC<br />
Title: Alternative splicing in hum<strong>an</strong> NaV1.7 alters modulation by accessory β1 subunits<br />
Authors: *C. E. FARMER 1 , J. J. COX 3 , C. G. WOODS 3 , J. N. WOOD 2 , S. SCHORGE 1 ;<br />
1 2 3<br />
Inst. of Neurol., WIBR, UCL, London, United Kingdom; Med. Genet., Univ. of Cambridge,<br />
Cambridge, United Kingdom<br />
Abstract: The voltage gated sodium ch<strong>an</strong>nel NaV1.7 plays <strong>an</strong> import<strong>an</strong>t role in nociception in<br />
peripheral sensory neurons. The primary pore-<strong>for</strong>ming α subunit of this ch<strong>an</strong>nel is encoded by<br />
<strong>the</strong> gene SCN9A which undergoes alternative splicing. We are interested in two alternative<br />
splicing sites in SCN9A, one in exon 5 <strong>an</strong>d one in exon 11. Splicing in exon 5 leads to<br />
incorporation of ei<strong>the</strong>r <strong>the</strong> adult (5A) or neonatal (5N) <strong>for</strong>m of <strong>the</strong> exon which differ at 2 amino<br />
acids, whereas splicing in exon 11 leads to ei<strong>the</strong>r a long (11L) or short (11S) <strong>for</strong>m of <strong>the</strong> exon.<br />
Voltage gated sodium ch<strong>an</strong>nel α subunits are modulated by accessory β subunits however it is<br />
currently unclear whe<strong>the</strong>r this modulation is affected by alternative splicing. We have<br />
investigated <strong>the</strong> effect of β subunit co-expression on sodium currents <strong>from</strong> NaV1.7 ch<strong>an</strong>nels<br />
derived <strong>from</strong> <strong>the</strong> four combinations of exon 5 <strong>an</strong>d exon 11 splice vari<strong>an</strong>ts.<br />
Whole-cell voltage clamp recordings were per<strong>for</strong>med on HEK293 cells tr<strong>an</strong>sfected with hum<strong>an</strong><br />
SCN9A splice vari<strong>an</strong>ts with or without β1 using a cesium chloride based intracellular recording<br />
solution. St<strong>an</strong>dard activation <strong>an</strong>d inactivation protocols were employed to assess sodium current<br />
properties. Coexpression of β1 with <strong>the</strong> splice vari<strong>an</strong>ts of NaV1.7 increased sodium current<br />
density <strong>for</strong> all vari<strong>an</strong>ts but had no effect on <strong>the</strong> voltage dependence of activation. However <strong>the</strong><br />
increase in current density was more pronounced in those ch<strong>an</strong>nels containing sequences<br />
encoded by exon 5N <strong>an</strong>d/or 11L suggesting that <strong>the</strong> surface expression of <strong>the</strong>se splice iso<strong>for</strong>ms<br />
may be more strongly regulated by β1. In agreement with previously published data we also<br />
showed that ch<strong>an</strong>nels containing exon 5A showed a slowed development of inactivation but we<br />
found that this was reduced by coexpression with β1. NaV1.7 ch<strong>an</strong>nels containing exon 5N are<br />
likely to be expressed predomin<strong>an</strong>tly in neonatal <strong>an</strong>d early postnatal tissues <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e <strong>the</strong><br />
strong interaction between 5N <strong>an</strong>d β1 may be particularly import<strong>an</strong>t in enh<strong>an</strong>cing ch<strong>an</strong>nel<br />
activity, <strong>an</strong>d subsequently cell excitation, during early development. We conclude that<br />
alternatively spliced NaV1.7 ch<strong>an</strong>nels c<strong>an</strong> be differentially modulated by <strong>the</strong> accessory β1<br />
subunit <strong>an</strong>d this selective modulation, possibly by increasing surface expression of some splice<br />
vari<strong>an</strong>ts, may affect peripheral sensory neuron excitability.<br />
Disclosures: C.E. Farmer, None; J.J. Cox, None; C.G. Woods, None; J.N. Wood, None; S.<br />
Schorge, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.15/C52<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: NIH gr<strong>an</strong>t NS55860<br />
NIH gr<strong>an</strong>t NS45594<br />
Title: Ch<strong>an</strong>ges in Na currents in sensory neurons early after nerve injury: Differences between<br />
axotomized <strong>an</strong>d adjacent intact g<strong>an</strong>glia <strong>an</strong>d possible influence of inflammation<br />
Authors: *R.-H. YANG 1 , J. A. STRONG 2 , J.-M. ZHANG 3 ;<br />
1 Univ. Cincinnati, Cincinnati, OH; 2 Dept. of Anes<strong>the</strong>siology,, Pain Res. Center, Univ. of<br />
Cincinnati Col. of Med., Cincinnati, OH; 3 Dept. of Anes<strong>the</strong>siology,, Pain Res. Center, Univ. of<br />
Cincinnati Col. of Medicine,, Cincinnati,, OH<br />
Abstract: Animal pain models have long been classified as neuropathic or inflammatory. More<br />
recently it has become apparent that inflammation plays some role even in nerve injury models.<br />
The spinal nerve ligation (SNL) model, in which <strong>the</strong> L5 spinal nerve is ligated near <strong>the</strong> L5 dorsal<br />
root g<strong>an</strong>glion (DRG), was originally developed as a nerve injury pain model in which<br />
axotomized neurons were separated <strong>from</strong> <strong>the</strong> nonaxotomized neurons in <strong>the</strong> adjacent L4 DRG<br />
that mediate enh<strong>an</strong>ced pain behavior. Since <strong>the</strong> ligation is quite close to <strong>the</strong> DRG in this model,<br />
it is plausible that inflammation in <strong>the</strong> DRG plays import<strong>an</strong>t roles, especially early after <strong>the</strong><br />
injury when pain behaviors are initiated. In support of this, we recently reported that<br />
proinflammatory cytokines increase in <strong>the</strong> axotomized DRG early after spinal nerve ligation, <strong>an</strong>d<br />
systemic <strong>an</strong>ti-inflammatory steroids given during this early period reversed <strong>the</strong>se cytokine<br />
ch<strong>an</strong>ges <strong>an</strong>d provided a long-term reduction in mech<strong>an</strong>ical pain behaviors. In this study, we<br />
examined ch<strong>an</strong>ges in Na currents in small DRG neurons <strong>from</strong> <strong>the</strong> axotomized L5 DRG <strong>an</strong>d <strong>from</strong><br />
<strong>the</strong> adjacent intact L4 DRG, early (24 hours) after <strong>the</strong> SNL injury. Na currents were recorded in<br />
neurons maintained in short term (2 to 5 hours). Cells were identified as IB4-positive (primarily<br />
nonpeptidergic) or IB4-negative (primarily peptidergic). Na currents were separated into TTXsensitive<br />
<strong>an</strong>d TTX-resist<strong>an</strong>t components based on holding potential. Our results suggested that<br />
some but not all of <strong>the</strong> Na current ch<strong>an</strong>ges in L5 neurons resembled ch<strong>an</strong>ges seen in<br />
inflammatory models. TTX-resist<strong>an</strong>t currents increased, especially in IB4-positive neurons. This<br />
is similar to effects seen in inflammatory models <strong>an</strong>d contrasts with <strong>the</strong> downregulation of most<br />
Na currents seen (at later times) in axotomy models. However, preliminary qPCR experiments<br />
suggested that upregulation of <strong>the</strong> TTX-sensitive Nav1.3 ch<strong>an</strong>nel could be detected at 24 hours;<br />
this is a well-described ch<strong>an</strong>ge seen in o<strong>the</strong>r axotomy but not inflammatory models. Na currents<br />
in L4 neurons were affected quite differently. In IB4-positive cells both TTX-sensitive <strong>an</strong>d TTXresist<strong>an</strong>t<br />
Na currents actually decreased. The robust increase in TTX-resist<strong>an</strong>t currents seen in<br />
inflammation models was not observed in L4. Our results suggest that Na currents in <strong>the</strong><br />
axotomized L5 g<strong>an</strong>glion in this model reflect a bal<strong>an</strong>ce between effects of inflammation <strong>an</strong>d of
axotomy, <strong>an</strong>d that inflammation plays a relatively import<strong>an</strong>t role early after <strong>the</strong> injury.<br />
(Supported in part by NIH gr<strong>an</strong>ts NS55860 <strong>an</strong>d NS45594)<br />
Disclosures: R. Y<strong>an</strong>g, None; J.A. Strong, None; J. Zh<strong>an</strong>g, None.<br />
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.16/C53<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: Americ<strong>an</strong> Heart Association<br />
UMKC Start-up fund<br />
Title: Characterization of acid-sensing ion ch<strong>an</strong>nels in medium spiny neurons of mouse striatum<br />
Authors: *X. CHU, Q. JIANG, C. J. PAPASIAN, J. Q. WANG;<br />
Basic Med. Sci., Univ. Missouri-K<strong>an</strong>sas City, K<strong>an</strong>sas City, MO<br />
Abstract: Acid-sensing ion ch<strong>an</strong>nels (ASICs) regulate synaptic activities <strong>an</strong>d play import<strong>an</strong>t<br />
roles in neurodegenerative diseases. They are highly expressed in <strong>the</strong> striatum, where medium<br />
spiny neurons (MSNs) are a major population. Given that <strong>the</strong> properties of ASICs in MSNs are<br />
unknown, in this study, we characterized ASICs in MSNs of <strong>the</strong> mouse striatum. A rapid drop in<br />
extracellular pH induced tr<strong>an</strong>sient inward currents in all MSNs. The pH value <strong>for</strong> half maximal<br />
activation (pH50) was 6.25, close to that obtained in homomeric ASIC1a ch<strong>an</strong>nels. Based on<br />
PcTX1 <strong>an</strong>d zinc sensitivity, ASIC1a (70.5% of neurons) <strong>an</strong>d heteromeric ASIC1a-2 ch<strong>an</strong>nels<br />
(29.5% of neurons) appeared responsible <strong>for</strong> <strong>the</strong> acid-induced currents in MSNs. ASIC currents<br />
were diminished in MSNs <strong>from</strong> ASIC1, but not ASIC2, null mice. Fur<strong>the</strong>rmore, a drop in pH<br />
induced calcium influx by activating homomeric ASIC1a ch<strong>an</strong>nels. Activation of ASICs<br />
increased <strong>the</strong> membr<strong>an</strong>e excitability of MSNs <strong>an</strong>d lowering extracellular Ca 2+ potentiated ASIC<br />
currents. Our data suggest that <strong>the</strong> homomeric ASIC1a ch<strong>an</strong>nel represents a majority of <strong>the</strong><br />
ASIC iso<strong>for</strong>m in MSNs. The potential function of ASICs in <strong>the</strong> striatum requires fur<strong>the</strong>r<br />
investigation.<br />
Disclosures: X. Chu , None; Q. Ji<strong>an</strong>g, None; C.J. Papasi<strong>an</strong>, None; J.Q. W<strong>an</strong>g, None.
Poster<br />
517. Sodium Ch<strong>an</strong>nels: Physiology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 517.17/C54<br />
Topic: B.04.a. Sodium ch<strong>an</strong>nels: Physiology<br />
Support: NS PPG P01-NS-056202<br />
GAANN<br />
UC Davis/NIH Neuromab Facility<br />
NSF GRFP<br />
Title: The interplay of calpain <strong>an</strong>d calpastatin in proteolysis of <strong>the</strong> voltage gated sodium ch<strong>an</strong>nel<br />
Authors: *C. R. VON REYN 1 , J. M. SPAETHLING 2 , M. N. MESFIN 1 , M. MA 3 , R. W.<br />
NEUMAR 3 , R. SIMAN 4 , D. H. SMITH 4 , D. F. MEANEY 1 ;<br />
1 Bioengineering, Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA; 2 Pharmacol., 3 Emergency Med.,<br />
4 Neurosurg., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Proteolysis of voltage gated sodium ch<strong>an</strong>nels (NaChs) in models of traumatic brain<br />
injury <strong>an</strong>d ischemia has led to <strong>the</strong> suggestion that this event plays a common role in <strong>the</strong><br />
progression of both diseases. The cause of ch<strong>an</strong>nel proteolysis <strong>an</strong>d resulting downstream<br />
consequences are still under investigation. Our previous work demonstrated a calpain dependent<br />
proteolysis of NaChs upon elevation of [Ca 2+ ]. In <strong>the</strong>se studies, we fur<strong>the</strong>r validated that NaCh<br />
proteolysis is calpain mediated using specific activation/inhibition of calpain. Direct addition of<br />
calpain to rat brain membr<strong>an</strong>e preparations led to proteolysis of <strong>the</strong> α-subunits of <strong>the</strong> ch<strong>an</strong>nel<br />
(n=3, p
proteolyzed NaChs appear <strong>an</strong>d remain in <strong>the</strong> membr<strong>an</strong>e early after injury, prior to <strong>the</strong> loss of<br />
plasma membr<strong>an</strong>e integrity, indicating that proteolysis may have a signific<strong>an</strong>t effect on ionic<br />
homeostasis, neurotr<strong>an</strong>smission, <strong>an</strong>d o<strong>the</strong>r pathophysiological processes following ischemic <strong>an</strong>d<br />
traumatic brain injury.<br />
Disclosures: C.R. von Reyn, None; J.M. Spaethling, None; M.N. Mesfin, None; M. Ma,<br />
None; R.W. Neumar, None; R. Sim<strong>an</strong>, None; D.H. Smith, None; D.F. Me<strong>an</strong>ey, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.1/C55<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: INTAS 97-32194<br />
WELCOME TRUST 064222<br />
Title: Local extracellular field potential may control <strong>the</strong> functional state of <strong>the</strong> fast-inactivating<br />
low-voltage-activated calcium ch<strong>an</strong>nels in rat laterodorsal thalamic neurons<br />
Authors: *A. N. TARASENKO, V. E. LUGOVSKOY, A. S. PALAGINA, E. V.<br />
KADOCHNIKOV, D. S. ISAEV;<br />
Bogomoletz Inst. Physiol, Kiev, Ukraine<br />
Abstract: We have previously shown that rat laterodorsal (LD) thalamic neurons near <strong>the</strong> end of<br />
<strong>the</strong> second postnatal week (postnatal day, (P)12), express only low-voltage-activated (LVA) Ca 2+<br />
ch<strong>an</strong>nels with fast kinetic decay. With 2 mM Ca in <strong>the</strong> external solutions, whole-cell voltageclamp<br />
experiments in situ have revealed that <strong>the</strong>se ch<strong>an</strong>nels become available <strong>for</strong> activation only<br />
if membr<strong>an</strong>e potential differences (MPDs) are r<strong>an</strong>ged <strong>from</strong> -75 to -100 mV. They are inactive at<br />
more positive MPD. To be activated again <strong>the</strong> MPD should increase to <strong>the</strong> values larger th<strong>an</strong> 75<br />
mV. However, <strong>for</strong> m<strong>an</strong>y decades, <strong>the</strong> mech<strong>an</strong>isms controlling MPD increase remain unclear. The<br />
number of reports suggested that <strong>the</strong> electrical field of <strong>the</strong> local surface charges might be<br />
involved in <strong>the</strong> control of <strong>the</strong> functional states of <strong>the</strong> ion ch<strong>an</strong>nels. Moreover, ch<strong>an</strong>ges of <strong>the</strong><br />
surface charge density might occur without affecting <strong>the</strong> resting membr<strong>an</strong>e potential (RMP).<br />
We now show that in isolated P12 LD thalamic neurons, <strong>the</strong> recovery r<strong>an</strong>ge of about 25 mV<br />
remains <strong>the</strong> same as was observed in situ. It was not ch<strong>an</strong>ged ei<strong>the</strong>r <strong>the</strong>se ch<strong>an</strong>nels were<br />
activated solely or toge<strong>the</strong>r with K + ch<strong>an</strong>nels. The unitary currents associated with LVA Ca 2+
ch<strong>an</strong>nels activity at 2 mM Ca 2+ revealed that <strong>the</strong> first 5% of <strong>the</strong> ch<strong>an</strong>nels availability was already<br />
achieved at 25 mV above <strong>the</strong> resting membr<strong>an</strong>e potential (if it was about -55 mV). The fur<strong>the</strong>r<br />
small local increase within <strong>the</strong> r<strong>an</strong>ge of 25 mV was sufficient to have full control over <strong>the</strong> fastinactivating<br />
LVA Ca 2+ ch<strong>an</strong>nels availability. Thus, local ch<strong>an</strong>ges of <strong>the</strong> external potential<br />
increase MPD across <strong>the</strong> ch<strong>an</strong>nel <strong>an</strong>d make more ch<strong>an</strong>nels available <strong>for</strong> activation.<br />
We believe that <strong>the</strong> promise c<strong>an</strong>didates that ch<strong>an</strong>ge <strong>the</strong> local surface potential through charge<br />
neutralization are <strong>the</strong> potassium ions. They may appear close to <strong>the</strong> mouse of <strong>the</strong> single LVA<br />
Ca 2+ ch<strong>an</strong>nel as <strong>the</strong> result of potassium ch<strong>an</strong>nel opening. The diffusion will destroy clouds of<br />
potassium ions as fast as <strong>the</strong> potassium ch<strong>an</strong>nels are closed. If <strong>the</strong> two types of <strong>the</strong> ch<strong>an</strong>nels, fastinactivating<br />
LVA Ca 2+ ch<strong>an</strong>nels <strong>an</strong>d potassium ch<strong>an</strong>nels, are well spatially distributed <strong>the</strong>n one<br />
may suggest <strong>an</strong> existence of <strong>the</strong> single functional unit allowing <strong>the</strong> generation of <strong>the</strong> local spike.<br />
Once activated it c<strong>an</strong> fur<strong>the</strong>r fire <strong>an</strong>o<strong>the</strong>r functional units if <strong>the</strong>y are well spatially org<strong>an</strong>ized.<br />
Here, we w<strong>an</strong>t to emphasize that in order to involve <strong>the</strong> fast-inactivating LVA Ca 2+ ch<strong>an</strong>nels into<br />
<strong>the</strong> rhythmic discharge patter observed in <strong>the</strong> thalamocortical network one may expect ch<strong>an</strong>ges<br />
within extracellular environment ra<strong>the</strong>r <strong>the</strong>n intracellular one as was previously expected <strong>for</strong> a<br />
long time.<br />
Disclosures: A.N. Tarasenko, None; V.E. Lugovskoy, None; A.S. Palagina, None; E.V.<br />
Kadochnikov, None; D.S. Isaev, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.2/C56<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: ANR-06-Neuro<br />
CNRS LEA528<br />
Wellcome Trust gr<strong>an</strong>ts 71436 & 78403<br />
Title: Effect of TTA-P2 a novel potent <strong>an</strong>d selective T-type calcium ch<strong>an</strong>nel blocker on thalamic<br />
cell excitability<br />
Authors: *N. LERESCHE 1 , F. M. DREYFUS 1 , A. TSCHERTER 1 , A. E. ERRINGTON 2 , J. J.<br />
RENGER 3 , H.-S. SHIN 4 , V. N. UEBELE 3 , V. CRUNELLI 2 , R. C. LAMBERT 1 ;<br />
1 UPMC-CNRS, Paris 75005, Fr<strong>an</strong>ce; 2 Sch. of Biosci., Cardiff Univ., Cardiff, United Kingdom;
3 4<br />
Merck Res. Labs., West Point, PA; Ctr. <strong>for</strong> Neural Sci., Korea Inst. of Sci. <strong>an</strong>d Technol., Seoul,<br />
Republic of Korea<br />
Abstract: Although it is well established that low-voltage activated T-type Ca 2+ ch<strong>an</strong>nels play a<br />
key role in m<strong>an</strong>y neurophysiological functions <strong>an</strong>d pathological states, <strong>the</strong> lack of selective <strong>an</strong>d<br />
potent <strong>an</strong>tagonists has so far hampered a detailed <strong>an</strong>alysis of <strong>the</strong> full impact that <strong>the</strong>se ch<strong>an</strong>nels<br />
might have on single cell <strong>an</strong>d neuronal network excitability as well as on calcium homeostasis.<br />
Here, using rat thalamic slices, we directly show that <strong>the</strong> novel piperidine-based molecule TTA-<br />
P2 exerts a specific, potent (IC50: 22nM) <strong>an</strong>d reversible inhibition of T-type Ca 2+ currents (IT) in<br />
both thalamocortical (TC) <strong>an</strong>d reticular thalamic nucleus (NRT) neurons without <strong>an</strong>y action on<br />
HVA Ca 2+ currents. Under current clamp conditions, 1µM TTA-P2, a concentration that fully<br />
blocks IT (96±1%, n=7), had no effect on tonic firing <strong>an</strong>d action potentials characteristics<br />
(threshold, half-width, amplitude, AHP), but abolished <strong>the</strong> low threshold Ca 2+ potential (LTCP)dependent<br />
high frequency burst firing of thalamic neurons. In addition, when TC <strong>an</strong>d NRT<br />
neurons were maintained at -60mV, application of 1µM TTA-P2 produced a tonic<br />
hyperpolarization of 3.1±0.5mV (n=11) <strong>an</strong>d 5±2.2mV (n=6), respectively. Such<br />
hyperpolarization was not observed when TC neurons were maintained at -70mV or in TC<br />
neurons <strong>from</strong> Cav3.1 KO mice that were recorded at -60mV (n=6). These data demonstrate that<br />
<strong>the</strong> TTA-P2 induced hyperpolarization is due to <strong>the</strong> block of <strong>the</strong> window component of IT <strong>an</strong>d<br />
that this current contributes to <strong>the</strong> resting membr<strong>an</strong>e potential of thalamic neurons. Finally, <strong>the</strong><br />
use of this novel compound allowed us to consolidate <strong>an</strong>d enlarge our current underst<strong>an</strong>ding of<br />
<strong>the</strong> contribution of IT to single TC neuron excitability. Thus, we provide <strong>the</strong> first direct<br />
demonstration that <strong>the</strong> window component of IT contributes to <strong>the</strong> intrinsically generated slow<br />
(
Title: The effect of 1-oct<strong>an</strong>ol on spont<strong>an</strong>eous brain activity in healthy adults<br />
Authors: *K. D. WALTON 1 , T. CARDOZO 2 , E. L. MAILLET 3 , R. LLINÁS 1 ;<br />
1 2 3<br />
Dept Physiol, Dept Pharmacol., New York Univ., New York, NY; Neurosci., Mount Sinai<br />
Sch. of Med., New York, NY<br />
Abstract: The aim of this study was to examine <strong>the</strong> effect of 1-Oct<strong>an</strong>ol on spont<strong>an</strong>eous brain<br />
activity. We are interested in oct<strong>an</strong>ol because of its ability to block T-type (Cav3.1) calcium<br />
ch<strong>an</strong>nels. These ch<strong>an</strong>nels are a key element in <strong>the</strong> generation of low frequency oscillations such<br />
as those seen in thalamocortical dysrhythmia (TCD). Oct<strong>an</strong>ol reversibly reduces <strong>the</strong> low<br />
threshold calcium currents in thalamic <strong>an</strong>d inferior olivary (IO) neurons, reduces IO oscillations<br />
in vitro, <strong>an</strong>d prevents harmaline-induced tremor. 1-oct<strong>an</strong>ol has also been used to reduce tremor in<br />
patients with essential tremor.<br />
Oct<strong>an</strong>ol, a straight-chain fatty alcohol with 8 carbon atoms naturally occurs in some pl<strong>an</strong>ts <strong>an</strong>d<br />
fruits, notably citrus. It has been approved by <strong>the</strong> FDA <strong>an</strong>d <strong>the</strong> Council of Europe, as a flavoring<br />
agent in food <strong>an</strong>d cosmetics; daily intak, 1mg/kg. 1-Oct<strong>an</strong>ol was <strong>for</strong>mulated in a potent oral<br />
mucosal delivery <strong>for</strong>mat with appropriate org<strong>an</strong>oleptics characteristics, <strong>an</strong>d self-delivered to<br />
subjects at < 1mg/kg/day. Spont<strong>an</strong>eous brain activity was recorded <strong>from</strong> > 30 healthy adults<br />
using a 275-ch<strong>an</strong>nel MEG (CTF). Recordings were made be<strong>for</strong>e <strong>an</strong>d at six points after oct<strong>an</strong>ol.<br />
Power spectra <strong>an</strong>alysis was done <strong>for</strong> all sensors <strong>for</strong> 6 frequency r<strong>an</strong>ges. Questionnaires were<br />
completed be<strong>for</strong>e, <strong>an</strong>d at 5 <strong>an</strong>d 120 minutes after oct<strong>an</strong>ol examining relaxation, concentration,<br />
worry, sleepiness, stress, pain <strong>an</strong>d <strong>the</strong> oct<strong>an</strong>ol taste.<br />
Oct<strong>an</strong>ol increased <strong>the</strong> power in low alpha r<strong>an</strong>ge (8-10.5Hz) after 15 min <strong>an</strong>d continued to<br />
increase throughout <strong>the</strong> recording period. From 15-90 min power increased in <strong>the</strong> high alpha<br />
r<strong>an</strong>ge (10.5-13Hz), decreased in <strong>the</strong> delta r<strong>an</strong>ge (2-4Hz) <strong>an</strong>d <strong>the</strong>re was no effect on beta (13-<br />
30Hz), <strong>the</strong>ta (4-8Hz), or gamma (35-55Hz) activity. At 120 min after oct<strong>an</strong>ol <strong>the</strong>re was <strong>an</strong><br />
increase in all frequency r<strong>an</strong>ges. No signific<strong>an</strong>t effect was reported <strong>for</strong> <strong>an</strong>y of <strong>the</strong> items on <strong>the</strong><br />
questioners <strong>an</strong>d <strong>the</strong> taste was reported as just above neutral (me<strong>an</strong> 4.7±/24, 1 = “good”).<br />
These results indicate that 1-Oct<strong>an</strong>ol increases normal, alpha r<strong>an</strong>ge activity while reducing<br />
abnormal, delta r<strong>an</strong>ge activity. It was reported as having no effect on general, self-reported<br />
measures of wellness. Reduction of abnormal oscillations surgically through thalamic lesion has<br />
been shown to reduce symptoms in patients with TCD such as neuropathic pain <strong>an</strong>d Parkinson’s<br />
disease. Oct<strong>an</strong>ol is a promising c<strong>an</strong>didate <strong>for</strong> a non-surgical approach to reduction of abnormal<br />
oscillations seen in TCD <strong>an</strong>d <strong>the</strong> relief of symptoms.<br />
Disclosures: K.D. Walton, None; T. Cardozo, None; E.L. Maillet, None; R. Llinás, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 518.4/C58<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t GM070726<br />
Title: Oct<strong>an</strong>ol reduces T-current in thalamocortical relay neurons<br />
Authors: *V.-S. ECKLE 1,2 , S. M. TODOROVIC 1 ;<br />
1 Dept. of Anes<strong>the</strong>siol., Univ. of Virginia, Charlottesville, VA; 2 Dept. of Anes<strong>the</strong>siol., Klinikum<br />
rechts der Isar, Techical Univ., Munich, Germ<strong>an</strong>y<br />
Abstract: Modulation of thalamic T-type calcium current might be pivotal <strong>for</strong> generating<br />
<strong>an</strong>es<strong>the</strong>tic loss of consciousness. Using patch clamp technique, we investigated <strong>the</strong> effect of<br />
<strong>an</strong>es<strong>the</strong>tic alcohol oct<strong>an</strong>ol on T-type currents in intact neurons in brain slices <strong>from</strong> ventrobasal<br />
thalamic nucleus of young rats. Peak amplitude of native T-current was signific<strong>an</strong>tly reduced by<br />
oct<strong>an</strong>ol in concentration-dependent m<strong>an</strong>ner yielding IC50 at 352±97 µM in thalamocortical relay<br />
neurons (n=20). By oct<strong>an</strong>ol application (300 µM, 1 mM), <strong>the</strong>re was no apparent shift in peak<br />
amplitude of I-V curves (n=15). Oct<strong>an</strong>ol (1 mM) caused a signific<strong>an</strong>t hyperpolarizing shift in<br />
steady state inactivation curve (10-mV leftward shift vs. control conditions, n=5, p
NIH Gr<strong>an</strong>t 1F31NS059190<br />
NIH Gr<strong>an</strong>t T32GM08328<br />
Title: Glycosylation alters gating <strong>an</strong>d inactivation of T-type calcium ch<strong>an</strong>nels in <strong>the</strong> pain<br />
pathway<br />
Authors: *S. M. TODOROVIC 1 , W. CHOE 3 , P. ORESTES 2 ;<br />
1 Dept Anes<strong>the</strong>siol, 2 Dept Anes<strong>the</strong>siol <strong>an</strong>d Neurosci Grad Program, Univ. Virginia Hlth. Syst.,<br />
Charlottesville, VA; 3 Anes<strong>the</strong>siol., InJe University, IIs<strong>an</strong> Paik Hosp., Goy<strong>an</strong>g-city, Republic of<br />
Korea<br />
Abstract: Recent data indicate that alterations of T-type calcium ch<strong>an</strong>nels (T-ch<strong>an</strong>nels) in<br />
<strong>an</strong>imal models of diabetic neuropathy contribute to increased excitability of sensory neurons.<br />
However, little is known about mech<strong>an</strong>isms underlying plasticity of T-ch<strong>an</strong>nels under<br />
hyperglycemic conditions. Using acutely dissociated dorsal root g<strong>an</strong>glion (DRG) cells <strong>from</strong><br />
diabetic leptin-deficient (ob/ob) mice <strong>an</strong>d recombin<strong>an</strong>t CaV3.2 ch<strong>an</strong>nels expressed in HEK 293<br />
cells cultured in high glucose media, we explored <strong>the</strong> hypo<strong>the</strong>sis that elevated glucose levels<br />
result in increased glycosylation of T-ch<strong>an</strong>nels.<br />
T-currents <strong>from</strong> small DRG neurons of ob/ob mice inactivated two times faster th<strong>an</strong> those <strong>from</strong><br />
wild-type (WT) mice (inactivation tau at -40mV: WT, 73.8±11ms; ob/ob, 37.8±7ms). <strong>When</strong><br />
neurons were incubated with 1.5U/ml neuraminidase (NEU), both WT <strong>an</strong>d ob/ob ch<strong>an</strong>nel<br />
inactivation slowed dramatically to similar rates (WT, 105.6±5.9ms; ob/ob, 107.3±10.7ms).<br />
Similarly, T-ch<strong>an</strong>nels <strong>from</strong> ob/ob mice activated two times faster th<strong>an</strong> WT <strong>an</strong>d after treatments<br />
of cells with NEU, both slowed to nearly <strong>the</strong> same level. In addition, NEU is able to rescue ob/ob<br />
ch<strong>an</strong>nels beyond normal WT kinetics, consistent with <strong>the</strong> idea that smaller numbers of WT<br />
ch<strong>an</strong>nels are also glycosylated.<br />
Next, we cultured HEK-293 cells expressing <strong>the</strong> CaV3.2 ch<strong>an</strong>nels in high glucose media. As<br />
expected, when <strong>the</strong> cells were incubated with NEU, ch<strong>an</strong>nel activation <strong>an</strong>d inactivation slowed<br />
signific<strong>an</strong>tly (inactivation tau at -30mV: untreated, 11.2±1.2ms; NEU, 39.4±9.5ms). In addition,<br />
NEU treatment induced large depolarizing shifts (about 14 mV) in midpoint of steady-state<br />
inactivation of CaV3.2 currents.<br />
In conclusion, exposure to <strong>the</strong> NEU, enzyme thought to be selective <strong>for</strong> extracellular sialic<br />
groups in <strong>the</strong> proteins, reverses m<strong>an</strong>y of <strong>the</strong> kinetic effects produced by a high glucose<br />
environment on native <strong>an</strong>d recombin<strong>an</strong>t CaV3.2 currents. These results provide novel evidence<br />
that hyperglycemia promotes increased glycosylation of T-ch<strong>an</strong>nels, <strong>an</strong>d that this induces<br />
signific<strong>an</strong>t ch<strong>an</strong>ges in <strong>the</strong> gating behavior of <strong>the</strong>se ch<strong>an</strong>nels. These ch<strong>an</strong>ges may result in<br />
increased cellular excitability of sensory neurons <strong>an</strong>d abnormal pain tr<strong>an</strong>smission associated with<br />
diabetic neuropathy.<br />
Disclosures: S.M. Todorovic, None; W. Choe, None; P. Orestes, None.<br />
Poster
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.6/C60<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH GM 070726<br />
NIH NINDS 1F31NS059190<br />
NIH NIGMS T32 GM08328<br />
Title: Free radical signaling mediates subunit-specific nitrous oxide inhibition of T-type calcium<br />
ch<strong>an</strong>nels in <strong>the</strong> pain pathway<br />
Authors: *P. ORESTES 1 , D. BOJADZIC 2 , S. M. TODOROVIC 1 ;<br />
1 Anes<strong>the</strong>siol & Neurosci Grad Program, 2 Anes<strong>the</strong>siol, Univ. Virginia, Charlottesville, VA<br />
Abstract: Nitrous oxide (N2O) has been used as <strong>an</strong> <strong>an</strong>es<strong>the</strong>tic <strong>an</strong>d <strong>an</strong>algesic <strong>for</strong> centuries, but its<br />
mech<strong>an</strong>ism of action remains unclear. Previous studies have shown that N2O inhibits native Ttype<br />
Ca 2+ current in sensory neurons <strong>an</strong>d recombin<strong>an</strong>t CaV3.2 ch<strong>an</strong>nels. Fur<strong>the</strong>rmore, N2O<br />
requires a key metal-binding histidine (H191) in <strong>the</strong> extracellular IS3-4 loop <strong>for</strong> its action on<br />
CaV3.2 ch<strong>an</strong>nels.<br />
Here, we show that N2O-mediated inhibition is also dependent on <strong>the</strong> presence of common trace<br />
metal ions. Diethylenetriamine pentaacetic acid (DTPA), a non-specific metal chelator,<br />
effectively reduces N2O block of calcium current through CaV3.2 ch<strong>an</strong>nels by 70%. We also<br />
implicate free radicals as mediators of this effect since free radical scavengers such as catalase<br />
<strong>an</strong>d alpha-tocopherol (vitamin E), are able to reduce <strong>the</strong> inhibitory effects of N2O to 7% <strong>an</strong>d 16%<br />
of control, respectively.<br />
Thus we hypo<strong>the</strong>size that N2O is able to generate free radicals in <strong>the</strong> presence of trace metal<br />
ions, similar to those produced by hydrogen peroxide (H2O2). In accord<strong>an</strong>ce, we show that N2O<br />
is unable to achieve additional block of CaV3.2 ch<strong>an</strong>nels over that of H2O2. Additionally, we<br />
show that N2O is able to initiate <strong>the</strong> free radical-mediated oxidation of epinephrine to<br />
adrenochrome in <strong>the</strong> presence of trace metals. The production of adrenochrome was qu<strong>an</strong>tified<br />
by measurements of absorb<strong>an</strong>ce ch<strong>an</strong>ges at 485nm.<br />
To definitively establish <strong>the</strong> role of CaV3.2 ch<strong>an</strong>nels in N2O <strong>an</strong>algesia, we injected <strong>for</strong>malin into<br />
<strong>the</strong> hindpaws of CaV3.2 knockout mice <strong>an</strong>d <strong>the</strong>ir wild-type littermates. Following injection, <strong>the</strong><br />
wild-type mice showed a signific<strong>an</strong>t decrease in licking/biting time in <strong>the</strong> presence of 20% N2O.<br />
In contrast, knockout <strong>an</strong>imals show little decrease in pain with N2O.<br />
Thus, we suggest that N2O indirectly inhibits CaV3.2 ch<strong>an</strong>nels via <strong>the</strong> free oxygen radicals that<br />
are produced in its presence. These highly unstable molecules <strong>the</strong>n react in <strong>the</strong> immediate<br />
neighborhood of tr<strong>an</strong>sition metals bound to H191, oxidizing <strong>the</strong> histidine to 2-oxo-histidine. This
ch<strong>an</strong>ge alters CaV3.2 gating <strong>an</strong>d activity. Since recent data strongly suggest a crucial role of<br />
Cav3.2 T-type Ca 2+ ch<strong>an</strong>nels in pain tr<strong>an</strong>smission, N2O block of <strong>the</strong>se ch<strong>an</strong>nels mediated by free<br />
radical signaling may contribute to part of nitrous oxide’s potent <strong>an</strong>algesic effects.<br />
Disclosures: P. Orestes, None; D. Bojadzic, None; S.M. Todorovic, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.7/C61<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Title: TRPV1 activation inhibits both high-threshold <strong>an</strong>d T-type calcium ch<strong>an</strong>nels in rat DRG<br />
neurons<br />
Authors: V. COMUNANZA 1 , D. URSU 2 , *R. ZWART 2 , E. CARBONE 1 , E. SHER 2 ;<br />
1 Dept. of Neurosci., University of Torino, Italy; 2 Eli Lilly Co Ltd, Windlesham, United Kingdom<br />
Abstract: TRPV1 (tr<strong>an</strong>sient receptor potential v<strong>an</strong>illoid 1) is a non-selective cation ch<strong>an</strong>nel<br />
localized predomin<strong>an</strong>tly in small-diameter peripheral sensory neurones. TRPV1 is<br />
physiologically activated by heat, low pH, <strong>an</strong>d endoc<strong>an</strong>nabinoids, but c<strong>an</strong> also c<strong>an</strong> be activated<br />
by various natural compounds (capsaicin, RTX, piperine) or syn<strong>the</strong>tic <strong>an</strong>alogues (olv<strong>an</strong>il,<br />
arv<strong>an</strong>il). Exposure to capsaicin, after a brief excitatory effect, desensitizes nociceptive sensory<br />
neurons <strong>an</strong>d results in long lasting pain relief. This has been suggested to occur via crossmodulation<br />
of o<strong>the</strong>r ion ch<strong>an</strong>nels. The aim of <strong>the</strong> work was to investigate <strong>the</strong> effects of TRPV1<br />
activation on High Voltage Activated (HVA; L, N, P/Q, R) as well as Low Voltage Activated<br />
(LVA, T-type) Ca 2+ ch<strong>an</strong>nel currents in rat DRG neurons. Calcium influx through HVA Ca 2+<br />
ch<strong>an</strong>nels plays a crucial role in neurotr<strong>an</strong>smitter release, while T-type Ca 2+ ch<strong>an</strong>nels play <strong>an</strong><br />
import<strong>an</strong>t role in modulating sensory neurons excitability, under normal conditions <strong>an</strong>d in pain<br />
states. Whole-cell patch clamp recordings were per<strong>for</strong>med in rat DRG neurons <strong>an</strong>d Ca 2+ currents<br />
elicited with ei<strong>the</strong>r step depolarisations or with voltage ramps, be<strong>for</strong>e <strong>an</strong>d after application of 1<br />
µM capsaicin. In capsaicin-sensitive neurons <strong>the</strong> agonist produced rapid inward currents (648.6<br />
± 137.2 pA) that resulted in a “secondary” large inhibition of HVA Ca 2+ currents (54.5 ± 6.5 %<br />
inhibition). Capsaicin did not cause direct inhibition of HVA Ca 2+ currents in neurons lacking<br />
TRPV1. A large proportion of cells (65%) also expressed LVA Ca 2+ currents (activation<br />
threshold -30.0 ± 1.7 mV) that were confirmed, by both biophysical <strong>an</strong>d pharmacological me<strong>an</strong>s,<br />
to be mediated by T-type Ca 2+ ch<strong>an</strong>nels with activation-inactivation kinetics similar to <strong>the</strong><br />
Cav3.2 iso<strong>for</strong>m, <strong>the</strong> most expressed Cav3 subunit in DRG neurons. We found that <strong>the</strong>se T-type
Ca 2+ currents are also inhibited by capsaicin application (41.0 ± 12.2 % inhibition). The degree<br />
of Ca 2+ ch<strong>an</strong>nel inhibition was proportional to <strong>the</strong> amplitude of <strong>the</strong> capsaicin-induced current <strong>an</strong>d<br />
<strong>the</strong> inhibition of T <strong>an</strong>d HVA Ca 2+ ch<strong>an</strong>nels was highly correlated. In conclusion, we<br />
demonstrated that TRPV1 activation by capsaicin inhibits not only HVA Ca 2+ ch<strong>an</strong>nels, with<br />
potential direct inhibitory consequences on nociceptive synaptic tr<strong>an</strong>smission, but also T-type<br />
(Cav3.2) Ca 2+ ch<strong>an</strong>nels that are reported to play a key role in neuronal excitability in pain states.<br />
Disclosures: V. Comun<strong>an</strong>za, None; D. Ursu, None; R. Zwart, None; E. Carbone, None; E.<br />
Sher, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.8/C62<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: CIHR MOP86473<br />
Title: Synaptic activation of T-type ch<strong>an</strong>nels via metabotropic glutamate receptors contributes to<br />
spike output of mitral cells<br />
Authors: *J. JOHNSTON, K. R. DELANEY;<br />
Univ. Victoria, Victoria, BC, C<strong>an</strong>ada<br />
Abstract: The 1st level of odor processing occurs in <strong>the</strong> olfactory bulb with mitral cells (MC) as<br />
<strong>the</strong> output to higher brain areas. The MC membr<strong>an</strong>e potential oscillates with <strong>the</strong> respiratory<br />
cycle; in response to overt odor <strong>the</strong> amplitude of <strong>the</strong>se oscillations increases resulting in MC<br />
firing. Imaging Ca 2+ in MCs in vivo in rat (Charpak et al., 2001) showed signific<strong>an</strong>t [Ca 2+ ]<br />
ch<strong>an</strong>ges in MC dendrites correlated with subthreshold voltage shifts in response to odors <strong>an</strong>d<br />
direct current injection. To determine <strong>the</strong> source of <strong>the</strong>se subthreshold Ca 2+ ch<strong>an</strong>ges <strong>an</strong>d study<br />
<strong>the</strong>ir implications <strong>for</strong> signaling we have used mouse olfactory bulb slices <strong>an</strong>d identified a low<br />
voltage activated (LVA) T-type current in MC dendrites. We show that subthreshold Ca 2+<br />
increases c<strong>an</strong> be generated in <strong>the</strong> absence of synaptic activity <strong>an</strong>d that <strong>the</strong>y are blocked by<br />
500µM Ni 2+ . In voltage-clamp we show that a LVA Ca 2+ current is active over <strong>the</strong> resting<br />
membr<strong>an</strong>e potential that is insensitive to <strong>the</strong> L-ch<strong>an</strong>nel blocker isradipine <strong>an</strong>d 50µM Ni 2+ but<br />
blocked by 500µM Ni 2+ <strong>an</strong>d NNC 55-0396. Low sensitivity to Ni 2+ is consistent with ei<strong>the</strong>r<br />
Cav3.1 or Cav3.3 T-type ch<strong>an</strong>nels. Anti Cav3.3 staining reveals <strong>the</strong>se ch<strong>an</strong>nels are concentrated<br />
in <strong>the</strong> apical <strong>an</strong>d distal apical tuft dendrite, absent <strong>from</strong> somata <strong>an</strong>d weakly present in <strong>the</strong>
secondary dendrites. Ratiometric imaging combined with voltage clamp confirmed T-ch<strong>an</strong>nels<br />
are located in <strong>the</strong> apical dendrite <strong>an</strong>d tuft <strong>an</strong>d are robustly up-regulated by a group1 mGluR<br />
agonist; 50µM DHPG which caused a 55 ±11% (P
Calgary, Calgary, AB, C<strong>an</strong>ada; 4 Dept. of Psychology, Wilfrid Laurier Univ., Waterloo, ON,<br />
C<strong>an</strong>ada<br />
Abstract: Cerebellar Purkinje cells (PCs) receive excitatory synaptic input <strong>from</strong> a large number<br />
of parallel fibers (PF). Underst<strong>an</strong>ding <strong>the</strong> factors that regulate summation of <strong>the</strong>se inputs is<br />
import<strong>an</strong>t <strong>for</strong> determining <strong>the</strong> control of PC spike output. Here we show that Ca2+ influx<br />
through T-type Ca2+ ch<strong>an</strong>nels during <strong>the</strong> PF EPSP activate IK K+ ch<strong>an</strong>nels that modulate <strong>the</strong><br />
frequency at which PF inputs begin to summate.<br />
Cerebellar slices were prepared <strong>from</strong> P18-30 rats <strong>an</strong>d patch recordings obtained <strong>from</strong> PC somata<br />
at 32-35°C. PFs were directly activated using a monopolar stimulating electrode in <strong>the</strong> molecular<br />
layer. Alternatively, <strong>the</strong> role of postsynaptic PC ion ch<strong>an</strong>nels were selectively tested by injecting<br />
simulated EPSCs to evoke PF sEPSPs (1 - 10 mV) at <strong>the</strong> soma.<br />
Both synaptically evoked <strong>an</strong>d simulated PF EPSPs below threshold <strong>for</strong> spike discharge were<br />
followed by <strong>an</strong> AHP of up to 2.5 mV <strong>an</strong>d 250 ms. The EPSP rate of decay increased linearly<br />
with <strong>the</strong> size of <strong>the</strong> depolarization while <strong>the</strong> time to peak of <strong>the</strong> AHP remained const<strong>an</strong>t. The<br />
EPSP rate of decay increased as <strong>the</strong> holding potential became more hyperpolarized, indicating a<br />
role <strong>for</strong> a low voltage-activated current. Activation of sEPSPs revealed that application of<br />
blockers against high voltage activated Ca2+ ch<strong>an</strong>nels (30 µM Cd2+, 200 nM Agatoxin IVA),<br />
SK ch<strong>an</strong>nels (100 nM apamin), or BK ch<strong>an</strong>nels (0.5 mM TEA, 200 nM iberiotoxin, 100 nM<br />
paxilline) did not signific<strong>an</strong>tly affect <strong>the</strong> rate of EPSP decay or <strong>the</strong> time to peak AHP. However,<br />
T-type Ca2+ ch<strong>an</strong>nel blockers (100 µM Ni2+, 1 µM Mibefradil) caused a ~35% decrease in <strong>the</strong><br />
rate of EPSP decay <strong>an</strong>d ~70% increase in <strong>the</strong> time to peak AHP. Moreover, <strong>the</strong>se effects were<br />
reproduced by application of <strong>an</strong> IK K+ ch<strong>an</strong>nel blocker (100 nM TRAM-34) but not by 5 mM<br />
TEA, a result also consistent with TEA-insensitive IK ch<strong>an</strong>nels. With direct activation of PF<br />
inputs, application of 100 µM Ni2+ or 100 nM TRAM-34 blocked <strong>an</strong> outward current that<br />
followed <strong>the</strong> EPSC. The outward current was fur<strong>the</strong>r blocked by BAPTA (10 mM) but not<br />
EGTA (10 mM) in <strong>the</strong> internal patch solution, indicating that <strong>the</strong> Ca2+-IK ch<strong>an</strong>nel interaction<br />
resides within a n<strong>an</strong>odomain. To examine <strong>the</strong> effect of this interaction on temporal summation,<br />
PFs were stimulated at varying frequencies. For frequencies up to 25 Hz (inclusive), no temporal<br />
summation was observed in control conditions, regardless of <strong>the</strong> amplitude of <strong>the</strong> EPSP.<br />
However, blocking ei<strong>the</strong>r <strong>the</strong> T-type Ca2+ current or IK current caused signific<strong>an</strong>t summation<br />
<strong>for</strong> 25 Hz stimulations. These results reveal a functional coupling between T-type Ca2+ ch<strong>an</strong>nels<br />
<strong>an</strong>d IK K+ ch<strong>an</strong>nels that controls <strong>the</strong> frequency at which PF summation c<strong>an</strong> occur in PCs.<br />
Disclosures: J.D. Engbers, None; D.M. Anderson, None; R. Rehak, None; W.H. Mehaffey,<br />
None; B.E. McKay, None; G.W. Zamponi, None; R.W. Turner, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 518.10/C64<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: CIHR, AHFMR, CRC Program<br />
Title: CCR2 receptor lig<strong>an</strong>ds inhibit Cav3.2 T-type calcium ch<strong>an</strong>nels<br />
Authors: H. YOU, C. ALTIER, *G. ZAMPONI;<br />
Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: Monocyte chemotactic protein-1 (MCP-1) is a cytochine that is involved in <strong>the</strong><br />
recruitment of monocytes to sites of injury. MCP-1 activates <strong>the</strong> CCR2 chemokine receptor, a 7<br />
tr<strong>an</strong>smembr<strong>an</strong>e helix G protein coupled receptor that has been implicated in inflammatory pain<br />
responses. Here we show that MCP-1 mediated activation of <strong>the</strong> CCR2 receptor inhibits N-type<br />
calcium ch<strong>an</strong>nels expressed in tsA-201 cells. Moreover, MCP-1 inhibits Cav3.2 calcium<br />
ch<strong>an</strong>nels, but not o<strong>the</strong>r members of <strong>the</strong> Cav3 calcium ch<strong>an</strong>nel family, directly, ra<strong>the</strong>r th<strong>an</strong> via a<br />
receptor <strong>an</strong>d G protein-independent pathway. Whole cell T-type calcium currents in acutely<br />
dissociated DRG neurons are effectively inhibited by MCP-1. CCR2 receptor <strong>an</strong>tagonists also<br />
regulate Cav3.2 ch<strong>an</strong>nel activity, suggesting that <strong>the</strong> Cav3.2 ch<strong>an</strong>nel shares structural feature<br />
with <strong>the</strong> CCR2 receptor lig<strong>an</strong>d binding pocket. Our findings may provide novel avenues <strong>for</strong> <strong>the</strong><br />
design of small org<strong>an</strong>ic inhibitors of T-type calcium ch<strong>an</strong>nels <strong>for</strong> <strong>the</strong> treatment of pain <strong>an</strong>d o<strong>the</strong>r<br />
T-type ch<strong>an</strong>nel linked disorders.<br />
Disclosures: H. You, None; C. Altier, None; G. Zamponi, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.11/C65<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: T32HL007381 (PB)<br />
RO1HL065680 (DH)<br />
Title: Phenylalkylamine block of T-type calcium ch<strong>an</strong>nels
Authors: *P. BERGSON, G. LIPKIND, D. A. HANCK;<br />
Univ. of Chicago, Chicago, IL<br />
Abstract: Verapamil, <strong>the</strong> prototypical phenylalkylamine, was <strong>the</strong> first calcium ch<strong>an</strong>nel blocker<br />
to be used clinically. Although <strong>for</strong> <strong>the</strong> most part its use has been eclipsed by longer lasting, more<br />
specific dihydropyridines, it remains useful in situations where a fast acting drug is preferred.<br />
Phenylalkylamines block both high voltage activated <strong>an</strong>d low voltage activated calcium<br />
ch<strong>an</strong>nels. In high voltage activated L-type ch<strong>an</strong>nels <strong>the</strong>y tonically block in <strong>the</strong> inner pore with<br />
micromolar affinity, <strong>an</strong>d display prominent state dependent block, with <strong>an</strong> affinity of tens of<br />
n<strong>an</strong>omolar <strong>for</strong> <strong>the</strong> inactivated state. We examined block in low voltage activated T-type calcium<br />
ch<strong>an</strong>nels to identify similarities <strong>an</strong>d differences in phenylalkylamines’ action compared to <strong>the</strong>ir<br />
action in L-type ch<strong>an</strong>nels. Three phenylalkylamines, Verapamil, D600, <strong>an</strong>d D888, demonstrate<br />
tonic block of T-type ch<strong>an</strong>nels similar to that in L-type ch<strong>an</strong>nels. They display only a modest<br />
degree of state dependent block, in contrast to <strong>the</strong> signific<strong>an</strong>t increase in affinity seen in L-type<br />
ch<strong>an</strong>nels. Several residues lining <strong>the</strong> inner pore of L-type ch<strong>an</strong>nels have been implicated in<br />
binding of D888. Although phenylalkylamines appear to bind in <strong>the</strong> pore of T-type ch<strong>an</strong>nels,<br />
residues implicated in drug binding in L-type ch<strong>an</strong>nels do not affect block of T-type ch<strong>an</strong>nels.<br />
We were surprised to find that intracellular phenylalkylamines blocked T-type ch<strong>an</strong>nels with<br />
much lower affinity th<strong>an</strong> expected based on diffusional limitations. Also surprisingly,<br />
perm<strong>an</strong>ently charged <strong>for</strong>ms of phenylalkylamines, which should not be able to cross <strong>the</strong> plasma<br />
membr<strong>an</strong>e, blocked <strong>the</strong> ch<strong>an</strong>nel <strong>from</strong> both <strong>the</strong> inside <strong>an</strong>d <strong>the</strong> outside. Thus drugs are able to<br />
access <strong>the</strong> binding site both <strong>from</strong> <strong>the</strong> intracellular <strong>an</strong>d <strong>the</strong> extracellular space. A structural model<br />
of <strong>the</strong> T-type ch<strong>an</strong>nel pore suggests that a space, large enough to accommodate <strong>the</strong>se bulky<br />
drugs, exists behind <strong>the</strong> selectivity filter of <strong>the</strong> ch<strong>an</strong>nel. Drugs might move through <strong>the</strong> relatively<br />
narrow, disordered stretch of membr<strong>an</strong>e between this space <strong>an</strong>d <strong>the</strong> extracellular surface, in order<br />
to access <strong>the</strong> binding site without passing through <strong>the</strong> selectivity filter.<br />
Disclosures: P. Bergson, None; G. Lipkind, None; D.A. H<strong>an</strong>ck, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.12/C66<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIAAA AA014106<br />
AA017056
AA016852<br />
AA017048<br />
Title: Acute effects of eth<strong>an</strong>ol on CaV3.2 ch<strong>an</strong>nels are mediated through PKC<br />
Authors: *H. SHAN, J. D. GRAEF, D. W. GODWIN;<br />
Wake Forest Univ. Sch. Med., Winston-Salem, NC<br />
Abstract: T-type calcium ch<strong>an</strong>nels are widely distributed in <strong>the</strong> brain. There are three<br />
functionally distinctive T-type ch<strong>an</strong>nel iso<strong>for</strong>ms(CaV3.1, CaV3.2 <strong>an</strong>d CaV3.3), each of which<br />
has been cloned <strong>an</strong>d expressed in hum<strong>an</strong> embryonic kidney (HEK) 293 cell lines (Perez-Reyes<br />
1998). Our preliminary results show that only one iso<strong>for</strong>m of <strong>the</strong>se ch<strong>an</strong>nels, CaV3.2 is<br />
selectively sensitive to acute eth<strong>an</strong>ol <strong>an</strong>d constitutes a novel target of eth<strong>an</strong>ol effects. However,<br />
<strong>the</strong> mech<strong>an</strong>isms underlying <strong>the</strong>se effects are pooly understood. The objective of our study was to<br />
investigate <strong>the</strong> mech<strong>an</strong>isms of acute eth<strong>an</strong>ol effects on CaV3.2 ch<strong>an</strong>nels <strong>an</strong>d to test whe<strong>the</strong>r <strong>the</strong>y<br />
are mediated through second messengers, particularly Protein Kinase C (PKC). Whole cell<br />
per<strong>for</strong>ated patch clamp recordings were per<strong>for</strong>med with CaV3.2 ch<strong>an</strong>nels on HEK 293 cells after<br />
two days in culture. Myristoylated Protein Kinase C Peptide (MPKCP) inhibitor (Promega<br />
Corporation; 50 µM) was delivered through <strong>the</strong> internal solution. The cells were treated with<br />
100mM eth<strong>an</strong>ol. Eth<strong>an</strong>ol treatment resulted in a hyperpolarizing shift in voltage dependence of<br />
inactivation, measured by a shift in <strong>the</strong> V50 <strong>from</strong> -85.77+3.122 mV to -114.9+5.780 mV (n=9;<br />
p< 0.001). This acute effect of eth<strong>an</strong>ol was totally abolished by co-application of <strong>the</strong> MPKCP<br />
inhibitor (n=12; p>0.05). In order to fur<strong>the</strong>r characterize <strong>the</strong> role of PKC in <strong>the</strong> modulation of<br />
CaV3.2 ch<strong>an</strong>nels by acute eth<strong>an</strong>ol, <strong>the</strong> cells were treated in <strong>the</strong> bath with 100nM PMA (phorbol<br />
12 - myristate 13- acetate, P8139-1mg. Sigma), a PKC activator. PMA replicated <strong>the</strong><br />
hyperpolarizing shift we observed in inactivation kinetics, <strong>an</strong>d <strong>the</strong> V50 was shifted <strong>from</strong> -86.79+<br />
3.024 mV to -108.5+ 2.403 mV after exposure to 100nM PMA, <strong>an</strong> effect which partially<br />
recovered. This suggests that acute eth<strong>an</strong>ol effects c<strong>an</strong> be replicated by 100nM PMA. Taken<br />
toge<strong>the</strong>r, <strong>the</strong>se results suggest that <strong>the</strong> acute effects of eth<strong>an</strong>ol on CaV3.2 ch<strong>an</strong>nels occur through<br />
eth<strong>an</strong>ol activation of <strong>the</strong> PKC pathway, which in turn leads to altered inactivation.<br />
We th<strong>an</strong>k Edward Perez-Reyes(UVA) <strong>for</strong> T-type ch<strong>an</strong>nel cell lines.<br />
Disclosures: H. Sh<strong>an</strong>, None; J.D. Graef, None; D.W. Godwin, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.13/C67
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: Allegheny College Neuroscience Program<br />
Title: The effects of specific Conus snail venoms on <strong>the</strong> T-type calcium ch<strong>an</strong>nel Cav3.2<br />
Authors: A. M. WELKO 1 , *L. B. FRENCH 2 ;<br />
1 Neurosci., 2 Allegheny Col., Meadville, PA<br />
Abstract: T-type calcium ch<strong>an</strong>nels are low voltage-activated ch<strong>an</strong>nels that are vital to a number<br />
of physiological processes. Cav3.2 is a splice vari<strong>an</strong>t of T-type calcium ch<strong>an</strong>nels that is linked to<br />
<strong>the</strong> etiology of several diseases, including insulin-dependent diabetes mellitus <strong>an</strong>d certain types<br />
of epilepsy. In this study, <strong>the</strong> venoms of predatory marine snails were tested on Cav3.2 ch<strong>an</strong>nels<br />
expressed in Xenopus laevis oocytes. Oocytes were injected with Cav3.2 cRNA, incubated, <strong>an</strong>d<br />
run through a series of depolarizing steps in a two-electrode voltage clamp setup. It was found<br />
that <strong>the</strong> crude venom of Conus striatus signific<strong>an</strong>tly decreased <strong>the</strong> current expressed by <strong>the</strong><br />
Cav3.2 ch<strong>an</strong>nels, <strong>an</strong>d through fractionation of <strong>the</strong> venom it may be possible to isolate <strong>the</strong> specific<br />
peptide that targets <strong>the</strong> ch<strong>an</strong>nel.<br />
Disclosures: A.M. Welko, None; L.B. French, None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.14/C68<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH gr<strong>an</strong>t NS061535-01<br />
Title: Iso<strong>for</strong>m-dependent modification of T-type calcium ch<strong>an</strong>nels by ProTxII<br />
Authors: *G. B. EDGERTON 1 , E. O. HALL 1 , K. M. BLUMENTHAL 2 , D. A. HANCK 1 ;<br />
1 Univ. Chicago, Chicago, IL; 2 SUNY Buffalo, Buffalo, NY<br />
Abstract: The three iso<strong>for</strong>ms of <strong>the</strong> T-type calcium (Ca) ch<strong>an</strong>nel display distinct distribution<br />
patterns both throughout <strong>the</strong> nervous system <strong>an</strong>d within individual neurons (Perez-Reyes 2003;<br />
McKay et al. 2006). Iso<strong>for</strong>m differences in gating properties have also been noted <strong>an</strong>d likely<br />
underlie unique physiological roles. Despite <strong>the</strong>se differences <strong>the</strong>re exist very few experimental
tools capable of distinguishing among <strong>the</strong> T-ch<strong>an</strong>nel iso<strong>for</strong>ms. Here, we show that ProTxII, a<br />
peptide toxin recently isolated <strong>from</strong> <strong>the</strong> venom of <strong>the</strong> tar<strong>an</strong>tula spider Thrixopelma pruriens,<br />
differentially modified <strong>the</strong> T-ch<strong>an</strong>nel iso<strong>for</strong>ms. Both CaV3.1 <strong>an</strong>d CaV3.3 ch<strong>an</strong>nels were<br />
sensitive to <strong>the</strong> toxin when expressed heterologously in HEK cells. For CaV3.1 we observed a<br />
dose-dependent positive shift in <strong>the</strong> voltage r<strong>an</strong>ge of activation <strong>an</strong>d decrease in macroscopic<br />
conduct<strong>an</strong>ce with a maximum of ~20mV shift <strong>an</strong>d ~50% decrease with 5µM toxin. Interestingly,<br />
<strong>for</strong> CaV3.3 ch<strong>an</strong>nels toxin decreased current amplitude (~60%), but minimally shifted activation<br />
(~6mV) at this concentration. CaV3.2 ch<strong>an</strong>nels were insensitive to ProTxII at <strong>the</strong>se<br />
concentrations. Consistent with charge on ProTxII electrostatically interacting with CaV3.1 to<br />
induce <strong>the</strong> shift in activation, extracellular barium (Ba) at 20mM <strong>an</strong>d 60mM concentrations dosedependently<br />
precluded <strong>the</strong> ability of ProTxII to shift activation gating in this ch<strong>an</strong>nel. These<br />
results highlight <strong>the</strong> potential of ProTxII as a tool <strong>for</strong> studying individual T-ch<strong>an</strong>nel iso<strong>for</strong>ms in<br />
isolation <strong>an</strong>d suggest a possible mech<strong>an</strong>ism (i.e. differences in extracellular electrostatic<br />
interaction patterns) by which unique toxin-ch<strong>an</strong>nel interactions are determined in this case.<br />
Disclosures: G.B. Edgerton, None; E.O. Hall, None; K.M. Blumenthal, None; D.A. H<strong>an</strong>ck,<br />
None.<br />
Poster<br />
518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 518.15/C69<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: Korea Science <strong>an</strong>d Engineering Foundation (KOSEF) gr<strong>an</strong>t (<strong>2009</strong>0063276)<br />
Title: Oxidation effects on T-type Ca 2+ ch<strong>an</strong>nels expressed in Xenopus oocytes<br />
Authors: *S.-S. LEE, H.-W. KANG, Y.-M. PARK, J.-H. LEE;<br />
Life Sci., Sogsng Univ., Seoul, Republic of Korea<br />
Abstract: Voltage-gated Ca 2+ ch<strong>an</strong>nels are found in excitable cells <strong>an</strong>d <strong>the</strong>y contribute to<br />
import<strong>an</strong>t physiological functions including muscular contraction, gene expression, neuronal<br />
excitation, or neurotr<strong>an</strong>smitter release. They c<strong>an</strong> be potently regulated by various signaling<br />
molecules including G-proteins, protein kinases, lipid derivatives, <strong>an</strong>d calcium binding proteins.<br />
Although redox (oxidation <strong>an</strong>d reduction) levels of numerous ion ch<strong>an</strong>nels were reported to be<br />
critical to determine <strong>the</strong>ir activity, gating, <strong>an</strong>d kinetics, it has not been closely examined how<br />
redox modulates <strong>the</strong> properties of T-type Ca 2+ ch<strong>an</strong>nels. In present study, <strong>the</strong>re<strong>for</strong>e, we examined
<strong>the</strong> redox modulation effects on Cav3.1 <strong>an</strong>d Cav3.2 ch<strong>an</strong>nels expressed in Xenopus oocytes by<br />
comparing <strong>the</strong>ir biophysical properties in <strong>the</strong> absence or <strong>the</strong> presence of <strong>an</strong> oxidizing agent<br />
(DTNB) or a reducing agent (DTT). Application of 0.1 mM DTT did not show <strong>an</strong>y signific<strong>an</strong>t<br />
alternation in current amplitude <strong>an</strong>d voltage-dependent biophysical properties of <strong>the</strong> two T-type<br />
ch<strong>an</strong>nel iso<strong>for</strong>ms. In contrast, application of 1 mM DTNB similarly reduced me<strong>an</strong> current<br />
amplitude of both ch<strong>an</strong>nels by ~50 % <strong>an</strong>d slowed down <strong>the</strong>ir activation <strong>an</strong>d inactivation kinetics.<br />
It is of interest that <strong>the</strong> oxidizing agent positively shifted both activation <strong>an</strong>d steady-state<br />
inactivation curves of Cav3.1, while it did not signific<strong>an</strong>tly shift those of Cav3.2. To localize<br />
structural region(s) responsible <strong>for</strong> <strong>the</strong> different shifts between Cav3.1 <strong>an</strong>d Cav3.2 ch<strong>an</strong>nels, we<br />
constructed chimeras (GGHH <strong>an</strong>d HHGG) by swapping two domains of <strong>the</strong> two ch<strong>an</strong>nels. The<br />
activation <strong>an</strong>d inactivation curves of HHGG ch<strong>an</strong>nel were positively shifted by DTNB, whereas<br />
those of GGHH were not shifted. These findings suggest that <strong>the</strong> domains III <strong>an</strong>d IV of Cav3.1<br />
contain structural elements responsible <strong>for</strong> <strong>the</strong> positive shifts caused by DTNB. To fur<strong>the</strong>r<br />
localize structural region(s) contributing to <strong>the</strong> positive shift effects, consequently, we<br />
constructed HHGH <strong>an</strong>d HHHG chimeras, of which electrophysiological properties remained to<br />
be characterized in <strong>the</strong> absence <strong>an</strong>d presence of DTNB.<br />
Disclosures: S. Lee, None; H. K<strong>an</strong>g, None; Y. Park, None; J. Lee, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.1/C70<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH gr<strong>an</strong>t #1R15NS048900-01A1<br />
Title: Signal tr<strong>an</strong>sduction mech<strong>an</strong>ism of L-type calcium current facilitation by GABAB receptor<br />
activation in <strong>the</strong> neonatal rat hippocampus<br />
Authors: *J. G. BRAY, M. MYNLIEFF;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: Previous studies in <strong>the</strong> laboratory have shown modulation of N-type <strong>an</strong>d L-type<br />
calcium current by baclofen, a GABAB agonist. In hippocampal cultures isolated <strong>from</strong> 5-7 day<br />
old rats, N-type current decreases <strong>an</strong>d L-type current increases in response to activation of<br />
GABAB receptors. Neuronal calcium ch<strong>an</strong>nels c<strong>an</strong> be directly regulated by both Gα <strong>an</strong>d Gβγ<br />
subunits or c<strong>an</strong> be indirectly regulated by G-proteins through second messenger systems. One of
<strong>the</strong> most common mech<strong>an</strong>isms of ch<strong>an</strong>nel modulation by GABAB receptor activation occurs<br />
through direct modulation by a Gi/o protein. To determine if Gi/o is involved in calcium ch<strong>an</strong>nel<br />
modulation by GABAB receptor activation, hippocampal cultures obtained <strong>from</strong> 7 day old rat<br />
pups were treated overnight with pertussis toxin (PTX, 200 ng/ml) <strong>an</strong>d <strong>the</strong> effects of 10 µM<br />
baclofen application on total calcium currents were determined using whole cell voltage clamp<br />
recording. The distribution of <strong>the</strong> responses differed in cultures treated with PTX in comparison<br />
to non-treated cultures (P=0.037). In cultures that were not treated with PTX, 25% of cells (8/32)<br />
demonstrated a decrease, 44% (14/32) of cells demonstrated no ch<strong>an</strong>ge, <strong>an</strong>d 31% (10/32) of cells<br />
demonstrated <strong>an</strong> increase in total calcium current in response to 10 µM baclofen. In cultures that<br />
were treated with PTX, no cells (0/20) demonstrated a decrease, 70% (14/20) of cells<br />
demonstrated no ch<strong>an</strong>ge, <strong>an</strong>d 30% (6/20) of cells demonstrated <strong>an</strong> increase in total calcium<br />
current in response to 10 µM baclofen. Facilitation of calcium current in response to baclofen<br />
was still seen in cells treated with PTX, which suggests that facilitation of L-type current is not<br />
mediated by a PTX sensitive G-protein. However, <strong>the</strong> data indicate that <strong>the</strong> inhibition of N-type<br />
current is mediated through a Gi/o protein, because following PTX treatment no cells<br />
demonstrated inhibition of <strong>the</strong>ir current. GABAB receptor activation of PKC has been shown to<br />
facilitate L-type current in salam<strong>an</strong>der retinal neurons (Shen <strong>an</strong>d Slaughter, 1999) <strong>an</strong>d activation<br />
of PKC within <strong>the</strong> hippocampus has been shown to facilitate L-type current (Doerner <strong>an</strong>d Alger,<br />
1992). There<strong>for</strong>e, <strong>the</strong> role of PKC in <strong>the</strong> mech<strong>an</strong>ism of facilitation of L-type calcium current by<br />
GABAB receptors was investigated. Preliminary electrophysiological data using <strong>the</strong> PKC<br />
inhibitor GF 109203X (500nM) suggest that PKC is involved in <strong>the</strong> mech<strong>an</strong>ism of L-type<br />
calcium current facilitation by GABAB receptor activation. Experiments using GF 109203X,<br />
o<strong>the</strong>r PKC inhibitors <strong>an</strong>d PKC activators will be used to confirm that PKC is involved in <strong>the</strong><br />
signal tr<strong>an</strong>sduction mech<strong>an</strong>ism of L-type current facilitation by GABAB receptor activation.<br />
Disclosures: J.G. Bray, None; M. Mynlieff, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.2/C71<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t1R15NS048900-01A1<br />
Title: Modulation of intracellular calcium in neonatal hippocampus by GABAB receptors
Authors: A. S. KARLS, E. R. WEH, *M. MYNLIEFF;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: Previous electrophysiological studies demonstrated that baclofen, a GABAB agonist,<br />
enh<strong>an</strong>ces L-type calcium current <strong>an</strong>d decreases N-type calcium current in rat hippocampal<br />
neurons. The facilitation of L-type current appears to be a developmental phenomenon as <strong>the</strong><br />
response peaks in <strong>the</strong> first postnatal week. While electrophysiological studies provide <strong>an</strong><br />
accurate way to rapidly measure currents, it is limited because data collection is relatively slow<br />
<strong>an</strong>d one is not measuring <strong>the</strong> amount of intracellular calcium. Ratiometric calcium imaging with<br />
FURA allows one to measure <strong>the</strong> intracellular calcium concentration in m<strong>an</strong>y cells<br />
simult<strong>an</strong>eously. In <strong>the</strong> present study, hippocampal cultures were prepared <strong>from</strong> <strong>the</strong> superior<br />
region of 7 day old rat pups <strong>an</strong>d kept in culture <strong>for</strong> 24 hours. Calcium influx through voltage<br />
dependent ch<strong>an</strong>nels was stimulated with 30mM KCl in <strong>the</strong> presence <strong>an</strong>d absence of 10µM<br />
baclofen. The increase in intracellular calcium in response to KCl alone was compared to <strong>the</strong><br />
increase in calcium in response to KCl in <strong>the</strong> presence of baclofen. The increase in intracellular<br />
calcium was higher in <strong>the</strong> presence of baclofen in 23.4% of <strong>the</strong> cells with <strong>an</strong> average facilitation<br />
of 43.8% ± 4.4% (N=304). The increase in intracellular calcium was lower in <strong>the</strong> presence of<br />
baclofen in 38.5% of <strong>the</strong> cells with <strong>an</strong> average attenuation of 31.7% ± 2.1%. These data correlate<br />
well with <strong>the</strong> results obtained with electrophysiology. While electrophysiological recording<br />
measures <strong>the</strong> current across <strong>the</strong> surface of <strong>the</strong> cell, calcium imaging results include ch<strong>an</strong>ges in<br />
intracellular calcium <strong>from</strong> all sources. In 70.4% of <strong>the</strong> cells <strong>the</strong> L-type ch<strong>an</strong>nel <strong>an</strong>tagonist<br />
nimodipine (5µM) completely blocked all KCl stimulated influx with more th<strong>an</strong> 50% blocked in<br />
<strong>the</strong> remaining 29.6% of <strong>the</strong> cells suggesting that a large component of <strong>the</strong> increase in<br />
intracellular calcium is due to influx through L-type ch<strong>an</strong>nels. Electrophysiological data suggest<br />
that GABAB receptors activate protein kinase C (PKC), leading to phosphorylation of L-type<br />
ch<strong>an</strong>nels, <strong>the</strong>reby increasing L-type current. Calcium may also be released <strong>from</strong> intracellular<br />
stores via <strong>the</strong> IP3 receptor, which would contribute to response seen with calcium imaging.<br />
Imaging experiments will be per<strong>for</strong>med in <strong>the</strong> presence of cadmium to block all of <strong>the</strong> high<br />
voltage activated calcium ch<strong>an</strong>nels to determine <strong>the</strong> contribution of calcium <strong>from</strong> intracellular<br />
stores. Additionally, depletion of intracellular stores with thapsigargin blocks <strong>an</strong>y signal coming<br />
<strong>from</strong> <strong>the</strong>se stores but may also interfere with <strong>the</strong> activation of PKC due to its calcium<br />
dependence. PKC could be activated directly with phorbol esters to bypass <strong>the</strong> GABAB receptors<br />
in <strong>the</strong>se experiments.<br />
Disclosures: A.S. Karls, None; E.R. Weh, None; M. Mynlieff, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 519.3/C72<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Title: Regulation of N-type voltage-gated calcium ch<strong>an</strong>nels (Cav2.2) <strong>an</strong>d tr<strong>an</strong>smitter release by<br />
collapsin response mediator protein-2 (CRMP-2) in sensory neurons<br />
Authors: *R. KHANNA, X. X. CHI, B. S. SCHMUTZLER, Y. WANG, C. M. HINGTGEN, G.<br />
D. NICOL;<br />
Indi<strong>an</strong>a Univ., Indi<strong>an</strong>apolis, IN<br />
Abstract: Collapsin response mediator proteins (CRMPs) mediate signal tr<strong>an</strong>sduction of neurite<br />
outgrowth <strong>an</strong>d axonal guid<strong>an</strong>ce during neuronal development. Voltage-gated Ca 2+ ch<strong>an</strong>nels <strong>an</strong>d<br />
interacting proteins are essential in neuronal signaling <strong>an</strong>d synaptic tr<strong>an</strong>smission during this<br />
period. We recently identified <strong>the</strong> presynaptic N-type voltage-gated Ca 2+ ch<strong>an</strong>nels (Cav2.2) as a<br />
CRMP-2-interacting partner. Here, we investigated <strong>the</strong> affects of a functional association of<br />
CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature<br />
synapses/growth cones, in mature synapses <strong>an</strong>d in cell bodies of dorsal root g<strong>an</strong>glion (DRG)<br />
neurons. Co-immunoprecipitation experiments showed CRMP-2 associates with Cav2.2 <strong>from</strong><br />
DRG lysates. Overexpression of CRMP-2 fused to enh<strong>an</strong>ced green fluorescent protein (EGFP) in<br />
DRG neurons, via nucleofection, resulted in a signific<strong>an</strong>t increase in Cav2.2 current density<br />
compared to EGFP expressing cells. Since CRMP-2 is localized to synaptophysin-positive<br />
puncta in dense DRG cultures, we tested if this CRMP-2-mediated alteration of Ca 2+ currents<br />
culminated in ch<strong>an</strong>ges in synaptic tr<strong>an</strong>smission. Following a brief high K + -induced stimulation,<br />
<strong>the</strong>se puncta loaded with FM4-64 dye. In EGFP <strong>an</strong>d CRMP-2-EGFP expressing neurons, similar<br />
densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG<br />
increased release of <strong>the</strong> neurotr<strong>an</strong>smitter, calcitonin gene-related peptide (iCGRP) by ~70%,<br />
whereas CRMP-2 siRNA signific<strong>an</strong>tly reduced release of iCGRP by ~54% compared to control<br />
cultures. Thus, <strong>the</strong>se findings support a novel role <strong>for</strong> CRMP-2 in regulation of N-type Ca 2+<br />
ch<strong>an</strong>nels <strong>an</strong>d in tr<strong>an</strong>smitter release.<br />
Disclosures: R. Kh<strong>an</strong>na, None; X.X. Chi, None; B.S. Schmutzler, None; Y. W<strong>an</strong>g,<br />
None; C.M. Hingtgen, None; G.D. Nicol, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.4/C73
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Title: An atypical role <strong>for</strong> CRMP-2 in neurotr<strong>an</strong>smitter release via interaction with presynaptic<br />
Ca2+ ch<strong>an</strong>nels<br />
Authors: *J. M. BRITTAIN, A. D. PIEKARZ, Y. WANG, A. S. GARCIA, T. R. CUMMINS,<br />
R. KHANNA;<br />
Indi<strong>an</strong>a Univ., Indi<strong>an</strong>apolis, IN<br />
Abstract: Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate <strong>an</strong>d axonal<br />
growth of neurons through protein-protein interactions. Their functions in presynaptic biology<br />
remain unknown. Here, we identify <strong>the</strong> presynaptic N-type Ca2+ ch<strong>an</strong>nel (CaV2.2) as a CRMP-<br />
2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions; <strong>the</strong> ch<strong>an</strong>nel domain I-II<br />
intracellular loop <strong>an</strong>d <strong>the</strong> distal C-terminus, but not to <strong>an</strong>y o<strong>the</strong>r regions. Both proteins colocalize<br />
within presynaptic sites in hippocampal neurons. Overexpression in hippocampal<br />
neurons of a CRMP-2 protein fused to EGFP caused a signific<strong>an</strong>t increase in Ca2+ ch<strong>an</strong>nel<br />
current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect.<br />
Interestingly, <strong>the</strong> increase in Ca2+ current density was not due to a ch<strong>an</strong>ge in ch<strong>an</strong>nel gating.<br />
Ra<strong>the</strong>r, cell surface biotinylation studies showed <strong>an</strong> increased number of CaV2.2 at <strong>the</strong> cell<br />
surface in CRMP-2-overexpressing neurons. These neurons also exhibited a signific<strong>an</strong>t increase<br />
in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-EGFP<br />
overexpressing neurons elicited a signific<strong>an</strong>t increase in release of glutamate compared to control<br />
neurons. Toxin block of Ca2+ entry via CaV2.2 abolished this stimulated release. Thus, <strong>the</strong><br />
CRMP-2-Ca2+ ch<strong>an</strong>nel interaction represents a novel mech<strong>an</strong>ism <strong>for</strong> modulation of Ca2+ influx<br />
into nerve terminals <strong>an</strong>d, hence, of synaptic strength.<br />
Disclosures: J.M. Brittain, None; A.D. Piekarz, None; Y. W<strong>an</strong>g, None; A.S. Garcia,<br />
None; T.R. Cummins, None; R. Kh<strong>an</strong>na, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.5/C74<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t NS051668
Title: Neurofibromin, CRMP-2 <strong>an</strong>d presynaptic calcium ch<strong>an</strong>nel proteins control synaptic<br />
tr<strong>an</strong>smission in a mouse model of neurofibromatosis type 1<br />
Authors: *C. M. HINGTGEN 1 , J. M. BRITTAIN 2 , B. S. SCHMUTZLER 3 , R. KHANNA 3 ;<br />
1 2 3<br />
Dept Neurol, Med. Neurosciences Program, Pharmacol. <strong>an</strong>d Toxicology, Indi<strong>an</strong>a Univ. Sch.<br />
Med., Indi<strong>an</strong>apolis, IN<br />
Abstract: Neurofibromatosis type I (NF1) is a common autosomal domin<strong>an</strong>t disease<br />
characterized by <strong>the</strong> <strong>for</strong>mation of multiple benign <strong>an</strong>d malign<strong>an</strong>t tumors. People with NF1 often<br />
have learning disabilities <strong>an</strong>d o<strong>the</strong>r cognitive symptoms. The mech<strong>an</strong>isms by which mutations of<br />
<strong>the</strong> neurofibromin gene (NF1) cause <strong>the</strong>se deficits are not known. Learning <strong>an</strong>d memory deficits<br />
also are observed in mice with a heterozygous mutation of <strong>the</strong> Nf1 gene (Nf1 +/- ) <strong>an</strong>d <strong>the</strong>se<br />
deficits c<strong>an</strong> be rescued by genetic <strong>an</strong>d pharmacological m<strong>an</strong>ipulations that decrease Ras<br />
function. Recently, direct interactions of collapsin response mediator protein 2 (CRMP-2) - a<br />
protein involved in neurite outgrowth, guid<strong>an</strong>ce <strong>an</strong>d axonogenesis - <strong>an</strong>d neurofibromin have<br />
been demonstrated. In addition, we have demonstrated that CRMP-2 associates with <strong>an</strong>d<br />
influences <strong>the</strong> function of presynaptic calcium ch<strong>an</strong>nels involved in tr<strong>an</strong>smitter release. We also<br />
have demonstrated that CRMP-2 influences axonal outgrowth <strong>an</strong>d synaptic connectivity of<br />
neurons in <strong>the</strong> brain. Based on <strong>the</strong>se results, we hypo<strong>the</strong>size that a lack of neurofibromin in NF1<br />
alters CRMP-2 function leading to altered axonal connections <strong>an</strong>d tr<strong>an</strong>smitter release that results<br />
in altered learning <strong>an</strong>d cognitive function. Immunoprecipitation studies <strong>from</strong> brains of wildtype<br />
<strong>an</strong>d Nf1 +/- mice reveal a direct protein-protein interaction between neurofibromin <strong>an</strong>d CRMP-2.<br />
In addition, both proteins are enriched in cholesterol <strong>an</strong>d sphingolipid rich, “lipid raft-like”<br />
microdomains within <strong>the</strong> plasma membr<strong>an</strong>e import<strong>an</strong>t <strong>for</strong> org<strong>an</strong>izing specialized signaling<br />
tr<strong>an</strong>sduction plat<strong>for</strong>ms within membr<strong>an</strong>es. A signific<strong>an</strong>tly lower percentage of total<br />
neurofibromin is raft-associated in brain tissue <strong>from</strong> Nf1 +/- mice compared to wildtype mice (31.4<br />
± 0.9% vs. 44 ± 1.1%). Treatment of wildtype sensory neurons with short interfering RNA<br />
(siRNA) to CRMP-2 causes <strong>an</strong> 80 ± 5% knockdown of protein expression <strong>an</strong>d a subsequent<br />
decrease in release of <strong>the</strong> peptide tr<strong>an</strong>smitter, calcitonin gene-related peptide, of 52 ± 4%<br />
compared to neurons treated with scramble siRNA. CRMP-2 knockdown also causes a 80 ± 5%<br />
reduction in Ca 2+ currents in hippocampal neurons compared to those treated with scramble<br />
siRNA. These data demonstrate <strong>an</strong> altered interaction of CRMP-2 <strong>an</strong>d neurofibromin in Nf1 +/-<br />
mice. Reduced raft sequestration of neurofibromin may impair CRMP-2 <strong>from</strong> freely interacting<br />
with calcium ch<strong>an</strong>nels resulting in altered calcium ch<strong>an</strong>nel density <strong>an</strong>d tr<strong>an</strong>smitter release. By<br />
underst<strong>an</strong>ding <strong>the</strong> molecular mech<strong>an</strong>isms underlying <strong>the</strong> signaling involved in NF1-CRMP-2-<br />
Ca 2+ ch<strong>an</strong>nel interactions, we hope to unravel new targets <strong>for</strong> treatment of learning deficits in<br />
NF1.<br />
Disclosures: C.M. Hingtgen, None; J.M. Brittain, None; B.S. Schmutzler, None; R. Kh<strong>an</strong>na,<br />
None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.6/C75<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research<br />
Title: Functional impact of <strong>the</strong> GAERS Cav3.2 R1854P mutation on T-type currents in <strong>the</strong><br />
thalamocortical system<br />
Authors: *S. M. CAIN 1 , J. R. TYSON 1 , E. TRINGHAM 2 , K. L. POWELL 3 , T. J. O'BRIEN 3,4 ,<br />
T. P. SNUTCH 1 ;<br />
1 Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada; 2 Neuromed Pharmaceuticals, V<strong>an</strong>couver,<br />
BC, C<strong>an</strong>ada; 3 The Royal Melbourne Hospital, Univ. of Melbourne, Melbourne, Australia; 4 The<br />
Univ. of Tasm<strong>an</strong>ia, Tasm<strong>an</strong>ia, Australia<br />
Abstract: The Genetic Absence Epilepsy Rats <strong>from</strong> Strasbourg (GAERS) model displays a well<br />
characterized absence epilepsy phenotype. We have recently described <strong>an</strong> arginine to proline<br />
missense mutation at position 1584 (R1584P) in <strong>the</strong> GAERS Cacna1h gene encoding <strong>the</strong> Cav3.2<br />
low-voltage activated (T-type) calcium ch<strong>an</strong>nel. The R1584P point mutation segregates codomin<strong>an</strong>tly<br />
with both <strong>the</strong> number of seizures <strong>an</strong>d <strong>the</strong> time spent in seizure activity in <strong>the</strong> progeny<br />
of <strong>an</strong> F1 intercross. In Cav3.2 ch<strong>an</strong>nels exogenously expressed in HEK cells <strong>the</strong> mutation both<br />
increases <strong>the</strong> rate of recovery <strong>from</strong> inactivation <strong>an</strong>d increases charge tr<strong>an</strong>sference during high<br />
frequency bursts in a Cav3.2 splice vari<strong>an</strong>t-specific m<strong>an</strong>ner. The impact of <strong>the</strong> R1584P mutation<br />
on thalamocortical T-type calcium currents has not yet been reported.<br />
Acute brain slices <strong>from</strong> neonatal GAERS <strong>an</strong>d non-epileptic control (NEC) rats were used <strong>for</strong><br />
electrophysiological recordings. Neurons of <strong>the</strong> reticular thalamic nucleus (nRT), which express<br />
mixed Cav3.2 / Cav3.3 T-type calcium currents were recorded under whole-cell voltage- <strong>an</strong>d<br />
current-clamp conditions at 33-35oC. Low voltage activated T-type currents were isolated<br />
pharmacologically <strong>an</strong>d electrophysiologically <strong>an</strong>d compared between <strong>the</strong> two rat strains (GAERS<br />
vs NEC). St<strong>an</strong>dard biophysical properties (voltage-dependent activation <strong>an</strong>d inactivation,<br />
conduct<strong>an</strong>ce, kinetics <strong>an</strong>d current density) as well as properties highly relev<strong>an</strong>t to T-type<br />
ch<strong>an</strong>nel-mediated neuronal burst firing (recovery <strong>from</strong> inactivation <strong>an</strong>d response to high<br />
frequency burst firing) were examined in <strong>the</strong> slices. Pharmacologically, a novel T-type blocking<br />
small org<strong>an</strong>ic molecule, NP169944, with good efficacy towards attenuating absence seizure<br />
activity in GAERS <strong>an</strong>imals demonstrated high potency against <strong>the</strong> nRT neuronal T-type currents<br />
<strong>an</strong>d also reduced burst firing in <strong>the</strong>se neurons. Supported by <strong>the</strong> C<strong>an</strong>adi<strong>an</strong> Institutes of Health<br />
Research.<br />
Disclosures: S.M. Cain, None; J.R. Tyson, None; E. Tringham, None; K.L. Powell,<br />
None; T.J. O'Brien, None; T.P. Snutch, None.
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.7/C76<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research<br />
Title: Calcium-dependent modulation of P/Q-type calcium ch<strong>an</strong>nels is altered by Familial<br />
Hemiplegic Migraine Type-1 mutations <strong>an</strong>d in a Cav2.1 splice-dependent m<strong>an</strong>ner<br />
Authors: P. J. ADAMS, E. GARCIA, *T. P. SNUTCH;<br />
Michael Smith Lab, UBC, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: Neurotr<strong>an</strong>smission is a dynamic process that requires <strong>the</strong> coordination of m<strong>an</strong>y pre-<br />
<strong>an</strong>d post-synaptic proteins in order to provide high fidelity communication between neurons.<br />
P/Q-type calcium ch<strong>an</strong>nels are highly localized to synaptic terminals <strong>an</strong>d <strong>the</strong>ir modulation via<br />
calmodulin mediated calcium-dependent signaling likely affects synaptic plasticity. It has been<br />
shown that disruption of calcium sensor binding to <strong>the</strong> P/Q-type ch<strong>an</strong>nel Cav2.1 subunit c<strong>an</strong><br />
prevent calcium dependent modulation <strong>an</strong>d result in a marked reduction in synaptic plasticity in a<br />
model system. We hypo<strong>the</strong>sized that naturally occurring mutations in <strong>the</strong> P/Q-type ch<strong>an</strong>nel<br />
might alter two <strong>for</strong>ms of calcium-dependent modulation: calcium dependent facilitation (CDF)<br />
<strong>an</strong>d calcium-dependent inactivation (CDI). In <strong>the</strong> present study we investigated how two<br />
mutations implicated in Familial Hemiplegic Migraine Type-1 (FHM-1) affect Cav2.1 CDF <strong>an</strong>d<br />
CDI. We find that both FHM-1 mutations signific<strong>an</strong>tly reduce <strong>the</strong> ability of P/Q-type ch<strong>an</strong>nels to<br />
undergo CDF, while only one mutation affects CDI. The ch<strong>an</strong>ges on CDF <strong>an</strong>d CDI were found<br />
to have profound effects on P/Q-type calcium currents during action potential wave<strong>for</strong>ms.<br />
Interestingly, <strong>the</strong> observed effects on CDF were Cav2.1 splice-dependent. Both FHM-1<br />
mutations nearly completely abolished CDF in <strong>the</strong> Aga IVA sensitive P-type splice-vari<strong>an</strong>t (-NP)<br />
while <strong>the</strong>y had little effect when expressed in <strong>the</strong> less sensitive Q-type vari<strong>an</strong>t (+NP).<br />
We hypo<strong>the</strong>size that <strong>the</strong> selective gain-of-function effects of FHM-1 mutations on certain Cav2.1<br />
splice vari<strong>an</strong>ts may underlie <strong>the</strong> increased glutamate release at central synapses relev<strong>an</strong>t to <strong>the</strong><br />
disease pathophysiology. Fur<strong>the</strong>r, <strong>the</strong> differential affects of FHM-1 mutations in <strong>the</strong> context of<br />
Cav2.1 splice variation may contribute to <strong>the</strong> temporal <strong>an</strong>d spatial aspects of FHM-1. Overall, <strong>the</strong><br />
results provide new insights into <strong>the</strong> possible disease mech<strong>an</strong>isms underlying FHM-1 <strong>an</strong>d also<br />
provide <strong>the</strong> first evidence <strong>for</strong> P/Q-type ch<strong>an</strong>nel calcium-dependent modulation as being<br />
potentially altered in a hum<strong>an</strong> disease state.<br />
Disclosures: P.J. Adams, None; E. Garcia, None; T.P. Snutch, None.
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.8/D1<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: Marie Curie Research Training Network “CAVNET” MRTN-CT-2006-035367<br />
Graduate Programme University of Tübingen<br />
Title: The role of <strong>the</strong> auxiliary Ca2+ ch<strong>an</strong>nel alpha2delta-3 subunit <strong>for</strong> signal tr<strong>an</strong>smission in <strong>the</strong><br />
auditory brainstem <strong>an</strong>d <strong>the</strong> acoustic startle reflex pathway<br />
Authors: A. PIRONE 1 , L. RÜTTIGER 1 , P. PILZ 2 , A. ZUCCOTTI 1 , C. FRANZ 1 , E. FRIAUF 3 ,<br />
M. KNIPPER 1 , *J. ENGEL 4 ;<br />
1 Dept of Otorhinolaryngology, Tübingen Hearing Res. Ctr., 2 Inst. of Zoology, Univ. of<br />
Tübingen, Tübingen, Germ<strong>an</strong>y; 3 Fac. of Biology, Dept Animal Physiol., Tech. Univ. of<br />
Kaiserslautern, Kaiserslautern, Germ<strong>an</strong>y; 4 Saarl<strong>an</strong>d Univ., Homburg, Germ<strong>an</strong>y<br />
Abstract: Voltage-activated Ca2+ ch<strong>an</strong>nels consist of a pore-<strong>for</strong>ming alpha1 subunit (SU), a<br />
beta <strong>an</strong>d <strong>an</strong> alpha2delta SU. Expression of alpha1 SUs is cell-specific, <strong>an</strong>d <strong>the</strong> co-assembly with<br />
auxiliary subunits beta <strong>an</strong>d alpha2delta may also be ra<strong>the</strong>r specific, giving rise to Ca2+ ch<strong>an</strong>nels<br />
with distinct cell-specific properties. The alpha2delta-SUs are required <strong>for</strong> trafficking <strong>an</strong>d<br />
stabilizing alpha1 SUs in <strong>the</strong> plasma membr<strong>an</strong>e, <strong>the</strong>reby defining <strong>the</strong> amplitude <strong>an</strong>d biophysical<br />
properties of Ca2+ currents. Mice deficient <strong>for</strong> <strong>the</strong> gene CACNA2D3 coding <strong>for</strong> <strong>the</strong> alpha2delta-<br />
3 SU showed <strong>an</strong> impared hearing phenotype with slightly increased ABR hearing thresholds over<br />
<strong>the</strong> entire frequency r<strong>an</strong>ge, reduced amplitudes of ABR wave<strong>for</strong>ms but normal DPOAE<br />
thresholds <strong>an</strong>d amplitudes. Additionally, <strong>the</strong> acoustic startle response was also reduced in <strong>the</strong><br />
alpha2delta-3-/- (ko) mice. To test if hearing <strong>an</strong>d acoustic startle deficits were caused by reduced<br />
presynaptic Ca2+ currents in <strong>the</strong> IHCs, Ba2+ currents were recorded in inner (IHC) cells pre <strong>an</strong>d<br />
post hearing onset. Ba2+ currents were slightly different between WT <strong>an</strong>d ko IHCs but this<br />
difference most likely does not account <strong>for</strong> <strong>the</strong> ko phenotype. Analysis of a2d3 expression in <strong>the</strong><br />
cochlea using lacZ reporter staining <strong>an</strong>d in-situ hybridization revealed its expression in spiral<br />
g<strong>an</strong>glion neurons <strong>an</strong>d cochlear root neurons. To investigate if <strong>the</strong> presynaptic ch<strong>an</strong>nels along <strong>the</strong><br />
hearing <strong>an</strong>d startle response pathways were affected, we per<strong>for</strong>med immunohistochemistry <strong>for</strong><br />
<strong>the</strong> fast P/Q <strong>an</strong>d N-type Ca2+ ch<strong>an</strong>nels. In brainstem nuclei <strong>an</strong>d gi<strong>an</strong>t pontine neurons, we found<br />
differences in <strong>the</strong> P/Q as well as N-type expression patterns between WT <strong>an</strong>d ko <strong>an</strong>imals. In<br />
conclusion, <strong>the</strong> hearing <strong>an</strong>d startle deficits of alpha2delta-3-/- mice are not due to malfunction of
IHCs or OHCs but may ra<strong>the</strong>r be caused by altered presynaptic Ca2+ ch<strong>an</strong>nels <strong>an</strong>d currents of<br />
spiral g<strong>an</strong>glion neurons, root neurons <strong>an</strong>d neurons fur<strong>the</strong>r up <strong>the</strong> hearing <strong>an</strong>d startle pathways.<br />
Supported by Marie Curie Research Training Network “CAVNET” (L-type Calcium Ch<strong>an</strong>nels in<br />
Health <strong>an</strong>d Disease; MRTN-CT-2006-035367) to AP, AZ, EF, MK, JE.<br />
Disclosures: A. Pirone, None; L. Rüttiger, None; P. Pilz, None; A. Zuccotti, None; C. Fr<strong>an</strong>z,<br />
None; E. Friauf, None; M. Knipper, None; J. Engel, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.9/D2<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: MH066198 (ETK)<br />
NS055251 (DL)<br />
Title: Activity-dependent trafficking of <strong>the</strong> voltage gated calcium ch<strong>an</strong>nel subunit CaVα2δ at<br />
synapses<br />
Authors: *J. RAINGO 1 , A. ANDRADE 2 , M. KHVOTCHEV 1 , D. LIPSCOMBE 2 , E. T.<br />
KAVALALI 1 ;<br />
1 Neurosci, UT Southwestern Med. Ctr., Dallas, TX; 2 Neurosci., Brown Univ., Providence, RI<br />
Abstract: The CaVα2δ subunit complexes with voltage-gated calcium ch<strong>an</strong>nel α1 subunits <strong>an</strong>d it<br />
is needed <strong>for</strong> proper surface membr<strong>an</strong>e targeting <strong>an</strong>d ch<strong>an</strong>nel activation. CaVα2δ subunits are<br />
present at presynaptic nerve terminals where <strong>the</strong>y are critical <strong>for</strong> calcium-dependent<br />
tr<strong>an</strong>smission. CaVα2δ levels increase following peripheral nerve injury <strong>an</strong>d this response is<br />
inhibited by <strong>the</strong> <strong>an</strong>algesic gabapentin. Here we report use of a fusion construct of CaVα2δ <strong>an</strong>d<br />
pH-sensitive GFP (pHluorin), pHT-CaVα2δ, to monitor its localization <strong>an</strong>d activity-dependent<br />
trafficking. We used <strong>an</strong> 11 aa linker to minimize <strong>the</strong> impact of pHluorin on CaVα2δ function. In<br />
tsA201 cells, pHT-CaVα2δ retained activity similar to wild type CaVα2δ, it augmented N-type<br />
currents <strong>an</strong>d increased rates of activation <strong>an</strong>d inactivation when co-expressed with CaV2.2 <strong>an</strong>d<br />
CaVβ3 (Peak current(pA/pF): Wild type CaVα2δ = -66.8± 11.0, pHT- CaVα2δ = -59.9 ± 13.0, (-<br />
)CaVα2δ = -8.1 ± 1.3). We next used lenitivirus to express pHT-CaVα2δ in cultured hippocampal<br />
neurons. pHT-CaVα2δ signal was widely distributed in somata, dendrites, <strong>an</strong>d axons. To test if<br />
surface CaVα2δ levels ch<strong>an</strong>ge with activity, we monitored fluorescence following extracellular
field stimulation (20 Hz-20 s). pHT-CaVα2δ fluorescence decreased (∆F= -562.6±74.4,n=7)<br />
consistent with internalization to acidic compartments. This effect was observed in presence of<br />
10 µM CNQX <strong>an</strong>d 50 µM AP-5 suggesting it does not require <strong>the</strong> activation of postsynaptic<br />
AMPA/NMDA receptors. The stimulus-dependent decrease in pHT-CaVα2δ fluorescence was<br />
prevented by <strong>the</strong> calcium ch<strong>an</strong>nel blocker CdCl2 (0.2 mM) (∆F=-46.4 ± 35.3, n=4) suggesting<br />
that calcium ch<strong>an</strong>nel activity is needed to trigger internalization. By co-expressing pHT-CaVα2δ<br />
<strong>an</strong>d mOr<strong>an</strong>ge-tagged synaptic vesicle protein synaptobrevin, we could selectively monitor<br />
trafficking of presynaptic CaVα2δ simult<strong>an</strong>eously with vesicle recycling induced by activity. In<br />
areas where <strong>the</strong>se two proteins were co-localized, we observed that pHT-CaVα2δ fluorescence<br />
was rapidly reduced in <strong>an</strong> activity-dependent m<strong>an</strong>ner in parallel with <strong>the</strong> increase of<br />
synaptobrevin signal. Finally, activity-dependent internalization of pHT-CaVα2δ was unaffected<br />
by dynasore (80 µM, ∆F=-673.5± 115.5, n=3) <strong>an</strong> inhibitor of <strong>the</strong> GTPase activity of dynamin.<br />
Our results suggest that <strong>the</strong> CaVα2δ subunit of voltage-gated calcium ch<strong>an</strong>nels c<strong>an</strong> be rapidly<br />
endocytosed in <strong>an</strong> activity-dependent m<strong>an</strong>ner in parallel with <strong>the</strong> trafficking of presynaptic<br />
synaptobrevin. We are investigating <strong>the</strong> mech<strong>an</strong>ism underlying internalization <strong>an</strong>d its functional<br />
impact on calcium ch<strong>an</strong>nel function. Supported by NIH gr<strong>an</strong>ts MH066198 (ETK), NS055251<br />
(DL).<br />
Disclosures: J. Raingo, None; A. Andrade, None; M. Khvotchev, None; D. Lipscombe,<br />
None; E.T. Kavalali, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.10/D3<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: BBSRC<br />
Title: Investigation of <strong>the</strong> association of voltage-gated calcium ch<strong>an</strong>nel α2δ subunits with lipid<br />
rafts<br />
Authors: *A. ALVAREZ LAVIADA, L. DOUGLAS, A. DAVIES, I. KADURIN, W. S.<br />
PRATT, A. C. DOLPHIN;<br />
Res. Dept. of Neuroscience, Physiol. <strong>an</strong>d Pharmacol., Univ. Col. London, London, United<br />
Kingdom
Abstract: Voltage-gated calcium (Cav) ch<strong>an</strong>nels are thought to exist in <strong>the</strong> plasma membr<strong>an</strong>e as<br />
heteromeric proteins, in which <strong>the</strong> α1 subunit <strong>for</strong>ms <strong>the</strong> pore <strong>an</strong>d determines <strong>the</strong> main functional<br />
<strong>an</strong>d pharmacological attributes of <strong>the</strong> ch<strong>an</strong>nel. For <strong>the</strong> high-voltage activated ch<strong>an</strong>nels, <strong>the</strong> α1<br />
subunit is associated with <strong>an</strong> intracellular β subunit <strong>an</strong>d <strong>an</strong> α2δ subunit, both of which influence<br />
<strong>the</strong> trafficking <strong>an</strong>d properties of <strong>the</strong> ch<strong>an</strong>nels. The molecular characterization <strong>an</strong>d topology of <strong>the</strong><br />
α2δ subunits was initially determined <strong>for</strong> skeletal muscle α2δ-1, but it has been suggested to<br />
generalize to all α2δ subunits. They are universally described as type I tr<strong>an</strong>s-membr<strong>an</strong>e proteins,<br />
since <strong>the</strong>y have <strong>an</strong> N-terminal signal peptide <strong>an</strong>d a C-terminal hydrophobic tr<strong>an</strong>s-membr<strong>an</strong>e<br />
region.<br />
We have examined <strong>the</strong> association of α2δ-1, -2 <strong>an</strong>d -3 with lipid rafts, both in native tissues <strong>an</strong>d<br />
following heterologous expression. We have also examined <strong>the</strong> ability of mutations in α2δ<br />
subunits to reduce expression of α2δ subunits in lipid rafts, <strong>an</strong>d concomit<strong>an</strong>tly showed that <strong>the</strong>se<br />
mutations affect cell surface expression <strong>an</strong>d <strong>the</strong> ability of <strong>the</strong> α2δ subunits to enh<strong>an</strong>ce calcium<br />
ch<strong>an</strong>nel currents. Fur<strong>the</strong>rmore depletion or supplementation of cholesterol also modified calcium<br />
ch<strong>an</strong>nel currents.<br />
Our data shed light on <strong>the</strong> processes involved in <strong>the</strong> association of α2δ subunits with lipid rafts.<br />
Disclosures: A. Alvarez Laviada, None; L. Douglas, None; A. Davies, None; I. Kadurin,<br />
None; W.S. Pratt, None; A.C. Dolphin, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.11/D4<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIMH RO1 MH065531<br />
NHLBI R37 HL076795<br />
NIDCD RO1 DC00276<br />
Title: The distal carboxy tail of Ca 2+ ch<strong>an</strong>nels retunes <strong>the</strong>ir calmodulin sensitivity into <strong>the</strong><br />
neurobiological r<strong>an</strong>ge<br />
Authors: X. LIU, P. S. YANG, W. YANG, V. WU, *D. T. YUE;<br />
Dept Biomed Engin Neurosci, Johns Hopkins Univ. Sch. Med., Baltimore, MD
Abstract: Crucial to numerous aspects of neurobiology is <strong>the</strong> exquisite Ca 2+ feedback inhibition<br />
of Ca 2+ ch<strong>an</strong>nels. Such Ca 2+ -dependent inactivation (CDI) of ch<strong>an</strong>nels is mediated by a single<br />
calmodulin (CaM) that is constitutively complexed with ch<strong>an</strong>nels. The Ca 2+ -free <strong>for</strong>m of CaM<br />
(apoCaM) is preassociated with <strong>the</strong> IQ domain on <strong>the</strong> ch<strong>an</strong>nel carboxy terminus, <strong>an</strong>d Ca 2+<br />
binding to this CaM drives subsequent con<strong>for</strong>mational ch<strong>an</strong>ges that triggers CDI. Ch<strong>an</strong>nels<br />
lacking <strong>an</strong> associated CaM fail to exhibit CDI, but <strong>the</strong> prevailing view has been that <strong>the</strong> strong<br />
ch<strong>an</strong>nel affinity <strong>for</strong> apoCaM more th<strong>an</strong> ensures saturation of ch<strong>an</strong>nels with CaM in <strong>the</strong> natural<br />
context. Here, we report that while shorter splice vari<strong>an</strong>ts of CaV1.3 <strong>an</strong>d CaV1.4 ch<strong>an</strong>nels<br />
con<strong>for</strong>m to this expectation, longer splice <strong>for</strong>ms that include a distal carboxy tail module (ICDI)<br />
exhibit a striking effect−massive retuning of apoCaM affinity into a r<strong>an</strong>ge where natural<br />
fluctuations in ambient CaM could affect <strong>the</strong> fraction of ch<strong>an</strong>nels preassociated with CaM, <strong>an</strong>d<br />
<strong>the</strong>reby <strong>the</strong> overall strength of CDI. Recent studies have reported that <strong>the</strong> ICDI module acts as a<br />
switch to essentially eliminate CDI (Wahl-Schott et al PNAS 2006; Singh et al Nature Neurosci<br />
2006; Singh et al J Biol Chem 2008), but <strong>the</strong> underlying mech<strong>an</strong>ism has remained controversial.<br />
Accordingly, to per<strong>for</strong>m definitive <strong>an</strong>d qu<strong>an</strong>titative CaM biochemical <strong>an</strong>alysis upon functional<br />
holoch<strong>an</strong>nels within individual cells, we combine simult<strong>an</strong>eous electrophysiological<br />
determination of CDI with FRET sensor determinations of free apoCaM concentration<br />
(BisCaMIQ, Black et al Biochemistry 2006). In so doing, we rigorously establish a competitive<br />
inhibitory mech<strong>an</strong>ism of ICDI action, whereby ICDI competes with apoCaM <strong>for</strong> binding to <strong>the</strong><br />
ch<strong>an</strong>nel IQ domain. Import<strong>an</strong>tly, ra<strong>the</strong>r th<strong>an</strong> altoge<strong>the</strong>r switching off CDI, this competitive<br />
mech<strong>an</strong>ism adjusts <strong>the</strong> overall ch<strong>an</strong>nel affinity <strong>for</strong> apoCaM into <strong>the</strong> neurobiological r<strong>an</strong>ge (~5-10<br />
µM), <strong>an</strong>d differing degrees of adjustment among various long splice <strong>for</strong>ms of CaV1.3 <strong>an</strong>d CaV1.4<br />
are qu<strong>an</strong>titatively explained by distinct ICDI affinities <strong>for</strong> IQ domains in <strong>the</strong>se various ch<strong>an</strong>nels.<br />
To illustrate <strong>the</strong> biological impact of such retuning, we demonstrate that CDI of long splice <strong>for</strong>m<br />
ch<strong>an</strong>nels c<strong>an</strong> be altoge<strong>the</strong>r suppressed by coexpressing neuromodulin, a ubiquitous synaptic<br />
growth/plasticity molecule with strong apoCaM buffering. As schizophrenia is linked to<br />
decreased neuromodulin, a resurgence of CDI might be expected. More broadly, numerous<br />
neurodegenerative diseases are potentially affiliated with perturbed CaM concentration, <strong>an</strong>d <strong>the</strong>ir<br />
pathogenesis may <strong>the</strong>re<strong>for</strong>e involve maldaptive ch<strong>an</strong>ges in ch<strong>an</strong>nel CDI with attend<strong>an</strong>t<br />
der<strong>an</strong>gement of Ca 2+ signaling.<br />
Disclosures: X. Liu, None; P.S. Y<strong>an</strong>g, None; W. Y<strong>an</strong>g, None; V. Wu, None; D.T. Yue, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.12/D5<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology
Support: NS029967<br />
Title: Iso<strong>for</strong>m-specific ubiquitination of <strong>the</strong> N-type voltage-gated calcium ch<strong>an</strong>nel<br />
Authors: *S. MARANGOUDAKIS 1 , T. HELTON 1 , D. LIPSCOMBE 2 ;<br />
1 Brown Univ., Providnece, RI; 2 Brown Univ., Providence, RI<br />
Abstract: N-type CaV2.2 calcium ch<strong>an</strong>nels are <strong>the</strong> molecular gatekeepers of excitationdependent<br />
calcium influx in m<strong>an</strong>y neurons. They are responsible <strong>for</strong> regulating neurotr<strong>an</strong>smitter<br />
release, neurite extension, <strong>an</strong>d synaptic stabilization. The function of <strong>the</strong>se ch<strong>an</strong>nels is regulated<br />
by numerous cellular mech<strong>an</strong>isms including post-tr<strong>an</strong>slational modification <strong>an</strong>d alternative<br />
splicing. In this study we examine <strong>the</strong> convergence of post-tr<strong>an</strong>slational modification <strong>an</strong>d<br />
alternative splicing on <strong>the</strong> modulation of N-type ch<strong>an</strong>nels- with specific focus on <strong>the</strong> mutually<br />
exclusive exon pair 37a <strong>an</strong>d 37b. Recent characterization of e37a <strong>an</strong>d e37b shows that <strong>the</strong>y<br />
encode sequences differing in 14 of <strong>the</strong> total 33 amino acids, <strong>an</strong>d that <strong>the</strong> CaV2.2e[37b] iso<strong>for</strong>m<br />
is ubiquitously expressed in <strong>the</strong> nervous system, while CaV2.2e[37a] is enriched in <strong>the</strong><br />
nociceptive neurons of dorsal root g<strong>an</strong>glia (DRG). Fur<strong>the</strong>r examination also shows that<br />
CaV2.2e[37a] ch<strong>an</strong>nels are expressed at signific<strong>an</strong>tly higher levels on <strong>the</strong> cell surface (Castiglioni<br />
et al., 2006, Journal of Physiology). To better underst<strong>an</strong>d <strong>the</strong> molecular basis of <strong>the</strong>se<br />
differences, we created CaV2.2-Gal4 DNA binding domain fusion constructs containing ei<strong>the</strong>r<br />
exon 37a or 37b, <strong>an</strong>d per<strong>for</strong>med yeast two-hybrid screens using rat DRG cDNA library in order<br />
to identify proteins that differentially associate with <strong>the</strong> protein sequences coded by e37a <strong>an</strong>d<br />
e37b. A select number of identified proteins were shown to interact exclusively with protein<br />
sequences derived <strong>from</strong> one or <strong>the</strong> o<strong>the</strong>r exon. Of <strong>the</strong> identified proteins, a ubiquitin-activating<br />
enzyme E1 was found to interact solely with constructs containing e37b. We <strong>the</strong>re<strong>for</strong>e<br />
hypo<strong>the</strong>sized that e37b iso<strong>for</strong>m ch<strong>an</strong>nels are more ubiquitinated th<strong>an</strong> e37a ch<strong>an</strong>nels, <strong>an</strong>d<br />
followed <strong>the</strong> screens with experiments aimed at determining <strong>the</strong> ubiquitination state of <strong>the</strong> fulllength<br />
ch<strong>an</strong>nels. In <strong>the</strong>se experiments we expressed both iso<strong>for</strong>ms of CaV2.2 in HEK293-derived<br />
cells, immunoprecipitating CaV2.2, <strong>an</strong>d blotting <strong>for</strong> ubiquitin. We found that <strong>the</strong> CaV2.2e[37b]<br />
iso<strong>for</strong>m associates with 0.8- to 2.2-fold more ubiquitination th<strong>an</strong> CaV2.2e[37a]. We also<br />
per<strong>for</strong>med whole-cell patch clamp on cells tr<strong>an</strong>sfected with ei<strong>the</strong>r iso<strong>for</strong>m <strong>an</strong>d found that after<br />
treating <strong>the</strong> cells with <strong>the</strong> potent proteasome inhibitor MG132, current densities increased more<br />
robustly when expressing CaV2.2e[37b] th<strong>an</strong> CaV2.2e[37a]. These results indicate more robust<br />
ubiquitination of CaV2.2e[37b] <strong>an</strong>d potentially higher rate of degradation of CaV2.2 [37b]<br />
through <strong>the</strong> ubiquitin proteasome system. These results also suggest a role <strong>for</strong> ubiquitination in<br />
controlling <strong>the</strong> intrinsic current densities associated with ch<strong>an</strong>nels of <strong>the</strong> CaV2.2e[37b] iso<strong>for</strong>m.<br />
Disclosures: S. Mar<strong>an</strong>goudakis, None; T. Helton, None; D. Lipscombe, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.13/D6<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: CIHR<br />
AHFMR<br />
Title: The Cav beta subunit prevents Nedd4 mediated ubiquitination <strong>an</strong>d proteasomal<br />
degradation of L-type calcium ch<strong>an</strong>nels via <strong>the</strong> Derlin-1/p97 ERAD protein complex<br />
Authors: *C. ALTIER, A. GARCIA-CABALLERO, B. SIMMS, J. WALCHER, H.<br />
TEDFORD, T. HERMOSILLA, G. ZAMPONI;<br />
Univ. Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: The calcium ch<strong>an</strong>nel beta (Cavb) subunit has a well documented role in increasing <strong>the</strong><br />
cell surface expression of voltage-gated calcium<br />
ch<strong>an</strong>nels, but <strong>the</strong> underlying molecular mech<strong>an</strong>isms have remained<br />
undefined. It has been suggested that <strong>the</strong> beta subunit masks <strong>an</strong> ER<br />
retention signal in <strong>the</strong> domain I-II linker, however, this has never been confirmed<br />
experimentally. Indeed, our data show that CD4 fusion constructs of <strong>the</strong> domain I-II linker<br />
regions of Cav1.2 <strong>an</strong>d Cav2.2 are trafficked efficiently to <strong>the</strong> plasma membr<strong>an</strong>e, ra<strong>the</strong>r th<strong>an</strong><br />
being retained. Instead, biochemical <strong>an</strong>d imaging data show that <strong>the</strong> calcium ch<strong>an</strong>nel beta<br />
subunit protects <strong>the</strong> Cav1.2 L-type calcium ch<strong>an</strong>nel <strong>from</strong> ubiquitination <strong>an</strong>d proteasomal<br />
degradation. The ubiquitin ligase, Nedd4, interacts with <strong>the</strong> calcium ch<strong>an</strong>nel complex<br />
independently of <strong>the</strong> calcium ch<strong>an</strong>nel beta subunit. Nedd4 is able to ubiquitinate <strong>the</strong> ch<strong>an</strong>nel<br />
only when <strong>the</strong> beta subunit is not present. The ubiquitinated ch<strong>an</strong>nel <strong>the</strong>n interacts with <strong>the</strong><br />
derlin-1/p97 ERAD complex en route to <strong>the</strong> proteasome. Interfering with proteasomal<br />
degradation via pharmacological me<strong>an</strong>s results in <strong>an</strong> increase in Cav1.2 ch<strong>an</strong>nel surface<br />
expression, indicating that <strong>the</strong> ch<strong>an</strong>nels do not require <strong>the</strong> beta subunit <strong>for</strong> membr<strong>an</strong>e trafficking.<br />
Collectively, our data show that <strong>the</strong> calcium ch<strong>an</strong>nel beta subunit serves to stabilize <strong>the</strong> ch<strong>an</strong>nel<br />
complex, <strong>an</strong>d increases plasma membr<strong>an</strong>e expression by preventing <strong>the</strong> activation of <strong>the</strong><br />
proteasomal degradation pathway.<br />
Disclosures: C. Altier, None; A. Garcia-Caballero, None; B. Simms, None; J. Walcher,<br />
None; H. Ted<strong>for</strong>d, None; T. Hermosilla, None; G. Zamponi, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.14/D7<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t MH080291<br />
AHA Predoctoral Fellowship 0815611G<br />
Title: Calcineurin <strong>an</strong>choring dynamics in a neuronal L-type ch<strong>an</strong>nel signaling complex promote<br />
tr<strong>an</strong>sduction of membr<strong>an</strong>e proximal calcium signals to <strong>the</strong> nucleus<br />
Authors: *M. D. PINK 1 , H. LI 2 , W. A. SATHER 1 , P. HOGAN 2 , M. L. DELL'ACQUA 1 ;<br />
1 2<br />
Neurosci. Program, Univ. Colorado Denver-School of Med., Aurora, CO; Immune Dis. Inst.,<br />
Harvard Med. Sch., Boston, MA<br />
Abstract: Neuronal L-type voltage-gated calcium ch<strong>an</strong>nels contribute to activity-dependent<br />
ch<strong>an</strong>ges in gene expression that influence synaptic plasticity. Phosphorylation-mediated<br />
enh<strong>an</strong>cement of L-type ch<strong>an</strong>nels containing <strong>the</strong> CaV1.2 pore-<strong>for</strong>ming subunit is promoted by Akinase<br />
<strong>an</strong>choring proteins (AKAPs) that target <strong>the</strong> cAMP-dependent protein kinase (PKA) to <strong>the</strong><br />
ch<strong>an</strong>nel. In hippocampal neurons, CaV1.2 L-ch<strong>an</strong>nels interact directly with <strong>the</strong> postsynaptic<br />
scaffold protein AKAP79/150, which binds both PKA <strong>an</strong>d <strong>the</strong> Ca2+/calmodulin-activated<br />
phosphatase, calcineurin (CaN). Co-targeting of PKA <strong>an</strong>d CaN by AKAP79/150 confers bidirectional<br />
regulation of L-type current amplitude in response to cAMP <strong>an</strong>d Ca2+. Local Ca2+<br />
influx through neuronal L-ch<strong>an</strong>nels has a privileged role in regulating gene expression through<br />
<strong>the</strong> tr<strong>an</strong>scription factor NFATc4. L-ch<strong>an</strong>nel opening creates a Ca2+ microdomain that initiates<br />
plasma membr<strong>an</strong>e localized activation of CaN resulting in dowstream NFAT dephosphorylation.<br />
A key question is how ch<strong>an</strong>nel localized signaling processes are regulated to respond to a highly<br />
compartmentalized membr<strong>an</strong>e Ca2+ source yet also promote tr<strong>an</strong>duction to <strong>the</strong> nucleus while<br />
maintaining high specificity. Using <strong>an</strong> RNAi knock-down <strong>an</strong>d rescue approach, we showed that<br />
CaN-dependent tr<strong>an</strong>slocation of NFATc4 to <strong>the</strong> nucleus in response to Ca2+ influx through<br />
neuronal L-type ch<strong>an</strong>nels requires AKAP79/150 <strong>an</strong>choring of CaN near <strong>the</strong> ch<strong>an</strong>nel. X-ray<br />
crystallographic studies revealed that AKAP-CaN <strong>an</strong>choring, like NFAT-CaN substrate<br />
recognition, requires binding of <strong>the</strong> CaN A subunit to a PxIxIT-type docking motif with <strong>the</strong><br />
AKAP having a non-consensus vari<strong>an</strong>t of this motif with I in place of P in <strong>the</strong> first position.<br />
These structural findings indicate that AKAP <strong>an</strong>d NFAT actually compete with each o<strong>the</strong>r <strong>for</strong><br />
CaN binding suggesting a fine bal<strong>an</strong>ce of dynamic CaN binding interactions must exist to<br />
promote CaN signaling <strong>from</strong> <strong>the</strong> L-ch<strong>an</strong>nel-AKAP complex to NFAT. Accordingly, FRAP,<br />
FRET, <strong>an</strong>d NFAT imaging <strong>an</strong>alysis of neurons expressing AKAP I to P mut<strong>an</strong>ts that increase<br />
CaN binding affinity <strong>an</strong>d decrease cellular <strong>an</strong>choring dynamics confirm that local binding <strong>an</strong>d<br />
unbinding of CaN to AKAP79/150 is central to <strong>the</strong> ability of CaN to respond to ch<strong>an</strong>nel<br />
proximal Ca2+ signals <strong>an</strong>d tr<strong>an</strong>sduce <strong>the</strong>m distally to NFATc4. Thus, our findings help explain<br />
how dynamic <strong>an</strong>choring of CaN to AKAP79/150 contributes to <strong>the</strong> privileged role of neuronal Lch<strong>an</strong>nels<br />
in signaling to <strong>the</strong> nucleus by promoting specific activation of CaN in <strong>the</strong> ch<strong>an</strong>nel Ca2+
microdomain in a m<strong>an</strong>ner that still allows <strong>the</strong> activated enzyme to leave <strong>the</strong> ch<strong>an</strong>nel complex <strong>an</strong>d<br />
engage in downstream signaling events.<br />
Disclosures: M.D. Pink, None; H. Li, None; W.A. Sa<strong>the</strong>r, None; P. Hog<strong>an</strong>, None; M.L.<br />
Dell'Acqua, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.15/D8<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: PAPIIT IN210708<br />
Conacyt 90443<br />
ICyTDF 2008<br />
Title: Regulation of L-type Ca 2+ ch<strong>an</strong>nel activity by RIM1<br />
Authors: *A. SANDOVAL 1,2 , R. GONZALEZ 2 , R. FELIX 2 ;<br />
1 2<br />
Med., FES Iztacala UNAM, Tlalnep<strong>an</strong>tla, Estado de Mexico, Mexico; Cell Biol., Cinvestav-<br />
IPN, Mexico City, Mexico<br />
Abstract: Recent studies have documented a functional interaction between two components of<br />
<strong>the</strong> presynaptic active zone: Rab3-interacting molecule 1 (RIM1), a protein that play a role in<br />
priming <strong>an</strong>d docking of synaptic vesicles, <strong>an</strong>d <strong>the</strong> β auxiliary subunit of <strong>the</strong> voltage-gated Ca 2+<br />
(CaV) ch<strong>an</strong>nels, that control Ca 2+ -dependent fusion of synaptic vesicles <strong>an</strong>d consequently<br />
neurotr<strong>an</strong>smitter release in mammali<strong>an</strong> neurons (Kiyonaka et al., Nat Neurosci 2007; 10: 691-<br />
701). This interaction have a dual physiological function on <strong>the</strong> release of <strong>the</strong> neurotr<strong>an</strong>smitters<br />
by <strong>an</strong>choring <strong>the</strong> vesicles to <strong>the</strong> ch<strong>an</strong>nels <strong>an</strong>d by increasing Ca 2+ influx through <strong>the</strong> regulation of<br />
CaV ch<strong>an</strong>nel inactivation. Like in neurons, in some endocrine cell types, <strong>the</strong> elevation of Ca 2+<br />
ions that triggers <strong>the</strong> exocytotic process is basically due to <strong>the</strong> opening of CaV ch<strong>an</strong>nels that are<br />
clustered at <strong>the</strong> vesicle release sites. There is now considerable consensus that <strong>the</strong> L-type CaV<br />
ch<strong>an</strong>nels are <strong>the</strong> major determin<strong>an</strong>t of Ca 2+ current in m<strong>an</strong>y of <strong>the</strong>se cells. In this study, we<br />
attempt to provide evidence that RIM1 affects <strong>the</strong> activity of L-type CaV ch<strong>an</strong>nels. By using <strong>the</strong><br />
patch clamp technique we characterized whole-cell Ba 2+ currents through L-type CaV ch<strong>an</strong>nels<br />
(CaV1.2α1/CaVβ2-3/α2δ-1 <strong>an</strong>d CaV1.3α1/CaVβ2-3/α2δ-1) heterologously expressed in HEK-293
cells. The most prominent RIM1 effect on <strong>the</strong> macroscopic currents was observed on inactivation<br />
parameters. Inactivation was signific<strong>an</strong>tly decelerated. Notably, RIM1 was unable to affect<br />
ch<strong>an</strong>nel activity in <strong>the</strong> absence of <strong>the</strong> CaVβ subunit indicating that <strong>the</strong> physiological association<br />
between RIM1 <strong>an</strong>d L-type Ca 2+ ch<strong>an</strong>nels is mediated through this auxiliary subunit. These<br />
results suggest that RIM1 may constitute a functional link between <strong>the</strong> L-type CaV ch<strong>an</strong>nels <strong>an</strong>d<br />
<strong>the</strong> machinery <strong>for</strong> exocytosis.<br />
Disclosures: A. S<strong>an</strong>doval, None; R. Gonzalez, None; R. Felix, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.16/D9<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: HSFO Gr<strong>an</strong>t NA 6284<br />
Title: Structure-function <strong>an</strong>alyses of drug binding to L-type calcium ch<strong>an</strong>nels<br />
Authors: A. SENATORE, S. LAM, *J. SPAFFORD;<br />
Biol., Univ. of Waterloo, Waterloo, ON, C<strong>an</strong>ada<br />
Abstract: Calcium ch<strong>an</strong>nel <strong>an</strong>tagonists have a signific<strong>an</strong>t clinical role in <strong>the</strong> treatment of <strong>an</strong>gina<br />
<strong>an</strong>d hypertension, especially in those with patients with diabetes, <strong>an</strong>d where o<strong>the</strong>r agents are<br />
contraindicated. The current world market <strong>for</strong> L-type calcium ch<strong>an</strong>nel blockers is dominated by<br />
amlodipine (Norvasc TM ), a vari<strong>an</strong>t based upon <strong>the</strong> 125 year old structure of 1,4 dihydroypyridine.<br />
Development of a new line of L-type ch<strong>an</strong>nel blockers requires a fundamental underst<strong>an</strong>ding of<br />
binding sites of L-type blockers <strong>an</strong>d taking adv<strong>an</strong>tage of new structures that will give us insights<br />
into <strong>the</strong> L-type ch<strong>an</strong>nel pharmacophore.<br />
We have a unique opportunity to explore <strong>the</strong> residues responsible <strong>for</strong> dihydropyridine (DHP)<br />
block using <strong>an</strong> mollusc<strong>an</strong> L-type calcium ch<strong>an</strong>nel homolog, LCav1 isolated <strong>from</strong> <strong>the</strong> pond snail,<br />
Lymnaea stagnalis. The invertebrate Cav1 ch<strong>an</strong>nel is nearly indistinguishable to <strong>the</strong> mammali<strong>an</strong><br />
Cav1.2 L-type ch<strong>an</strong>nel in biophysical features <strong>an</strong>d yet is almost insensitive to DHPs such as<br />
isradipine (> 100 fold less sensitive). Our strategy has been to strategically swap residues <strong>from</strong><br />
<strong>the</strong> Cav1.2 ch<strong>an</strong>nel onto <strong>the</strong> equivalent invertebrate surrogate ch<strong>an</strong>nel (LCav1) until we create <strong>the</strong><br />
high affinity binding site found in mammali<strong>an</strong> Cav1.2 ch<strong>an</strong>nels with <strong>the</strong> minimum number of<br />
residue ch<strong>an</strong>ges. Recent modeling suggests eighteen key amino acid contacts <strong>for</strong> stabilization of<br />
DHPs in <strong>the</strong> L-type calcium ch<strong>an</strong>nel pore (Tikhonov <strong>an</strong>d Zhorov, J. Biol. Chem., accepted May
<strong>2009</strong>). We have been exploring three key residue differences of <strong>the</strong> eighteen contacts uncovered<br />
by modeling that may be critical <strong>for</strong> <strong>the</strong> DHP-insensitivity of <strong>the</strong> snail LCav1 ch<strong>an</strong>nel. These<br />
residues include one residue ch<strong>an</strong>ge in each of tr<strong>an</strong>smembr<strong>an</strong>e helices segments 5 <strong>an</strong>d 6 of<br />
Domain III <strong>an</strong>d in segment 6 of Domain IV.<br />
We also report mollusk-hunting cone snail toxins which bear sensitivity <strong>for</strong> L-type calcium<br />
ch<strong>an</strong>nels. Recombin<strong>an</strong>t conotoxin have been engineered in bacteria <strong>for</strong> exploration of <strong>the</strong><br />
structural features of <strong>the</strong> toxin responsible <strong>for</strong> drug block. Exploration of <strong>the</strong> L-type ch<strong>an</strong>nel<br />
pharmacophore using a number of different structures will assist in <strong>the</strong> design of a new<br />
generation of L-type ch<strong>an</strong>nel blockers. This research is supported by <strong>the</strong> Heart <strong>an</strong>d Stroke<br />
Foundation of Ontario, Gr<strong>an</strong>t-In-Aid NA 6284.<br />
Disclosures: A. Senatore, None; S. Lam, None; J. Spaf<strong>for</strong>d, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.17/D10<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t NS08174<br />
Title: Differential modulation of high voltage-activated Ca 2+ ch<strong>an</strong>nels by membr<strong>an</strong>e<br />
phosphatidylinositol 4,5-bisphosphate (PIP2)<br />
Authors: B. C. SUH, *B. HILLE;<br />
Physiol. & Biophysics, Univ. Washington Sch. Med., Seattle, WA<br />
Abstract: Membr<strong>an</strong>e lipid phosphatidylinositol 4,5-bisphosphate (PIP2) molecules are believed<br />
to be <strong>an</strong> import<strong>an</strong>t cofactor <strong>for</strong> <strong>the</strong> function of m<strong>an</strong>y ion ch<strong>an</strong>nels, including voltage-gated Ca 2+<br />
(Cav) ch<strong>an</strong>nels. Previous studies showed that, in inside-out excised patches of Cav2.1 (P/Q-type)<br />
<strong>an</strong>d Cav2.2 (N-type) ch<strong>an</strong>nels, <strong>the</strong> application of DiC8-PIP2 or Mg-ATP to <strong>the</strong> inner side of<br />
membr<strong>an</strong>e attenuates <strong>the</strong> rundown of Ca 2+ currents, whereas <strong>the</strong> application of PIP2 <strong>an</strong>tibody<br />
accelerates ch<strong>an</strong>nel closure (J. Y<strong>an</strong>g <strong>an</strong>d M.S. Shapiro labs). However, in intact cells, it is not<br />
clearly established whe<strong>the</strong>r a loss of PIP2 is sufficient <strong>for</strong> Cav ch<strong>an</strong>nel inhibition. Moreover, little<br />
is known about <strong>the</strong> electrophysiological properties of N-type ch<strong>an</strong>nel modulation by PIP2<br />
turnover, such as <strong>the</strong> kinetics <strong>for</strong> inhibition/recovery <strong>an</strong>d activation/inactivation of ch<strong>an</strong>nels, <strong>an</strong>d<br />
<strong>the</strong> functions of PIP2 in L-type Cav1 ch<strong>an</strong>nel modulation. Here we used two methods to deplete<br />
PIP2 in living cell membr<strong>an</strong>e within seconds without activating PLC or producing <strong>an</strong>y second
messengers. One is <strong>the</strong> chemically modified rapamycin (iRap)-induced protein dimerization<br />
scheme (Lyn11-FRB <strong>an</strong>d CFP-FKBP) that rapidly tr<strong>an</strong>slocates a C-terminal truncated, soluble<br />
PIP2 specific 5-phosphatase (Inp54p) to <strong>the</strong> plasma membr<strong>an</strong>e. The o<strong>the</strong>r is <strong>the</strong> membr<strong>an</strong>e<br />
protein, voltage-sensitive phosphatase (Dr-VSP), which removes 5-phosphate <strong>from</strong> PIP2 in<br />
response to membr<strong>an</strong>e depolarization. With both techniques, we found that PIP2 depletion in <strong>the</strong><br />
plasma membr<strong>an</strong>e triggers <strong>the</strong> inhibition of Cav1.2 <strong>an</strong>d Cav1.3 ch<strong>an</strong>nels by ~35% <strong>an</strong>d Cav2.2<br />
ch<strong>an</strong>nels by ~55%, without signific<strong>an</strong>t ch<strong>an</strong>ges in <strong>the</strong> voltage-activated ch<strong>an</strong>nel gating. The<br />
current depression by iRap/Inp54p is irreversible <strong>an</strong>d produces voltage-independent inhibition.<br />
With Dr-VSP, we found that <strong>the</strong> Cav ch<strong>an</strong>nel inhibition <strong>an</strong>d recovery are tightly controlled by<br />
membr<strong>an</strong>e PIP2. The activation of Dr-VSP by pulses to 120 mV decreased <strong>the</strong> membr<strong>an</strong>e PIP2<br />
<strong>an</strong>d inhibited <strong>the</strong> Cav currents simult<strong>an</strong>eously, both with τ = 100 - 150 ms. The Cav2.2 ch<strong>an</strong>nel<br />
inhibition was Gβγ-independent. PIP2 depletion speeded <strong>the</strong> inactivation of Cav2.2 currents seen<br />
with longer test pulses. Recovery of Ca 2+ currents <strong>from</strong> <strong>the</strong> Dr-VSP-induced inhibition takes 8<br />
<strong>an</strong>d 17 s <strong>for</strong> Cav1.3 <strong>an</strong>d Cav2.2 ch<strong>an</strong>nels, respectively, <strong>an</strong>d requires intracellular hydrolysable<br />
ATP <strong>an</strong>d PIP2 resyn<strong>the</strong>sis. Toge<strong>the</strong>r, <strong>the</strong>se results suggest that L- <strong>an</strong>d N-type Ca 2+ ch<strong>an</strong>nels need<br />
PIP2 <strong>for</strong> full activation, but are differentially regulated by <strong>the</strong> depletion of membr<strong>an</strong>e PIP2. Our<br />
data suggest that <strong>the</strong> N-type Cav2.2 ch<strong>an</strong>nels enter inactive states when <strong>the</strong> PIP2 level goes down<br />
<strong>an</strong>d thus <strong>the</strong> recovery <strong>from</strong> inactivation becomes slower th<strong>an</strong> <strong>the</strong> PIP2 resyn<strong>the</strong>sis.<br />
Disclosures: B.C. Suh, None; B. Hille, NIH gr<strong>an</strong>t NS08174, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.18/D11<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: AHFMR<br />
HSF<br />
CIHR<br />
Title: Sc<strong>an</strong>ning mutagenesis identifies residues Arg376 <strong>an</strong>d Val416 in <strong>the</strong> N-type calcium<br />
ch<strong>an</strong>nel I-II linker as import<strong>an</strong>t sites <strong>for</strong> voltage-dependent G protein inhibition
Authors: *L. B. VIEIRA 1 , W. TEDFORD 2 , L. CHEN 2 , A. E. KISILEVSKY 2 , G. W.<br />
ZAMPONI 2 ;<br />
1 2<br />
Univ. Federal de Minas Gerais, Belo Horizonte, Brazil; Dept. of Physiol. <strong>an</strong>d Pharmacol.,<br />
Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: The direct interaction of <strong>the</strong> N-type calcium ch<strong>an</strong>nel with <strong>the</strong> G protein beta gamma<br />
subunit mediates voltage-dependent inhibition, crucially contributing to <strong>an</strong>algesia induced by<br />
opioid drugs. Previous work by our <strong>an</strong>d o<strong>the</strong>r laboratories has identified <strong>the</strong> domain I-II linker<br />
region of <strong>the</strong> ch<strong>an</strong>nel as a key site <strong>for</strong> G protein inhibition, revealing that <strong>the</strong>re are two distinct<br />
regions within <strong>the</strong> I-II linker that are capable of interacting with G beta gamma. Here, using<br />
al<strong>an</strong>ine mutagenesis within <strong>the</strong>se two regions, we identify residues Arg376 <strong>an</strong>d Val416 as<br />
import<strong>an</strong>t sites <strong>for</strong> G protein action.To fur<strong>the</strong>r characterize <strong>the</strong> molecular determin<strong>an</strong>ts of this<br />
interaction, we per<strong>for</strong>med secondary mutagenesis of residues 376 <strong>an</strong>d 416, <strong>an</strong>d coexpressed <strong>the</strong><br />
resulting mut<strong>an</strong>ts with wild type G beta1 gamma2 subunits, <strong>an</strong>d with several iso<strong>for</strong>ms of <strong>the</strong><br />
auxiliary beta subunit of <strong>the</strong> N-type ch<strong>an</strong>nel.Voltage-depend<strong>an</strong>t, G protein-mediated inhibition of<br />
<strong>the</strong> ch<strong>an</strong>nels was assessed using patch clamp recordings. The results i) confirm that Arg376, a<br />
residue which is surface-exposed in <strong>the</strong> presence of <strong>the</strong> calcium ch<strong>an</strong>nel beta subunit, contributes<br />
signific<strong>an</strong>tly to modulatory interaction with G beta 1,<strong>an</strong>d ii) suggest that G protein-mediated<br />
inhibition involves complex interactions that depend in part on residue Arg376 <strong>an</strong>d on <strong>the</strong><br />
calcium ch<strong>an</strong>nel beta subunit iso<strong>for</strong>m.<br />
Disclosures: L.B. Vieira, None; W. Ted<strong>for</strong>d, None; L. Chen, None; A.E. Kisilevsky,<br />
None; G.W. Zamponi, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.19/D12<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: Wellcome Trust Project Gr<strong>an</strong>t H503 6200<br />
Title: Probing <strong>the</strong> molecular determin<strong>an</strong>ts of G protein beta gamma subunit-mediated<br />
modulation of presynaptic calcium ch<strong>an</strong>nels in superior cervical g<strong>an</strong>glion neurons<br />
Authors: *G. BUCCI 1 , S. MOCHIDA 2 , G. STEPHENS 1 ;<br />
1 Reading Univ., Reading, United Kingdom; 2 Tokyo Med. Univ., Tokyo, Jap<strong>an</strong>
Abstract: A domin<strong>an</strong>t <strong>for</strong>m of presynaptic inhibition of neurotr<strong>an</strong>smitter release is mediated by<br />
<strong>the</strong> binding of neurotr<strong>an</strong>smitters to G protein-coupled receptors (GPCRs). A key underlying<br />
mech<strong>an</strong>ism is <strong>the</strong> interaction between G protein beta gamma subunits <strong>an</strong>d voltage-dependent<br />
calcium (Ca 2+ ) ch<strong>an</strong>nels. Several G beta gamma interaction sites have been identified on <strong>the</strong><br />
presynaptic CaV2.2 Ca 2+ ch<strong>an</strong>nel subunit, including sites on <strong>the</strong> I-II loop <strong>an</strong>d <strong>the</strong> amino terminal.<br />
We have shown that presynaptic inhibition of tr<strong>an</strong>smitter release in superior cervical g<strong>an</strong>glion<br />
neurones (SCGNs) is mediated by G beta gamma subunits (Stephens & Mochida, 2005). To<br />
investigate <strong>the</strong> specific molecular determin<strong>an</strong>ts <strong>for</strong> G protein inhibition of Ca 2+ ch<strong>an</strong>nels, we<br />
have studied <strong>the</strong> effects of peptides based on G beta gamma/Ca 2+ ch<strong>an</strong>nel interaction sites on<br />
synaptic tr<strong>an</strong>smission in SCGNs.<br />
SCGNs maintained in long-term culture <strong>for</strong>m synaptically-coupled cells amenable to <strong>the</strong> study of<br />
presynaptic protein function (Ma & Mochida, 2007). Using dual microelectrode recording, we<br />
injected current into <strong>the</strong> presynaptic cell to evoke acetylcholine-mediated excitatory postsynaptic<br />
potentials (EPSPs). Presynaptic injection of peptides based on <strong>the</strong> alpha interaction domain of<br />
<strong>the</strong> CaV2.2 I-II loop (AID peptide) caused a decrease in EPSP amplitude (100 microM: 16 ± 8 %,<br />
n = 5; 1 mM: 36 ± 11 %, n = 6). Similarly, <strong>an</strong> amino terminal peptide (NT peptide) caused a<br />
decrease in EPSP amplitude (1 mM: 24 ± 8 %, n = 6). A scrambled AID peptide showed no clear<br />
ch<strong>an</strong>ge in EPSP amplitude.<br />
These data suggest that <strong>the</strong> CaV2.2 amino terminal <strong>an</strong>d I-II loop, previously shown to interact<br />
with G beta gamma subunits, contribute molecular determin<strong>an</strong>ts involved in modulation of<br />
neurotr<strong>an</strong>smitter release in SCGNs.<br />
Disclosures: G. Bucci, None; S. Mochida, None; G. Stephens, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.20/D13<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t NS29967<br />
Sidney Fr<strong>an</strong>k Predoctoral Fellowship<br />
Title: G protein-coupled receptor inhibition of N-type calcium ch<strong>an</strong>nel splice-vari<strong>an</strong>ts<br />
Authors: *C. G. PHILLIPS, D. LIPSCOMBE;<br />
Brown Univ., Providence, RI
Abstract: M<strong>an</strong>y G protein-coupled receptors (GPCRs) modulate neurotr<strong>an</strong>smitter release by<br />
inhibiting presynaptic CaV2.2 N-type calcium ch<strong>an</strong>nels. However, <strong>the</strong> molecular rules governing<br />
tissue-specific coupling of GPCRs to N-type ch<strong>an</strong>nels are not fully understood. We have shown<br />
that cell-specific expression of particular CaV2.2 alternative exons c<strong>an</strong> control <strong>the</strong> coupling of<br />
GPCRs to N-type ch<strong>an</strong>nels (Raingo et al., 2007, Nature Neuroscience). This suggests that <strong>the</strong><br />
receptor-ch<strong>an</strong>nel interaction is specified by <strong>the</strong> target ch<strong>an</strong>nel. Here we explore how patterns of<br />
N-type ch<strong>an</strong>nel inhibition are influenced by receptor identity. We show that <strong>the</strong> relative levels of<br />
voltage-dependent (VD) <strong>an</strong>d voltage-independent (VI) inhibition vary with GPCR subtype. The<br />
<strong>an</strong>giotensin II receptor type 1 (AT1R), <strong>the</strong> muscarinic receptor type 1 (M1R) <strong>an</strong>d <strong>the</strong> bradykinin<br />
receptor type 2 (B2R) all couple to Gq G proteins, <strong>an</strong>d inhibit <strong>the</strong> N-type ch<strong>an</strong>nel in neurons. We<br />
studied inhibition of N-type currents by co-expressing M1R, B2R, or AT1R with N-type<br />
ch<strong>an</strong>nels in tsA201 cells. We found that M1R <strong>an</strong>d AT1R inhibit <strong>the</strong> ch<strong>an</strong>nel in a mostly VI<br />
m<strong>an</strong>ner, while <strong>the</strong> B2R inhibits <strong>the</strong> ch<strong>an</strong>nel in a strongly VD m<strong>an</strong>ner. These two <strong>for</strong>ms of<br />
inhibition use distinct signaling pathways. Our data <strong>the</strong>n suggest that although all of <strong>the</strong>se<br />
receptors depend on Gq, <strong>the</strong>y differentially activate downstream pathways. We have also<br />
explored <strong>the</strong> coupling of <strong>the</strong>se GPCRs to different splice-iso<strong>for</strong>ms of <strong>the</strong> N-type ch<strong>an</strong>nel.<br />
Specifically, we have studied <strong>the</strong> effect of inclusion of <strong>the</strong> cassette exon 18a (e18a) in <strong>the</strong><br />
intracellular II-III loop of <strong>the</strong> ch<strong>an</strong>nel. We previously showed that inclusion of e18a renders <strong>the</strong><br />
N-type ch<strong>an</strong>nel susceptible to GTPγS-induced, PTX-insensitive, VI inhibition (Raingo, Phillips,<br />
<strong>an</strong>d Lipscombe, poster 783.11/G5, SfN 2007). We hypo<strong>the</strong>sized that Gq activation was<br />
responsible <strong>for</strong> this inhibition. However, when we tested this hypo<strong>the</strong>sis using Gq-coupled M1R<br />
<strong>an</strong>d AT1R, we found that <strong>the</strong>y both mediate VI inhibition of N-type currents independent of e18a<br />
inclusion. We <strong>the</strong>re<strong>for</strong>e speculated that GTPγS might inhibit N-type currents by activating Gs G<br />
proteins. Our preliminary data suggest that Gs G proteins are in fact responsible <strong>for</strong> <strong>the</strong><br />
inhibition: both RGS2 <strong>an</strong>d cholera toxin occlude <strong>the</strong> GTPγS-induced VI inhibition of e18acontaining<br />
N-type ch<strong>an</strong>nels. Overall, we conclude that <strong>the</strong> inhibitory signaling between GPCRs<br />
<strong>an</strong>d N-type calcium ch<strong>an</strong>nels is controlled both by specific receptor characteristics <strong>an</strong>d by <strong>the</strong><br />
alternative splicing of ch<strong>an</strong>nel pre-mRNA.<br />
Disclosures: C.G. Phillips, None; D. Lipscombe, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.21/D14<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: UNAM-DGAPA-PAPIIT
CONACyT<br />
Title: Gating charges per ch<strong>an</strong>nel of CaV2.2 ch<strong>an</strong>nels are modified by G protein activation in rat<br />
sympa<strong>the</strong>tic neurons<br />
Authors: S. REBOLLEDO-ANTÚNEZ, J. M. FARÍAS, I. ARENAS, *D. E. GARCIA-DIAZ;<br />
Univ. Nacional Autonoma Mexico, Mexico DF 04510, Mexico<br />
Abstract: It has been suggested that voltage-dependent G protein modulation of CaV2.2 ch<strong>an</strong>nels<br />
is carried out at closed states of <strong>the</strong> ch<strong>an</strong>nel. Our purpose was to estimate <strong>the</strong> number of gating<br />
charges of CaV2.2 ch<strong>an</strong>nel in control <strong>an</strong>d G protein-modulated conditions. By using a Cole-<br />
Moore protocol we observed a signific<strong>an</strong>t delay in CaV2.2 ch<strong>an</strong>nel activation according to a<br />
tr<strong>an</strong>sit of <strong>the</strong> ch<strong>an</strong>nel through a series of closed states be<strong>for</strong>e ch<strong>an</strong>nel opening. If G protein<br />
voltage-dependent modulation were carried out at <strong>the</strong>se closed states, <strong>the</strong>n we would have<br />
expected a greater Cole-Moore lag in <strong>the</strong> presence of a neurotr<strong>an</strong>smitter. This prediction was<br />
confirmed <strong>for</strong> noradrenaline, while no ch<strong>an</strong>ge was observed in <strong>the</strong> presence of <strong>an</strong>giotensin II, a<br />
voltage-insensitive G protein modulator. We used <strong>the</strong> limiting slope method <strong>for</strong> calculation of<br />
<strong>the</strong> gating charge per ch<strong>an</strong>nel. Effective charge z was 6.32+/-0.65 <strong>for</strong> CaV2.2 ch<strong>an</strong>nels in<br />
unmodulated conditions (facilitated currents), while gu<strong>an</strong>osine 5'-O-[gamma-thio]triphosphate<br />
(GTPγS) reduced elementary charge by approximately 4 e0. Accordingly, increased<br />
concentration of noradrenaline induced a gradual decrease on z in a reversible m<strong>an</strong>ner, indicating<br />
that this decrement was due to a G protein voltage-sensitive modulation. This work shows <strong>for</strong> <strong>the</strong><br />
first time a signific<strong>an</strong>t <strong>an</strong>d reversible decrease in charge tr<strong>an</strong>sfer of CaV2.2 ch<strong>an</strong>nels under G<br />
protein modulation, which might depend on <strong>the</strong> activated G protein inhibitory pathway.<br />
Disclosures: S. Rebolledo-Antúnez, None; J.M. Farías, None; I. Arenas, None; D.E. Garcia-<br />
Diaz, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.22/D15<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH R01NS043394<br />
AHA 0755071Y
Title: Role of phosphatidylinositol (PI) kinase stimulation in modulation of N-type Ca 2+<br />
ch<strong>an</strong>nels by Gq/11-coupled receptors<br />
Authors: O. ZAIKA, J. ZHANG, *M. S. SHAPIRO;<br />
Univ. Texas Hlth. Sci. Ctr., S<strong>an</strong> Antonio, TX<br />
Abstract: We hypo<strong>the</strong>size specificity in regulation of N-type Ca 2+ ch<strong>an</strong>nels by Gq/11-coupled<br />
receptors in superior cervical g<strong>an</strong>glion (SCG) sympa<strong>the</strong>tic neurons to arise <strong>from</strong> receptorspecific<br />
stimulation of PI4- <strong>an</strong>d PI(4)5-kinases that compensates <strong>for</strong> <strong>an</strong>y reduction of [PIP2] by<br />
phospholipase C activity. One pathway involves acceleration of PI4-kinases by Ca 2+ -bound<br />
neuronal calcium sensor-1 (NCS-1), However, NCS-1 has been shown to act specifically via <strong>the</strong><br />
PI4-kinase IIIβ iso<strong>for</strong>m, which has been localized not to <strong>the</strong> plasma membr<strong>an</strong>e, but to <strong>the</strong> Golgi<br />
(Balla <strong>an</strong>d Balla, Trends Cell Biol 16:351-61). We tested <strong>the</strong> specific PI4-kinase IIIβ blocker,<br />
PIK93 (Toth et al., JBC, 281: 36369-77) on suppression of ICa in SCG neurons by three types of<br />
receptors under per<strong>for</strong>ated-patch voltage clamp. Whereas <strong>the</strong>re was no effect of PIK93 (300 nM)<br />
on muscarinic suppression of ICa, <strong>the</strong> drug bestowed upon bradykinin B2 <strong>an</strong>d purinergic P2Y<br />
receptors robust suppression of ICa, similar to <strong>the</strong> effect of <strong>the</strong> broad-spectrum PI4-K inhibitor<br />
wortm<strong>an</strong>nin previously reported. Our modeling also suggests that receptor-specific stimulation<br />
of PI(4)5-kinase must also be involved, possibly via phosphatidic acid that is produced<br />
downstream of diacylglycerol (DAG) by DAG-kinase. Indeed, application of DAG-kinase<br />
inhibitor II (Calbiochem, 20 µM, 1 hr pre-treatment) likewise bestowed suppression of ICa by<br />
P2Y <strong>an</strong>d B2-receptor agonists, whereas stimulation of nei<strong>the</strong>r receptor normally alters ICa in <strong>the</strong>se<br />
neurons (Zaika et al., J. Neurosci., 27: 8914-8926). Thus, both tests implicate stimulation of PI4-<br />
<strong>an</strong>d PI(4)-5 kinases in receptor specificity towards modulation of N-type Ca 2+ ch<strong>an</strong>nels.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> experiments with PIK93 suggest <strong>the</strong> PI4-kinase IIIβ iso<strong>for</strong>m to be specifically<br />
involved. We are currently using <strong>the</strong> PI(4)P-specific probe, OSH2T-PH, to localize <strong>the</strong> site of<br />
PIP syn<strong>the</strong>sis during receptor stimulation. Supported by NIH gr<strong>an</strong>t RO1 NS043394 <strong>an</strong>d by <strong>an</strong><br />
Americ<strong>an</strong> Heart Association gr<strong>an</strong>t in aid 0755071Y to M.S.S.<br />
Disclosures: O. Zaika, None; J. Zh<strong>an</strong>g, None; M.S. Shapiro, None.<br />
Poster<br />
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.23/D16<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t AG09796
US-Israel Binational Sci Fnd #2005370<br />
NIH Gr<strong>an</strong>t MH071316<br />
NAAR<br />
NARSAD<br />
AHA 0825719G<br />
Title: CaV1.3 protein expression level is signific<strong>an</strong>tly reduced in CA1 region after trace eyeblink<br />
conditioning<br />
Authors: F. L. NUNEZ-SANTANA, M. M. OH, D. P. SRIVASTAVA, P. PENZES, *J. F.<br />
DISTERHOFT;<br />
Northwestern Univ. Med. Sch., Chicago, IL<br />
Abstract: L-type Ca 2+ ch<strong>an</strong>nels are a signific<strong>an</strong>t source of Ca 2+ that activates <strong>the</strong> slow postburst<br />
afterhyperpolarization (AHP), which is mediated by a yet to be discovered Ca 2+ -dependent K +<br />
ch<strong>an</strong>nel (Power et al., J Neurosci 2002). Import<strong>an</strong>tly, <strong>the</strong> slow AHP in CA1 pyramidal neurons<br />
has been repeatedly shown to be reduced in hippocampal slices <strong>from</strong> <strong>an</strong>imals that have<br />
successfully learned a hippocampus-dependent task (Disterhoft & Oh, TiNs 2006). Thus, we<br />
examined <strong>the</strong> potential learning-related alterations in <strong>the</strong> expression level of CaV1.3 subunits that<br />
comprise <strong>the</strong> L-type ch<strong>an</strong>nels.<br />
Young adult male Fischer 344 x Brown Norway rats (3 mo) were r<strong>an</strong>domly assigned to one of 3<br />
groups: trained, pseudoconditioned (pseudo), <strong>an</strong>d naïve. The trained group received 30 paired<br />
tone CS <strong>an</strong>d corneal airpuff US separated by a 250ms trace interval. Pseudo rats were given 30<br />
CS <strong>an</strong>d 30 US in <strong>an</strong> unpaired m<strong>an</strong>ner. Both groups had 5 training sessions (2sessions/day).<br />
Hippocampal slices were cut in ice-cold aCSF 1 d after <strong>the</strong> last training session using a<br />
vibratome. The CA1 region was isolated <strong>from</strong> <strong>the</strong> hippocampal slice, <strong>an</strong>d CaV1.3 L-type Ca 2+<br />
ch<strong>an</strong>nel protein levels were determined by western blotting. Here we report a decrease in CaV1.3<br />
levels in CA1 region <strong>from</strong> rats that learned as compared to naïve <strong>an</strong>d pseudo rats. These data<br />
strongly suggest that <strong>the</strong> reduction in <strong>the</strong> amount of CaV1.3 is one source of <strong>the</strong> learning-related<br />
AHP reduction observed in CA1 neurons after trace EBC. Previously we have reported a<br />
signific<strong>an</strong>t reduction in CaV1.2 levels in <strong>the</strong> CA1 region after trace eyeblink conditioning (Oh et<br />
al., SFN Abstract 2008). Thus, <strong>the</strong>se data suggest that <strong>the</strong> learning-related postburst AHP<br />
reductions in CA1 neurons may in part be mediated by <strong>the</strong> reductions in <strong>the</strong> CaV1.2 <strong>an</strong>d CaV1.3<br />
subunits that comprise <strong>the</strong> L-type Ca 2+ ch<strong>an</strong>nels.<br />
Disclosures: F.L. Nunez-S<strong>an</strong>t<strong>an</strong>a, None; M.M. Oh, None; D.P. Srivastava, None; P. Penzes,<br />
None; J.F. Disterhoft, None.<br />
Poster
519. Voltage-Dependent Calcium Ch<strong>an</strong>nel Modulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 519.24/D17<br />
Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />
Support: NIH gr<strong>an</strong>t NS23022<br />
NIH gr<strong>an</strong>t NS33091<br />
Postdoctoral Fellowship <strong>from</strong> <strong>the</strong> National Multiple Sclerosis <strong>Society</strong>, FG1723<br />
Title: Regulation of calcium entry into oligodendrocytes by <strong>the</strong> golli products of <strong>the</strong> myelin<br />
basic protein gene<br />
Authors: *P. M. PAEZ, D. J. FULTON, V. SPREUR, V. HANDLEY, C. W. CAMPAGNONI,<br />
A. T. CAMPAGNONI;<br />
Semel Inst. <strong>for</strong> Neurosci, UCLA, Los Angeles, CA<br />
Abstract: Genetic ablation of golli, a product of <strong>the</strong> myelin basic protein gene, results in severe<br />
hypomyelination, which appears to result in part <strong>from</strong> a disruption of Ca ++ homeostasis in<br />
oligodendrocytes (OLs). Cell membr<strong>an</strong>e depolarization by high K + activates voltage operated<br />
Ca ++ ch<strong>an</strong>nels (VOCCs), while depletion of intracellular stores triggers influx via store operated<br />
Ca ++ ch<strong>an</strong>nels (SOCCs). To characterize <strong>the</strong> mech<strong>an</strong>isms of Ca ++ influx regulated by golli we<br />
examined VOCC <strong>an</strong>d SOCC entry in isolated OL precursor cells (OPCs) <strong>an</strong>d acute brain slice<br />
preparations <strong>from</strong> golli knock-out (KO) <strong>an</strong>d J37-golli overexpressing (JOE) mice. Initially, we<br />
examined <strong>the</strong> effect of golli on VOCCs. Both, KO <strong>an</strong>d JOE cells exhibited Ca ++ signaling<br />
following stimulation with high K + , but <strong>the</strong>se Ca ++ signals were signific<strong>an</strong>tly weaker in KO cells,<br />
<strong>an</strong>d signific<strong>an</strong>tly stronger in JOE cells. This effect was abolished in <strong>the</strong> presence of zero<br />
[Ca ++ ]ext, <strong>an</strong>d by verapamil <strong>an</strong>d nifedipine confirming that this rise in [Ca ++ ]int involves influx via<br />
L-type VOCC. Fur<strong>the</strong>rmore, this phenomenon disappeared with OPC differentiation, indicating<br />
that golli modulates VOCC in a developmentally regulated fashion. To examine SOCC activity,<br />
intracellular Ca ++ stores were depleted by thapsigargin in <strong>the</strong> absence of extracellular Ca ++ .<br />
Under <strong>the</strong>se conditions we found that Ca ++ uptake following Ca ++ re-addition were signific<strong>an</strong>tly<br />
stronger in golli-overexpressing cells. This increased Ca ++ response was sensitive to SOCCs<br />
<strong>an</strong>tagonists 2-APB <strong>an</strong>d MRS-1845 <strong>an</strong>d using a specific <strong>an</strong>tibody against TRPC1 (Tr<strong>an</strong>sient<br />
Receptor Potential C<strong>an</strong>onical Type 1) we were able to block <strong>the</strong> golli effect on store-operated<br />
Ca ++ influx. Toge<strong>the</strong>r our data indicate a key role <strong>for</strong> golli protein in <strong>the</strong> regulation of both<br />
VOCC- <strong>an</strong>d SOCC-mediated Ca ++ influx, a finding that that is of signific<strong>an</strong>ce <strong>for</strong> regulation of<br />
multiple processes in oligodendroglial cells.
Disclosures: P.M. Paez, None; D.J. Fulton, None; V. Spreur, None; V. H<strong>an</strong>dley, None; C.W.<br />
Campagnoni, None; A.T. Campagnoni, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.1/D18<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Title: Aβ1-42-induced supression of BK ch<strong>an</strong>nel is counteracted by Homer 1a expression in<br />
neocortical pyramidal neurons<br />
Authors: *K. YAMAMOTO 1,2 , Y. UETA 2 , R. YAMAMOTO 2 , N. INOUE 3 , K. INOKUCHI 3 ,<br />
N. KATO 2 ;<br />
1 Dept. of Neurol., Ut<strong>an</strong>o Natl. Hosp., Kyoto, Jap<strong>an</strong>; 2 Dept. of Phisiology, K<strong>an</strong>azawa Med. Univ.,<br />
Uchinada, Jap<strong>an</strong>; 3 Mitsubishi Kagaku Inst. of Life Sci., Machida, Jap<strong>an</strong><br />
Abstract: Recent studies have indicated that intracellular amyloid-β (Aβ), prior to accumulating<br />
extracellularly, c<strong>an</strong> induce cognitive defect in <strong>the</strong> early stage of Alzheimer disease (AD), but its<br />
pathophysiological basis remained to be examined. Here we report <strong>the</strong> functional signific<strong>an</strong>ce of<br />
intracellular Aβ by applying Aβ protein into rat <strong>an</strong>d mouse neocortical pyramidal neurons<br />
through whole-cell patch pipettes. Intracellular Aβ1-42, but not Aβ1-40, broadened spike width <strong>an</strong>d<br />
augmented Ca 2+ influx via voltage-dependent Ca 2+ ch<strong>an</strong>nel in rat neocortical neurons. However,<br />
this class of ch<strong>an</strong>nel turned out to elude direct modulation by Aβ1-42. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d,<br />
charybdotoxin, a blocker of large-conduct<strong>an</strong>ce Ca 2+ -activated K + (BK) ch<strong>an</strong>nel, mimicked <strong>an</strong>d<br />
occluded <strong>the</strong>se effects of Aβ1-42. Moreover, isopimaric acid, a BK ch<strong>an</strong>nel opener, <strong>an</strong>d<br />
electroconvulsive stimulation (ECS), which we previously showed to facilitate BK ch<strong>an</strong>nel<br />
opening via Homer 1a/Vesl-1S expression, blocked <strong>the</strong> effects of Aβ1-42. These results suggest<br />
that intracellular Aβ1-42 caused <strong>the</strong> suppression of BK ch<strong>an</strong>nel <strong>an</strong>d Homer 1a may counteract Aβinduced<br />
suppression of this class of ch<strong>an</strong>nel. To test this hypo<strong>the</strong>sis, we examined <strong>the</strong> actions of<br />
intracellular Aβ in wild-type mice, Homer 1a knockout mice <strong>an</strong>d 3xTg AD model mice. Both<br />
Aβ1-42-injected neurons of wild-type mice <strong>an</strong>d neurons of 3xTg mice at 4 months of age, which<br />
are reported to have intracellular Aβ without extracellular Aβ accumulation, showed spike width<br />
broadening <strong>an</strong>d attenuation of spike peak amplitude during spike trains. Just as in <strong>the</strong> wild rat,<br />
charybdotoxin mimicked <strong>an</strong>d occluded <strong>the</strong>se actions of Aβ1-42, <strong>an</strong>d both isopimaric acid <strong>an</strong>d ECS<br />
blocked <strong>the</strong> actions of Aβ1-42 in <strong>the</strong> wild mice. Consistent data were obtained <strong>from</strong> <strong>the</strong> 3xTg mice<br />
with intracellular Aβ overexpression. By contrast, Homer 1a knockout mice failed to show <strong>the</strong><br />
same blocking effects of ECS on Aβ1-42. Thus, we showed that suppression of BK ch<strong>an</strong>nel by
intracellular Aβ1-42, which we suppose is a possible mech<strong>an</strong>ism <strong>for</strong> early dysfunction in <strong>the</strong> AD<br />
brain, is counteracted by activity-dependent expression of Homer 1a during ECS.<br />
(Acknowledgement: 3xTg mice have been kindly provided by Dr LaFerla, University of<br />
Cali<strong>for</strong>nia, Irvine.)<br />
Disclosures: K. Yamamoto, None; Y. Ueta, None; R. Yamamoto, None; N. Inoue, None; K.<br />
Inokuchi, None; N. Kato, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.2/D19<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Director’s Pioneer Award<br />
NIH AG9900<br />
Title: Splice vari<strong>an</strong>t-specific intron retention facilitates <strong>the</strong> structural diversity of BKCa ch<strong>an</strong>nel<br />
mRNAs <strong>an</strong>d proteins<br />
Authors: *T. J. BELL 1 , K. Y. MIYASHIRO 1 , P. T. BUCKLEY 1,2 , J. EBERWINE 1,2 ;<br />
1 Dept Pharmacol, Univ. Pennsylv<strong>an</strong>ia Sch. Med., Philadelphia, PA; 2 Penn Genome Frontiers<br />
Inst., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Neuronal dendrites <strong>an</strong>d cytoplasm have <strong>the</strong> capacity to splice retained introns <strong>from</strong><br />
cytoplasmically localized RNAs (Gl<strong>an</strong>zer et al., 2005). In a recent study <strong>from</strong> our lab, we<br />
reported a functional role <strong>for</strong> a retained intron in BKCa ch<strong>an</strong>nel mRNAs (also called introncontaining<br />
BKCa mRNAs) that were located in <strong>the</strong> cytoplasm of hippocampal neurons (Bell et<br />
al., 2008). Here, we <strong>an</strong>alyzed <strong>the</strong> subcellular localization, alternative splicing patterns <strong>an</strong>d<br />
protein products of two intron-containing BKCa ch<strong>an</strong>nel mRNAs in hippocampal neurons. In<br />
situ hybridization (ISH) <strong>an</strong>alysis was used to identify <strong>the</strong> subcellular localization of each introncontaining<br />
mRNA. The ISH signals of each retained intron extended <strong>from</strong> <strong>the</strong> cell body into <strong>the</strong><br />
distal dendrites, however, <strong>the</strong>ir distribution patterns were distinct. One intron-containing mRNA<br />
showed distinct puncta <strong>an</strong>d clustering along <strong>the</strong> dendrites. In contrast, <strong>the</strong> o<strong>the</strong>r intron-containing<br />
mRNA showed a more even distribution pattern throughout <strong>the</strong> dendrites. The alternative<br />
splicing patterns of each retained intron were determined by MALDI-TOF MS base extension.<br />
Remarkably, each retained intron was limited to a subset of <strong>the</strong> BKCa ch<strong>an</strong>nel mRNA splice
vari<strong>an</strong>ts. There<strong>for</strong>e, <strong>the</strong> alternative splicing of intron-containing BKCa ch<strong>an</strong>nel mRNAs is a<br />
highly regulated process. Next, by combining siRNA-treatment <strong>an</strong>d immunostaining with splice<br />
vari<strong>an</strong>t-specific <strong>an</strong>tibodies, we determined whe<strong>the</strong>r each intron-containing mRNA could indeed<br />
generate <strong>the</strong>ir respective splice vari<strong>an</strong>t proteins. In agreement with our splicing <strong>an</strong>alysis, <strong>the</strong><br />
selective reduction of each intron-containing mRNA by siRNA-treatment reduced <strong>the</strong> levels of<br />
BKCa ch<strong>an</strong>nel protein in splice vari<strong>an</strong>t-dependent m<strong>an</strong>ner. These results show retained introns<br />
play a role in generating <strong>the</strong> splice vari<strong>an</strong>t populations of BKCa ch<strong>an</strong>nel mRNAs <strong>an</strong>d proteins in<br />
neurons. Analogous to BKCa ch<strong>an</strong>nel splice vari<strong>an</strong>t regulation by different 3’-untr<strong>an</strong>slated<br />
regions (Pietrzykowski et. el., 2008), retained introns in BKCa ch<strong>an</strong>nel mRNAs could per<strong>for</strong>m a<br />
similar role. Their restricted splicing linkages allow <strong>the</strong>m to selectively regulate a small number<br />
of BKCa ch<strong>an</strong>nel splice vari<strong>an</strong>ts. This is <strong>the</strong> first report showing that both intron retention <strong>an</strong>d<br />
alternative splicing contribute to <strong>the</strong> structural diversity of mRNAs <strong>an</strong>d proteins in neurons. This<br />
mech<strong>an</strong>ism provides a new layer of post-tr<strong>an</strong>scriptional control that regulates <strong>the</strong> functional<br />
complexity of neurons. Fur<strong>the</strong>r, it appears as though intron retention is a general mech<strong>an</strong>ism<br />
utilized by neurons to influence synaptic tr<strong>an</strong>smission <strong>an</strong>d plasticity.<br />
Disclosures: T.J. Bell, None; K.Y. Miyashiro, None; P.T. Buckley, None; J. Eberwine, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.3/D20<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: NIH GM066215<br />
Title: Modification of BK ch<strong>an</strong>nels by n<strong>an</strong>omolar concentrations of Ag+<br />
Authors: Y. ZHOU 1 , X.-M. XIA 1 , *C. J. LINGLE 2 ;<br />
1 Anes<strong>the</strong>siol., 2 Washington Univ. Schl Med., St. Louis, MO<br />
Abstract: BK ch<strong>an</strong>nels (Slo1) are found in a variety of cell types <strong>an</strong>d participate in m<strong>an</strong>y<br />
import<strong>an</strong>t body functions. Here we examined <strong>the</strong> effects of silver, a widely distributed element,<br />
on wild type BK ch<strong>an</strong>nels. The results show that Ag+ strongly inhibits BK ch<strong>an</strong>nel activity at<br />
n<strong>an</strong>omolar concentrations by modifying <strong>an</strong> intracellular cysteine. The modification is<br />
independent of membr<strong>an</strong>e potential <strong>an</strong>d is not delayed by BK ch<strong>an</strong>nel pore block. The results<br />
indicate that <strong>the</strong> target <strong>for</strong> modification is a cytosolically accessible residue in <strong>the</strong> C-terminus or<br />
<strong>the</strong> intracellular linkers connecting tr<strong>an</strong>smembr<strong>an</strong>e segments, but not those in <strong>the</strong> tr<strong>an</strong>smembr<strong>an</strong>e
segments. This key cysteine is inaccessible to <strong>the</strong> thiolate-reagent MTSET since modification by<br />
Ag+ is insensitive to pre-treatment with MTSET <strong>an</strong>d a mutation C430S that removes <strong>the</strong> MTS<br />
reagent sensitivity of BK ch<strong>an</strong>nels. As <strong>an</strong> attempt to determine <strong>the</strong> key cysteine, we generated a<br />
construct with 16 cysteine residues modified to serine which retained gating properties similar to<br />
wild type BK ch<strong>an</strong>nels, but with much lower Ag+ sensitivity (Ag+ modification rate was<br />
decreased by about 20-fold). This construct is of potential use as a background ch<strong>an</strong>nel <strong>for</strong><br />
accessibility studies in <strong>the</strong> narrow regions of <strong>the</strong> BK ch<strong>an</strong>nel pore that only c<strong>an</strong> be accessed by a<br />
small thiolate-reagent, such as Ag+.<br />
Disclosures: Y. Zhou, None; X. Xia, None; C.J. Lingle, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.4/D21<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: HFSP gr<strong>an</strong>t (RGP0049/2002) to L.J.GRAHAM<br />
Agence Nationale pour la Recherche, gr<strong>an</strong>t (FUNVISYNIN) to L.J.GRAHAM<br />
Title: The precise timing of a BK-like hyperpolarizing current modulates <strong>the</strong> neuronal tr<strong>an</strong>sfer<br />
function to current <strong>an</strong>d visual stimuli in vivo - An experimental/<strong>the</strong>oretical study<br />
Authors: *A. SCHRAMM, D. MARINAZZO, L. J. GRAHAM;<br />
Lab. of Neurophysics <strong>an</strong>d Physiology, CNRS, Univ. Paris Descartes, Paris, Fr<strong>an</strong>ce<br />
Abstract: Potassium currents are usually considered as counteracting neuronal excitation<br />
because of <strong>the</strong>ir hyperpolarizing action. Never<strong>the</strong>less, simulations show (Borg-Graham, 1987,<br />
1999) that <strong>the</strong> voltage <strong>an</strong>d Ca++ dependent BK current (I_BK) may act in <strong>the</strong> opposite way :<br />
because of its size <strong>an</strong>d fast kinetics I_BK tends to dominate o<strong>the</strong>r K+ currents as well as<br />
accelerating repolarization to allow <strong>the</strong> neuron to fire a subsequent spike sooner. This prediction<br />
has been supported by in vitro studies (Gu et al. 2007) <strong>an</strong>d we have now confirmed it with in<br />
vivo dynamic clamp recordings in rat <strong>an</strong>d cat cortex, under both artificial (current steps) <strong>an</strong>d<br />
functional (visual) stimuli. Based on this non-intuitive role <strong>for</strong> I_BK, we asked how details of<br />
such a tr<strong>an</strong>sient current could affect <strong>the</strong> neuron's input-output tr<strong>an</strong>sfer function. We are<br />
specifically interested in how <strong>the</strong> precise timing of this current during a spike affects <strong>the</strong> overall<br />
output, reminiscent to a phase-response curve but on a fine time scale. Thus, <strong>for</strong> a given input,
<strong>the</strong> prediction is that a BK-like current triggered just after spike initiation inhibits firing <strong>an</strong>d<br />
consequently increases <strong>the</strong> threshold input (e.g. rheobase). <strong>When</strong> a BK-like current is triggered<br />
just after <strong>the</strong> spike peak, <strong>the</strong> normal time <strong>for</strong> I_BK, spike output is increased, as mentioned<br />
above. For trigger times between <strong>the</strong>se two moments, <strong>the</strong> overall firing rate will be reduced. This<br />
last point is supported by our in vivo dynamic clamp experiments with shunting inhibition<br />
conduct<strong>an</strong>ce steps, which in part injects a brief hyperpolarizing current during <strong>the</strong> spike that is<br />
similar to but occurs earlier th<strong>an</strong> I_BK. Finally, <strong>for</strong> even more delayed trigger times, <strong>the</strong><br />
hyperpolarization due to <strong>the</strong> BK-like current increases <strong>the</strong> refractory period, thus again reducing<br />
<strong>the</strong> firing rate. In summary, our <strong>the</strong>oretical <strong>an</strong>d experimental results show how I_BK, or<br />
<strong>an</strong>alogous hyperpolarizing currents, c<strong>an</strong> act as gain <strong>an</strong>d offset controls <strong>for</strong> <strong>the</strong> input-output<br />
tr<strong>an</strong>sfer function. In particular our results suggest a novel role <strong>for</strong> I_BK in shaping <strong>the</strong> neural<br />
code during tr<strong>an</strong>sient responses that are domin<strong>an</strong>t in natural vision.<br />
Disclosures: A. Schramm, None; D. Marinazzo, None; L.J. Graham, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.5/D22<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Title: Investigation of BK currents produced by <strong>the</strong> gain-of-function mutation R207Q<br />
Authors: J. R. MONTGOMERY, *A. MEREDITH;<br />
Dept. of Physiol., Univ. Maryl<strong>an</strong>d Sch. Med., Baltimore, MD<br />
Abstract: The large conduct<strong>an</strong>ce Ca 2+ - activated BK K + ch<strong>an</strong>nel plays <strong>an</strong> import<strong>an</strong>t role in a<br />
variety of excitable <strong>an</strong>d non-excitable tissues in mouse. We have previously shown that BK<br />
ch<strong>an</strong>nels are essential regulators of <strong>the</strong> circadi<strong>an</strong> rhythm in neuronal activity in <strong>the</strong> brain’s<br />
internal clock, <strong>the</strong> suprachiasmatic nucleus (SCN). BK ch<strong>an</strong>nels shape <strong>the</strong> daily oscillation in
SCN firing rate by suppressing spont<strong>an</strong>eous action potentials at night, when BK expression is <strong>the</strong><br />
highest in SCN neurons. Loss of BK ch<strong>an</strong>nels prolongs repolarization, reduces<br />
afterhyperpolarizations, <strong>an</strong>d increases <strong>the</strong> active:silent neuron ratio. In this study, to fur<strong>the</strong>r<br />
underst<strong>an</strong>d how BK ch<strong>an</strong>nels shape circadi<strong>an</strong> rhythms in neuronal activity, we made a tr<strong>an</strong>sgenic<br />
mouse expressing a gain-of-function mut<strong>an</strong>t of <strong>the</strong> BK ch<strong>an</strong>nel, R207Q, under <strong>the</strong> control of 6.8<br />
kb of regulatory sequence <strong>from</strong> <strong>the</strong> mPer1 locus. Tr<strong>an</strong>sgenic mice (Per-R207Q) express higher<br />
levels of BK ch<strong>an</strong>nels in <strong>the</strong> SCN during <strong>the</strong> day compared to wild-type (WT). In order to<br />
underst<strong>an</strong>d <strong>the</strong> cellular mech<strong>an</strong>isms underlying effects on excitability in Per-R207Q SCNs, <strong>the</strong><br />
properties of R207Q ch<strong>an</strong>nels were examined in a heterologous expression system. The R207Q<br />
mutation is located in <strong>the</strong> S4 voltage-sensing region of <strong>the</strong> ch<strong>an</strong>nel, <strong>an</strong>d previous studies in<br />
oocytes showed that <strong>the</strong> R207Q mutation increased open probability <strong>an</strong>d shifted <strong>the</strong> voltagedependence<br />
of activation towards more negative potentials compared to WT (Diaz et al, JBC,<br />
1998). However, little is known about <strong>the</strong> properties of R207Q currents in mammali<strong>an</strong> cells<br />
under physiological conditions. There<strong>for</strong>e, we per<strong>for</strong>med a characterization of WT (mBR5) <strong>an</strong>d<br />
R207Q BK ch<strong>an</strong>nels expressed in HEK cells. Compared to WT, macroscopic R207Q currents<br />
were shifted in <strong>the</strong>ir voltage-dependence of activation to lower voltages, currents could be<br />
evoked at nominal concentrations of intracellular Ca 2+ , <strong>an</strong>d activation was faster at all voltages.<br />
These data show that R207Q retains gain-of-function properties in HEK cells under<br />
physiological conditions <strong>an</strong>d suggests that Per-R207Q SCN neurons will have hyperactive BK<br />
currents.<br />
Disclosures: J.R. Montgomery, None; A. Meredith, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.6/D23<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: Louisi<strong>an</strong>a BORSF<br />
NINDS Gr<strong>an</strong>t NS049427<br />
Title: Modulation of <strong>the</strong> BK-mediated fahp by Phosphoinositol-3 kinase<br />
Authors: *S. J. SPRINGER 1,2 , L. A. SCHRADER 3,1 ;<br />
2 Neurosci. Program, 3 Cell <strong>an</strong>d Mol. Biol., 1 Tul<strong>an</strong>e Univ., New Orle<strong>an</strong>s, LA
Abstract: Modulation of ion ch<strong>an</strong>nels <strong>an</strong>d <strong>the</strong> currents that <strong>the</strong>y underlie by kinase activation<br />
c<strong>an</strong> be a powerful mech<strong>an</strong>ism to regulate neuronal excitability. Regulation of <strong>the</strong><br />
afterhyperpolarization (AHP) has been described in multiple inst<strong>an</strong>ces of plasticity, such as<br />
learning <strong>an</strong>d memory-dependent ch<strong>an</strong>ges <strong>an</strong>d also in pathological states such as epilepsy.<br />
Previous studies have shown that activation of phosphoinositol-3 kinase (PI3K) modulates BK<br />
ch<strong>an</strong>nels in cultured neurons. In this study we characterized IAHP in adult <strong>an</strong>imals <strong>an</strong>d<br />
investigated regulation of <strong>the</strong> IAHP by PI3K. We used whole-cell patch clamp electrophysiology<br />
in CA1 pyramidal cells in slices <strong>from</strong> adult mice to study modulation of IAHP by PI3K. Cells<br />
included in <strong>the</strong> study had a resting membr<strong>an</strong>e potential less th<strong>an</strong> or equal to -60 <strong>an</strong>d <strong>an</strong> input<br />
resist<strong>an</strong>ce between 100 <strong>an</strong>d 300 megaohms. Access resist<strong>an</strong>ce was less th<strong>an</strong> 20 megaohms <strong>an</strong>d<br />
did not ch<strong>an</strong>ge more th<strong>an</strong> 15% over <strong>the</strong> course of <strong>the</strong> recording. Depolarizing pulses of<br />
increasing duration (20 to 1000 ms) resulted in increased amplitude <strong>an</strong>d duration of <strong>the</strong> AHP.<br />
Short pulses (20 - 100 ms) yielded a tail current that was blocked by <strong>the</strong> M current blocker<br />
linopirdine. This suggests that this current is mediated by KCNQ ch<strong>an</strong>nels as previously shown.<br />
Longer pulses (100 -1000 ms) elicited a current that was reduced 60-70% by blockade of basal<br />
PI3K. This effect was mimicked by <strong>the</strong> BK ch<strong>an</strong>nel blockers paxilline (10 uM) <strong>an</strong>d iberiotoxin<br />
(10 uM). Fur<strong>the</strong>rmore, <strong>the</strong> reduction of <strong>the</strong> AHP by paxilline was occluded by prior incubation<br />
with wortm<strong>an</strong>nin. These results show that basal activation of PI3K is necessary <strong>for</strong> <strong>the</strong><br />
mainten<strong>an</strong>ce of BK ch<strong>an</strong>nel activity in CA1 pyramidal cells. Future studies will investigate <strong>the</strong><br />
ch<strong>an</strong>nels that underlie IAHP in young <strong>an</strong>imals <strong>an</strong>d regulation by PI3K.<br />
Disclosures: S.J. Springer, None; L.A. Schrader, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.7/D24<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: CIHR<br />
Ontario Neurotrauma Foundation<br />
Heart <strong>an</strong>d Stroke Foundation of C<strong>an</strong>ada<br />
Title: Molecular <strong>an</strong>d electrophysiological evidence <strong>for</strong> <strong>the</strong> expression of BK ch<strong>an</strong>nels in<br />
oligodendroglial lineage cells
Authors: *J. A. BUTTIGIEG, E. EFTEKHARPOUR, S. KARIMI-ABDOLREZAEE, M.<br />
FEHLINGS;<br />
Toronto Western Hospital, Univ. Hlth. Network, Toronto, ON, C<strong>an</strong>ada<br />
Abstract: A common defining feature of glial cells has been <strong>the</strong>ir inability to generate action<br />
potentials. However, recent evidence suggests that oligodendroglial precursor cells (OPC) may<br />
express voltage-gated sodium <strong>an</strong>d potassium ch<strong>an</strong>nels, <strong>an</strong>d generate spont<strong>an</strong>eous action<br />
potentials. If this is indeed <strong>the</strong> case, some mech<strong>an</strong>isms must be put into action to control Ca 2+<br />
influx, <strong>an</strong>d stop or dampen <strong>the</strong> physiological effects of <strong>the</strong> ensuing increase in cytoplasmic Ca 2+ .<br />
In m<strong>an</strong>y cases, this is accomplished by one of <strong>the</strong> most broadly expressed K + ch<strong>an</strong>nels in<br />
mammals: <strong>the</strong> large-conduct<strong>an</strong>ce, voltage- <strong>an</strong>d Ca 2+ -activated K + ch<strong>an</strong>nel, also known as <strong>the</strong><br />
Maxi K + (or BK) ch<strong>an</strong>nel. Here we investigated whe<strong>the</strong>r cells of <strong>the</strong> oligodendroglial lineage do<br />
indeed express BK ch<strong>an</strong>nels <strong>an</strong>d what potential role <strong>the</strong>y may play in <strong>the</strong> normal physiology of<br />
<strong>the</strong>se cells. We found that in oligodendrocytes derived <strong>from</strong> differentiated adult neural precursor<br />
cells (APC) (in vitro) <strong>an</strong>d endogenous oligodendrocytes of <strong>the</strong> spinal cord (in situ) express<br />
outward currents that were sensitive to <strong>the</strong> BK ch<strong>an</strong>nel specific blocker iberiotoxin (IbTx).<br />
Expression of this IbTx sensitive current was observed in both precursor cells (PDGFRα<br />
positive) <strong>an</strong>d mature oligodendrocytes (CNPase positive) as determined via<br />
immunocytochemistry. Western blot <strong>an</strong>alysis <strong>an</strong>d electrophysiological difference currents<br />
suggest expression levels of <strong>the</strong> BK ch<strong>an</strong>nel decreases with developmental age, indicating a<br />
possible role of this ch<strong>an</strong>nel in <strong>the</strong> maturation of OPCs. Of <strong>the</strong> possible pre-mRNA splice<br />
vari<strong>an</strong>ts, it appears that <strong>the</strong> predomin<strong>an</strong>t splice iso<strong>for</strong>m is <strong>the</strong> stress regulated exon (STREX),<br />
which is known to lead to cell depolarization <strong>an</strong>d Ca 2+ influx during ischemia, in o<strong>the</strong>r cell<br />
system. The presence of <strong>the</strong> STREX BK ch<strong>an</strong>nel may explain, in part, why oligodendrocytes are<br />
acutely sensitive to ischemia, <strong>an</strong>d may lead to a possible pharmacological intervention in <strong>the</strong><br />
treatment of spinal cord injury or stroke.<br />
Disclosures: J.A. Buttigieg, None; E. Eftekharpour, None; S. Karimi-Abdolrezaee,<br />
None; M. Fehlings, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.8/D25<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Title: Inhibition of BK ch<strong>an</strong>nel by ASIC1a ch<strong>an</strong>nel <strong>an</strong>d ASIC1a peptide
Authors: *E. PETROFF;<br />
Biol. <strong>an</strong>d Mol. Biol., Montclair State Univ., Montclair, NJ<br />
Abstract: Acid Sensing Ion Ch<strong>an</strong>nels (ASICs) contain a sequence that is identical to scorpion<br />
toxins blocking potassium ch<strong>an</strong>nels, <strong>an</strong>d this sequence is critical <strong>for</strong> ASICs’ inhibition of large<br />
conduct<strong>an</strong>ce calcium- <strong>an</strong>d voltage-activated potassium ch<strong>an</strong>nels (BK), <strong>an</strong>d voltage-activated<br />
potassium ch<strong>an</strong>nels (Kv) (Petroff et al., PNAS 2008, 118(4), 1571). A 14 amino acid long<br />
peptide containing <strong>the</strong> ASIC1a toxin-like sequence also reduces BK current amplitude. ASIC<br />
peptide inhibition of BK ch<strong>an</strong>nel expressed in stably tr<strong>an</strong>sfected HEK293 cells was reversible<br />
<strong>an</strong>d dose-dependent. A known BK ch<strong>an</strong>nel blocker tetraethylammonium (TEA) competed with<br />
<strong>the</strong> peptide <strong>for</strong> BK ch<strong>an</strong>nel block. Similarly, competition between TEA <strong>an</strong>d ASIC1a ch<strong>an</strong>nels<br />
expressed in <strong>the</strong> cells with BK was also observed. Extracellular K + is known to reduce<br />
iberiotoxin <strong>an</strong>d charybdotoxin block of potassium ch<strong>an</strong>nels. Raising <strong>the</strong> extracellular K + <strong>an</strong>d Rb +<br />
concentrations, but not Na + , Cs + , Li + or Ca 2+ , reduced BK inhibition by <strong>the</strong> ASIC1a ch<strong>an</strong>nel <strong>an</strong>d<br />
by <strong>the</strong> ASIC1a peptide. In addition, <strong>the</strong> ASIC1a ch<strong>an</strong>nel <strong>an</strong>d <strong>the</strong> ASIC1a peptide inhibition of<br />
BK ch<strong>an</strong>nels was voltage-dependent only in <strong>the</strong> presence of intracellular K + . These results<br />
indicate that ASIC1a peptide, <strong>an</strong>d ASIC1a ch<strong>an</strong>nel interact with <strong>the</strong> K + conduction pathway in<br />
<strong>the</strong> BK ch<strong>an</strong>nel <strong>an</strong>d may act as a BK ch<strong>an</strong>nel blocker.<br />
Disclosures: E. Petroff, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.9/D26<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t R37 DK-37963 to DCE<br />
Title: A putative arachidonic acid binding site in BK-ch<strong>an</strong>nels<br />
Authors: *D. DENSON 1 , J. LI 2 , O. AL-KHALILI 2 , D. C. EATON 2 ;<br />
1 Anes<strong>the</strong>siol., 2 Physiol., Emory Univ. Sch. Med., Atl<strong>an</strong>ta, GA<br />
Abstract: Like m<strong>an</strong>y o<strong>the</strong>r potassium ch<strong>an</strong>nels, BK ch<strong>an</strong>nels occur in different cells as m<strong>an</strong>y<br />
different splice vari<strong>an</strong>ts. Altering <strong>the</strong> splice vari<strong>an</strong>t composition of BK ch<strong>an</strong>nels c<strong>an</strong> alter <strong>the</strong>ir<br />
activity <strong>an</strong>d apparent sensitivity to calcium <strong>an</strong>d o<strong>the</strong>r regulators of activity. For example, BK<br />
ch<strong>an</strong>nels in GH3 cells are sensitive to arachidonic acid (AA), but BK ch<strong>an</strong>nels in a sub-clone of
GH3 cells, GH4-C1, are not. We have reported that a 27 amino acid difference in <strong>the</strong> C terminus<br />
of <strong>the</strong> α-subunit of BK ch<strong>an</strong>nels in GH3 <strong>an</strong>d GH4 cells is responsible <strong>for</strong> <strong>the</strong> different<br />
responsiveness to AA. In this work we used a stable CHO cell line expressing ei<strong>the</strong>r rSlo(27)<br />
with or rSlo(0) without <strong>the</strong> 27 amino acid motif, (obtained <strong>from</strong> C. Park) to test <strong>the</strong> hypo<strong>the</strong>sis<br />
that AA would bind to <strong>the</strong> cells expressing rSlo(27) but not to <strong>the</strong> cells expressing rSlo(0). T-75<br />
culture flasks containing untreated CHO cells, CHO cells expressing rSlo(0) or CHO cells<br />
expressing rSlo(27) were allowed to grow to 80-90% confluency. Cells were lysed <strong>an</strong>d incubated<br />
with <strong>the</strong> appropriate primary <strong>an</strong>tibody overnight in <strong>the</strong> cold .After allowing <strong>the</strong> lysates to mix<br />
with <strong>the</strong> protein A beads <strong>for</strong> 1 hr, <strong>the</strong> mixtures were centrifuged <strong>an</strong>d <strong>the</strong> supernat<strong>an</strong>ts removed.<br />
Each set of beads was exposed to PBS containing 1% BSA <strong>an</strong>d 3 H-AA (10µCi/mL) <strong>for</strong> 15min.<br />
Each of <strong>the</strong> tubes was centrifuged, supernat<strong>an</strong>ts removed <strong>an</strong>d replaced with ‘cold’ PBS<br />
containing 1% BSA. Each mixture was gently mixed <strong>for</strong> 15min, centrifuged, <strong>the</strong> supernat<strong>an</strong>t<br />
removed, counted <strong>an</strong>d ‘cold’ buffer added. This procedure was done at 15, 30, 60, 120 <strong>an</strong>d 180<br />
min. After removal of <strong>the</strong> supernat<strong>an</strong>t at <strong>the</strong> 3hr time point, <strong>the</strong> beads were dispersed in 0.1 mL<br />
of cold buffer, removed <strong>an</strong>d counted. The rates <strong>an</strong>d half-lives of unbinding were determined <strong>for</strong><br />
each condition using nonlinear iterative regression. Estimates of <strong>the</strong> individual half-lives <strong>for</strong> each<br />
of <strong>the</strong> five replicates <strong>for</strong> each of <strong>the</strong> three conditions were compared using <strong>an</strong> ANOVA followed<br />
by a Holm-Sidak multiple comparison test. The me<strong>an</strong> ½ lives (± SD) <strong>for</strong> binding of AA to<br />
rSlo(27), rSlo(0) <strong>an</strong>d GAPDH (Control) were 0.89±0.15, 0.55±0.09 <strong>an</strong>d 0.52±0.11 hr,<br />
respectively. The unbinding half-life <strong>for</strong> rSlo(27) was signific<strong>an</strong>tly longer (p
DNASTAR, Inc<br />
Title: Differential tr<strong>an</strong>script regulation of <strong>the</strong> calcium-activated potassium ion ch<strong>an</strong>nel in flight<br />
muscle <strong>an</strong>d <strong>the</strong> CNS by <strong>the</strong> steroid hormone ecdysone<br />
Authors: *S. L. GARRISON, J. L. WITTEN;<br />
Biol Sci., Univ. Wisconsin, Milwaukee, WI<br />
Abstract: The large conduct<strong>an</strong>ce calcium-activated, voltage-gated potassium ion ch<strong>an</strong>nel, BK or<br />
slowpoke, plays a central role cell excitability. One mech<strong>an</strong>ism to modify tissue excitability<br />
during development is to alter ch<strong>an</strong>nel expression. The hawkmoth, M<strong>an</strong>duca sexta, is <strong>an</strong><br />
adv<strong>an</strong>tageous model system to study slowpoke (msslo) tr<strong>an</strong>script regulation. During late adult<br />
development, msslo mRNA levels are abruptly upregulated in <strong>the</strong> dorsal longitudinal flight<br />
muscles (DLMs) <strong>an</strong>d a smaller, signific<strong>an</strong>t upregulation in <strong>the</strong> CNS. The upregulation in both<br />
tissues temporally correlates to <strong>the</strong> decline in steroid hormone (ecdysteroid) titers. We propose<br />
that msslo tr<strong>an</strong>scription is repressed by <strong>an</strong> ecdysteroid gene regulatory cascade <strong>an</strong>d tr<strong>an</strong>script<br />
levels are upregulated when titers decline. To test this, we delayed titer decline by locally<br />
injecting ecdysteroids to create heterochronic mosaic pupae. Tr<strong>an</strong>script levels were qu<strong>an</strong>tified<br />
using qPCR <strong>an</strong>d fold ch<strong>an</strong>ge calculated between treatment groups. Statistical <strong>an</strong>alysis was<br />
per<strong>for</strong>med using one-way ANOVA <strong>an</strong>d Tukey’s post-hoc test.<br />
Ecdysteroid regulation of msslo tr<strong>an</strong>script levels requires that a receptor complex must be<br />
present. Iso<strong>for</strong>m specific receptor complexes consist of <strong>the</strong> ecdysteroid receptor (EcR) <strong>an</strong>d<br />
ultraspiracle protein (USP), each heterodimeric pair member with two iso<strong>for</strong>ms. Our results<br />
reveal that receptor complex member tr<strong>an</strong>script levels are present in both <strong>the</strong> DLMs <strong>an</strong>d CNS at<br />
times when msslo mRNA levels are ch<strong>an</strong>ging. This suggests that <strong>an</strong> ecdysteroid receptor<br />
complex is present <strong>an</strong>d may be involved in gene regulation during mid- to late- adult<br />
development.<br />
Ecdysteroids appear to differentially regulate msslo tr<strong>an</strong>script levels in <strong>the</strong> DLMs <strong>an</strong>d CNS. In<br />
<strong>the</strong> DLMs, ecdysteroid treated <strong>an</strong>imals have signific<strong>an</strong>tly lower msslo tr<strong>an</strong>script expression<br />
levels th<strong>an</strong> non-injected <strong>an</strong>d saline-injected controls (p0.05)<br />
. To fur<strong>the</strong>r support our argument that ecdysteroids act to repress msslo levels in <strong>the</strong> DLMs, local<br />
injection of ecdysteroids when msslo expression was high appeared to cause a rapid <strong>an</strong>d<br />
signific<strong>an</strong>t drop in levels (p
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.11/D28<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: F.R.S-FNRS (Belgium) Gr<strong>an</strong>t 9.4560.03<br />
Belgi<strong>an</strong> Science Policy Gr<strong>an</strong>t IAP 6/31<br />
Title: An apamin-sensitive SK current underlies <strong>the</strong> medium duration afterhyperpolarization in<br />
dorsal raphe neurons<br />
Authors: P. ALIX, *J. J. MOREAU, V. SEUTIN;<br />
Univ. Liege, Liege, Belgium<br />
Abstract: The Dorsal Raphe (DR) nucleus is implicated in various physiological functions<br />
(affect, memory <strong>an</strong>d learning...) <strong>an</strong>d has possible pathophysiological roles in depression <strong>an</strong>d<br />
Alzheimer's disease. A majority of DR neurons are serotonergic <strong>an</strong>d have two<br />
electrophysiological characteristics: a long duration (2 ms) action potential due to <strong>the</strong> presence of<br />
a shoulder on its falling phase <strong>an</strong>d a prominent medium-duration afterhyperpolarization (mAHP)<br />
following <strong>the</strong> action potential. Moreover, recent in vivo experiments suggest that SK ch<strong>an</strong>nel<br />
blockade increases bursting in serotonergic neurons of <strong>the</strong> DR. The purpose of our study was<br />
<strong>the</strong>re<strong>for</strong>e to identify <strong>the</strong> current(s) responsible <strong>for</strong> <strong>the</strong> mAHP in <strong>the</strong>se neurons. We used wholecell<br />
recordings of infra-red visualised neurons in acute slices <strong>an</strong>d elicited <strong>an</strong>d recorded AHP<br />
currents under voltage-clamp conditions with repetitive short (20 ms) unclamped depolarizations<br />
(holding <strong>an</strong>d recording potential, -60 mV; steps to -10 to +100 mV. After pulses to +100 mV, a<br />
putative mAHP current appeared as <strong>an</strong> outward current peaking at ~100 ms after <strong>the</strong> depolarizing<br />
pulse. This current had kinetics comparable with IAHP. It was sensitive to supramaximal<br />
concentrations of two SK ch<strong>an</strong>nel blockers: apamin (300 nM) <strong>an</strong>d (-)-bicuculline methiodide<br />
(BMI; 100 µM). The effect of BMI on <strong>the</strong> mAHP was reversible <strong>an</strong>d mimicked by a subsequent<br />
application of apamin. An inward current was unmasked by <strong>the</strong>se two agents as previously<br />
observed in hippocampal neurons. This inward current was sensitive to cobalt (1 mM).<br />
Moreover, cobalt also blocked <strong>the</strong> outward currents generated in <strong>the</strong> absence of BMI <strong>an</strong>d<br />
apamin. Block of <strong>the</strong> mAHP by apamin, BMI <strong>an</strong>d cobalt suggests that, in <strong>the</strong>se neurons, <strong>the</strong><br />
mAHP is induced by SK ch<strong>an</strong>nel activation following <strong>the</strong> influx of Ca 2+ via voltage-depend<strong>an</strong>t<br />
Ca 2+ ch<strong>an</strong>nels.<br />
Disclosures: P. Alix, None; J.J. Moreau, None; V. Seutin, None.<br />
Poster
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.12/D29<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: 3.4566.03 (F.R.S.-FNRS)<br />
IAP P6/31 (Belgi<strong>an</strong> Science Policy)<br />
Title: The SK blocker AG525E1 modulates bursting in subgroups of midbrain dopamine<br />
neurons in awake rats<br />
Authors: S. KOULCHITSKY 1 , *J.-F. LIEGEOIS 2 , V. SEUTIN 1 ;<br />
1 2<br />
Univ. of Liège GIGA Neurosciences-Pharmacology, Liège, Belgium; Univ. Liège, Liège,<br />
Belgium<br />
Abstract: Small conduct<strong>an</strong>ce, Ca 2+ -activated K + ch<strong>an</strong>nels (SK ch<strong>an</strong>nels) are known to underlie<br />
<strong>the</strong> medium duration afterhyperpolarization in various types of CNS neurons. Reduction of SK<br />
currents should enh<strong>an</strong>ce bursting. Indeed, previous experiments of our group demonstrated that<br />
local SK ch<strong>an</strong>nel blockade increases bursting in dopaminergic (DA) <strong>an</strong>d serotonergic, but not<br />
noradrenergic neurons in vivo in <strong>an</strong>aes<strong>the</strong>tized rats. In <strong>the</strong> present study, we recorded <strong>from</strong><br />
midbrain dopaminergic neurons in awake rats using a telemetric system (Alpha Omega, Israel).<br />
Male Wistar rats (250-350 g) were impl<strong>an</strong>ted with microelectrode arrays (8 electrodes per<br />
<strong>an</strong>imal) in <strong>the</strong> ventral tegmental area. Neural activity was recorded in four sessions: one baseline<br />
session, without <strong>an</strong>y experimental m<strong>an</strong>ipulation, <strong>an</strong>d three sessions in which <strong>the</strong> rats received<br />
intraperitoneal injections of saline, 100 µg/kg of D2 agonist quinpirole or 10 mg/kg of <strong>the</strong> novel<br />
tertiary SK blocker AG525E1. In total, 30 neurons were recorded. Based on <strong>the</strong> spike duration<br />
(>1.5 ms) <strong>an</strong>d reaction to <strong>the</strong> systemic injection of D2 agonist quinpirole (>50% decrease of<br />
firing rate within 15 min after <strong>the</strong> injection), 21 out of 30 units were assumed to be DA. Their<br />
average baseline activity varied between 0.1 <strong>an</strong>d 7 Hz, with most of <strong>the</strong> units firing between 0.1<br />
<strong>an</strong>d 2 Hz. In control conditions, presumed DA neurons exhibited mostly irregular tonic firing<br />
with some bursting periods. Injection of saline did not lead to <strong>an</strong>y signific<strong>an</strong>t ch<strong>an</strong>ge in firing<br />
rate or pattern in <strong>an</strong>y of <strong>the</strong> recorded neurons. In DA neurons, <strong>the</strong> effect of AG525E1 correlated<br />
with <strong>the</strong> initial firing rate <strong>an</strong>d percentage of spikes in bursts. Indeed, 11 units with a low fraction<br />
of spikes in bursts (8 ± 3 %) <strong>an</strong>d firing rate of 0.45 ± 0.04 Hz underwent <strong>an</strong> increase in bursting<br />
activity up to 26 ± 5 % of spikes in bursts <strong>an</strong>d <strong>an</strong> increase in <strong>the</strong> firing rate to 0.93 ± 0.05 Hz.<br />
Units with a high baseline fraction of spikes in bursts (44 ± 7 %) <strong>an</strong>d firing rate of 1.85 ± 0.13<br />
Hz, demonstrated decrease in <strong>the</strong> percentage of spikes in bursts down to 23 ± 5 %, <strong>an</strong>d slight<br />
increase in firing rate up to 2.13 ± 0.13 Hz (n=10). The effects of AG525E1 were reversible 40-<br />
45 min after <strong>the</strong> injection. In 9 presumed non-DA neurons, nei<strong>the</strong>r quinpirole nor AG525E1 had<br />
<strong>an</strong>y signific<strong>an</strong>t effect.
Additional experiments are needed to check that <strong>the</strong> increase in bursting produced by AG525E1<br />
in a subgroup of DA neurons is due specifically to its blockade of SK ch<strong>an</strong>nels. The mech<strong>an</strong>ism<br />
of <strong>the</strong> opposite effect that is seen in <strong>the</strong> o<strong>the</strong>r subgroup remains to be investigated.<br />
Disclosures: S. Koulchitsky, None; J. Liegeois, None; V. Seutin, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.13/D30<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: F.R.S.-FNRS (Belgium) gr<strong>an</strong>t 3.4566.03<br />
Belgi<strong>an</strong> Science Policy IAP Gr<strong>an</strong>t P6/31<br />
Title: A hypo<strong>the</strong>sis to explain <strong>the</strong> effect of <strong>the</strong> SK blocker apamin on <strong>the</strong> firing of dopaminergic<br />
neurons in vitro<br />
Authors: G. DRION, A. COLLARD, *V. M. SEUTIN, R. SEPULCHRE;<br />
Univ. Liege, Sart Tilm<strong>an</strong> / Liege, Belgium<br />
Abstract: Midbrain dopaminergic (DA) neurons profoundly influence behavior by promulgating<br />
a reward signal in critical striatal <strong>an</strong>d <strong>for</strong>ebrain structures. In physiological conditions, <strong>the</strong>se cells<br />
c<strong>an</strong> switch between tonic, irregular, <strong>an</strong>d burst firing. The latter is a critical component of <strong>the</strong><br />
signalling.<br />
It has been shown that <strong>an</strong> inhibition of a calcium-activated potassium current (carried by SK<br />
ch<strong>an</strong>nels) in vitro induces irregularity in <strong>the</strong> firing pattern of DA neurons, whereas <strong>an</strong> IK,SK<br />
inhibition in vivo facilitates burst firing in <strong>the</strong>se cells. This suggests that SK ch<strong>an</strong>nels act as a<br />
protection against <strong>the</strong> noise in vitro, <strong>an</strong>d reduce <strong>the</strong> sensitivity of <strong>the</strong> neuron to <strong>the</strong> synaptic<br />
afferents in vivo.<br />
In <strong>the</strong> most detailed model of DA neurons, which was developed by C<strong>an</strong>avier’s group, setting<br />
<strong>the</strong> SK conduct<strong>an</strong>ce to 0 does not induce irregularity in <strong>the</strong> absence of synaptic inputs. We<br />
<strong>the</strong>re<strong>for</strong>e hypo<strong>the</strong>sized that noise <strong>from</strong> ion ch<strong>an</strong>nels or o<strong>the</strong>r sources could contribute to <strong>the</strong><br />
effect which is observed experimentally in vitro. The noise was modeled through a Poisson<br />
stochastic process. It was applied ei<strong>the</strong>r on <strong>the</strong> global ionic current or on <strong>the</strong> intracellular<br />
concentration of calcium.<br />
The application of a noise on <strong>the</strong> global ionic current only induced irregularity during blockade
of SK ch<strong>an</strong>nels <strong>an</strong>d had little effect on <strong>the</strong> firing frequency. Stochastic variations of [Ca 2+ ]i<br />
strongly affected <strong>the</strong> firing frequency both when SK ch<strong>an</strong>nels were present <strong>an</strong>d absent. These<br />
variations had variable effects on <strong>the</strong> firing pattern. Based on <strong>the</strong> similarities between <strong>the</strong> effect<br />
of apamin <strong>an</strong>d of noise induced in <strong>the</strong> ionic current, <strong>the</strong>se simulations suggest that <strong>the</strong><br />
irregularities in <strong>the</strong> firing rate induced by apamin in vitro could be generated by stochastic<br />
activation of ion ch<strong>an</strong>nels. IK,SK would oppose <strong>the</strong> irregularity of firing induced by this noise.<br />
Disclosures: G. Drion, None; A. Collard, None; V.M. Seutin, None; R. Sepulchre, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.14/D31<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: NIH Gr<strong>an</strong>t R21<br />
NS 055896<br />
Title: Regulation of excitability in spinal dorsal horn neurons by calcium-dependent potassium<br />
conduct<strong>an</strong>ces<br />
Authors: *K. YANG, S. M. THOMPSON;<br />
Dept. of Physiol., Univ. of Maryl<strong>an</strong>d, Baltimore, Baltimore, MD<br />
Abstract: Neuronal excitability is defined as action potential output <strong>from</strong> a given input signal<br />
<strong>an</strong>d/or spont<strong>an</strong>eous spike bursting. A principal determin<strong>an</strong>t of neuronal excitability is <strong>the</strong><br />
afterhyperpolarizaiton (AHP) that follows individual action potentials <strong>an</strong>d action potential bursts.<br />
The AHP is mediated by K + ch<strong>an</strong>nels opened in response to elevated intracellular Ca 2+ . The<br />
excitability of spinal cord dorsal horn neurons plays a pivotal role in nociceptive in<strong>for</strong>mation<br />
modulation <strong>an</strong>d tr<strong>an</strong>smission <strong>from</strong> periphery to central. Small <strong>an</strong>d intermediate conduct<strong>an</strong>ce<br />
Ca 2+ -activated K + (SK <strong>an</strong>d IK, respectively) ch<strong>an</strong>nels on dorsal horn neurons are particularly<br />
import<strong>an</strong>t because <strong>the</strong>y supply strong negative feedback control of neuronal excitability by<br />
affecting AHP.<br />
Here, we examined ch<strong>an</strong>ges in neuronal excitability in response to intracellular <strong>an</strong>d extracellular<br />
Ca 2+ , by employing whole-cell recording on a subset (tonic firing) of lamina II neurons <strong>from</strong><br />
acutely prepared young adult rat spinal cord slices. The number of current injection-induced APs<br />
was greatly decreased reversibly by elevating <strong>the</strong> concentration of calcium in <strong>the</strong> artificial
cerebrospinal fluid (ACSF) <strong>from</strong> 2 mM Ca 2+ to 7 mM ACSF, as was spont<strong>an</strong>eous bursting, in<br />
parallel with <strong>an</strong> increase in <strong>the</strong> amplitude of AHP. These effects were mimicked by perfusion of<br />
1-ethyl-2-benzimidazolinone (EBIO) (1 mM), <strong>an</strong> activator of SK/IK ch<strong>an</strong>nels, <strong>an</strong>d reversed after<br />
washout. EBIO had no effect on <strong>the</strong> paired-pulse ratio of dorsal root-evoked EPSPs, consistent<br />
with a postsynaptic action of EBIO. Ano<strong>the</strong>r SK ch<strong>an</strong>nel opener, NS309 (50 µM), produced a<br />
similar action. In contrast, apamin (100 nM), a specific blocker of SK ch<strong>an</strong>nels, increased<br />
neuronal excitability by decreasing AHPs <strong>an</strong>d shortening interspike interval. This effect was<br />
mimicked by Ca 2+ -free ACSF. Surprisingly, EBIO decreased excitability even in <strong>the</strong> presence of<br />
apamin or when intracellular Ca 2+ was chelated by intracellular BAPTA (30 mM), indicating that<br />
EBIO acts via some pathway o<strong>the</strong>r th<strong>an</strong> apamin-sensitive SK ch<strong>an</strong>nels.<br />
Taken toge<strong>the</strong>r, our findings suggest a previously uncharacterized mech<strong>an</strong>ism <strong>for</strong> m<strong>an</strong>ipulating<br />
spinal dorsal horn neuronal excitability involving Ca 2+ -dependent AHPs, mediated by apaminsensitive<br />
SK ch<strong>an</strong>nels. Since lamina II neurons in dorsal horn are involved in regulating<br />
nociceptive in<strong>for</strong>mation, m<strong>an</strong>ipulating neuronal excitability by Ca 2+ provides evidence of a novel<br />
<strong>the</strong>rapeutic target <strong>for</strong> <strong>an</strong>algesic drugs.<br />
Disclosures: K. Y<strong>an</strong>g, None; S.M. Thompson, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.15/D32<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: R01 NS 40338<br />
Title: SK ch<strong>an</strong>nels regulate synaptic signaling, dendritic integration <strong>an</strong>d induction of LTP at<br />
temporoammonic-CA1 cell synapses<br />
Authors: *X. CAI, K. YANG, A. J. KALLARACKAL, S. M. THOMPSON;<br />
Univ. Maryl<strong>an</strong>d Baltimore, Baltimore, MD<br />
Abstract: Apamin-sensitive, small-conduct<strong>an</strong>ce, Ca 2+ -activated K + ch<strong>an</strong>nels (SK ch<strong>an</strong>nels) play<br />
<strong>an</strong> import<strong>an</strong>t role in regulating <strong>the</strong> excitability of CA1 neurons by Schaffer collateral inputs. SK<br />
ch<strong>an</strong>nels contribute to spike frequency adaptation <strong>an</strong>d are also localized at dendritic spines where<br />
<strong>the</strong>ir activity reduces EPSPs in a NMDA receptor-dependent m<strong>an</strong>ner. There are three SK ch<strong>an</strong>nel<br />
subtypes (SK1-SK3) expressed in mammali<strong>an</strong> brain, although SK3 ch<strong>an</strong>nels is present in CA1<br />
area in signific<strong>an</strong>tly lower density th<strong>an</strong> SK1 <strong>an</strong>d SK2 ch<strong>an</strong>nels. The temporo-ammonic (TA)
pathway, consisting of axons <strong>from</strong> layer III neurons in <strong>the</strong> entorhinal cortex, <strong>for</strong>ms excitatory<br />
synapses with <strong>the</strong> distal apical dendrites of CA1 pyramidal neurons <strong>an</strong>d provides <strong>the</strong> main source<br />
of specific sensory in<strong>for</strong>mation <strong>for</strong> area CA1 <strong>an</strong>d is critical <strong>for</strong> memory consolidation. In <strong>the</strong><br />
current study, we wished to determine <strong>the</strong> function of SK ch<strong>an</strong>nels located on <strong>the</strong> distal apical<br />
dendrites on TA-CA1 synaptic tr<strong>an</strong>smission <strong>an</strong>d dendritic integration in area CA1. We found that<br />
blocking SK ch<strong>an</strong>nels by apamin (100 nM) enh<strong>an</strong>ced field EPSPs or whole-cell EPSPs (both<br />
slope <strong>an</strong>d amplitude) <strong>an</strong>d facilitated population spikes initiation evoked by TA stimulation in<br />
acutely prepared hippocampal brain slices, whereas <strong>the</strong> SK ch<strong>an</strong>nel openers NS309 (50 µM) <strong>an</strong>d<br />
EBIO (1 mM) greatly decreased TA-CA1 field EPSPs or whole-cell recorded EPSPs. EBIO was<br />
equally effective in mice lacking SK2 ch<strong>an</strong>nels, in rats <strong>an</strong>d mice in <strong>the</strong> presence of apamin, <strong>an</strong>d<br />
in rats in <strong>the</strong> presence of AP5, however, suggesting that EBIO may exert actions at targets o<strong>the</strong>r<br />
th<strong>an</strong> SK ch<strong>an</strong>nels. The effect of apamin required NMDA receptor <strong>an</strong>d sodium ch<strong>an</strong>nel activation,<br />
since AP5 blocked <strong>an</strong>d intracellular dialysis of QX-314 attenuated <strong>the</strong> potentiating action of<br />
apamin on TA-CA1 fEPSPs or EPSPs. Block of SK ch<strong>an</strong>nels with apamin facilitated induction<br />
of LTP with weak tet<strong>an</strong>ic stimulation at TA-CA1 synapses <strong>an</strong>d LTP induction was facilitated in<br />
SK2 knockout mice. We conclude that activation of SK ch<strong>an</strong>nels in response to NMDAR<br />
activation <strong>an</strong>d depolarization limits synaptic excitation of CA1 cells by TA inputs <strong>an</strong>d regulates<br />
<strong>the</strong>ir plasticity.<br />
Disclosures: X. Cai, None; K. Y<strong>an</strong>g, None; A.J. Kallarackal, None; S.M. Thompson, None.<br />
Poster<br />
520. Calcium-Activated Potassium Ch<strong>an</strong>nels<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 520.16/D33<br />
Topic: B.04.c. Potassium ch<strong>an</strong>nels: Physiology<br />
Support: NSF Gr<strong>an</strong>t IBN 0630522 to RWS<br />
Title: Differential effects of <strong>the</strong> small conduct<strong>an</strong>ce calcium-activated potassium (SK, KCA2)<br />
ch<strong>an</strong>nel activators 1-ethyl-2-benzimidazolinone (EBIO) <strong>an</strong>d 6,7-dichloro-1H-indole-2,3-dione 3oxime<br />
(NS309) on learning, memory <strong>an</strong>d motor activity in C57BL/6NHsd mice<br />
Authors: *K. A. VICK, IV, M. GUIDI, R. STACKMAN, Jr.;<br />
Interdisciplinary Program in Neurosci. <strong>an</strong>d Dept. of Psychology, Florida Atl<strong>an</strong>tic Univ., Boca<br />
Raton, FL
Abstract: Small-conduct<strong>an</strong>ce Ca2+-activated K+ (SKCa1-3; KCa2.1-2.3) ch<strong>an</strong>nels are voltage<br />
insensitive, but open in response to micromolar increases of intracellular Ca2+. Three subunits of<br />
SK ch<strong>an</strong>nels have been identified (SK1-SK3) <strong>an</strong>d are located ubiquitously throughout <strong>the</strong><br />
mammali<strong>an</strong> brain. Both 1-ethyl-2-benzimidazolinone (EBIO) <strong>an</strong>d 6,7-dichloro-1H-indole-2,3dione<br />
3-oxime (NS309) activate SK ch<strong>an</strong>nels by influencing <strong>the</strong> sensitivity of <strong>the</strong> constitutively<br />
bound calmodulin to Ca2+. Strobaek et al. (2004) characterized NS309 as a 1000 times more<br />
potent activator of SK ch<strong>an</strong>nels th<strong>an</strong> EBIO; while both compounds increase <strong>the</strong> amplitude of <strong>the</strong><br />
apamin-sensitive mIAHP, NS309 increases <strong>the</strong> duration of <strong>the</strong> mIAHP as well. EBIO also<br />
increases <strong>the</strong> amplitude of <strong>the</strong> longer lasting apamin-insensitive sIAHP current; NS309, in<br />
contrast, does not alter <strong>the</strong> sIAHP. SK ch<strong>an</strong>nel blockade, by systemic injection of <strong>the</strong> peptide<br />
apamin, facilitates spatial <strong>an</strong>d object memory encoding in mice. Likewise SK ch<strong>an</strong>nel activation,<br />
by systemic injection of EBIO, impairs object memory encoding in mice. SK2+/T mice, that<br />
overexpress <strong>the</strong> SK2 subunit, exhibit impaired cued <strong>an</strong>d contextual fear memory. Interestingly,<br />
SK ch<strong>an</strong>nel blockade with systemic apamin enh<strong>an</strong>ced only hippocampal-dependent contextual<br />
fear, sparing amygdala-dependent cued memories. Here, we compared <strong>the</strong> behavioral influence<br />
of <strong>the</strong> two SK ch<strong>an</strong>nel activators, EBIO <strong>an</strong>d NS309, in C57BL/6NHsd mice using four wellknown<br />
tasks. Briefly, male C57BL/6NHsd mice received <strong>an</strong> injection of 17.5 mg/kg EBIO in 1%<br />
DMSO, 10 mg/kg NS309 in 10 % DMSO, 1% or 10% DMSO <strong>an</strong>d were immediately placed into<br />
<strong>an</strong> open field <strong>for</strong> one-hour. Results indicate that EBIO, but not NS309, signific<strong>an</strong>tly depressed<br />
locomotor activity of <strong>the</strong> mice only during <strong>the</strong> first 15 min after injection. For <strong>the</strong> remaining<br />
behavioral tasks, testing was delayed <strong>for</strong> 20 min after <strong>the</strong> injection. Nei<strong>the</strong>r EBIO nor NS309<br />
affected measures of <strong>an</strong>xiety in <strong>the</strong> elevated plus maze. However, both EBIO <strong>an</strong>d NS309<br />
impaired <strong>the</strong> encoding of object memory to ch<strong>an</strong>ce levels, supporting a SK ch<strong>an</strong>nel modulatory<br />
role in object memory. Interestingly, activation of SK ch<strong>an</strong>nels with EBIO does not influence <strong>the</strong><br />
encoding of fear memory regardless of session or number of CS-US pairs. Using a three session<br />
delay fear conditioning protocol (Pre-exposure, Conditioning, Test) with 24 hr between sessions,<br />
EBIO injected be<strong>for</strong>e ei<strong>the</strong>r <strong>the</strong> pre-exposure or conditioning session did not influence contextual<br />
or cued fear memories, regardless of st<strong>an</strong>dard 3CS-US or weakened 1CS-US pairings. Additional<br />
experimentation using NS309 in place of EBIO will fur<strong>the</strong>r <strong>the</strong> underst<strong>an</strong>ding of <strong>the</strong> influence<br />
SK ch<strong>an</strong>nels have on aversive learning.<br />
Disclosures: K.A. Vick, None; M. Guidi, None; R. Stackm<strong>an</strong>, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.1/D34<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease
Title: Loss of dendritic Ih results in prolonged thalamic oscillations associated with absence<br />
seizures<br />
Authors: *S.-W. YING 1 , J. HUGUENARD 2 , A. LUDWIG 3 , F. HOFMANN 3 , P. A.<br />
GOLDSTEIN 1 ;<br />
1 Dept Anes<strong>the</strong>siol, Weill Med. Col, Cornell Univ., New York, NY; 2 Dept. Neurol. & Neurolog.<br />
Sci., St<strong>an</strong><strong>for</strong>d Univ., St<strong>an</strong><strong>for</strong>d, CA; 3 Inst. für Experimentelle und Klinische Pharmakologie und<br />
Toxikologie, Friedrich-Alex<strong>an</strong>der-Universität Erl<strong>an</strong>gen-Nürnberg, Erl<strong>an</strong>gen, Germ<strong>an</strong>y<br />
Abstract: Our previous studies have shown that HCN2 ch<strong>an</strong>nels are <strong>the</strong> primary functional HCN<br />
iso<strong>for</strong>m in thalamic neurons, <strong>an</strong>d are predomin<strong>an</strong>tly expressed in dendrites of presynaptic<br />
GABAergic reticular thalamic nucleus (RTN) neurons <strong>an</strong>d somata of postsynaptic<br />
thalamocortical relay neurons. Mice lacking HCN2 exhibit generalized absence seizures, <strong>an</strong>d<br />
RTN neurons <strong>from</strong> <strong>the</strong>se mice showed enh<strong>an</strong>ced GABAergic output to neurons in ventrobasal<br />
complex (VB). Here we investigated spont<strong>an</strong>eous rhythmic burst patterns in brain slices <strong>from</strong><br />
wild-type (WT) <strong>an</strong>d HCN2 knockout (KO) mice (P70-120) using whole-cell recording<br />
techniques. WT RTN neurons spont<strong>an</strong>eously fired 70-80 s periodic groups of spikes composed<br />
of both rhythmic low-threshold calcium spikes (LTSs) <strong>an</strong>d tonic fast spikes. HCN2 KO RTN<br />
neurons, however, spont<strong>an</strong>eously fired a distinct rhythmic oscillation pattern: bursts were<br />
org<strong>an</strong>ized into groups of 3-7; such rhythmic grouped oscillations could last <strong>for</strong> ~30 min be<strong>for</strong>e<br />
shifting to a tonic firing pattern. The durations of such spont<strong>an</strong>eous oscillations <strong>an</strong>d <strong>the</strong> interburst<br />
frequencies in KO RTN neurons were much greater th<strong>an</strong> those of WT neurons. The<br />
grouped LTS bursts could be induced by injection of hyperpolarizing current pulses in KO RTN<br />
neurons, suggesting that grouped rhythmic bursts appear to be <strong>an</strong> intrinsic mech<strong>an</strong>ism, while <strong>the</strong><br />
longer (~30 min) bouts of activity are likely <strong>the</strong> result of enh<strong>an</strong>ced intrinsic excitability on<br />
network oscillations. We have previously observed (Ying et al. J Neurosci, 27:8719, 2007) that<br />
pronounced oscillations in spont<strong>an</strong>eous large-amplitude multi-peaked GABAergic inhibitory<br />
currents occur in postsynaptic VB neurons <strong>from</strong> KO mice. Deletion of HCN2 results in <strong>the</strong><br />
removal of RTN dendritic shunting, which facilitates <strong>the</strong> summation of dendritic subthreshold<br />
potentials. Here we show that lasting oscillations occur in KO RTN neurons, which likely<br />
generate profound GABAergic inhibition of postsynaptic VB neurons, <strong>an</strong>d would effectively deinactivate<br />
low-threshold calcium currents <strong>an</strong>d facilitate thalamocortical rebound bursts. This<br />
cascade of events may contribute to <strong>the</strong> generation of a hyper-synchronization or excessive<br />
oscillations in thalamocortical circuits. Our results suggest that Ih expressed in RTN dendrites<br />
plays a central role in maintaining normal intra-RTN excitability <strong>an</strong>d firing pattern; <strong>an</strong> imbal<strong>an</strong>ce<br />
of Ih <strong>an</strong>d GABAergic conduct<strong>an</strong>ces in thalamic network may underlie <strong>the</strong> generation of spike<strong>an</strong>d-wave<br />
discharges characteristic of absence epilepsy.<br />
Disclosures: S. Ying, None; J. Huguenard, None; A. Ludwig, None; F. Hofm<strong>an</strong>n, None; P.A.<br />
Goldstein, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.2/D35<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIAAA AA014106<br />
NIAAA AA017056<br />
NIAAA AA016852<br />
NIAAA AA017048<br />
Title: Acquired ch<strong>an</strong>nelopathies involving T-type Ca 2+ ch<strong>an</strong>nel CaV3.2 in CNS models of<br />
seizure<br />
Authors: *J. D. GRAEF 1 , D. W. GODWIN 2 ;<br />
1 Neurosci. Program, 2 Neurobio. <strong>an</strong>d Anat., Wake Forest Univ. Sch. of Med., Winston Salem, NC<br />
Abstract: Ictal activity in <strong>the</strong> brain results <strong>from</strong> pathological hyperexcitability within specific<br />
neuronal populations. While some seizure disorders have been linked to inherited alterations in<br />
ion ch<strong>an</strong>nel properties, a few have been shown to arise through acquired ch<strong>an</strong>ges in intrinsic<br />
membr<strong>an</strong>e properties as a result <strong>from</strong> <strong>an</strong> initial insult. In our lab, we are using two mouse models<br />
of CNS seizure disorders - a chemically-induced model of temporal lobe epilepsy <strong>an</strong>d a model of<br />
alcohol withdrawal following intermittent exposures to eth<strong>an</strong>ol - to demonstrate a similar<br />
acquired ch<strong>an</strong>nelopathy involving thalamic T-type Ca 2+ ch<strong>an</strong>nels. We have focused on a specific<br />
midline thalamic nucleus, <strong>the</strong> reuniens (RE), because of its strong connections with both <strong>the</strong><br />
hippocampus <strong>an</strong>d medial pre-frontal cortex. Our results show that a particular T-type calcium<br />
ch<strong>an</strong>nel iso<strong>for</strong>m, CaV3.2, is selectively upregulated at key time points following chemicallyinduced<br />
seizures that correspond to both epileptogenesis <strong>an</strong>d a chronic period of spont<strong>an</strong>eous<br />
recurrent seizures. CaV3.2 is also upregulated during withdrawal <strong>from</strong> repeated, intermittent<br />
exposures to eth<strong>an</strong>ol. Additionally, in both models <strong>the</strong>se ch<strong>an</strong>ges produce a functional<br />
upregulation of <strong>the</strong> overall T-type Ca 2+ current, including longer decay kinetics <strong>an</strong>d depolarizing<br />
shifts in <strong>the</strong> voltage-dependent properties, which lead to a greater tendency <strong>for</strong> RE neurons to<br />
participate in burst activity. These ch<strong>an</strong>ges in ch<strong>an</strong>nel function leading to increased bursts have<br />
been demonstrated experimentally <strong>an</strong>d computationally modeled. We conclude that ch<strong>an</strong>ges in<br />
RE burst activity are due to alterations in T-type Ca 2+ ch<strong>an</strong>nel properties that result <strong>from</strong> <strong>the</strong><br />
increased expression of <strong>an</strong> inducible T-type iso<strong>for</strong>m, CaV3.2. This increase in burst activity plays<br />
a key role in pathological hyperexcitability within thalamo-hippocampal circuitry, which may<br />
lead to ei<strong>the</strong>r mild impairments in decision-making <strong>an</strong>d working memory, or seizures through <strong>the</strong><br />
generation or propagation of ictal activity <strong>from</strong> limbic regions.<br />
Disclosures: J.D. Graef, None; D.W. Godwin, None.
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.3/D36<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: SR is funded by a Clinical Research Training Fellowship <strong>from</strong> <strong>the</strong> Wellcome Trust<br />
TDG held <strong>an</strong> Action Medical Research Training Fellowship <strong>an</strong>d was previously<br />
funded by <strong>the</strong> Guar<strong>an</strong>tors of Brain<br />
TDG, SR <strong>an</strong>d MGH receive support <strong>from</strong> CINCH (NIH RU54 RR019482,<br />
NINDS/ORD)<br />
SS is funded by <strong>the</strong> Worshipful Comp<strong>an</strong>y of Pewterers<br />
MRC<br />
Title: Genetic variation in Cav2.1 linked to episodic ataxia <strong>an</strong>d epilepsy<br />
Authors: *S. RAJAKULENDRAN, T. D. GRAVES, D. M. KULLMANN, S. SCHORGE, M.<br />
G. HANNA;<br />
Inst. of Neurol., London, United Kingdom<br />
Abstract: Background: The genetic basis of idiopathic generalized epilepsy remains poorly<br />
understood. Although <strong>the</strong> loci responsible <strong>for</strong> <strong>the</strong> heritability of common epilepsy are not known,<br />
genes encoding ion ch<strong>an</strong>nel subunits remain <strong>the</strong> best c<strong>an</strong>didates, mainly because <strong>the</strong>y underlie<br />
several rare Mendeli<strong>an</strong> epilepsies. CACNA1A encodes <strong>the</strong> α1-subunit of <strong>the</strong> Cav2.1 (P/Q-type)<br />
calcium ch<strong>an</strong>nel, which plays <strong>an</strong> import<strong>an</strong>t role in fast synaptic tr<strong>an</strong>smission. Mutations of<br />
CACNA1A underlie Episodic Ataxia type 2 <strong>an</strong>d Familial Hemiplegic Migraine type 1. Cav2.1<br />
has also been implicated in <strong>the</strong> pathogenesis of idiopathic generalized epilepsy, mainly on <strong>the</strong><br />
basis of inbred strains of mice with Cav2.1 mutations that exhibit spike-wave epilepsy.<br />
Objective: To investigate <strong>the</strong> prevalence <strong>an</strong>d functional consequences of genetic vari<strong>an</strong>ts in<br />
CACNA1A in patients with a combination of episodic ataxia <strong>an</strong>d epilepsy. Method: We<br />
identified 18 patients with a clinical phenotype consisting of episodic ataxia <strong>an</strong>d generalized<br />
epilepsy (EA+E). We sequenced CACNA1A in all 18 patients to determine <strong>the</strong> frequency of nonsynonymous<br />
polymorphisms. Genetic vari<strong>an</strong>ts, both novel <strong>an</strong>d those present in online control<br />
data, but enriched in our patients were introduced into a CACNA1A cDNA <strong>an</strong>d expressed in HEK
cells to allow biophysical characterization with whole-cell patch clamp. Results: We identified 7<br />
non-synonymous variations in CACNA1A in <strong>the</strong> 18 patients, of which three were not present in<br />
databases of single nucleotide polymorphisms. Individual vari<strong>an</strong>ts did not segregate with ei<strong>the</strong>r<br />
ataxia or epilepsy in <strong>the</strong> families of affected individuals. However, when <strong>an</strong>alyzed as a group,<br />
patients with EA+E had a signific<strong>an</strong>tly higher frequency of overall non-synonymous<br />
polymorphisms in CACNA1A compared with control populations (p =0.00014). The frequency of<br />
synonymous polymorphisms was however comparable between <strong>the</strong> two groups, suggesting a<br />
disease-specific increase in non-synonymous variation in CACNA1A. Functional expression<br />
studies of <strong>the</strong> non-synonymous vari<strong>an</strong>ts revealed subtle effects on current density <strong>an</strong>d/or voltage<br />
dependence of activation. Conclusion: These data suggest that rare vari<strong>an</strong>ts in CACNA1A with<br />
subtle effects on ch<strong>an</strong>nel function contribute to <strong>the</strong> risk of developing epilepsy <strong>an</strong>d ataxia.<br />
Disclosures: S. Rajakulendr<strong>an</strong>, None; T.D. Graves, None; D.M. Kullm<strong>an</strong>n, None; S.<br />
Schorge, None; M.G. H<strong>an</strong>na, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.4/D37<br />
Topic: C.01.a. Ch<strong>an</strong>nelopathies: Functional consequences<br />
Support: MIUR gr<strong>an</strong>t PRIN2007<br />
Telethon gr<strong>an</strong>t GGP06234<br />
EU gr<strong>an</strong>t EUROHEAD LSHM-CT-2004-504837<br />
Title: Excitatory synaptic tr<strong>an</strong>smission at cortical pyramidal neuron synapses in CAV2.1 knockin<br />
mice carrying a mutation that causes a severe hemiplegic migraine syndrome with cerebral<br />
edema <strong>an</strong>d epilepsy<br />
Authors: D. VECCHIA 1 , A. TOTTENE 1 , A. VAN DEN MAAGDENBERG 2 , *D.<br />
PIETROBON 1 ;<br />
1 Univ. Padova, Padova 35100, Italy; 2 Leiden Univ. Med. Ctr., Leiden, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: In patients, mutation S218L in <strong>the</strong> CaV2.1 (P/Q-type) calcium ch<strong>an</strong>nel gives rise to a<br />
dramatic hemiplegic migraine syndrome that is associated with seizures, coma <strong>an</strong>d severe<br />
cerebral edema often triggered by mild head trauma. In comparison with mutation R192Q, that
causes pure familial hemiplegic migraine (FHM1), mutation S218L produces a larger shift of<br />
activation of recombin<strong>an</strong>t hum<strong>an</strong> CaV2.1 ch<strong>an</strong>nels towards more negative membr<strong>an</strong>e potentials<br />
<strong>an</strong>d, accordingly, a larger gain of function of neuronal Ca 2+ influx at low voltages <strong>an</strong>d a larger<br />
facilitation of cortical spreading depression (CSD) in FHM1 knockin mice (Tottene et al., 2002,<br />
2005; v<strong>an</strong> den Maagdenberg et al., 2004, Pietrobon, 2007). Here we show gain of function of<br />
cortical excitatory neurotr<strong>an</strong>smission due to increased action potential-evoked Ca 2+ influx <strong>an</strong>d<br />
increased probability of glutamate release at cortical pyramidal cell synapses of heterozygous<br />
S218L knockin mice. In fact, in single cortical pyramidal cells <strong>for</strong>ming autapses <strong>from</strong> mut<strong>an</strong>t<br />
mice i) <strong>the</strong> amplitude of <strong>the</strong> evoked excitatory postsynaptic current (EPSC) <strong>an</strong>d <strong>the</strong> contribution<br />
of P/Q-type Ca 2+ ch<strong>an</strong>nels to synaptic tr<strong>an</strong>smission were both increased, ii) saturation of <strong>the</strong><br />
EPSC occurred at lower Ca 2+ concentration <strong>an</strong>d iii) <strong>the</strong> paired pulse ratio (PPR) was decreased.<br />
The ch<strong>an</strong>ges in EPSC amplitude, Ca 2+ -dependence of <strong>the</strong> EPSC <strong>an</strong>d PPR in heterozygous S218L<br />
knockin mice were qu<strong>an</strong>titatively similar to those measured in homozygous R192Q knockin<br />
mice. There<strong>for</strong>e, <strong>the</strong> S218L mutation produces a larger increase in presynaptic Ca 2+ influx <strong>an</strong>d<br />
glutamate release at cortical pyramidal cell synapses th<strong>an</strong> <strong>the</strong> mild FHM1 mutation. Given our<br />
recent evidence of a causative link between enh<strong>an</strong>ced glutamate release <strong>an</strong>d CSD facilitation<br />
(Tottene et al, <strong>2009</strong>), this may explain <strong>the</strong> greater susceptibility to CSD induced by <strong>the</strong> S218L<br />
mutation, <strong>an</strong>d possibly its dramatic clinical phenotype.<br />
We also report <strong>the</strong> novel finding that cortical excitatory tr<strong>an</strong>smission in FHM1 knockin mice is<br />
less susceptible to G-protein-coupled presynaptic inhibition. In fact, <strong>the</strong> fraction of <strong>the</strong> EPSC<br />
inhibited by <strong>the</strong> GABAB receptor agonist baclofen was lower in mut<strong>an</strong>t mice. As a consequence,<br />
excitatory neurotr<strong>an</strong>smission was fur<strong>the</strong>r facilitated in <strong>the</strong> presence of baclofen. Heterozygous<br />
S218L <strong>an</strong>d homozygous R192Q mice showed a similar reduction in presynaptic inhibition. Our<br />
data suggest that hyperactivity of cortical circuits due to both enh<strong>an</strong>ced CaV2.1-dependent<br />
glutamate release <strong>an</strong>d reduced presynaptic inhibition of glutamate release during G proteincoupled<br />
neuromodulation may render <strong>the</strong> cortex of FHM patients vulnerable to CSD ignition in<br />
response to migraine triggers.<br />
Disclosures: D. Vecchia, None; A. Tottene, None; A. v<strong>an</strong> den Maagdenberg, None; D.<br />
Pietrobon, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.5/D38<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t NS061505
Title: Hippocampal hyperexcitability <strong>an</strong>d enh<strong>an</strong>ced long-term potentiation in familial<br />
hemiplegic migraine 1 (R192Q) mut<strong>an</strong>t mice, in vivo<br />
Authors: *E. DILEKOZ 1 , K. EIKERMANN-HAERTER 1 , M. FERRARI 2 , A. VAN DEN<br />
MAAGDENBERG 3 , C. AYATA 1 ;<br />
1 Stroke <strong>an</strong>d Neurovascular Reg. Lab., Massachusetts Gen. Hosp., Charlestown, MA; 2 Neurol.,<br />
3 Hum<strong>an</strong> Genet., Leiden Univ. Med. Ctr., Leiden, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Familial hemiplegic migraine (FHM) is a severe migraine vari<strong>an</strong>t with tr<strong>an</strong>sient<br />
unilateral motor deficits. FHM1 has been linked to missense gain-of-function mutations in <strong>the</strong><br />
CACNA1A gene encoding <strong>the</strong> α1-subunit of neuronal Cav2.1 ch<strong>an</strong>nels. In vitro, FHM1 mutations<br />
augment evoked presynaptic Ca 2+ influx <strong>an</strong>d enh<strong>an</strong>ce excitatory tr<strong>an</strong>smission at pyramidal<br />
synapses. We studied hippocampal neurotr<strong>an</strong>smission <strong>an</strong>d long-term potentiation (LTP) in<br />
R192Q knockin mice, in vivo, <strong>an</strong>d compared this to <strong>the</strong> wild type. Field potentials were recorded<br />
<strong>from</strong> hippocampal CA1 region evoked by stimulation of fimbria/commissure (100 µsec sq<br />
pulse), in spont<strong>an</strong>eously breathing, isoflur<strong>an</strong>e-<strong>an</strong>es<strong>the</strong>tized, physiologically monitored, female<br />
mice. The stimulus intensity-response curves were obtained using field excitatory postsynaptic<br />
potential (fEPSP) slopes in stratum radiatum, <strong>an</strong>d population spike amplitudes in stratum<br />
pyramidale. LTP was induced by 3 stimulus trains (1 sec, 100 Hz, 20 sec intertrain interval) at a<br />
stimulus intensity yielding 50% of maximum fEPSP slope. In R192Q mut<strong>an</strong>ts, <strong>the</strong> stimulus<br />
intensity-response curves were signific<strong>an</strong>tly shifted to <strong>the</strong> left <strong>for</strong> both fEPSP slope <strong>an</strong>d spike<br />
amplitude compared to wild type, suggesting enh<strong>an</strong>ced glutamatergic tr<strong>an</strong>smission (two-way<br />
ANOVA, p
may involve spont<strong>an</strong>eously occuring hippocampal spreading depressions in this susceptible<br />
mut<strong>an</strong>t mouse model.<br />
Disclosures: E. Dilekoz, None; K. Eikerm<strong>an</strong>n-Haerter, None; M. Ferrari, None; A. v<strong>an</strong> den<br />
Maagdenberg, None; C. Ayata, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.6/E1<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: DFG Gr<strong>an</strong>t Ha5085/1-1<br />
NS061505<br />
Title: Facilitated subcortical propagation of cortical spreading depression in mice expressing <strong>the</strong><br />
S218L, R192Q mutations causing familial hemiplegic migraine type 1<br />
Authors: K. EIKERMANN-HAERTER 1 , Y. WANG 1 , I. YUZAWA 1 , C. WAEBER 1 , U.<br />
HOFFMANN 1 , M. FERRARI 2 , A. VAN DEN MAAGDENBERG 2 , *M. A. MOSKOWITZ 3 , C.<br />
AYATA 1 ;
1 Massachusetts Gen. Hosp., Charlestown, MA; 2 Univ. Med. Ctr. Leiden, Leiden, Ne<strong>the</strong>rl<strong>an</strong>ds;<br />
3 Massachusetts Gen. Hosp, Charlestown, MA<br />
Abstract: Familial hemiplegic migraine (FHM) is a severe migraine vari<strong>an</strong>t. Characteristic are<br />
headaches, sometimes accomp<strong>an</strong>ied by tr<strong>an</strong>sient motor deficits. FHM type 1 has been linked to<br />
mutations (S218L, R192Q) in <strong>the</strong> CACNA1A gene encoding <strong>the</strong> α1-subunit of Cav2.1. S218L<br />
confers a more severe phenotype compared to R192Q, with seizures <strong>an</strong>d coma. Cortical<br />
spreading depression (CSD), <strong>the</strong> electrophysiological event underlying migraine, is a tr<strong>an</strong>sient<br />
disruption of membr<strong>an</strong>e ionic gradients that slowly propagates across cerebral cortex. FHM1<br />
mut<strong>an</strong>t mice have increased susceptibility to CSD, with a stronger phenotype in S218L th<strong>an</strong><br />
R192Q. We postulated that <strong>the</strong> severe S218L phenotype was caused by widespread subcortical<br />
propagation of CSD. We examined subcortical propagation pattern of CSD in FHM1 mice. CSD<br />
was induced by topical KCl in ventilated female R192Q or S218L mut<strong>an</strong>t mice. SDs were<br />
recorded <strong>from</strong> cortex, striatum, hippocampus, <strong>an</strong>d thalamus. In S218L, CSDs propagated into all<br />
structures, with <strong>an</strong> allele-dosage effect on SD occurrence <strong>an</strong>d latency. Majority of thalamic SDs<br />
were associated with tr<strong>an</strong>sient increases in blood pressure (~30-50%), <strong>an</strong>d spread to contralateral<br />
thalamus. In R192Q mice, CSD propagated only into striatum. c-Fos was upregulated ipsilateral<br />
to CSD in cortex, striatum, <strong>an</strong>d hippocampus in S218L, <strong>an</strong>d in cortex <strong>an</strong>d striatum in R192Q. In<br />
conclusion, thalamic <strong>an</strong>d hippocampal propagation of CSD may underlie coma <strong>an</strong>d seizures in<br />
severe FHM1.<br />
Disclosures: K. Eikerm<strong>an</strong>n-Haerter, None; Y. W<strong>an</strong>g, None; I. Yuzawa, None; C. waeber,<br />
None; U. hoffm<strong>an</strong>n, None; M. Ferrari, None; A. v<strong>an</strong> den Maagdenberg, None; M.A.<br />
Moskowitz, None; C. ayata, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.7/E2<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t NS053792<br />
Title: Fine mapping of a seizure susceptibility locus on mouse Chromosome 11<br />
Authors: N. A. HAWKINS 1 , *J. A. KEARNEY 2 ;<br />
1 Neurosci., 2 Med., V<strong>an</strong>derbilt Univ., Nashville, TN
Abstract: Mutations in voltage-gated sodium ch<strong>an</strong>nels are responsible <strong>for</strong> several types of<br />
hum<strong>an</strong> epilepsy. Variable expressivity among family members is a common feature of <strong>the</strong>se<br />
inherited epilepsies, suggesting that genetic modifiers may influence clinical severity. The mouse<br />
model Scn2a Q54 has <strong>an</strong> epilepsy phenotype caused by a mutation in Scn2a that slows ch<strong>an</strong>nel<br />
inactivation. The Scn2a Q54 phenotype is influenced by genetic background. On <strong>the</strong> C57BL/6J<br />
strain background, Scn2a Q54 mice (B6.Q54) exhibit low spont<strong>an</strong>eous seizure frequency with<br />
delayed onset <strong>an</strong>d increased survival, compared with (C57BL/6JxSJL/J)F1.Q54 mice. This<br />
indicates that strain specific modifier alleles influence <strong>the</strong> severity of <strong>the</strong> epilepsy phenotype. We<br />
identified two modifier loci responsible <strong>for</strong> <strong>the</strong> strain difference in seizure susceptibility on<br />
Chromosomes 11 <strong>an</strong>d 19, designated Moe1 (Modifier of Epilepsy 1) <strong>an</strong>d Moe2 (Bergren et al,<br />
Mamm Genome 16:683, 2005).<br />
To confirm <strong>an</strong>d fur<strong>the</strong>r refine <strong>the</strong> map position of <strong>the</strong> Moe1 locus on Chromosome 11, we<br />
generated <strong>an</strong> interval specific congenic (ISC) strain that carries B6 alleles in <strong>the</strong> Moe1 interval<br />
on <strong>an</strong> SJL background. Offspring are maintained by continued backcrossing to SJL <strong>an</strong>d<br />
monitored <strong>for</strong> retention of B6 alleles in <strong>the</strong> Moe1 interval <strong>from</strong> D11Mit188 (45 Mb) to D11Mit<br />
360 (103 Mb). Recombin<strong>an</strong>t <strong>an</strong>imals with smaller B6-derived intervals were used to establish 3<br />
subcongenic lines that subdivide <strong>the</strong> Moe1 interval. Hemizygous B6.Q54 were crossed with ISC<br />
heterozygotes to generate Q54 tr<strong>an</strong>sgenic offspring carrying heterozygous or homozygous B6<br />
alleles in <strong>the</strong> Moe1 interval. Tr<strong>an</strong>sgenic mice were video-taped <strong>for</strong> 30 minute sessions at 3, 4.5<br />
<strong>an</strong>d 6 weeks age. The number of spont<strong>an</strong>eous partial motor seizures was qu<strong>an</strong>titated by offline<br />
<strong>an</strong>alysis of video-taped sessions. Comparison of seizure number between genotype groups<br />
supports that a modifier locus influencing seizure frequency was captured within <strong>the</strong> B6introgressed<br />
interval between D11Mit36-D11Mit360 (84-103 Mb). Fur<strong>the</strong>r refinement of <strong>the</strong><br />
Moe1 interval is in progress. We also per<strong>for</strong>med <strong>an</strong>alysis of ISC lines <strong>for</strong> susceptibility to<br />
induced seizures independent of <strong>the</strong> Q54 tr<strong>an</strong>sgene. Thresholds to flurothyl-induced seizures<br />
were determined in ISC backcross <strong>an</strong>d intercross offspring. Analysis of seizure thresholds in<br />
<strong>the</strong>se lines suggests <strong>the</strong> presence of multiple seizure susceptibility genes on Chromosome 11<br />
between D11Mit188 <strong>an</strong>d D11Mit360.<br />
Identification of genes that influence seizure susceptibility <strong>an</strong>d disease progression will provide<br />
insight into <strong>the</strong> molecular events of epileptogenesis, <strong>an</strong>d may identify novel <strong>the</strong>rapeutic targets<br />
<strong>for</strong> treatment of hum<strong>an</strong> patients.<br />
Disclosures: N.A. Hawkins, None; J.A. Kearney, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.8/E3
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t 5P01DC004732<br />
NIH Gr<strong>an</strong>t 5R01DC000086<br />
Title: Disruption of signal processing in oblique dendrites in a model of early Alzheimer's<br />
Authors: *T. M. MORSE 1 , P. G. MUTALIK 2 , M. MIGLIORE 3,4 , G. M. SHEPHERD 3 ;<br />
2 Yale Ctr. <strong>for</strong> Med. In<strong>for</strong>matics, 3 Neurobio., 1 Yale Univ. Sch. Med., New Haven, CT; 4 Inst. of<br />
Biophysics, Natl. Res. Council, Palermo, Italy<br />
Abstract: Most work on early Alzheimer's disease (AD) has focused on cellular degeneration,<br />
including effects of amyloid beta (Abeta) on dendrites <strong>an</strong>d dendritic spines. Disruption of<br />
electrical signalling in <strong>the</strong> dendrites in early AD has received very little attention. Abeta block of<br />
<strong>the</strong> A-type K+ ch<strong>an</strong>nels leads to enh<strong>an</strong>cement of CA1 pyramidal neuron backpropagating action<br />
potentials (bAPs) in hippocampal slices (Chen, 2005), which could interfere with synaptic<br />
plasticity <strong>an</strong>d lead to Ca2+ influx <strong>an</strong>d excitotoxicity (Good et al, 1996; Good <strong>an</strong>d Murphy, 1996;<br />
Chen, 2005). We wished to evaluate <strong>the</strong> disruption of this signalling beyond <strong>the</strong> accessibility of<br />
current recording methods, to test if it might cause ch<strong>an</strong>ges that could underlie <strong>the</strong> neural basis of<br />
<strong>the</strong> decline in cognitive function in early AD. We used a realistic computational model of a CA1<br />
pyramidal cell <strong>an</strong>d its dendritic tree (Migliore et al. 2005) in which we simulated <strong>the</strong> block of Atype<br />
K+ ch<strong>an</strong>nels by Abeta. The results showed that a bAP strongly propagated hundreds of<br />
microns fur<strong>the</strong>r into <strong>the</strong> dendritic arbor <strong>an</strong>d invaded <strong>the</strong> distal tuft when 1uM aBeta was applied,<br />
compared to normal controls. Oblique dendrites arising <strong>from</strong> <strong>the</strong> main apical trunk were weakly<br />
invaded by <strong>the</strong> bAP in <strong>the</strong> normal case, but strongly invaded during <strong>the</strong> K-block, <strong>an</strong> effect<br />
especially pronounced during simulated Abeta application restricted to <strong>the</strong> oblique br<strong>an</strong>ches.<br />
These results suggest that <strong>the</strong> thin oblique dendrites, with <strong>the</strong>ir large surface-to-volume ratios,<br />
would be especially vulnerable to <strong>the</strong> large-amplitude depolarizations of bAPs during K-block.<br />
On <strong>the</strong> basis of <strong>the</strong>se results, we hypo<strong>the</strong>size, building on Good <strong>an</strong>d Murphy (1996) <strong>an</strong>d Chen<br />
(2005), that <strong>the</strong> effect of Abeta in CA1 dendrites would be especially severe in <strong>the</strong> oblique<br />
dendrites, suggesting that <strong>the</strong>se are potential sites of <strong>the</strong> earliest electrical dendritic signalling<br />
dysfunction in early AD. This would be consistent with <strong>an</strong>atomical observations that <strong>the</strong> CA1<br />
pyramidal cell oblique <strong>an</strong>d basal dendrites are lost be<strong>for</strong>e <strong>the</strong> apical tuft in AD (Scheibel, 1979).<br />
Disclosures: T.M. Morse, None; P.G. Mutalik, None; M. Migliore, None; G.M. Shepherd,<br />
None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.9/E4<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: MEXT<br />
JSPS<br />
Naito Foundation<br />
CREST, JSTC<br />
21st Century COE program<br />
Title: Identification of <strong>the</strong> molecules determining cell death vulnerability in a cellular model of<br />
spinocerebellar ataxia type 6<br />
Authors: *T. TANABE, L. LI, H. SAEGUSA;<br />
Dept Pharmacol/Neurobiol, Tokyo Med. & Dent. Univ. Grad Sch. Med., Tokyo, Jap<strong>an</strong><br />
Abstract: Spinocerebellar ataxia type 6 (SCA6) is caused by <strong>the</strong> polyglutamine (polyQ)<br />
exp<strong>an</strong>sion of P/Q-type Ca 2+ ch<strong>an</strong>nels (Cav2.1) <strong>an</strong>d is characterized by <strong>the</strong> predomin<strong>an</strong>t<br />
degeneration of cerebellar Purkinje cells. PolyQ exp<strong>an</strong>sion in SCA6 is within <strong>the</strong> normal r<strong>an</strong>ge<br />
in o<strong>the</strong>r polyQ diseases. Thus, <strong>an</strong> idea that <strong>the</strong> functional alteration of <strong>the</strong> Cav2.1 is causally<br />
related to pathophysiology of SCA6 has emerged. However, our recent studies using SCA6<br />
knock-in mice suggested pathogenic mech<strong>an</strong>ism underlying SCA6 is likely to be independent of<br />
<strong>the</strong> alteration of <strong>the</strong> ch<strong>an</strong>nel properties (Saegusa et al. 2007). The carboxyl-terminus was shown<br />
to be cleaved <strong>from</strong> <strong>the</strong> full length <strong>for</strong>m of <strong>the</strong> Cav2.1 (Kubodera et al. 2003) <strong>an</strong>d tr<strong>an</strong>slocated<br />
into <strong>the</strong> nucleus (Kordasiewicz et al. 2006). Both groups found <strong>the</strong> tr<strong>an</strong>sient expression of<br />
truncated C-terminus with <strong>the</strong> disease r<strong>an</strong>ge of polyQ is toxic to cells. SCA6 is known to be a<br />
late onset chronic disease. Thus <strong>the</strong> acute toxicity observed in <strong>the</strong> tr<strong>an</strong>sient expression system<br />
might not be relev<strong>an</strong>t to <strong>the</strong> disease state. There<strong>for</strong>e, we have established <strong>the</strong> HEK 293 cell lines<br />
that express perm<strong>an</strong>ently <strong>the</strong> Cav2.1 carboxy terminal fragment (Cav2.1CT) with 13 polyQ<br />
repeat (S13 cells, normal r<strong>an</strong>ge) <strong>an</strong>d with 24 polyQ repeats (L24 cells, disease r<strong>an</strong>ge). We<br />
confirmed nuclear localization of <strong>the</strong> Cav2.1CT in S13 <strong>an</strong>d L24 cells. However, we found no<br />
apparent difference <strong>for</strong> cell viability between <strong>the</strong>se two cell lines in <strong>the</strong> normal culture<br />
conditions. Thus <strong>the</strong> cells became toler<strong>an</strong>t against <strong>the</strong> toxicity of Cav2.1CT with 24 polyQ under<br />
<strong>the</strong> environment of stable expression. Interestingly carboxyl-terminal region of Cav2.1 contains<br />
unique poly-histidine stretch near <strong>the</strong> polyQ site. We found Cav2.1CT binds Cd 2+ . To test<br />
whe<strong>the</strong>r <strong>the</strong> oxidative stress by heavy metal is import<strong>an</strong>t <strong>for</strong> triggering <strong>the</strong> onset of cell death<br />
o<strong>the</strong>r th<strong>an</strong> <strong>the</strong> exp<strong>an</strong>ded polyQ, we have <strong>an</strong>alyzed <strong>the</strong> effect of Cd 2+ on cell survival <strong>an</strong>d found<br />
that <strong>the</strong> L24 cells are more sensitive to Cd 2+ toxicity th<strong>an</strong> S13 cells. To underst<strong>an</strong>d <strong>the</strong> molecular<br />
basis of <strong>the</strong> vulnerability of L24 cells, four sets of microarray <strong>an</strong>alyses were conducted: 1) L24<br />
with vs without Cd 2+ , 2) S13 with vs without Cd 2+ , 3) L24 vs S13 in <strong>the</strong> presence of Cd 2+ , 4) L24
vs S13 under control conditions. To test whe<strong>the</strong>r <strong>the</strong> possible defect of defensive reaction via<br />
gene expression after Cd 2+ exposure is responsible <strong>for</strong> <strong>the</strong> vulnerability of L24 cells, we searched<br />
<strong>for</strong> <strong>the</strong> genes, whose expression was almost <strong>the</strong> same in S13 <strong>an</strong>d L24 cells under normal<br />
conditions <strong>an</strong>d showed different patterns in both cells when treated with Cd 2+ <strong>an</strong>d identified 21<br />
c<strong>an</strong>didate genes. We are currently examining whe<strong>the</strong>r <strong>the</strong>se selected genes are causally related to<br />
<strong>the</strong> increased cell death in L24 cells.<br />
Disclosures: T. T<strong>an</strong>abe, None; L. Li, None; H. Saegusa, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.10/E5<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t NS019865<br />
UW Medical School Research Committee<br />
Title: Surface-associated astrocytes in posterior piri<strong>for</strong>m <strong>an</strong>d amygdaloid cortex of rat have<br />
unique <strong>an</strong>atomical features <strong>an</strong>d distribution of Kir2.1 <strong>an</strong>d Kir4.1 subunits consistent with spatial<br />
buffering of potassium<br />
Authors: *S. L. FEIG, L. B. HABERLY;<br />
Anat. Dept., UW Sch. of Med. <strong>an</strong>d Publ. Hlth., Madison, WI<br />
Abstract: This study examined “surface-associated astrocytes” (SAAs) previously described in<br />
posterior piri<strong>for</strong>m cortex (PPC), which are unique by virtue of direct apposition to <strong>the</strong> cortical<br />
surface <strong>an</strong>d large caliber Kir2.1+ processes that descend into layer I (Howe et al., 2007).<br />
Subsequent study (unpublished) has shown that somata <strong>an</strong>d superficial processes of SAAs <strong>for</strong>m a<br />
glial limiting membr<strong>an</strong>e like that <strong>for</strong>med by endfeet in neocortex.<br />
The presence of SAAs with large Kir2.1+ descending processes suggests that <strong>the</strong>se cells might<br />
“siphon” K + <strong>from</strong> brain interstitial fluid (ISF) to CSF using mech<strong>an</strong>isms demonstrated in Müller<br />
cells (Kofuji <strong>an</strong>d Newm<strong>an</strong>, 2004). Processes of Müller cells near neuronal elements in retina<br />
express strong inwardly rectifying ch<strong>an</strong>nels with Kir2.1 subunits that are well-suited <strong>for</strong> taking<br />
up excess [K + ]o, whereas <strong>the</strong>ir endfeet express weak inwardly rectifying ch<strong>an</strong>nels with Kir4.1<br />
subunits that are well suited <strong>for</strong> release. Kir2.1 ch<strong>an</strong>nels are also found in “intermediate” regions<br />
of Müller cells where <strong>the</strong>ir high resist<strong>an</strong>ce at normal [K + ]o could leng<strong>the</strong>n <strong>the</strong> space const<strong>an</strong>t <strong>an</strong>d
facilitate efflux at a dist<strong>an</strong>ce <strong>from</strong> uptake. A prediction based on Müller cells is that SAAs<br />
express Kir4.1 subunits exclusively on <strong>the</strong>ir glial limiting membr<strong>an</strong>e that contacts CSF, <strong>an</strong>d<br />
Kir2.1 subunits in plasma membr<strong>an</strong>e of descending processes <strong>an</strong>d perhaps elsewhere.<br />
This prediction was tested by immunoperoxidase staining <strong>for</strong> Kir2.1 <strong>an</strong>d Kir4.1 subunits. The<br />
results showed that, in addition to PPC, amygdaloid cortex has SAAs with large Kir2.1+<br />
descending processes. SAAs were not observed in neocortex. EM study confirmed that Kir4.1<br />
subunits are restricted to somata <strong>an</strong>d superficial processes where <strong>the</strong>y are concentrated in <strong>the</strong><br />
glial limiting membr<strong>an</strong>e. In contrast, Kir2.1 subunits are present throughout <strong>the</strong> cell.<br />
Thus, SAAs have a spatially differential distribution of ch<strong>an</strong>nels containing Kir4.1 <strong>an</strong>d Kir 2.1<br />
subunits consistent with siphoning of K + <strong>from</strong> ISF to CSF. This could occur under normal <strong>an</strong>d<br />
pathological conditions including seizure to which piri<strong>for</strong>m <strong>an</strong>d amygdaloid cortex are highly<br />
susceptible.<br />
Disclosures: S.L. Feig, None; L.B. Haberly, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.11/E6<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Title: The electroneutral K + -Cl - cotr<strong>an</strong>sport is involved in <strong>the</strong> impulse conduction of peripheral<br />
nerve
Authors: *Y.-T. SUN 1 , M.-R. SHEN 2 ;<br />
1 2<br />
Natl. Cheng Kung University, Institude of Clin. Med., Tain<strong>an</strong>, Taiw<strong>an</strong>; Pharmacol., Natl.<br />
Cheng-Kung Univ., Tain<strong>an</strong>, Taiw<strong>an</strong><br />
Abstract: On peripheral nerve, a normal propagation of action potential requires fine tune of<br />
threshold <strong>an</strong>d current through <strong>the</strong> delicate interactions between axon <strong>an</strong>d Schw<strong>an</strong>n cells (SCs). It<br />
has been reported that <strong>the</strong> electroneurtral K + -Cl - cotr<strong>an</strong>sporter 3 (KCC3) contributes to <strong>the</strong><br />
mainten<strong>an</strong>ce of <strong>the</strong> nerve function in patients with agenesis of corpus callosum <strong>an</strong>d peripheral<br />
neuropathy(ACCPN) <strong>an</strong>d gene-deficient <strong>an</strong>imal model. KCC plays a major role in cell volume<br />
regulation which was quite different <strong>from</strong> <strong>the</strong> dysfunctional ch<strong>an</strong>nels or structural molecules<br />
determining <strong>the</strong> stability of axo-glial apparatus in <strong>the</strong> pathogenesis of neuropathy. Its function on<br />
peripheral nerve remains puzzled. To clarify <strong>the</strong> characteristics of KCC on peripheral nerve, <strong>the</strong><br />
expression pattern of KCC iso<strong>for</strong>ms (KCC1-4) on mouse sciatic nerve was examined <strong>an</strong>d <strong>the</strong><br />
subcellular distribution was studied. Excluding KCC1, o<strong>the</strong>r KCC iso<strong>for</strong>ms expressed in SCs as<br />
well as axons. The clustering pattern in <strong>the</strong> microvilli of SCs <strong>an</strong>d <strong>the</strong> node suggested its potential<br />
role in nodal firing. In terms of function, <strong>the</strong> electrophysiological parameters were investigated<br />
with applying <strong>the</strong> KCC blocker, (dihydroindenyl)oxy alk<strong>an</strong>oic acid (DIOA). Intr<strong>an</strong>eural injection<br />
of DIOA produced reductions of compound motor action potential (CMAP) amplitude <strong>an</strong>d area<br />
in a dose-dependent m<strong>an</strong>ner but had no effect on <strong>the</strong> time domains of nerve conduction. The<br />
tendency of recovery <strong>from</strong> conduction block in a long-period recording implied <strong>the</strong> tr<strong>an</strong>sient<br />
effect of KCC blockade on nodal firing. This study first reported <strong>the</strong> detail subcellular location of<br />
KCC on peripheral nerve <strong>an</strong>d showed a different view on KCC function.<br />
Disclosures: Y. Sun, None; M. Shen, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.12/E7<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH gr<strong>an</strong>t R0NS051833.<br />
Title: Reduced acetylcholine release at motor nerve terminals of lethargic mice<br />
Authors: *E. MOLINA CAMPOS 1 , W. D. ATCHISON 2 ;<br />
2 Pharm/Tox, 1 Michig<strong>an</strong> St Univ., East L<strong>an</strong>sing, MI
Abstract: Voltage gated calcium ch<strong>an</strong>nels are comprised of α1, β, <strong>an</strong>d α2δ subunits, where <strong>the</strong> α1<br />
subunits make up <strong>the</strong> selective pore <strong>for</strong> Ca 2+ <strong>an</strong>d determine most of <strong>the</strong> subtype-specific<br />
attributes of Ca 2+ ch<strong>an</strong>nels. This subunit contains binding sites <strong>for</strong> various pharmacological<br />
agents as well as <strong>the</strong> gating regions of <strong>the</strong> ch<strong>an</strong>nel. The β subunit regulates <strong>the</strong> assembly <strong>an</strong>d<br />
membr<strong>an</strong>e localization of <strong>the</strong> α1 subunits. The β subunit also strongly influences <strong>the</strong><br />
physiological features of <strong>the</strong> protein. A mutation in <strong>the</strong> β4 subunit of <strong>the</strong> P/Q-type (Cav2.1) Ca 2+<br />
ch<strong>an</strong>nel present in lethargic (lh) homozygous mice causes ataxia <strong>an</strong>d lethargic behavior at 15<br />
days of age. To determine how acetylcholine (ACh) is released at motor nerve terminals affected<br />
by <strong>the</strong> alteration of <strong>the</strong> β subunit, electrophysiological recordings at neuromuscular junctions<br />
(NMJs) were compared <strong>from</strong> adult lh mice with wild type (WT) mice of <strong>the</strong> same age. Different<br />
buffers were used <strong>for</strong> <strong>the</strong> recordings <strong>an</strong>d <strong>the</strong> Qu<strong>an</strong>tal content of <strong>the</strong> lh mice was compared to that<br />
of controls using CaCl2 2mM, 4mM <strong>an</strong>d 8mM;<strong>an</strong>d SrCl2 2mM <strong>an</strong>d 4mM. lh responses were<br />
reduced by: 68%, 60%, 35%, 46% <strong>an</strong>d 56%, respectively. The spont<strong>an</strong>eous release of ACh,<br />
measured as <strong>the</strong> frequency of occurrence of miniature end plate potentials (MEPPs) remained<br />
unch<strong>an</strong>ged <strong>for</strong> <strong>the</strong> CaCl2 buffer, but a 58% <strong>an</strong>d 53% reduction was observed when using 2mM<br />
<strong>an</strong>d 4mM SrCl2, respectively. A 55% reduction in <strong>the</strong> spont<strong>an</strong>eous release of ACh was observed<br />
when using BaCl2 0.5mM in <strong>the</strong> lh mice as compared to control <strong>an</strong>imals. Even though, lh mice<br />
have a reduction in <strong>the</strong> β4 protein levels, <strong>the</strong>ir α1A protein expression levels were <strong>the</strong> same as WT<br />
<strong>an</strong>imals, this could be explain due to <strong>the</strong> 3.5 fold increase in <strong>the</strong> protein expression level of <strong>the</strong> β3<br />
subunit that was seen in <strong>the</strong>se same <strong>an</strong>imals through western blot <strong>an</strong>alysis done on cerebellar<br />
proteins. Inasmuch as <strong>the</strong> β4 subunit is normally associated with <strong>the</strong> α1A at mammali<strong>an</strong> NMJs its<br />
disruption in lh mice might be <strong>an</strong>ticipated to result in aberr<strong>an</strong>t neuromuscular tr<strong>an</strong>smission.<br />
Disclosures: E. Molina Campos, None; W.D. Atchison, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.13/E8<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Title: Selective down-regulation of lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels expressed in <strong>the</strong> autonomic MPG<br />
neurons in culture<br />
Authors: *H.-G. KIM 1 , Y.-J. WON 4 , K.-H. SONG 5 , H.-C. JEONG 2 , B.-H. CHA 3 , S.-W.<br />
JEONG 1 ;<br />
1 Dept. of Phsiology, 2 Dept. of Urology, 3 Dept. of Pediatrics, Yonsei Univ.Wonju Cllg Med.,
Wonju K<strong>an</strong>g-Won, Republic of Korea; 4 NIH/NIAAA, Washington D.C, WA; 5 Dept. of Urology,<br />
Chungnam Natl.Univ.Hos, Deajeon, Republic of Korea<br />
Abstract: M<strong>an</strong>y studies had suggested that nerve injury affects expression <strong>an</strong>d distribution of<br />
functional membr<strong>an</strong>e proteins such as ion ch<strong>an</strong>nels <strong>an</strong>d receptors. Recently, we observed that<br />
lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels such as nicotinic <strong>an</strong>d GABAA receptors were signific<strong>an</strong>tly downregulated<br />
in autonomic major pelvic g<strong>an</strong>glia (MPG) neurons 1-day after enzymatic dissociation<br />
which mimics <strong>the</strong> nerve injury. Unlike <strong>the</strong> lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels, voltage-gated ion ch<strong>an</strong>nels<br />
were relatively well preserved in <strong>the</strong> same time-period of culturing. In <strong>the</strong> present study, thus,<br />
ef<strong>for</strong>ts were made to define mech<strong>an</strong>isms underlying <strong>the</strong>se phenomena. First of all, we tested if<br />
nicotinic currents could be protected by different neurotropic factors <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
which might be released <strong>from</strong> target tissues <strong>an</strong>d/or g<strong>an</strong>glion itself. However, percentage of<br />
current reduction in 1-day culture with NGF(100ng/ml), NT-3(300uM), CNTF(100ng/ml),<br />
GDNF(100uM), BDNF(100ng/ml), Ach(10nM), NE(10uM), <strong>an</strong>d enkephaline(100uM) was<br />
comparable to <strong>the</strong> control values. Interestingly, org<strong>an</strong>otypic culture of <strong>the</strong> MPG prevented downregulation<br />
of nicotinic currents in not parasympa<strong>the</strong>ic but sympa<strong>the</strong>tic MPG neurons. These data<br />
suggest that factors involved in regulation of lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels may arise <strong>from</strong> different<br />
sources <strong>for</strong> sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic MPG neurons.<br />
Disclosures: H. Kim, None; Y. Won, None; K. Song, None; H. Jeong, None; B. Cha, None; S.<br />
Jeong, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.14/E9<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Title: Impaired innate fear responses in TRPC5 null mice<br />
Authors: A. RICCIO 1 , Y. LI 2 , J. MOON 2 , K.-S. KIM 2 , K. S. SMITH 2 , U. RUDOLPH 2 , S.<br />
GAPON 1 , G. YAO 3 , E. TSVETKOV 2 , A. VAN'TVEER 2 , E. G. MELONI 2 , W. A. CARLEZON,<br />
Jr. 2 , V. Y. BOLSHAKOV 2 , *D. E. CLAPHAM 4 ;<br />
1 2 4 3<br />
Children's Hosp. Boston, Boston, MA; McLe<strong>an</strong> Hosp., Belmont, MA; Neurobio., Harvard<br />
Med. Sch., Boston, MA<br />
Abstract: Tr<strong>an</strong>sient receptor potential ch<strong>an</strong>nel 5 (TRPC5) functions as a nonselective cation<br />
ch<strong>an</strong>nel that is activated by Gq/11 family GPCRs via phospholipase C. In vitro studies indicate
that this ch<strong>an</strong>nel regulates <strong>the</strong> morphology of growth cones as well as neurite extension rates. To<br />
explore <strong>the</strong> role of TRPC5 in vivo, we generated mice in which <strong>the</strong> TRPC5 gene was ablated.<br />
Using a targeting construct we deleted <strong>the</strong> exon 5 genomic region within <strong>the</strong> putative 4th<br />
tr<strong>an</strong>smembr<strong>an</strong>e domain. TRPC1 <strong>an</strong>d TRPC4, which c<strong>an</strong> <strong>for</strong>m heteromeric cationic ch<strong>an</strong>nels with<br />
TRPC5, mRNA levels were unaltered in both <strong>the</strong> whole brain <strong>an</strong>d amygdala of TRPC5-/- mice<br />
compared to control <strong>an</strong>imals suggesting that <strong>the</strong>re were no compensatory ch<strong>an</strong>ges in expression.<br />
In situ hybridization in control mice showed that TRPC5-mRNA was abund<strong>an</strong>t in fear-related<br />
brain areas such as <strong>the</strong> amygdala, auditory cortex, somatosensory cortex <strong>an</strong>d hippocampus. To<br />
test <strong>the</strong> role of TRPC5 in <strong>an</strong>xiety, we per<strong>for</strong>med behavioral assays using wt <strong>an</strong>d TRPC5-/- mouse<br />
littermates. In elevated plus-maze experiments, knockout mice had a signific<strong>an</strong>tly increased<br />
number of open, but not closed-arm, entries suggesting reduced <strong>an</strong>xiety. In open field studies,<br />
TRPC5-/- mice spent signific<strong>an</strong>tly more time in <strong>the</strong> center of <strong>the</strong> arena <strong>an</strong>d also entered into <strong>the</strong><br />
aversive center of <strong>the</strong> field more frequently, confirming that TRPC5 ablation interfered with<br />
<strong>an</strong>xiety-related behaviors. In <strong>the</strong> social interaction test, TRPC5-/- mice spent more time in <strong>the</strong><br />
compartment where <strong>the</strong> novel mouse was confined <strong>an</strong>d exhibited increased number of <strong>the</strong> noseto-nose<br />
contacts with <strong>the</strong> novel mouse compared to control mice. Mut<strong>an</strong>t mice exhibited<br />
signific<strong>an</strong>t reductions in responses mediated by synaptic activation of Group I metabotropic<br />
glutamate <strong>an</strong>d cholecystokinin 2 receptors in neurons of <strong>the</strong> amygdala. These results are<br />
consistent with previous findings that blockade of <strong>the</strong>se two types of receptors have <strong>an</strong>xiolytic<br />
effects. Baseline synaptic tr<strong>an</strong>smission, membr<strong>an</strong>e excitability, <strong>an</strong>d spike timing-dependent longterm<br />
potentiation at cortical <strong>an</strong>d thalamic inputs to <strong>the</strong> amygdala were largely normal. Thus,<br />
<strong>the</strong>se experiments provide genetic evidence that TRPC5, activated via G protein-coupled<br />
neuronal receptors, has <strong>an</strong> essential function in fear-related behaviors.<br />
Disclosures: A. Riccio, None; Y. Li, None; J. Moon, None; K. Kim, None; K.S. Smith,<br />
None; U. Rudolph, None; S. Gapon, None; G. Yao, None; E. Tsvetkov, None; A. V<strong>an</strong>'tVeer,<br />
None; E.G. Meloni, None; W.A. Carlezon, None; V.Y. Bolshakov, None; D.E. Clapham ,<br />
None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.15/E10<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH GM055437-16
Title: Deafness mutation A88S induces cell death due to impairment of <strong>the</strong> slow gating of<br />
hCx26 hemich<strong>an</strong>nel<br />
Authors: J. XU 1 , *R. BRENNER 2 , B. J. NICHOLSON 3 ;<br />
1 Physiol., 3 Biochem., 2 UT Hlth. Sci. Ctr, S<strong>an</strong> Antonio, S<strong>an</strong> Antonio, TX<br />
Abstract: Connexin gap junctions c<strong>an</strong> be gated by tr<strong>an</strong>sjunctional voltage (Vj), pH, intracellular<br />
[Ca 2+ ] <strong>an</strong>d phosphorylation. Vj gating has two components, fast <strong>an</strong>d slow, which tr<strong>an</strong>sit <strong>the</strong><br />
ch<strong>an</strong>nels <strong>from</strong> <strong>the</strong> open to a subconduct<strong>an</strong>ce (residual state) or fully closed state, respectively.<br />
These same fast <strong>an</strong>d slow gating components are also seen in hemich<strong>an</strong>nels in response to<br />
ch<strong>an</strong>ges in membr<strong>an</strong>e potential. However, both <strong>the</strong> molecular mech<strong>an</strong>isms <strong>an</strong>d physiological<br />
signific<strong>an</strong>ce of <strong>the</strong>se two gating modes remains to be elucidated. Here, we show that a<br />
sensorineural mutation of hum<strong>an</strong> Cx 26, A88S, specifically impairs <strong>the</strong> slow gate responsible <strong>for</strong><br />
<strong>the</strong> depolarization induced opening <strong>an</strong>d hyperpolarization induced closing when expressed in<br />
Xenopus oocytes. Consistent with this, A88S hemich<strong>an</strong>nels have a persistent current that WT<br />
hemich<strong>an</strong>nels do not have after longer voltage pulses, due to <strong>the</strong> inability to fully close. In<br />
addition, <strong>the</strong> expression of A88S hemich<strong>an</strong>nels could induce more cell death th<strong>an</strong> those<br />
expressing WT hemich<strong>an</strong>nels, which could be inhibited by incubation in 2 mM extracellular<br />
[Ca 2+ ] solution, suggesting <strong>the</strong> persistent current generated by A88S hemich<strong>an</strong>nel could induce<br />
cell death. The mutation does not affect fast voltage gating, irrespective of extracellular [Ca 2+ ],<br />
or Vj gating of gap junction ch<strong>an</strong>nels, which is dominated by <strong>the</strong> fast gating phenotype. This is<br />
<strong>the</strong> first implication of a role <strong>for</strong> <strong>the</strong> slow voltage gating of hemich<strong>an</strong>nels in <strong>the</strong> etiology of<br />
deafness. The A88S site is adjacent to <strong>the</strong> strictly conserved proline in M2, <strong>an</strong>d is itself relatively<br />
conserved among isotypes. However, <strong>the</strong> deafness mutation at this position in Cx26 is actually<br />
<strong>the</strong> wild-type residue in Cx50, which <strong>for</strong>ms hemich<strong>an</strong>nels with no depolarization induced<br />
activation, <strong>an</strong>d a persistent sub-conduct<strong>an</strong>ce state. Fur<strong>the</strong>rmore, a Cx50 S89A mut<strong>an</strong>t shows<br />
depolarization activation <strong>an</strong>d a slow gating response that completely closes <strong>the</strong> ch<strong>an</strong>nel,<br />
comparable to WT Cx26 behavior. Thus, a disease associated mut<strong>an</strong>t phenotype in <strong>the</strong> ear<br />
(Cx26) has actually been “captured” as <strong>the</strong> wild-type function in <strong>the</strong> lens (Cx50). Location of<br />
this residue in <strong>the</strong> recently published crystal structure of Cx26 indicates that substitution of A88<br />
with serine could result in a hydrogen bond with R143 on <strong>the</strong> third tr<strong>an</strong>smembr<strong>an</strong>e segment,<br />
potentially limiting a critical movement required <strong>for</strong> slow gating of hemich<strong>an</strong>nels. The inability<br />
to close hemich<strong>an</strong>nels completely could lead to cell death of supporting cells in inner ear, a<br />
characteristic of connexin-associated deafness in mice, but this same property may play <strong>an</strong> as yet<br />
undefined role in <strong>the</strong> eye lens related to <strong>the</strong> normal role of connexins in micro-circulation.<br />
Disclosures: J. Xu, None; R. Brenner, None; B.J. Nicholson, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.16/E11<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: MICINN (SAF2008-01415)<br />
CICE de la Junta de Andalucía (PAI05-CTS-00844 <strong>an</strong>d PAI07-CTS-02606)<br />
Mutua Madrileña Foundation<br />
Title: The long-term autocrine nitric oxide/protein kinase g/Rho kinase signalling increases<br />
intrinsic excitability of injured neurons through inhibition of resting potassium currents<br />
Authors: *D. GONZALEZ-FORERO, L. GÓMEZ, C. CASTRO, F. PORTILLO, B.<br />
MORENO-LÓPEZ;<br />
Area de Fisiología. Facultad de Medicina. Univ. de Cadiz, Cadiz, Spain<br />
Abstract: Traumatic injury of a peripheral motor nerve induces hyperexcitability <strong>an</strong>d de novo<br />
expression of <strong>the</strong> neuronal iso<strong>for</strong>m of nitric oxide (NO) synthase (NOS-I) in <strong>the</strong> lesioned<br />
motoneurons. Risk factors by which neurons become susceptible to excitotoxic death in m<strong>an</strong>y<br />
neurodegenerative diseases also involve increases in intrinsic excitability <strong>an</strong>d NO syn<strong>the</strong>sis. The<br />
aim of this study was to investigate whe<strong>the</strong>r <strong>the</strong> sustained production of NO following injury is<br />
causally related to <strong>the</strong> enh<strong>an</strong>ced excitability of motoneurons <strong>an</strong>d to examine <strong>the</strong> underlying<br />
cellular tr<strong>an</strong>sduction pathways. Modulation of resting K + currents has a profound influence on<br />
neuronal excitability. Among <strong>the</strong> KCNK family of two-pore-domain K + ch<strong>an</strong>nels, <strong>the</strong> pHsensitive<br />
subunits TWIK-related acid-sensitive K + -1 (TASK-1) <strong>an</strong>d TASK-3 are highly<br />
expressed in motoneurons. Our preliminary hypo<strong>the</strong>sis was that NO regulates neuronal<br />
excitability by interfering with <strong>the</strong> function of TASK-like pH-sensitive “leak” K + ch<strong>an</strong>nels. The<br />
functional expression of TASK subunits was <strong>an</strong>alyzed by using whole cell patch clamp<br />
electrophysiological recordings <strong>an</strong>d western blot techniques. We found that long-lasting<br />
activation of <strong>the</strong> NO/cGMP/PKG cascade in in vitro slices induced a robust increase in<br />
motoneuron excitability that was related to <strong>the</strong> inhibition of TASK-like pH-sensitive K + currents.<br />
Western blot <strong>an</strong>alysis revealed no ch<strong>an</strong>ges in <strong>the</strong> expression levels of TASK subunits in <strong>the</strong><br />
hypoglossal nucleus following chronic stimulation of <strong>the</strong> pathway. Instead, NO/PKG-action<br />
involved downstream phosphorylation of <strong>the</strong> ATF-1 tr<strong>an</strong>scription factor, up-regulation of RhoA<br />
protein levels <strong>an</strong>d required RhoA/Rho-kinase (ROCK) activation. Finally, we found evidence <strong>for</strong><br />
<strong>the</strong> contribution of this pathway in motoneuron hyperexcitability induced by axon trauma. Given<br />
that TASK ch<strong>an</strong>nels are widely expressed throughout brain, this long-term mech<strong>an</strong>ism regulating<br />
neuronal excitability could be a neuron-sensitizing pathway <strong>for</strong> glutamate-induced excitotoxicity<br />
in pathological conditions.<br />
Disclosures: D. Gonzalez-Forero, None; L. Gómez, None; C. Castro, None; F. Portillo,<br />
None; B. Moreno-López, None.
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.17/E12<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Title: Stress alters excitability of autonomic neurons innervating <strong>the</strong> urogenital system<br />
Authors: C.-K. LEE 1 , K.-H. SONG 3 , H.-S. SONG 1 , H.-K. KIM 1 , K. WHANG 2 , *S.-W.<br />
JEONG 1 ;<br />
1 Dept Physiol, 2 Dept Neurosurg., Yonsei Univ. Wonju Cllg Med., Wonju K<strong>an</strong>g-Won, Republic<br />
of Korea; 3 Dept. of Urology, Chungnam Natl. Univ. Hosp., Deajeon, Republic of Korea<br />
Abstract: Major pelvic g<strong>an</strong>glia (MPG), containing both sympa<strong>the</strong>tic <strong>an</strong>d parasympa<strong>the</strong>tic<br />
neurons, are responsible <strong>for</strong> autonomic neural control of <strong>the</strong> urogenital system. The sympa<strong>the</strong>tic<br />
<strong>an</strong>d parasympa<strong>the</strong>tic neurons show tonic <strong>an</strong>d phasic firing patterns in response to prolonged<br />
current injections, respectively. Several studies suggest that stress c<strong>an</strong> impact on <strong>the</strong> urogenital<br />
functions including voiding although mech<strong>an</strong>isms underlying this are poorly understood. In <strong>the</strong><br />
present study, thus, we addressed whe<strong>the</strong>r stress c<strong>an</strong> alter excitability of <strong>the</strong> MPG neurons. In<br />
this regard, we immobilized male Sprague-Dawley rats daily <strong>for</strong> 2 hr. After 7 days, serum level<br />
of corticosterone was measured <strong>an</strong>d MPG neurons were enzymatically dissociated <strong>for</strong> patchclamp<br />
study. In control groups, sympa<strong>the</strong>tic MPG neurons showed tonic firing at rates of 12, 16,<br />
<strong>an</strong>d 20 Hz in response to 1X, 2X, <strong>an</strong>d 3X threshold stimulation, respectively. However, MPG<br />
neurons of <strong>the</strong> stressed rats became less excitable as <strong>the</strong> firing rates were reduced to 10, 12, <strong>an</strong>d<br />
14 Hz when <strong>the</strong> same sequence of stimulation was applied. Moreover, <strong>the</strong> restraint stress<br />
signific<strong>an</strong>tly inhibited generation of nickel-sensitive low threshold spike (LTS) in sympa<strong>the</strong>tic<br />
MPG neurons. Qu<strong>an</strong>titative real-time PCR <strong>an</strong>alysis revealed that <strong>the</strong> restraint stress decreased<br />
expression of Cav3.2 <strong>an</strong>d BK beta 4 being responsible <strong>for</strong> LTS <strong>an</strong>d repolarizing phase of <strong>the</strong><br />
action potentials, respectively with a liitle affecting SK2 <strong>an</strong>d SK3 ch<strong>an</strong>nels involved in<br />
generation of afterhyperpolarization. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest a neural mech<strong>an</strong>ism<br />
by which stress c<strong>an</strong> alter <strong>the</strong> urogenital functions.<br />
Disclosures: C. Lee, None; K. Song, None; H. Song, None; H. Kim, None; K. Wh<strong>an</strong>g,<br />
None; S. Jeong, None.<br />
Poster
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.18/E13<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH NS 44062<br />
NSF 0447192<br />
Title: Glutamate receptor 2 subunit containing AMPA receptors are neuroprotective<br />
Authors: *K. A. COLLINS 1 , K. R. ERICKSON 2 , J. R. DAY 3 ;<br />
2 Dept. of Biol. Sci., 3 Deparment of Biol. Sci., 1 Cali<strong>for</strong>nia State Univ. Chico, Chico, CA<br />
Abstract: In vivo studies have revealed that susceptibility to excitotoxic cell death is determined<br />
genetically in mice. We have shown that strain-specific differences in excitotoxin susceptibility<br />
result <strong>from</strong> <strong>an</strong> intrinsic neuronal vulnerability. Using glutamate induced excitotoxicity, we asked<br />
whe<strong>the</strong>r <strong>the</strong> genetic resist<strong>an</strong>ce to injury is due to a differential response of specific glutamate<br />
receptor subtypes. After 3 days in vitro, C57BL/6 (B6) <strong>an</strong>d FVB/N (FVB) neuron cultures were<br />
treated with glutamate (Glu) <strong>an</strong>tagonists followed by Glu. Neurons were tr<strong>an</strong>sferred into<br />
neuronal mainten<strong>an</strong>ce medium with various concentrations of glutamate agonists <strong>for</strong> 15 min <strong>an</strong>d<br />
<strong>the</strong>n returned to <strong>the</strong> cultures. Following a 24 hour recovery period, neuronal viability was<br />
assessed using <strong>the</strong> LIVE/DEAD staining assay (Molecular Probes, Eugene, OR). Four fields<br />
were photographed <strong>from</strong> each coverslip; <strong>an</strong>d, <strong>the</strong> numbers of live <strong>an</strong>d dead cells were determined<br />
by m<strong>an</strong>ual counting. Inhibiting <strong>the</strong> metabotropic receptors did not affect FVB neuron’s aability<br />
to survive excitotoxic insult. All ionotropic receptors alone were sufficient enough to induce<br />
excitotoxicity in <strong>the</strong> FVB neurons. Only when all three receptors were inhibited did <strong>the</strong> FVB<br />
neurons survive <strong>the</strong> excitotoxic insult. The data suggests that <strong>the</strong> strain difference in excitotoxic<br />
susceptibility was influenced by fast acting ionotropic receptors. In this experiment, Glu receptor<br />
subunit 2 (GluR2) containg AMPA receptors were not blocked with <strong>the</strong> <strong>an</strong>tagonists used. Since<br />
<strong>the</strong> GluR2 containing AMPA receptors are impermeable to Ca2+, a second experiment<br />
investigated <strong>the</strong> receptor subunit composition of AMPA receptors using immunofluorescence.<br />
FVB <strong>an</strong>d B6 neuron cultures were fixed with 4% para<strong>for</strong>maldehyde after 3 days in vitro. The<br />
cells were stained with immunofluorescence <strong>for</strong> GluR2. Four fields <strong>from</strong> each of six cover-slips<br />
were <strong>an</strong>alyzed. On a single neuron process, <strong>the</strong> immunoreactive punctae were m<strong>an</strong>ually counted<br />
along <strong>the</strong> dendrite. The B6 neurons had <strong>an</strong> average of 2.880 IR/10µm, while <strong>the</strong> FVB neurons<br />
had <strong>an</strong> average of 2.298 IR/10µm (p = 0.0005). The GluR2 data suggests that it is <strong>the</strong> AMPA<br />
receptor responsible <strong>for</strong> resist<strong>an</strong>ce of <strong>the</strong> B6. Westernblot data showed <strong>the</strong> same increase in<br />
GluR2 in B6 mice. These data suggest that GluR2 has neuroprotective properties in neurons.<br />
Disclosures: K.A. Collins, None; K.R. Erickson, None; J.R. Day, None.
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.19/E14<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: EU Biomed Project GRIPANNT<br />
DFG Gr<strong>an</strong>t SFB773<br />
(NFR/ SFF/ FUGE)<br />
Title: Neuronal Ca 2+ -activated K + ch<strong>an</strong>nels limit brain infarction <strong>an</strong>d promote survival<br />
Authors: *Y. LIAO 1,2 , A. M. KRISTIANSEN 3 , C. P. OKSVOLD 3 , F. A. TUVNES 3 , N. GU 3 , E.<br />
R. PRAN 3 , P. RUTH 2 , M. SAUSBIER 2 , J. F. STORM 3 ;<br />
1 Tongji Hosp. of Huazhong Univ. of Sci. & Technol., Wuh<strong>an</strong>, China; 2 Pharmacol. &<br />
Toxicology, Inst. of Pharmacy, Univ. of Tübingen, Tübingen, Germ<strong>an</strong>y; 3 Ctr. of Mol. Biol. <strong>an</strong>d<br />
Neuroscience, <strong>an</strong>d Inst. of Basal Med. Sciences,University of Oslo, Oslo, Norway<br />
Abstract: Cerebral ischemia is often caused by thrombotic occlusions of a cerebral artery <strong>an</strong>d<br />
may lead to neuronal death <strong>an</strong>d brain infarction. Neuronal calcium-activated potassium ch<strong>an</strong>nels<br />
of <strong>the</strong> BK type (KCa1.1/SLO1/Maxi-K), which are activated by membr<strong>an</strong>e depolarization <strong>an</strong>d<br />
elevations of intracellular Ca 2+ , may contribute signific<strong>an</strong>ty to neuroprotection during <strong>an</strong>d after<br />
brain ischemia. Here, we have used BK ch<strong>an</strong>nel deficient (BK -/- ) mice to test this hypo<strong>the</strong>sis.<br />
We found that tr<strong>an</strong>sient middle cerebral artery occlusion produced signific<strong>an</strong>tly larger infarct<br />
volume, larger post-ischemic mortality, <strong>an</strong>d more severe neurological deficits, in BK -/- mice<br />
compared to wild-type (WT) littermates. However, regional cerebral blood flow (rCBF)<br />
measurements by laser Doppler were not signific<strong>an</strong>tly different between <strong>the</strong>se genotypes.<br />
Fur<strong>the</strong>rmore, lesions induced by intracerebral injection of NMDA, reflecting NMDA-induced<br />
neurotoxicity, were larger in BK -/- mice compared to WT. Whole-cell patch clamp recordings<br />
<strong>from</strong> CA1 pyramidal cells in org<strong>an</strong>otypic hippocampal slice cultures showed BK ch<strong>an</strong>neldependent<br />
action potential repolarization. Thus, <strong>the</strong> spikes were broader in CA1 pyramidal cells<br />
<strong>from</strong> BK -/- mice, as compared to cells <strong>from</strong> WT mice; <strong>an</strong>d <strong>the</strong> selective BK ch<strong>an</strong>nel blocker<br />
IbTX induced spike-broadening only in WT cells, not in cells <strong>from</strong> BK -/- mice. Exposure of <strong>the</strong>se<br />
cultures to ischemia-like conditions induced signific<strong>an</strong>tly more neuronal death (in both areas<br />
CA1, CA3 <strong>an</strong>d <strong>the</strong> dentate gyrus), in cultures <strong>from</strong> BK -/- mice th<strong>an</strong> in cultures <strong>from</strong> WT mice.
These results indicate that neuronal BK ch<strong>an</strong>nels are import<strong>an</strong>t <strong>for</strong> protection against ischemic<br />
brain damage.<br />
Disclosures: Y. Liao, None; A.M. Kristi<strong>an</strong>sen, None; C.P. Oksvold, None; F.A. Tuvnes,<br />
None; N. Gu, None; E.R. Pr<strong>an</strong>, None; P. Ruth, None; M. Sausbier, None; J.F. Storm, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.20/E15<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t NS058792<br />
Title: TRPM2 ch<strong>an</strong>nels contribute to male-specific hippocampal neuron cell death following<br />
oxygen-glucose deprivation<br />
Authors: S. VERMA, Y.-F. YANG, *P. S. HERSON;<br />
Anes<strong>the</strong>siol, Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: Oxidative stress is a major contributor to ischemia-induced neuronal cell death in vitro<br />
<strong>an</strong>d in vivo. Data <strong>from</strong> our group <strong>an</strong>d o<strong>the</strong>rs have demonstrated that oxidative stress activates <strong>the</strong><br />
tr<strong>an</strong>sient receptor potential-M2 (TRPM2) non-selective cation ch<strong>an</strong>nel resulting in cell death (1,<br />
2), suggesting that TRPM2 participates in ischemia-induced neuronal cell death. However, no<br />
direct link between TRPM2 activation <strong>an</strong>d ischemic neuronal death has been established.<br />
Exposure to <strong>the</strong> TRP inhibitor 2-Aminoethoxy diphenyl borate (2-APB) (100 µM) signific<strong>an</strong>tly<br />
protected male hippocampal neurons <strong>from</strong> injury, decreasing cell death <strong>from</strong> 55.1±8.6% (n=5) to<br />
26.6±8.3 % (n=5; P
completely abolished following <strong>the</strong> application of 20 µM FFA. Current clamp recordings<br />
obtained <strong>from</strong> cells immediately upon re-oxygenation, following OGD, exhibited a rapid <strong>an</strong>d<br />
complete depolarization. The delay to depolarization varied greatly (15-85 minutes), but <strong>the</strong><br />
kinetics of depolarization were remarkably similar in all cells, exhibiting complete depolarization<br />
within 2 minutes (n=3). Following OGD-induced depolarization, I-V relations revealed <strong>the</strong><br />
presence of voltage-independent cation current inhibited by 20 µM FFA, indicative of TRPM2<br />
current. Consistent with our neuroprotection assays, we did not observe OGD-induced TRPM2<br />
ch<strong>an</strong>nel activation <strong>an</strong>d depolarization in female hippocampal neurons. In conclusion, we report a<br />
sex-specific role <strong>for</strong> TRPM2 ch<strong>an</strong>nel activation in neuronal damage following in vitro ischemia<br />
<strong>an</strong>d that inhibition of TRPM2 ch<strong>an</strong>nels may provide a novel neuroprotective strategy <strong>for</strong><br />
improving stroke outcomes in men.<br />
References:<br />
1. Herson PS & Ash<strong>for</strong>d ML (1997) J. Physiol. 501.1, 59-66<br />
2. McNulty S & Fonfria E (2005) Pflugers Arch. 451, 235-42<br />
Disclosures: S. Verma, None; Y. Y<strong>an</strong>g, None; P.S. Herson, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.21/E16<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: Telethon Gr<strong>an</strong>t GGP030159<br />
Title: Mutations in KCNA1 affect stochiometry <strong>an</strong>d fast inactivation of heteromeric K + ch<strong>an</strong>nels<br />
Authors: P. IMBRICI 1 , M. D'ADAMO 1 , J. MAYLIE 2 , *M. PESSIA 1 ;<br />
1 Univ. of Perugia, Perugia, Italy; 2 Oregon Hlth. <strong>an</strong>d Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: Episodic ataxia type 1 (EA1) is <strong>an</strong> autosomal domin<strong>an</strong>t disorder characterized by<br />
continuous myokymia, attacks of ataxia <strong>an</strong>d uncontrolled movements. Several mutations<br />
responsible <strong>for</strong> EA1 have been identified in <strong>the</strong> gene KCNA1 that codes <strong>the</strong> voltage-gated<br />
potassium ch<strong>an</strong>nel Kv1.1 <strong>an</strong>d haplotype insufficiency has been implicated in <strong>the</strong><br />
aetiopathogenesis of this disease (Zerr et al., 1998). In several brain areas Kv1.4 assembles with<br />
Kv1.1 <strong>an</strong>d confers N-type inactivating properties to heteromeric ch<strong>an</strong>nels, by providing <strong>the</strong><br />
inactivation particle. We propose that <strong>the</strong> EA1 mutations which have reduced expression, give
ise to a reduced number of Kv1.1 subunits in hetero-tetramer Kv1.1-Kv1.4 ch<strong>an</strong>nels, resulting<br />
in <strong>an</strong> increased rate of N-type inactivation. To test this hypo<strong>the</strong>sis, we injected equal amounts of<br />
Kv1.1 EA1 <strong>an</strong>d Kv1.4 cRNAs into Xenopus laevis oocytes which, presumably, freely assemble<br />
<strong>the</strong> ch<strong>an</strong>nel with a relative stochiometry determined by <strong>the</strong> relative amounts of each subunit <strong>an</strong>d<br />
trafficking of <strong>the</strong> ch<strong>an</strong>nel to <strong>the</strong> membr<strong>an</strong>e. Under this experimental setting, we found that EA1<br />
mutations, which had normal (V404I <strong>an</strong>d V408A) or low (I177N <strong>an</strong>d E325D) expression, as<br />
determined by homomeric current amplitudes, resulted in approximately similar or faster N-type<br />
inactivation, respectively, compared to WT Kv1.1 <strong>an</strong>d Kv1.4 coexpression. The effects of <strong>the</strong>se<br />
mutations on <strong>the</strong> amount of current undergoing N-type inactivation, <strong>the</strong> rate of current decay<br />
after a train of stimuli <strong>an</strong>d <strong>the</strong> recovery rate <strong>from</strong> inactivation also appeared to be strictly<br />
dependent on <strong>the</strong> number of EA1 subunits present in <strong>the</strong> tetramer. To determine <strong>the</strong> relative<br />
stochiometry <strong>for</strong> each ch<strong>an</strong>nel complex, we exploited <strong>the</strong> different Ki values <strong>for</strong> TEA block of<br />
Kv1.1 <strong>an</strong>d Kv1.4 ch<strong>an</strong>nels, which were 0.60±0.03 mM <strong>an</strong>d 900±50 mM <strong>for</strong> homomeric<br />
ch<strong>an</strong>nels, respectively. Application of <strong>the</strong> binomial equation to <strong>the</strong> TEA dose-response curve<br />
showed that I177N, E325D, V404I or V408A contributed respectively 0.16, 0.04, 0.33 <strong>an</strong>d 0.35<br />
to <strong>the</strong> total number of subunits available <strong>for</strong> ch<strong>an</strong>nel <strong>for</strong>mation. Whereas, WT Kv1.1 <strong>an</strong>d Kv1.4<br />
contributed approximately 0.5 each. These results are consistent with <strong>the</strong> possibility that fewer<br />
mutated Kv1.1 subunits (I177N, E325D) are available <strong>for</strong> co-assembly with Kv1.4. Thus, <strong>the</strong><br />
number of Kv1.4 subunits <strong>an</strong>d of inactivating particles increases, giving rise to faster N-type<br />
inactivation. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>for</strong> EA1 mutations that cause ei<strong>the</strong>r little (V404I, V408A) or no<br />
ch<strong>an</strong>ge in ch<strong>an</strong>nel expression, fast inactivation would be only slightly affected or slowed down<br />
(Imbrici et al., 2006).<br />
Disclosures: P. Imbrici, None; M. D'Adamo, None; J. Maylie, None; M. Pessia, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.22/E17<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> scientific Research (B) <strong>from</strong> Jap<strong>an</strong>ese <strong>Society</strong> <strong>for</strong> <strong>the</strong> Promotion of<br />
Science 19390066<br />
Title: Methamphetamine regulates GIRK ch<strong>an</strong>nels expression through dopamine system in <strong>the</strong><br />
brain
Authors: *S. HONDA, F. SOEDA, T. SHIRASAKI, K. TAKAHAMA;<br />
Envrn. <strong>an</strong>d molecular health sciences, Fac. of Med. <strong>an</strong>d Pharmaceut. Sciences, Kumamoto Univ.,<br />
Kumamoto, Jap<strong>an</strong><br />
Abstract: G-protein coupled inwardly rectifying K+ (GIRK) ch<strong>an</strong>nels couple with various Gprotein<br />
coupled receptors such as GABAB, dopamine D2 <strong>an</strong>d 5-HT1A receptors in <strong>the</strong> central<br />
nervous system. Activation of GIRK ch<strong>an</strong>nels causes membr<strong>an</strong>e hyperpolarization, playing <strong>an</strong><br />
import<strong>an</strong>t role in <strong>the</strong> inhibitory regulation of neuronal excitability <strong>an</strong>d heart rate. Several studies<br />
showed that gene expression of GIRK ch<strong>an</strong>nels increased in pathological states such as ischemia,<br />
c<strong>an</strong>cer <strong>an</strong>d <strong>the</strong> model of Down syndrome. Methamphetamine (MAP) has abuse potential <strong>an</strong>d is a<br />
powerful stimul<strong>an</strong>t of <strong>the</strong> brain. In this study, we investigated effect of MAP on expression of<br />
GIRK ch<strong>an</strong>nels mRNA in <strong>the</strong> brain. Male ddY mice (5 weeks-old) were used. Following<br />
injection of MAP 0.5 mg/kg, s.c., locomotor activity was measured <strong>for</strong> 30 min <strong>an</strong>d <strong>the</strong>n <strong>the</strong><br />
striatum was dissected to measure mRNA of GIRK ch<strong>an</strong>nel subunits at 10 min after <strong>the</strong><br />
behavioral test. mRNA expression was measured by RT-qPCR method. MAP dose-dependently<br />
increased locomotor activity but did not impair working memory. MAP at doses of 0.5 <strong>an</strong>d 2.0<br />
mg/kg, s.c. increased expression level of GIRK ch<strong>an</strong>nels mRNA but not Kir 2, <strong>an</strong>o<strong>the</strong>r types of<br />
inward rectifying potassium ch<strong>an</strong>nels. However <strong>the</strong> increasing effect was not dose-dependent.<br />
Interestingly, Girk2 subunit mRNA level correlated well with <strong>the</strong> level of locomotor activity.<br />
SCH23390, a selective dopamine D1 <strong>an</strong>tagonist, signific<strong>an</strong>tly inhibited MAP-induced<br />
hyperlocomotion. Raclopride, a dopamine D2 <strong>an</strong>tagonist, inhibited to some extent MAP-induced<br />
hyperlocomotion <strong>an</strong>d expression of Girk2 subunit mRNA, SCH23390 signific<strong>an</strong>tly inhibited<br />
Girk2 subunit mRNA expression but did not Girk1, 3 <strong>an</strong>d 4 subunit mRNA expression. O<strong>the</strong>r<br />
central stimul<strong>an</strong>t, scopolamine at 1.0 mg/kg s.c. enh<strong>an</strong>ced locomotor activity, but decreased<br />
expression of GIRK ch<strong>an</strong>nels mRNA. The results suggest that expression of GIRK ch<strong>an</strong>nels may<br />
be regulated by dopamine D1 receptor, fur<strong>the</strong>rmore at least partly by dopamine D2 receptor.<br />
Disclosures: S. Honda, None; F. Soeda, None; T. Shirasaki, None; K. Takahama, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.23/E18<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: CIHR
Title: Cholesterol elevation impairs glucose-mediated Ca 2+ signaling in mouse p<strong>an</strong>creatic beta<br />
cells<br />
Authors: V. YEUNG-YAM-WAH 1 , A. K. LEE 2 , F. W. TSE 2,1 , *A. TSE 2,1 ;<br />
1 Ctr. <strong>for</strong> Neurosci., 2 Pharmacol., Univ. Alberta, Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: Elevation of cholesterol in p<strong>an</strong>creatic islets is associated with a reduction in glucosemediated<br />
insulin secretion. We examined <strong>the</strong> effects of cholesterol elevation in β cells isolated<br />
<strong>from</strong> C57BL/6J mice by incubating <strong>the</strong> cells with 1 mg/ml of soluble cholesterol at 37 o C <strong>for</strong> 1<br />
hour. Cytosolic [Ca 2+ ] ([Ca 2+ ]i) was monitored with fura-2 digital imaging at ~35 o C. In<br />
controls, ~80% (45/56) of <strong>the</strong> β cells (identified by <strong>the</strong>ir Ca 2+ response to <strong>the</strong> KATP ch<strong>an</strong>nel<br />
blocker, tolbutamide; 100 µM) exhibited a [Ca 2+ ]i rise when exposed to glucose (20 mM).<br />
Cholesterol treatment reduced <strong>the</strong> fraction of glucose-responding β cells to 19% (9/47) but<br />
treatment with cholesterol plus excessive cholesterol chelator, methyl-β-cyclodextrin (MβCD; 10<br />
or 20 mM) did not affect <strong>the</strong> fraction of glucose-responding β cells (~80%; 21/26). There was no<br />
signific<strong>an</strong>t difference in <strong>the</strong> resting potentials (per<strong>for</strong>ated-patch recording) between <strong>the</strong><br />
cholesterol-overload cells (-67 + 1 mV; n = 5) <strong>an</strong>d <strong>the</strong> controls (-71 + 2 mV; n = 5).<br />
Never<strong>the</strong>less, glucose (20 mM) triggered only a small depolarization (2 + 1 mV) in <strong>the</strong><br />
cholesterol-overloaded cells (versus 46 + 4 mV in controls). In β cells, <strong>the</strong> KATP <strong>an</strong>d <strong>the</strong> delayed<br />
rectifier K + ch<strong>an</strong>nels are <strong>the</strong> major determin<strong>an</strong>ts of cell excitability. We examined whe<strong>the</strong>r <strong>the</strong><br />
poor glucose response in <strong>the</strong> cholesterol-overloaded cells was related to <strong>an</strong> increase in <strong>the</strong> KATP<br />
or delayed rectifier current. The me<strong>an</strong> density of KATP current (normalized to cell capacit<strong>an</strong>ce) at<br />
-60 mV was 1.1 + 0.4 pA/pF (n = 5) in <strong>the</strong> cholesterol-overloaded cells (per<strong>for</strong>ated patch<br />
recording), which was ~1/4 that of <strong>the</strong> controls (4.4 + 1.2 pA/pF; n = 5). The current density of<br />
<strong>the</strong> delayed rectifier (whole-cell recording) in <strong>the</strong> cholesterol-overload cells was 25 + 5 pA/pF at<br />
+20 mV (n = 8), which was ~1/2 of that in <strong>the</strong> controls (56 + 6 pA/pF; n = 10) <strong>an</strong>d in cells<br />
treated with cholesterol plus 10 mM MβCD (63 + 12 pA/pF; n = 6). Thus, cholesterol overload<br />
reduced <strong>the</strong> density of both <strong>the</strong> KATP current <strong>an</strong>d <strong>the</strong> delayed rectifier. Cholesterol-overload also<br />
reduced <strong>the</strong> density of <strong>the</strong> voltage-gated Ca 2+ current (VGCC). The VGCC density at +10 mV in<br />
cholesterol-overload cells was 5 + 1 pA/pF (n = 8) which was only ~1/3 of <strong>the</strong> value in <strong>the</strong><br />
controls (14 + 2 pA/pF; n = 11) <strong>an</strong>d in cells treated with cholesterol plus MβCD (14 + 2 pA/pF;<br />
n = 7). Our results indicate that cholesterol elevation in β cells has inhibitory effect on <strong>the</strong> KATP<br />
ch<strong>an</strong>nels, delayed rectifier <strong>an</strong>d VGCC. A reduction in voltage-gated Ca 2+ entry in conjunction<br />
with a decrease in <strong>the</strong> ability of glucose to evoke depolarization contribute to <strong>the</strong> impairment of<br />
<strong>the</strong> glucose-mediated Ca 2+ signalling <strong>an</strong>d insulin secretion in cholesterol-elevated β cells.<br />
Disclosures: V. Yeung-Yam-Wah, None; A.K. Lee, None; F.W. Tse, None; A. Tse, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.24/E19<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH R0-1 gr<strong>an</strong>t GM075229 (to S.M.T)<br />
NIH P0-1 gr<strong>an</strong>t GM47969 (to D.F.C.)<br />
Dr. Harold Carron’s endowment (to V.J-T.)<br />
Title: Cav3.2 is necessary <strong>for</strong> <strong>the</strong>rmal hyperalgesia but attenuates hypoalgesia in experimental<br />
diabetic neuropathy: A possible dual role <strong>for</strong> T-ch<strong>an</strong>nels in disease<br />
Authors: *J. R. LATHAM 1 , K. KRISHNAN 2 , D. F. COVEY 2 , V. JEVTOVIC-TODOROVIC 1 ,<br />
S. M. TODOROVIC 1 ;<br />
1 Dept. of Anes<strong>the</strong>siol., Univ. of Virginia, Charlottesville, VA; 2 Dept. of Developmental Biol.,<br />
Washington Univ. Sch. of Med., St. Louis, MO<br />
Abstract: Early stages of diabetic neuropathy are characterized by <strong>the</strong>rmal hyperalgesia which<br />
tr<strong>an</strong>sitions to hypoalgesia as <strong>the</strong> disease progresses. Although diabetic neuropathy is a common<br />
complication, <strong>the</strong> mech<strong>an</strong>isms underlying <strong>the</strong> mainten<strong>an</strong>ce of <strong>the</strong> early painful symptoms <strong>an</strong>d <strong>the</strong><br />
later loss of sensation are still poorly understood. It has been relatively well established that Tch<strong>an</strong>nels<br />
are signal amplifiers in <strong>the</strong> pain pathway. Previously we have shown that up-regulation<br />
in T-type calcium currents (T-currents), particularly through <strong>the</strong> α1H subunit (Cav3.2), may<br />
contribute to increased excitability of sensory neurons <strong>an</strong>d hyperalgesia in type 1 diabetic rats<br />
(Jagodic et al., 2007). In <strong>the</strong> current study, Cav3.2 knockout mice (α1H-/-) <strong>an</strong>d <strong>the</strong>ir wild-type<br />
littermates (α1H+/+) were made diabetic by a single injection of streptozotocin intraperitoneally.<br />
Weekly paw withdrawal latencies to <strong>the</strong>rmal stimuli <strong>an</strong>d blood glucose levels were measured <strong>for</strong><br />
7 weeks following <strong>the</strong> induction of diabetes. After 1-2 weeks of diabetes, α1H+/+ mice exhibited<br />
<strong>the</strong>rmal hyperalgesia, however α1H-/- mice maintained <strong>the</strong>ir original <strong>the</strong>rmal latencies.<br />
Intraperitoneal administration of <strong>the</strong> T-ch<strong>an</strong>nel selective <strong>an</strong>tagonist ECN ((3β,5α,17β)-17hydroxyestr<strong>an</strong>e-3-carbonitrile)<br />
alleviated hyperalgesia in α1H+/+ mice <strong>an</strong>d did not alter α1H-/-<br />
<strong>the</strong>rmal latencies, fur<strong>the</strong>r proving that hyperalgesia is T-ch<strong>an</strong>nel mediated. While α1H+/+ mice<br />
developed hypoalgesia after 5 weeks of diabetes, α1H-/- mice exhibited a greater degree of<br />
hyposensitivity. These results suggest a dual role <strong>for</strong> T-ch<strong>an</strong>nels in diabetic neuropathy, with Tcurrents<br />
being problematic in <strong>the</strong> early hyperalgesic stage but attenuating later hypoalgesic<br />
symptoms. Thus, by increasing or decreasing <strong>the</strong> function of T-ch<strong>an</strong>nels at <strong>the</strong> appropriate time,<br />
T-ch<strong>an</strong>nel selective compounds could be <strong>the</strong>rapeutic throughout <strong>the</strong> progression of diabetic<br />
neuropathy.<br />
Disclosures: J.R. Latham, None; K. Krishn<strong>an</strong>, None; D.F. Covey, None; V. Jevtovic-<br />
Todorovic, None; S.M. Todorovic, None.
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.25/E20<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH RO1 NS48564-01<br />
St<strong>an</strong><strong>for</strong>d Bio-X Gr<strong>an</strong>t<br />
Term<strong>an</strong> Scholar Award<br />
St<strong>an</strong><strong>for</strong>d Neurosciences Program Training Gr<strong>an</strong>t<br />
NINDS Gr<strong>an</strong>t RO1 NS48564<br />
Title: The Timothy syndrome mutation in CaV1.2 alters differentiation <strong>an</strong>d migration of cortical<br />
progenitors<br />
Authors: *G. PANAGIOTAKOS 1 , J. F. KREY 1 , T. D. PALMER 2 , R. E. DOLMETSCH 1 ;<br />
1 Neurobio., 2 Neurosurg., St<strong>an</strong><strong>for</strong>d Univ., St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: L-type voltage gated calcium ch<strong>an</strong>nels (LTCs) such as CaV1.2 convert electrical<br />
signals into long-term biochemical <strong>an</strong>d tr<strong>an</strong>scriptional events in developing neurons. A point<br />
mutation in CaV1.2 associated with loss of voltage dependent inactivation gives rise to autism in<br />
a multi-systemic disorder called Timothy Syndrome. While <strong>the</strong> effects of this mutation on<br />
ch<strong>an</strong>nel biophysics have been described, little is known about its effects on <strong>the</strong> signaling or<br />
developmental functions of CaV1.2. Our lab has previously demonstrated that <strong>the</strong> TS mutation in<br />
CaV1.2 causes dendritic retraction through ectopic activation of <strong>the</strong> RhoA pathway. Here we<br />
show that <strong>the</strong> TS mutation alters proliferation <strong>an</strong>d differentiation of mouse neural progenitor<br />
cells in vitro <strong>an</strong>d in vivo <strong>an</strong>d disrupts neuronal migration in developing cortex. Introduction of<br />
CaV1.2 ch<strong>an</strong>nels bearing <strong>the</strong> TS mutation (CaV1.2-TS) into differentiating embryonic mouse<br />
neural progenitor cells results in a signific<strong>an</strong>t decrease in <strong>the</strong> number of neurons generated in<br />
vitro (31.9% in YFP-tr<strong>an</strong>sfected cells versus 6.5% in CaV1.2-TS tr<strong>an</strong>sfected cells, p=0.004). In<br />
vivo <strong>an</strong>alyses of post-natal day 1 (P1) TS knock-in mice reveal a marked increase in <strong>the</strong> number<br />
of proliferating progenitor cells at <strong>the</strong> ventricular zone, subtle ch<strong>an</strong>ges in <strong>the</strong> total number <strong>an</strong>d<br />
distribution of parvalbumin-expressing cortical interneurons, <strong>an</strong>d alterations in <strong>the</strong> migration of a<br />
subset of layer 2/3 cortical projection neurons. We have fur<strong>the</strong>r explored this phenotype by<br />
electroporating CaV1.2-TS ch<strong>an</strong>nels into developing ferret cortex, where we observed a marked<br />
reduction in <strong>the</strong> number of migrating layer 2/3 neurons expressing CaV1.2-TS that reach <strong>the</strong>ir
appropriate laminar position. These studies shed light on <strong>the</strong> role of CaV1.2 in <strong>the</strong> developing<br />
brain <strong>an</strong>d provide insights into <strong>the</strong> cellular mech<strong>an</strong>isms that might cause autism in patients with<br />
Timothy Syndrome as well as o<strong>the</strong>r autism spectrum disorders.<br />
Disclosures: G. P<strong>an</strong>agiotakos, None; J.F. Krey, None; T.D. Palmer, None; R.E. Dolmetsch,<br />
None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.26/E21<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: RR&D <strong>an</strong>d Medical Research Services of <strong>the</strong> VA<br />
NIH<br />
NMSS<br />
Title: Alterations of NaV1.7 expression <strong>an</strong>d localization in <strong>the</strong> tr<strong>an</strong>sected sciatic nerve <strong>an</strong>d DRG<br />
Authors: *E. J. ARROYO, C. RADTKE, J. D. KOCSIS;<br />
Neurol., Yale Univ. Sch. of Med., West Haven, CT<br />
Abstract: Nerve injury through nerve tr<strong>an</strong>section c<strong>an</strong> lead to <strong>the</strong> <strong>for</strong>mation of neuromas that<br />
result in painful neuropathy. A contributing factor is <strong>an</strong> alteration in <strong>the</strong> ion ch<strong>an</strong>nels expressed<br />
by afferent cut<strong>an</strong>eous sensory neurons in <strong>the</strong> dorsal root g<strong>an</strong>glion (DRG) which derails <strong>the</strong>ir<br />
electrophysiological bal<strong>an</strong>ce. The voltage-gated sodium ch<strong>an</strong>nel, NaV1.7, is one of <strong>the</strong> ch<strong>an</strong>nels<br />
involved in nociception <strong>an</strong>d its expression is altered in different neuroma models. In this study,<br />
we ligated <strong>an</strong>d tr<strong>an</strong>sected rat sciatic nerve to allow <strong>for</strong> neuroma <strong>for</strong>mation. Three weeks after<br />
ligation we examined <strong>the</strong> DRG <strong>an</strong>d <strong>the</strong> resulting neuroma <strong>for</strong> NaV1.7 expression.<br />
Immunofluorescence was used to assess ch<strong>an</strong>ges in NaV1.7 expression <strong>an</strong>d localization, with<br />
<strong>an</strong>ti-caspr staining to indicate <strong>the</strong> presence of axonal par<strong>an</strong>odes, <strong>an</strong>d <strong>an</strong>ti-MAG staining <strong>for</strong><br />
myelin sheaths. In normal DRG, NaV1.7 is highly expressed by <strong>the</strong> small DRG neuron<br />
population (C-cells) <strong>an</strong>d its expression progressively diminishes with increasing cell size<br />
(C>Aδ>Αβ/α). Three weeks after neuroma <strong>for</strong>mation <strong>the</strong>re was <strong>an</strong> increase in NaV 1.7 in<br />
subpopulations of medium(Aδ) to large(Aβ/α) DRG neurons with a decrease in KV1.2<br />
expression. NaV1.7 clustered at nodes in small myelinated axons at <strong>the</strong> neuroma. These
alterations in ion ch<strong>an</strong>nel expression could contribute to <strong>the</strong> increase in hyperexcitability<br />
observed in DRG neurons after neuroma <strong>for</strong>mation. Keratinocyte injection into <strong>the</strong> neuroma end<br />
of a ligated nerve results in increases in NGF <strong>an</strong>d extreme hyperexcitability (Radtke, et. al. 2007<br />
SFN Abst. 778.19). Ongoing work is examining <strong>the</strong> effect of keratinocytes engrafted into nerve<br />
on NaV1.7 expression.<br />
Disclosures: E.J. Arroyo, None; C. Radtke, None; J.D. Kocsis, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.27/E22<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: Rehabilitation Research Service <strong>an</strong>d Medical Research Service, Department of<br />
Veter<strong>an</strong>s Affairs<br />
Ery<strong>the</strong>rmalgia Association<br />
Title: Alternative splicing underlies time-dependent m<strong>an</strong>ifestation of a sodium ch<strong>an</strong>nelopathy<br />
Authors: *J. CHOI 1,2,3 , X. CHENG 1,2,3 , L. TYRRELL 1,2,3 , R. TE MORSCHE 4 , E. M.<br />
EASTMAN 1,2,3 , H. J. JANSEN 4 , S. D. DIB-HAJJ 1,2,3 , J. P. DRENTH 4 , S. G. WAXMAN 1,2,3 ;<br />
1 Dept. of Neurol., Yale Univ. Sch. of Med., New Haven, CT; 2 Ctr. <strong>for</strong> Neurosci. <strong>an</strong>d<br />
Regeneration Research, Yale Univ. Sch. of Med., New Haven, CT; 3 Rehabil. Res. Center,<br />
Veter<strong>an</strong>s Affairs Connecticut Healthcare Syst., West Haven, CT; 4 Dept. of Gastroenterology <strong>an</strong>d<br />
Hepatology, Radboud Univ. Nijmegen Med. Ctr., Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Inherited erythromelalgia (IEM) is <strong>an</strong> autosomal domin<strong>an</strong>t ch<strong>an</strong>nelopathy<br />
characterized by episodic burning pain <strong>an</strong>d skin redness in <strong>the</strong> extremities triggered by mild<br />
warmth or moderate exercise, with variable age-of-onset. The average age of early-onset IEM is<br />
3 years, while adult-onset IEM has been reported in <strong>the</strong> third decade of life. Fully penetr<strong>an</strong>t,<br />
gain-of-function mutations in Nav1.7 sodium ch<strong>an</strong>nel, which is preferentially expressed in<br />
nociceptive <strong>an</strong>d sympa<strong>the</strong>tic neurons, have been linked to early-onset IEM, whereas <strong>the</strong> etiology<br />
of familial adult-onset has yet to be determined.<br />
Nav1.7 undergoes alternative splicing at two loci (exon 5 <strong>an</strong>d exon/intron 11), thus producing<br />
four iso<strong>for</strong>ms that have been termed neonatal-short (NS), neonatal-long (NL), adult-short (AS)<br />
<strong>an</strong>d adult-long (AL). Thus far, all early-onset IEM mutations in <strong>the</strong> NS iso<strong>for</strong>m have been shown
to cause a hyperpolarizing shift of activation of Nav1.7, <strong>an</strong>d induce hyperexcitability in<br />
tr<strong>an</strong>sfected DRG neurons. We now report a new mutation of Nav1.7 (G616R), in loop1, in a<br />
family with a vari<strong>an</strong>t of erythromelalgia in which <strong>the</strong>re is adult-onset of pain in some family<br />
members. The mutation shifts steady-state inactivation in <strong>the</strong> G616R/AL but not G616R/NS<br />
splicing iso<strong>for</strong>m of Nav1.7 following expression in DRG neurons, but no effect of <strong>the</strong> mutation<br />
in ei<strong>the</strong>r iso<strong>for</strong>m was observed in HEK293 cells. Altered inactivation does not depend on <strong>the</strong> age<br />
of <strong>the</strong> DRG neurons in which <strong>the</strong> mut<strong>an</strong>t is expressed. Expression of G616R/AL, but not<br />
G616R/NS renders DRG neurons hyperexcitable. The switch <strong>from</strong> a neonatal to <strong>an</strong> adult splicing<br />
iso<strong>for</strong>m is a prerequisite <strong>for</strong> m<strong>an</strong>ifestation of <strong>the</strong> functional phenotype of this mutation, <strong>an</strong>d<br />
provides a basis <strong>for</strong> its link to a family that includes members with adult-onset clinical<br />
phenotype.<br />
Disclosures: J. Choi, None; X. Cheng, None; L. Tyrrell, None; R. te Morsche, None; E.M.<br />
Eastm<strong>an</strong>, None; H.J. J<strong>an</strong>sen, None; S.D. Dib-Hajj, None; J.P. Drenth, None; S.G. Waxm<strong>an</strong>,<br />
None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.28/E23<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: Dept. Veter<strong>an</strong>s Affairs<br />
National Multiple Sclerosis <strong>Society</strong><br />
Erythromelalgia Association<br />
Title: C<strong>an</strong> robots patch-clamp as well as hum<strong>an</strong>s? Characterization of <strong>the</strong> novel NaV1.7<br />
Ery<strong>the</strong>romelalgia mutation S211P<br />
Authors: *M. R. ESTACION 1,2,3 , J. S. CHOI 1,2,3 , L. J. TYRRELL 1,2,3 , Z. LIN 4 , Y. LI 4 , E. M.<br />
EASTMAN 1,2,3 , S. D. DIB-HAJJ 1,2,3 , Y. YANG 4 , S. G. WAXMAN 1,2,3 ;<br />
1 2<br />
Neurol, Ctr. <strong>for</strong> Neurosci. <strong>an</strong>d Regeneration Res., Yale Univ. Sch. of Med., New Haven, CT;<br />
3 4<br />
Rehabil. Res. Ctr., VA CT Healthcare Syst., West Haven, CT; Dermatol., Peking Univ. First<br />
Hosp., Beijing, China
Abstract: Over <strong>the</strong> past 5 years <strong>an</strong> increasing number of ion ch<strong>an</strong>nel mutations linked to<br />
disorders of excitation have been found; in m<strong>an</strong>y cases <strong>the</strong>se mutations cause disease by<br />
ch<strong>an</strong>ging biophysical properties of <strong>the</strong> ch<strong>an</strong>nel. As additional mutations have been identified, <strong>the</strong><br />
work load <strong>for</strong> patch-clamp electrophysiologists has increased. Here we describe <strong>the</strong> use of a<br />
robotic patch-clamp plat<strong>for</strong>m to profile a novel mutation of sodium ch<strong>an</strong>nel NaV1.7 associated<br />
with inherited erythromelalgia (IEM), a pain disorder characterized by burning pain in <strong>the</strong><br />
extremities. The mutation substitutes serine 211 with proline (S211P) in domain I S4. We present<br />
<strong>the</strong> physiological profiling of this mutation by a robotic patch-clamp instrument <strong>an</strong>d compare its<br />
results to those obtained by hum<strong>an</strong> electrophysiologists. Both modes of patch-clamp <strong>an</strong>alysis<br />
show that S211P hyperpolarizes (-8 mV by hum<strong>an</strong> profiling, -12 mV by robotic profiling) <strong>the</strong><br />
voltage-dependence of activation <strong>an</strong>d enh<strong>an</strong>ces slow ramp responses (~2-fold by hum<strong>an</strong>, ~1.4fold<br />
by robotic) compared to wild-type. Steady-state fast-inactivation <strong>for</strong> S211P required <strong>an</strong><br />
additional Boltzm<strong>an</strong> that accounted <strong>for</strong> a fraction (~20% by hum<strong>an</strong>, ~40-60% by robotic) of <strong>the</strong><br />
total current. Slow-inactivation gating of S211P ch<strong>an</strong>nels was strongly enh<strong>an</strong>ced, showing a<br />
hyperpolarizing shift (-15 mV by hum<strong>an</strong>, -10 mV by robotic) compared to wild-type. The robotic<br />
system achieved a 3 to 10-fold increase in <strong>the</strong> number of usable cells/day. We conclude that<br />
robotic patch-clamp plat<strong>for</strong>ms c<strong>an</strong> also be used to speed <strong>the</strong> biophysical characterization of<br />
naturally occurring <strong>an</strong>d experimentally induced ion ch<strong>an</strong>nel mutations.<br />
Disclosures: M.R. Estacion, None; J.S. Choi, None; L.J. Tyrrell, None; Z. Lin, None; Y. Li,<br />
None; E.M. Eastm<strong>an</strong>, None; S.D. Dib-Hajj, None; Y. Y<strong>an</strong>g, None; S.G. Waxm<strong>an</strong>, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.29/E24<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Title: Positive modulation of <strong>the</strong> glycine receptor by me<strong>an</strong>s of glycine receptor binding aptamers<br />
Authors: *K. SANDBERG 1 , N. DEKKI WENNA 1 , E. ANEIROS 1 , M. BLANK 2 , J.<br />
MEULLER 3 , E. NYMAN 1 , D. MICHAEL DABROWSKI 1 , C. V. ANDERSSON 1 ;<br />
1 AstraZeneca R & D Sodertalje, Södertälje, Sweden; 2 Inst. of Immunol., Helmholtz Zentrum<br />
München, Germ<strong>an</strong> Res. Ctr. <strong>for</strong> Envrn. Hlth., Munich, Germ<strong>an</strong>y; 3 AstraZeneca R&D Mölndal,<br />
Mölndal, Sweden<br />
Abstract: According to <strong>the</strong> gate control <strong>the</strong>ory of pain, <strong>the</strong> glycine receptors (GlyR) are<br />
potentially interesting targets <strong>for</strong> <strong>the</strong>rapeutic development to treat peripheral
inflammatory pain. Both GlyRα1 <strong>an</strong>d GlyRα3 are expressed in lamina II neurons <strong>an</strong>d have<br />
<strong>the</strong> potential to reduce peripheral input <strong>from</strong> Aδ <strong>an</strong>d C-fibers. Hence, a possible approach<br />
<strong>for</strong> <strong>the</strong> development of <strong>an</strong>algesics could be a positive modulator of <strong>the</strong> glycine activated Cl - -<br />
ch<strong>an</strong>nels by enh<strong>an</strong>cing or potentiating <strong>the</strong> inward Cl - flux. Un<strong>for</strong>tunately, <strong>the</strong>re has been<br />
limited success to develop drug-like, sub-type selective small molecules to study <strong>the</strong> impact<br />
of agonists or positive modulators of GlyR. In this study, 2’F stabilized RNA aptamers<br />
binding to <strong>the</strong> alpha 1 subunit of GlyR were generated. The pharmacological properties of<br />
<strong>the</strong>se aptamers were <strong>an</strong>alysed. Eight aptamers with low n<strong>an</strong>omolar affinity to GlyRα1 were<br />
identified. Five of <strong>the</strong>se aptamers were shown to be positive modulators of <strong>the</strong> GlyRα1 ion<br />
ch<strong>an</strong>nel by potentiating <strong>the</strong> chloride influx in GlyRα1 expressing L(tk) cells using a FLIPR<br />
assay. Cytochemistry using fluorescein-labelled aptamers demonstrated GlyRα1 dependent<br />
binding to <strong>the</strong> plasma membr<strong>an</strong>e but also intracellular binding with a pattern similar to<br />
immunofluorescence per<strong>for</strong>med with <strong>an</strong> <strong>an</strong>tibody directed against GlyR. The positive<br />
modulation of one of <strong>the</strong> aptamers, “C2” was confirmed by patch clamp electrophysiology<br />
in promoting whole cell Cl - -currents on L(tk) cells expressing recombin<strong>an</strong>t GlyRα1 as well<br />
as cells expressing recombin<strong>an</strong>t GlyRα1β. We conclude that <strong>the</strong>se GlyR binding aptamers<br />
are unique tools <strong>for</strong> use in target validation studies, mode of action studies <strong>an</strong>d potentially<br />
also as tools <strong>for</strong> assay development.<br />
Disclosures: K. S<strong>an</strong>dberg, Kristi<strong>an</strong> S<strong>an</strong>dberg, A. Employment (full or part-time); N. Dekki<br />
Wenna, None; E. Aneiros, None; M. Bl<strong>an</strong>k, None; J. Meuller, None; E. Nym<strong>an</strong>, None; D.<br />
Michael Dabrowski, None; C.V. Andersson, None.<br />
Poster<br />
521. Ion Ch<strong>an</strong>nels <strong>an</strong>d Disease<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 521.30/E25<br />
Topic: B.04.d. Ion ch<strong>an</strong>nels <strong>an</strong>d disease<br />
Support: NIH Gr<strong>an</strong>t NS031234<br />
NIH Gr<strong>an</strong>t NS063688-01<br />
Title: TRPC1 is essential <strong>for</strong> store-operated calcium entry in gliomas<br />
Authors: *V. C. BOMBEN, H. SONTHEIMER;<br />
Neurobio., Univ. Alabama-Birmingham, Birmingham, AL
Abstract: Glial-derived primary brain tumors, gliomas, proliferate <strong>an</strong>d migrate extensively<br />
throughout <strong>the</strong> brain often killing patients within months of diagnosis. In m<strong>an</strong>y cells,<br />
including c<strong>an</strong>cer cells, ion ch<strong>an</strong>nels have been suggested to play <strong>an</strong> import<strong>an</strong>t role in cell<br />
cycle progression. Recent attention has focused on <strong>the</strong> tr<strong>an</strong>sient receptor potential (TRP)<br />
family of cation ch<strong>an</strong>nels which are activated downstream of <strong>the</strong> phospholipase C cascade.<br />
The TRP c<strong>an</strong>onical (TRPC) subfamily is a 7-member, non-selective cation ch<strong>an</strong>nel family<br />
that is thought to have high selectivity <strong>for</strong> calcium. We previously demonstrated that TRPC<br />
ch<strong>an</strong>nel subunits are expressed in glioma cells <strong>an</strong>d that pharmacological inhibition of<br />
TRPC ch<strong>an</strong>nels impairs store-operated calcium entry as well as proliferation in D54 glioma<br />
cells. Store operated calcium entry is also impaired with <strong>an</strong> <strong>an</strong>tibody generated specifically<br />
against <strong>an</strong> extracellular domain of <strong>the</strong> TRPC1 ch<strong>an</strong>nel, implicating TRPC1 as a component<br />
of store operated calcium entry. In this study we show that TRPC1 colocalizes with <strong>the</strong><br />
mitotic spindle during cell division <strong>an</strong>d immunoprecipitations demonstrate <strong>an</strong> affinity of<br />
TRPC1 <strong>for</strong> tubulin. We utilized <strong>an</strong> shRNA knockdown approach to selectively reduce<br />
TRPC1 expression by tr<strong>an</strong>sient tr<strong>an</strong>sfection of two constitutively active TRPC1 shRNA<br />
plasmids that target different sequences of <strong>the</strong> gene. This led to a signific<strong>an</strong>t reduction in<br />
TRPC1 protein expression as determined by Western blot <strong>an</strong>d signific<strong>an</strong>tly reduced<br />
TRPC1 mediated currents as determined by whole cell patch-clamp recordings.<br />
Fur<strong>the</strong>rmore, FURA-2 imaging studies show that store-operated calcium entry was<br />
impaired in cells that have been tr<strong>an</strong>sfected with <strong>the</strong>se TRPC1 shRNA plasmids. Finally,<br />
tr<strong>an</strong>sfection with TRPC1 shRNA also slowed glioma proliferation <strong>an</strong>d a proportion of<br />
tr<strong>an</strong>sfected cells became large <strong>an</strong>d multinucleated as we had previously observed with<br />
pharmacological inhibition of TRPC1 ch<strong>an</strong>nels. To evaluate <strong>the</strong> role of TRPC1 ch<strong>an</strong>nels<br />
fur<strong>the</strong>r, we generated stable cell lines containing doxycycline inducible shRNA plasmids<br />
directed against TRPC1. Using a non-silencing plasmid control <strong>an</strong>d TRPC1 plasmid<br />
shRNA in <strong>the</strong> presence or absence of doxycycline, we are able to m<strong>an</strong>ipulate expression of<br />
TRPC1 protein. After several days of doxycycline treatment, cells have impaired storeoperated<br />
calcium entry <strong>an</strong>d proliferate slower. These studies suggest that TRPC1<br />
constitutes a molecularly defined component of <strong>the</strong> store-operated Ca 2+ entry pathway<br />
required by glioma cells to undergo normal cell divisions (supported by NIH 5RO1<br />
NS031234 & 1 F31 NS063688-01).<br />
Disclosures: V.C. Bomben, None; H. Son<strong>the</strong>imer, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.1/E26
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: Graduate school GK1188<br />
Title: Perturbing exocyst in <strong>the</strong> calyx of Held: Altered presynaptic morphology coexists with<br />
normal synaptic tr<strong>an</strong>smission<br />
Authors: *D. SCHWENGER, T. KUNER;<br />
Inst. of Anat. <strong>an</strong>d Cell Biol. - Univ. of Heidelberg, Heidelberg, Germ<strong>an</strong>y<br />
Abstract: The exocyst complex is a multiprotein te<strong>the</strong>ring complex mediating directed vesicular<br />
tr<strong>an</strong>sport. Here, we investigated <strong>the</strong> involvement of exocyst in synaptic vesicle trafficking at <strong>the</strong><br />
calyx of Held. Recombin<strong>an</strong>t adeno-associated viral particles expressing a domin<strong>an</strong>t-negative<br />
truncation construct of <strong>the</strong> Exo70 subunit (Exo70∆C, disrupts membr<strong>an</strong>e targeting of <strong>the</strong> exocyst<br />
complex <strong>an</strong>d diminishes incorporation of proteins into <strong>the</strong> plasma membr<strong>an</strong>e) were injected into<br />
<strong>the</strong> ventral cochlear nucleus of 2-day-old Sprague Dawley rats to acutely perturb exocyst<br />
function in <strong>the</strong> calyx of Held. Synaptic tr<strong>an</strong>smission was assessed electrophysiologically <strong>an</strong>d<br />
calyceal morphology was reconstructed <strong>from</strong> confocal image stacks.<br />
M<strong>an</strong>ual 3D reconstruction of ~160 calyces of Held <strong>an</strong>d subsequent volume <strong>an</strong>alysis revealed that<br />
overexpression of Exo70∆C signific<strong>an</strong>tly decreased calyx volume in P13 rats, indicating that<br />
perturbed exocyst function caused a defect in membr<strong>an</strong>e addition. This effect was not found in<br />
mature P21 calyces suggesting that exocyst function is necessary <strong>for</strong> membr<strong>an</strong>e addition during<br />
early maturation of calyces, but dispensable at later maturational stages.<br />
Despite <strong>the</strong> hypomorphic nature of P13 calyces overexpressing Exo70∆C, low frequency (0.1<br />
Hz) stimulation did not affect EPSC kinetics, suggesting that <strong>the</strong> fusion machinery of synaptic<br />
vesicles remained unaffected. Decay of EPSC amplitudes upon repetitive stimulation (0.3 - 100<br />
Hz) was indistinguishable <strong>from</strong> wild type, suggesting that exocyst is not involved in <strong>the</strong><br />
molecular mech<strong>an</strong>isms underlying short-term depression. Similarly, readily releasable pool size<br />
<strong>an</strong>d kinetics of <strong>the</strong> recovery <strong>from</strong> depression, frequency of spont<strong>an</strong>eous release, qu<strong>an</strong>tal size <strong>an</strong>d<br />
mEPSC kinetics remained unaffected by Exo70∆C overexpression. Thus, perturbation of exocyst<br />
function by overexpression of a domin<strong>an</strong>t-negative truncation construct of Exo70 did not affect<br />
synaptic tr<strong>an</strong>smission, suggesting that exocyst does not contribute to <strong>the</strong> synaptic vesicle cycle<br />
<strong>an</strong>d neurotr<strong>an</strong>smitter release.<br />
In summary, our findings show that <strong>the</strong> exocyst complex is required <strong>for</strong> presynaptic membr<strong>an</strong>e<br />
addition at early stages of calyx maturation, while a role as a te<strong>the</strong>ring factor of synaptic vesicles<br />
in local vesicle recycling remains unlikely. Thus, <strong>the</strong> exocyst complex may be required <strong>for</strong> <strong>the</strong><br />
delivery of cargo <strong>from</strong> <strong>the</strong> cell soma to synaptic terminals, but not <strong>for</strong> trafficking steps in <strong>the</strong><br />
synaptic vesicle cycle.<br />
Disclosures: D. Schwenger, None; T. Kuner, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.2/E27<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NIH R01-NS38129<br />
NIH T32-DA007268<br />
NIH F32-GM086169<br />
Title: Plasma membr<strong>an</strong>e topological ch<strong>an</strong>ges associated with exocytosis visualized with<br />
polarized-TIRFM<br />
Authors: *A. ANANTHARAM 1 , M. BITTNER 1 , D. AXELROD 2 , R. W. HOLZ 1 ;<br />
1 Pharmacol., 2 Physics <strong>an</strong>d LSA Biophysics, Univ. of Michig<strong>an</strong> Med. Sch., Ann Arbor, MI<br />
Abstract: The topological dynamics of <strong>the</strong> plasma membr<strong>an</strong>e were visualized in bovine adrenal<br />
chromaffin cells using polarized total internal reflection fluorescence microscopy (TIRFM). This<br />
method is based on monitoring <strong>the</strong> fluorescence of <strong>an</strong> oriented membr<strong>an</strong>e probe (<strong>the</strong><br />
carbocy<strong>an</strong>ine dye, DiI) excited by a polarized ev<strong>an</strong>escent field created by TIR illumination. DiI<br />
has been shown to embed in <strong>the</strong> membr<strong>an</strong>e with its tr<strong>an</strong>sition dipole moments nearly in <strong>the</strong> pl<strong>an</strong>e<br />
of <strong>the</strong> membr<strong>an</strong>e. Thus, by monitoring <strong>the</strong> pixel-by-pixel ratio of <strong>the</strong> membr<strong>an</strong>e-embedded DiI<br />
fluorescence excited by <strong>the</strong> two polarizations (p - perpendicular to substrate; s - parallel to<br />
substrate) over time, regions of membr<strong>an</strong>e curvature are vividly highlighted. To relate <strong>the</strong><br />
orientation of <strong>the</strong> membr<strong>an</strong>e with exocytosis, gr<strong>an</strong>ules were labeled with <strong>the</strong> marker<br />
neuropeptide (NPY) - cerule<strong>an</strong>. A variety of topological ch<strong>an</strong>ges in <strong>the</strong> plasma membr<strong>an</strong>e (given<br />
by monitoring <strong>the</strong> ch<strong>an</strong>ge in p/s with time) were observed at <strong>the</strong> sites of exocytosis. Sometimes,<br />
<strong>the</strong> increase in p/s was tr<strong>an</strong>sitory, declining over seconds to pre-fusion levels. In o<strong>the</strong>r inst<strong>an</strong>ces,<br />
p/s increased with fusion <strong>an</strong>d remained elevated <strong>for</strong> tens of seconds, providing evidence <strong>for</strong><br />
longer-lasting membr<strong>an</strong>e curvature. In approximately 40% of cases, a ch<strong>an</strong>ge in p/s with fusion<br />
was not detected, likely reflecting events occurring more quickly th<strong>an</strong> our time resolution (~ 2.5<br />
Hz). <strong>When</strong> endocytosis was inhibited with strontium replacing bath calcium, or Dynasore<br />
(inhibitor of dynamin 1 <strong>an</strong>d 2), membr<strong>an</strong>e p/s ch<strong>an</strong>ges were detected more frequently <strong>an</strong>d were<br />
more resist<strong>an</strong>t to decay. These experiments demonstrate that polarized-TIRFM is able to detect<br />
rapid, submicron ch<strong>an</strong>ges in plasma membr<strong>an</strong>e topology as a result of exocytosis <strong>an</strong>d that <strong>the</strong>re<br />
is a r<strong>an</strong>ge of possible outcomes <strong>for</strong> <strong>the</strong> fused gr<strong>an</strong>ule/plasma membr<strong>an</strong>e complex. They also<br />
demonstrate that <strong>the</strong> gr<strong>an</strong>ule/plasma membr<strong>an</strong>e complex <strong>for</strong>med upon exocytosis, is immediately<br />
shaped by dynamin be<strong>for</strong>e endocytosis.<br />
Disclosures: A. An<strong>an</strong>tharam, None; M. Bittner, None; D. Axelrod, None; R.W. Holz, None.
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.3/E28<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: AHA<br />
Whitehall Foundation<br />
NSF<br />
<strong>the</strong> Jap<strong>an</strong> <strong>Society</strong> of <strong>the</strong> Promotion of Science<br />
Title: Optical detection of glutamate released <strong>from</strong> a single nerve terminal of hippocampal<br />
neurons<br />
Authors: *N. C. HARATA 1 , Y. KAKAZU 1 , K. M. GOODMAN 1,2 , J.-Y. KOH 1 , S. NAMIKI 3 ,<br />
K. HIROSE 3 , J. LIM 4 , Y. LI 4 ;<br />
1 Dept Mol Physiol & Biophys, Univ. Iowa Col. Med., Iowa City, IA; 2 Univ. Bath, Bath, United<br />
Kingdom; 3 Dept Neurobiol, Univ. Tokyo, Tokyo, Jap<strong>an</strong>; 4 Dept Math, Univ. Iowa, Iowa City, IA<br />
Abstract: The average extracellular concentration of <strong>the</strong> neurotr<strong>an</strong>smitter glutamate following<br />
its release has been estimated in <strong>the</strong> past, but it has been technically difficult to address to what<br />
extent this concentration varies <strong>from</strong> synapse to synapse, <strong>an</strong>d even <strong>from</strong> one release event to<br />
<strong>an</strong>o<strong>the</strong>r within a single synapse. We have addressed this issue using a “glutamate (E) optical<br />
sensor” (EOS), a hybrid molecule in which <strong>the</strong> thiol-reactive fluorescent dye Oregon Green is<br />
linked to <strong>the</strong> extracellular S1S2 domain of <strong>the</strong> AMPA subtype of glutamate receptor (GluR2) as<br />
a consequence of a single amino-acid mutation (Namiki et al, Eur J Neurosci, 2007). In this<br />
protein, glutamate binding leads to <strong>an</strong> increase in <strong>the</strong> fluorescence intensity of <strong>the</strong> dye. In this<br />
study, we have improved EOS by introducing <strong>an</strong> additional mutation, which leads to a 1.5-fold<br />
increase in <strong>the</strong> fluorescence ch<strong>an</strong>ge over that generated in <strong>the</strong> first-generation EOS. In live, rat<br />
hippocampal cultures that had been biotinylated <strong>an</strong>d treated with streptavidin-conjugated EOS,<br />
<strong>the</strong> sensor was immobilized on <strong>the</strong> plasma membr<strong>an</strong>es of both neurons <strong>an</strong>d astrocytes, failed to<br />
internalize at room temperature over <strong>the</strong> course of 30 min, <strong>an</strong>d showed minimal lateral<br />
movement along <strong>the</strong> plasma membr<strong>an</strong>e. Biotinylation did not affect presynaptic parameters such<br />
as <strong>the</strong> size of recycling pool of synaptic vesicles <strong>an</strong>d <strong>the</strong> release probability. Const<strong>an</strong>t application<br />
of exogenous glutamate led to a steady-state increase in fluorescence with a rise time const<strong>an</strong>t of<br />
much less th<strong>an</strong> 50 msec. Moreover, <strong>the</strong> response was not affected by application of<br />
cyclothiazide, <strong>an</strong> inhibitor of AMPAR desensitization. Thus, EOS is a spatially locked
fluorescent sensor that responds to rapid ch<strong>an</strong>ges in extracellular glutamate concentration<br />
without going through a desensitized state. <strong>When</strong> neurons were stimulated by field stimulation,<br />
<strong>the</strong> evoked EOS responses were similar in amplitude to <strong>the</strong> spont<strong>an</strong>eous events. Signific<strong>an</strong>t<br />
variation was noted in <strong>the</strong> rise times of EOS events. One adv<strong>an</strong>tage of this system is its high<br />
signal-to-noise ratio, which allows us to detect single EOS events without having to average<br />
multiple traces. It is expected to reveal heterogeneity in <strong>the</strong> concentration of synaptically<br />
released glutamate.<br />
Disclosures: N.C. Harata, None; Y. Kakazu, None; K.M. Goodm<strong>an</strong>, None; J. Koh, None; S.<br />
Namiki, None; K. Hirose, None; J. Lim, None; Y. Li, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.4/E29<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NSERC 326821-06<br />
NSHRF 2006-2100<br />
Title: Rapid synapse-specific structural ch<strong>an</strong>ges of <strong>the</strong> active zone cytomatrix regulate<br />
neurotr<strong>an</strong>smitter release<br />
Authors: J. MATZ, A. KOLAR, T. MCCARVILL, *S. R. KRUEGER;<br />
Dept Physiol & Biophysics, Dalhousie Univ., Halifax, NS, C<strong>an</strong>ada<br />
Abstract: The likelihood with which a presynaptic action potential elicits <strong>the</strong> release of<br />
neurotr<strong>an</strong>smitter, <strong>the</strong> release probability (pr), is <strong>an</strong> import<strong>an</strong>t component of synaptic strength.<br />
Regulatory mech<strong>an</strong>isms controlling several steps of synaptic vesicle (SV) exocytosis may affect<br />
release probability, yet <strong>the</strong>ir relative import<strong>an</strong>ce in determining pr <strong>an</strong>d eli<strong>citing</strong> temporal ch<strong>an</strong>ges<br />
in neurotr<strong>an</strong>smitter release at individual synapses is largely unknown. We have addressed <strong>the</strong><br />
hypo<strong>the</strong>sis that <strong>the</strong> size of <strong>the</strong> active zone, by limiting <strong>the</strong> number of release-competent synaptic<br />
vesicles, is a major determin<strong>an</strong>t of pr <strong>an</strong>d that ch<strong>an</strong>ges in <strong>the</strong> size of <strong>the</strong> active zone cytomatrix<br />
lead to corresponding alterations in neurotr<strong>an</strong>smitter release. To this end, we have employed a<br />
fluorescent sensor of SV exocytosis, synaptophysin-pHluorin, to qu<strong>an</strong>tify pr at individual<br />
synapses with high accuracy, <strong>an</strong>d utilized a fluorescently labeled cytomatrix protein, tdTomato-<br />
Bassoon, to obtain measures of active zone size. We find that, <strong>for</strong> synapses made by a visually
identified presynaptic neuron, active zone size correlates very well with release probability,<br />
suggesting that pr indeed is to a large extent determined by this structural parameter. Intriguingly,<br />
active zone cytomatrices are frequently subject to synapse-specific ch<strong>an</strong>ges in size on a time<br />
scale of minutes. These spont<strong>an</strong>eous alterations in active zones size lead to corresponding<br />
ch<strong>an</strong>ges in neurotr<strong>an</strong>smitter release. Our results suggest that <strong>the</strong> reliability of synaptic<br />
tr<strong>an</strong>smission is largely determined by <strong>the</strong> size of <strong>the</strong> presynaptic specialization <strong>an</strong>d implicate<br />
mech<strong>an</strong>isms leading to rapid alterations in active zone size in synapse-specific <strong>for</strong>ms of synaptic<br />
plasticity.<br />
Disclosures: J. Matz, None; A. Kolar, None; T. McCarvill, None; S.R. Krueger, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.5/E30<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NIH Gr<strong>an</strong>t MH64070<br />
Title: Three-dimensional n<strong>an</strong>ometer accuracy tracking of synaptic vesicles trafficking <strong>from</strong><br />
distinct vesicular pools<br />
Authors: *H. PARK, Y. LI, R. W. TSIEN;<br />
St<strong>an</strong><strong>for</strong>d Univ., St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Movements of presynaptic vesicles are critical <strong>for</strong> <strong>the</strong> continued supply of<br />
neurotr<strong>an</strong>smitter qu<strong>an</strong>ta at nerve terminals, but proper study of <strong>the</strong>ir dynamics requires real-time<br />
optical imaging with high spatial resolution. Using dual-focus imaging microscopy, we were able<br />
to track single Qdot-loaded synaptic vesicles in three dimensions at up to video frame rate, with<br />
<strong>an</strong> accuracy of a few nm in <strong>the</strong> X-Y pl<strong>an</strong>e <strong>an</strong>d of tens of nm along <strong>the</strong> Z-axis. Taking adv<strong>an</strong>tage<br />
of a biotinylated <strong>an</strong>tibody against a luminal synaptotagmin epitope, <strong>an</strong>d bright streptavidin-Qdots<br />
(Invitrogen), we directly observed exocytotic events of single synaptic vesicles during electrical<br />
stimulation of hippocampal synapses. Tryp<strong>an</strong> blue was included in <strong>the</strong> external medium to detect<br />
<strong>the</strong> exocytosis, via quenching of Qdots exposed to <strong>the</strong> dye to varying degrees following kiss-<strong>an</strong>drun<br />
or full collapse fusion. By using a combination of spectrally separable live markers,<br />
presynaptic (FM 4-64) <strong>an</strong>d postsynaptic (PSD95-GFP), we were able to localize exocytotic<br />
events as occurring between <strong>the</strong> centroids of FM 4-64 <strong>an</strong>d PSD 95 GFP. Using this high<br />
resolution imaging technique, we also observed that some subsets of synaptic vesicles moved to
adjacent synapses <strong>an</strong>d were subsequently released, confirming a report that synaptic vesicles c<strong>an</strong><br />
be shared between synapses. Using various loading protocols, we could track <strong>the</strong> dynamics <strong>an</strong>d<br />
fate of synaptic vesicles derived <strong>from</strong> ei<strong>the</strong>r readily releasable pools or total recycling pools. In<br />
<strong>the</strong> lead-up to fusion, synaptic vesicles <strong>from</strong> <strong>the</strong> total recycling pool moved by a larger net<br />
dist<strong>an</strong>ce th<strong>an</strong> vesicles <strong>from</strong> <strong>the</strong> readily releasable pool. This disparity is not mutually exclusive<br />
of o<strong>the</strong>r possible sources of functional differences, including <strong>the</strong> vesicle’s molecular identity.<br />
Disclosures: H. Park, None; Y. Li, None; R.W. Tsien, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.6/E31<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: MPG T<strong>an</strong>dem-Project<br />
Alex<strong>an</strong>der von Humboldt Foundation<br />
DFG, Center of <strong>the</strong> Molecular Physiology of <strong>the</strong> Brain<br />
Boehringer Ingeheim Fond<br />
NIH<br />
Title: Otoferlin is required <strong>for</strong> efficient synaptic vesicle replenishment in hair cells<br />
Authors: *T. MOSER 1,2 , T. PANGRSIC 2 , L. LASAROW 2 , D. KHIMICH 2 , D. RIEDEL 3 , M.<br />
SCHWANDER 4 , K. REUTER 2 , E. REISINGER 2,5 , N. STRENZKE 2 , N. BROSE 5 , U.<br />
MÜLLER 4 ;<br />
2 InnerEarLab, Dept. Otolaryng., 1 Univ. Goettingen Med. Sch., Goettingen, Germ<strong>an</strong>y; 3 Lab. of<br />
Electron Microscopy,, Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Biophysical Chem., Goettingen, Germ<strong>an</strong>y; 4 Dept. of<br />
Cell Biology, Inst. <strong>for</strong> Childhood <strong>an</strong>d Neglected Dis., The Scripps Res. Inst., La Jolla, CA; 5 Max<br />
Pl<strong>an</strong>ck Inst. <strong>for</strong> Exptl. Med., Goettingen, Germ<strong>an</strong>y<br />
Abstract: The multi-C2 domain protein otoferlin has previously been implicated in a late step of<br />
exocytosis, probably fusion, at <strong>the</strong> inner hair cell (IHC) ribbon synapse. Its absence causes<br />
profound deafness <strong>an</strong>d near-complete loss of calcium-dependent exocytosis.
Here, we provide evidence that otoferlin is crucial <strong>for</strong> efficient vesicle replenishment at <strong>the</strong> hair<br />
cell ribbon synapse. In vivo, pach<strong>an</strong>ga mut<strong>an</strong>ts, which carry a point mutation in <strong>the</strong> C2F domain<br />
of otoferlin, show normal distortion product otoacoustic emission but no auditory brainstem<br />
responses during typical stimulation protocols. Some auditory brainstem responses c<strong>an</strong> be<br />
achieved when <strong>the</strong> stimulus rate is reduced. Sound-evoked single neuron activity in <strong>the</strong> region of<br />
<strong>the</strong> cochlear nucleus is observed only at very high sound pressure levels <strong>an</strong>d shows low spike<br />
rates <strong>an</strong>d poor onset coding. Onset coding <strong>an</strong>d rates of <strong>the</strong> sound-evoked spiking both improve<br />
upon increasing <strong>the</strong> interstimulus intervals (providing a longer period <strong>for</strong> recovery). In vitro,<br />
IHCs of mut<strong>an</strong>t mice exhibit unimpaired exocytosis of <strong>the</strong> readily releasable pool of vesicles<br />
(RRP) when sufficient time <strong>for</strong> RRP recovery is provided. However, <strong>the</strong> RRP recovery is slowed<br />
<strong>an</strong>d <strong>the</strong> sustained exocytosis is strongly diminished. While <strong>the</strong>re is only a minor reduction of<br />
ribbon-containing synapses, <strong>the</strong> number of docked vesicles at ribbons appears subst<strong>an</strong>tially<br />
decreased in electron micrographs.<br />
Immunohistochemical qu<strong>an</strong>tification showed <strong>an</strong> approximately 70% reduction of otoferlin<br />
protein levels.<br />
We conclude that <strong>the</strong> pach<strong>an</strong>ga mutation, due to reduced abund<strong>an</strong>ce of otoferlin <strong>an</strong>d/or a specific<br />
alteration of protein function, impairs replenishment of synaptic vesicles to <strong>the</strong> hair cell active<br />
zone. This leads to fatigue of tr<strong>an</strong>smitter release during physiological activity, as a st<strong>an</strong>ding RRP<br />
c<strong>an</strong>not be sustained, causing a profound hearing impairment. In summary, this work identifies a<br />
specific function of otoferlin in <strong>an</strong> early step of exocytosis, most likely in vesicle docking.<br />
Disclosures: T. Moser, Center <strong>for</strong> Molecular Physiology of <strong>the</strong> Brain, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); T<strong>an</strong>dem Project Gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); T. P<strong>an</strong>grsic, None; L.<br />
Lasarow, None; D. Khimich, None; D. Riedel, None; M. Schw<strong>an</strong>der, None; K. Reuter,<br />
Boehringer Ingelheim Fund PhD Fellowship, C. O<strong>the</strong>r Research Support (receipt of drugs,<br />
supplies, equipment or o<strong>the</strong>r in-kind support); E. Reisinger, None; N. Strenzke, None; N.<br />
Brose, None; U. Müller, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.7/E32<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: EY14990
Title: Photodamaging <strong>the</strong> ribbon disrupts coordination of multivesicular release <strong>an</strong>d blocks<br />
vesicle replenishment at <strong>the</strong> rod bipolar cell synapse in mouse<br />
Authors: *J. SNELLMAN, D. ZENISEK;<br />
Cell. <strong>an</strong>d Mol. Physiol., Yale Univ. Shool of Med., New Haven, CT<br />
Abstract: To investigate synaptic ribbon function, we used fluorophore-assisted light<br />
inactivation (FALI) to photodamage <strong>the</strong> synaptic ribbon in retinal rod bipolar cells (RBCs). For<br />
this purpose, we photobleached a fluorescein-tagged peptide that binds to <strong>the</strong> B-domain of<br />
ribeye, a protein abund<strong>an</strong>t in synaptic ribbons. We used FALI to photodamage ribbons in RBCs<br />
loaded with <strong>the</strong> peptide via a patch pipette in a mouse retinal slice preparation. Synaptic release<br />
was elicited by depolarizing RBCs to -10 mV every 60 seconds, <strong>an</strong>d neurotr<strong>an</strong>smitter release<br />
was monitored by recording postsynaptic currents in AII amacrine cells. Integration of <strong>the</strong> AII<br />
EPSCs showed that prior to FALI, bipolar cells loaded with peptide exhibited two components of<br />
release: a phasic component with a time const<strong>an</strong>t of 6.1 (+/- 0.9) ms <strong>an</strong>d a tonic component with<br />
a time const<strong>an</strong>t of 217 (+/- 2.5) ms. With <strong>the</strong> peptide in <strong>the</strong> pipette, but without illumination, <strong>the</strong><br />
amplitudes of <strong>the</strong> phasic (1.57+/-0.69 pC) <strong>an</strong>d tonic 5.34 (+/- 1.06) pC components were not<br />
different <strong>from</strong> control cells loaded with a scrambled peptide (phasic: 1.78 (+/- 0.84) pC;<br />
tonic:4.91 (+/- 1.05) pC). Photobleaching of ribeye-binding-peptide irreversibly compromised<br />
synaptic release <strong>from</strong> rod bipolar cells while leaving <strong>the</strong> RBC calcium current intact. The first<br />
response after FALI showed only a modest decrease in <strong>the</strong> phasic component (12%), but a near<br />
complete lack of tonic release. In response to subsequent stimuli, however, both <strong>the</strong> tonic<br />
component (89 % decrease +/- 2.9) <strong>an</strong>d phasic component (66.4 % decrease +/- 12.3) were<br />
subst<strong>an</strong>tially reduced, indicating that a pool of vesicles is initially resist<strong>an</strong>t to photobleach, but<br />
fails to replenish. The size of this FALI resist<strong>an</strong>t pool is approximately 10 vesicles per synapse,<br />
similar to <strong>the</strong> number of vesicles thought to reside at <strong>the</strong> base of <strong>the</strong> ribbon. Import<strong>an</strong>tly,<br />
photobleaching of <strong>the</strong> scrambled peptide had no effect on ei<strong>the</strong>r <strong>the</strong> phasic (1.2 +/- 6.8 %<br />
increase) or tonic (28 +/- 16.4 % decrease) components. Spont<strong>an</strong>eous events were decreased by<br />
63%, suggesting that spont<strong>an</strong>eous release also originates at ribbon sites. Lastly, <strong>the</strong> size of<br />
mEPSCs were reduced during <strong>the</strong> tonic component of release in a m<strong>an</strong>ner consistent with a loss<br />
of multivesicular release. These data suggest a role <strong>for</strong> <strong>the</strong> ribbon in mainten<strong>an</strong>ce of continuous<br />
release <strong>an</strong>d in coordinating multivesicular release.<br />
Disclosures: J. Snellm<strong>an</strong>, None; D. Zenisek, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.8/E33
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Title: The role of tomosyn in Drosophila<br />
Authors: K. CHEN, *D. E. FEATHERSTONE, J. RICHMOND;<br />
Biol. Sci., Univ. of Illinois At Chicago, Chicago, IL<br />
Abstract: Synaptic plasticity c<strong>an</strong> result <strong>from</strong> ch<strong>an</strong>ges in neurotr<strong>an</strong>smitter secretion.<br />
Neurotr<strong>an</strong>smitter secretion depends on synaptic vesicle fusion, which relies on assembly of<br />
fusogenic SNARE complexes comprised of <strong>the</strong> vesicle protein synaptobrevin, <strong>an</strong>d plasma<br />
membr<strong>an</strong>e proteins syntaxin <strong>an</strong>d SNAP-25. Accumulating evidence indicates that SNARE<br />
complex <strong>for</strong>mation draws synaptic vesicles into close apposition with <strong>the</strong> plasma membr<strong>an</strong>e,<br />
rendering <strong>the</strong>m primed <strong>an</strong>d fusion competent. Tomosyn c<strong>an</strong> inhibit priming by binding syntaxin,<br />
<strong>the</strong>reby influencing synaptic strength. The interaction between tomosyn <strong>an</strong>d syntaxin is regulated<br />
by protein kinase A (PKA), <strong>an</strong> enzyme implicated in learning <strong>an</strong>d memory. We hypo<strong>the</strong>size that<br />
tomosyn may be <strong>an</strong> import<strong>an</strong>t effector in PKA-dependent synaptic plasticity. Several Drosophila<br />
mut<strong>an</strong>ts affecting <strong>the</strong> PKA pathway are known to be learning-defective, making flies <strong>an</strong><br />
excellent model to test whe<strong>the</strong>r tomosyn plays a role in learning. Since <strong>the</strong>re are currently no<br />
Drosophila tomosyn mut<strong>an</strong>ts, we used RNA interference to reduce tomosyn levels. Tomosyn<br />
levels in <strong>the</strong>se RNAi-expressing flies are 30% of wild-type, based on qu<strong>an</strong>titative RT-PCR <strong>an</strong>d<br />
reduced tomosyn immunoreactivity. As expected, tomosyn knockdown caused signific<strong>an</strong>t<br />
enh<strong>an</strong>cement of synaptic strength at <strong>the</strong> Drosophila larval NMJ, m<strong>an</strong>ifest as slower EJC decay<br />
time <strong>an</strong>d a 50% increase in evoked charge integral. This enh<strong>an</strong>ced release was not due to altered<br />
synaptic morphology as <strong>the</strong> NMJ size <strong>an</strong>d synaptic bouton density were unaffected following<br />
tomosyn RNAi. We next asked whe<strong>the</strong>r tomosyn RNAi impacted short-term synaptic plasticity<br />
<strong>an</strong>d learning. We saw no ch<strong>an</strong>ge in paired-pulsed depression at <strong>the</strong> larval NMJ over a r<strong>an</strong>ge of<br />
interstimulus intervals (20-200ms) in normal (1.8 mM) calcium. But olfactory associativelearning<br />
assays in tomosyn RNAi-expressing larvae were dramatically affected. Specifically, <strong>the</strong><br />
learning index after tomosyn knockdown was reduced to <strong>the</strong> level of sham controls, indicating<br />
that tomosyn is required <strong>for</strong> learning. We are currently testing whe<strong>the</strong>r <strong>the</strong> learning phenotype of<br />
tomosyn mut<strong>an</strong>ts is epistatic to mut<strong>an</strong>ts in <strong>the</strong> PKA pathway. These data indicate that tomosyn<br />
has a conserved role in <strong>the</strong> regulation of synaptic tr<strong>an</strong>smission <strong>an</strong>d provide <strong>the</strong> first behavioral<br />
evidence that tomosyn may be involved in learning.<br />
Disclosures: K. Chen, None; D.E. Fea<strong>the</strong>rstone, None; J. Richmond, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 522.9/E34<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: Wellcome project gr<strong>an</strong>t 083199/Z/07/Z<br />
BBSRC Joint MRes/PhD studentship (to JS)<br />
Title: High frequency kiss-<strong>an</strong>d-run exocytosis stimulated by LTX N4C at <strong>the</strong> mammali<strong>an</strong><br />
neuromuscular junction<br />
Authors: J. SUCKLING 1 , K. VOLYNSKI 1 , D. THOMSON 2 , V. LELIANOVA 1 , R. R.<br />
RIBCHESTER 2 , *Y. A. USHKARYOV 1 ;<br />
1 Div. of Cell & Mol. Biol., Imperial Col. London, London, United Kingdom; 2 Eu<strong>an</strong> MacDonald<br />
Ctr. <strong>for</strong> MND Research, Sch. of Biomed. Sci., Univ. of Edinburgh, Edinburgh, United Kingdom<br />
Abstract: At <strong>the</strong> mammali<strong>an</strong> neuromuscular junction (mNMJ; flexor digitorum brevis muscle),<br />
in <strong>the</strong> absence of Ca 2+ , α-latrotoxin (LTX) gradually increases <strong>the</strong> frequency of miniature end<br />
plate potentials (mepps) <strong>from</strong> 0.1 Hz to ~300 Hz. This high-frequency secretion <strong>the</strong>n slowly<br />
subsides <strong>an</strong>d ceases completely.<br />
In <strong>the</strong> presence of Ca 2+ , <strong>the</strong> toxin has two overlapping effects: (1) <strong>the</strong> slow increase <strong>an</strong>d gradual<br />
cessation of mepps frequency (same as in 0 Ca 2+ ) <strong>an</strong>d (2) abrupt bursts of high frequency (~250<br />
Hz) exocytosis (specifically caused by <strong>the</strong> addition of Ca 2+ ).<br />
The Ca 2+ -independent activity of LTX requires its insertion into <strong>the</strong> membr<strong>an</strong>e. Consistently, <strong>the</strong><br />
mut<strong>an</strong>t latrotoxin LTX N4C (which does not <strong>for</strong>m tetramers, nor insert itself into <strong>the</strong> membr<strong>an</strong>e)<br />
only acts in <strong>the</strong> presence of Ca 2+ . This effect of LTX N4C closely resembles <strong>the</strong> burst-like type 2<br />
exocytosis caused by <strong>the</strong> LTX in <strong>the</strong> presence of Ca 2+ . Import<strong>an</strong>tly, LTX N4C does not cause <strong>the</strong><br />
type 1 (gradual <strong>an</strong>d detrimental) increase in mepps frequency, so <strong>the</strong> burst-like type 2 release<br />
continues unabated <strong>for</strong> m<strong>an</strong>y hours.<br />
Studies of <strong>the</strong> LTX N4C -induced exocytosis demonstrate that <strong>the</strong> bursts occur r<strong>an</strong>domly, with<br />
bouts of high frequency release interspersed by periods of basal mepps frequency (1 Hz). On<br />
average, each burst lasts <strong>for</strong> 4 sec <strong>an</strong>d corresponds to <strong>the</strong> release of ~250 vesicles, or 5-10 % of<br />
all vesicles contained in <strong>an</strong> average bouton. To underst<strong>an</strong>d <strong>the</strong> source of this burst-like secretion<br />
<strong>an</strong>d compare <strong>the</strong> behaviour of individual boutons, we studied vesicle exocytosis using FM-dyes.<br />
Our data suggest that <strong>the</strong> bursts of secretion represent exocytosis of <strong>the</strong> readily releasable<br />
vesicles <strong>from</strong> individual boutons <strong>an</strong>d that LTX N4C is able to evoke at least some amount of kiss<strong>an</strong>d-run<br />
exocytosis, while KCl mostly induces full fusion.<br />
To investigate <strong>the</strong> modes of exocytosis at <strong>the</strong> mNMJ fur<strong>the</strong>r, we developed a novel method of<br />
specifically detecting kiss-<strong>an</strong>d-run exocytosis in cases where only a small proportion of vesicles<br />
undergo exocytosis at <strong>an</strong>y given time. This protocol involves loading vesicles with FM-dyes<br />
toge<strong>the</strong>r with a soluble fluorescence quencher. Initially, vesicles are non-fluorescent <strong>an</strong>d if<br />
exocytosis results in full fusion, no increase in fluorescence occurs, as both quencher <strong>an</strong>d FM<br />
dye are released. However, if vesicles undergo kiss-<strong>an</strong>d-run exocytosis, quencher completely<br />
escapes <strong>from</strong> <strong>the</strong> tr<strong>an</strong>siently fusing vesicles whilst <strong>the</strong> FM dye partially remains in <strong>the</strong> vesicles.<br />
This leads to <strong>an</strong> increase in FM fluorescence.<br />
Using this method, we have shown that high frequency kiss-<strong>an</strong>d-run exocytosis does occur at <strong>the</strong>
mNMJ <strong>an</strong>d that <strong>the</strong> mode of exocytosis c<strong>an</strong> be shifted between full fusion <strong>an</strong>d kiss-<strong>an</strong>d-run,<br />
depending on <strong>the</strong> type of stimulus applied.<br />
Disclosures: J. Suckling, None; K. Volynski, None; D. Thomson, None; V. Leli<strong>an</strong>ova,<br />
None; R.R. Ribchester, None; Y.A. Ushkaryov, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.10/E35<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Title: Genetic dissection of release states modulated by synaptotagmin <strong>an</strong>d complexin<br />
Authors: *R. A. JORQUERA 1,2 , R. W. CHO 1,2 , S. HUNTWORK 1,2 , Y. SONG 2 , J.<br />
LITTLETON 2,1 ;<br />
1 Dept. of Brain <strong>an</strong>d Cognitive Sci., 2 Dept. of Biol., MIT, Cambridge, MA<br />
Abstract: Neurotr<strong>an</strong>smitter release is a specialized <strong>for</strong>m of vesicle fusion that shares a common<br />
SNARE-mediated fusion mech<strong>an</strong>ism with o<strong>the</strong>r vesicle trafficking pathways within cells.<br />
Unique SNARE interactors that regulate SNARE complex-mediated fusion have evolved during<br />
<strong>the</strong> evolution of <strong>the</strong> synapse, including complexin <strong>an</strong>d synaptotagmin-1. How complexin <strong>an</strong>d<br />
synaptotagmin contribute to <strong>the</strong> unique requirements of synaptic vesicle release is still debated.<br />
In addition, <strong>the</strong> role of complexin <strong>an</strong>d synaptotagmin in short-term synaptic plasticity is poorly<br />
understood. Here, we investigate <strong>the</strong>se proteins in neurotr<strong>an</strong>smitter release <strong>an</strong>d short-term<br />
synaptic plasticity using <strong>the</strong> Drosophila neuromuscular junction (NMJ). We present a thorough<br />
<strong>an</strong>alysis of synaptic tr<strong>an</strong>smission at NMJs of mut<strong>an</strong>t <strong>an</strong>d overexpression <strong>an</strong>imals using high<br />
fidelity current recordings of postsynaptic evoked <strong>an</strong>d spont<strong>an</strong>eous currents using voltage-clamp.<br />
We combine qu<strong>an</strong>tal <strong>an</strong>alysis with immunocytochemical determination of <strong>the</strong> number of release<br />
sites, allowing a robust description of release properties regulated by complexin <strong>an</strong>d<br />
synaptotagmin. Kinetic <strong>an</strong>alysis of evoked currents reveals that synchronous <strong>an</strong>d asynchronous<br />
release critically depends on <strong>the</strong> level of complexin <strong>an</strong>d synaptotagmin expression. Moreover,<br />
spont<strong>an</strong>eous current <strong>an</strong>alysis indicates that spont<strong>an</strong>eous release is tightly regulated by<br />
synaptotagmin <strong>an</strong>d complexin. Additionally, synaptotagmin <strong>an</strong>d complexin alter short-term<br />
synaptic plasticity. Analysis of complexin <strong>an</strong>d synaptotagmin nulls, <strong>an</strong>d <strong>the</strong>ir genetic<br />
interactions, reveals <strong>the</strong> existence of different release sates depending on <strong>the</strong> availability or levels<br />
of <strong>the</strong> two proteins. We propose that synaptotagmin minimizes <strong>the</strong> energy of <strong>the</strong> tr<strong>an</strong>sition-state<br />
<strong>for</strong> SNARE complex fusion, while complexin decreases <strong>the</strong> free energy <strong>from</strong> SNARE complex
assembly, creating <strong>an</strong> energetic barrier at a late step of synaptic vesicle fusion that clamps<br />
synaptic vesicles in <strong>the</strong> immediately releasable pool<br />
Disclosures: R.A. Jorquera, None; R.W. Cho, None; S. Huntwork, None; Y. Song, None; J.<br />
Littleton, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.11/E36<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NIH Gr<strong>an</strong>t NS050655 to C.R.<br />
NIH Gr<strong>an</strong>t NS037200 to J.R.<br />
NIH Gr<strong>an</strong>t F31MH078678 to H.T.C.<br />
Welch Foundation Gr<strong>an</strong>t I-1304 to J.R.<br />
Title: Binding of <strong>the</strong> Complexin N terminus to <strong>the</strong> SNARE complex C terminus potentiates<br />
synaptic vesicle fusogenicity<br />
Authors: *M. XUE 1 , T. K. CRAIG 3 , J. XU 3 , H.-T. CHAO 1 , J. RIZO 3 , C. ROSENMUND 2 ;<br />
1 Dept of Neurosci, 2 Dept. of Neurosci. <strong>an</strong>d Dept. of Mol. <strong>an</strong>d Hum<strong>an</strong> Genet., Baylor Col. of<br />
Med., Houston, TX; 3 Dept. of Biochem. <strong>an</strong>d Dept. of Pharmacol., Univ. of Texas Southwestern<br />
Med. Ctr., Dallas, TX<br />
Abstract: The exquisite temporal <strong>an</strong>d spatial precision of SNAREs (soluble N-ethylmaleimidesensitive<br />
factor-attachment protein receptors)-mediated synaptic vesicle exocytosis requires a<br />
diversity of specialized proteins including Complexins, a family of SNARE complex-binding<br />
proteins. Complexins contain a central α-helix <strong>an</strong>d <strong>an</strong> accessory α-helix in <strong>the</strong> middle, <strong>an</strong>d <strong>the</strong> N-<br />
<strong>an</strong>d C-terminal sequences. Biophysical <strong>an</strong>d physiological studies indicate that Complexins play<br />
dual roles in neurotr<strong>an</strong>smitter release by activating <strong>an</strong>d inhibiting vesicle fusion via distinct<br />
protein domains. Previous structure-function <strong>an</strong>alyses show that binding of <strong>the</strong> central α-helix to<br />
<strong>the</strong> SNARE complex is essential <strong>for</strong> Complexin function. The accessory α-helix between <strong>the</strong> N<br />
terminus <strong>an</strong>d <strong>the</strong> central α-helix inhibits release, possibly by replacing <strong>the</strong> C-terminal portion of<br />
<strong>the</strong> Synaptobrevin-2 SNARE motif in <strong>the</strong> tr<strong>an</strong>s-SNARE complex, thus preventing <strong>the</strong> C-terminal
assembly of <strong>the</strong> SNARE complex. The Complexin N terminus is a strong stimulator of<br />
neurotr<strong>an</strong>smitter release, but <strong>the</strong> underlying molecular mech<strong>an</strong>ism remains unknown.<br />
Fur<strong>the</strong>rmore, a widespread model proposes that Complexins clamp vesicles at <strong>the</strong> primed state<br />
<strong>an</strong>d <strong>the</strong> Ca 2+ sensor Synaptotagmin-1 releases <strong>the</strong> inhibition upon Ca 2+ influx. However, <strong>the</strong><br />
nature of <strong>the</strong> interplay between Complexins <strong>an</strong>d Synaptotagmin-1 is still under debate <strong>an</strong>d it is<br />
unclear whe<strong>the</strong>r Complexin function is strictly coupled to Synaptotagmin-1.<br />
To gain fur<strong>the</strong>r insight into <strong>the</strong> mech<strong>an</strong>ism of Complexin function, we per<strong>for</strong>med physiological<br />
<strong>an</strong>d biophysical <strong>an</strong>alyses of Complexin I, a major paralog in <strong>the</strong> mouse brain. We now uncover a<br />
novel interaction between <strong>the</strong> Complexin I N terminus <strong>an</strong>d <strong>the</strong> SNARE complex C terminus, <strong>an</strong>d<br />
show that disrupting this binding abolishes <strong>the</strong> facilitatory function of Complexin I in mouse<br />
hippocampal neurons. This interaction appears to increase synaptic vesicle fusogenicity in a step<br />
that precedes <strong>the</strong> Ca 2+ triggering step. Moreover, genetic experiments crossing Complexin <strong>an</strong>d<br />
Synaptotagmin-1 null mice demonstrate that Complexins facilitate neurotr<strong>an</strong>smitter release even<br />
in <strong>the</strong> absence of Synaptotagmin-1. We propose that <strong>the</strong> Complexin N terminus stabilizes <strong>the</strong><br />
SNARE complex C terminus <strong>an</strong>d helps to release <strong>the</strong> inhibitory function of Complexins, <strong>the</strong>reby<br />
activating <strong>the</strong> fusion machinery in a m<strong>an</strong>ner that may cooperate with Synaptotagmin-1, but is not<br />
strictly dependent on Synaptotagmin-1.<br />
Disclosures: M. Xue, None; T.K. Craig, None; J. Xu, None; H. Chao, None; J. Rizo,<br />
None; C. Rosenmund, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.12/E37<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Title: Two SNARE complexes are sufficient to drive synaptic vesicle fusion<br />
Authors: R. SINHA, *J. KLINGAUF;<br />
Max Pl<strong>an</strong>ck Inst. Biophysical Chem, Goettingen 37077, Germ<strong>an</strong>y<br />
Abstract: Exocytosis of synaptic vesicles is mediated by <strong>the</strong> assembly of low-energy SNARE<br />
complexes <strong>for</strong>med by <strong>the</strong> coil-coiling of three proteins: SNAP-25, syntaxin, <strong>an</strong>d synaptobrevin 2<br />
(syb 2). However it is unknown how m<strong>an</strong>y SNARE complexes are minimally needed <strong>for</strong><br />
synaptic vesicle (SV) priming <strong>an</strong>d fusion. It was suggested that SNARE complexes might<br />
assemble toge<strong>the</strong>r in rosette-shaped super-complexes.<br />
Here we show that only two SNARE complexes are sufficient <strong>for</strong> mediating stimulation-
dependent fusion of a SV. We optically measured single vesicle fusion events in real time using<br />
a genetically encoded probe synaptopHluorin (spH), a pH-sensitive vari<strong>an</strong>t of GFP, pHluorin,<br />
fused to <strong>the</strong> luminal domain of syb 2. Based on qu<strong>an</strong>titative single molecule experiments we<br />
found that only 2-3 spH molecules are incorporated per SV upon tr<strong>an</strong>sient overexpression.<br />
Surprisingly, when spH was overexpressed on a null background, this low copy number was<br />
sufficient to fully rescue evoked SV fusion. SVs expressing only one copy of spH, however,<br />
were not able to fuse upon stimulation. Thus, two SNARE complexes are necessary <strong>an</strong>d<br />
sufficient to drive SV fusion during fast synaptic tr<strong>an</strong>smission.<br />
Disclosures: R. Sinha, None; J. Klingauf, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.13/E38<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: Association Fr<strong>an</strong>çaise contre les myopathies (AFM) FRANCE<br />
Ministère de l’enseignement superieur et de la recherche - Action Concertée Incitative<br />
« Biologie cellulaire, moléculaire et structurale (BCMS)» FRANCE<br />
Gr<strong>an</strong>ts-in aid <strong>for</strong> Scientific Research B (JAPAN)<br />
Title: V-ATPase membr<strong>an</strong>e sector associates with synaptobrevin to mediate neurotr<strong>an</strong>smitter<br />
release<br />
Authors: O. EL FAR 1 , J. DI GIOVANNI 1 , S. BODKKAZI 1 , S. MOCHIDA 2 , N. SAMARI 1 , C.<br />
LEVEQUE 1 , F. YOUSSOUF 1 , A. BRECHET 1 , C. IBORRA 1 , Y. MAULET 1 , D. DEBANNE 1 ,<br />
*M. J. SEAGAR 1 ;<br />
1 Inserm U641, 13916 Marseille 20, Fr<strong>an</strong>ce; 2 Dept. of Physiol., Tokyo Med. Univ., Tokyo, Jap<strong>an</strong><br />
Abstract: The vacuolar proton ATPase (V-ATPase) is a major actor in cellular physiology by its<br />
proton tr<strong>an</strong>sport activity. Earlier findings in Torpedo suggested <strong>an</strong> implication of <strong>the</strong> membr<strong>an</strong>e<br />
V0 sector of V-ATPase in acetylcholine release. Recent data have revitalized interest in a direct<br />
role <strong>for</strong> V0 in membr<strong>an</strong>e fusion but <strong>the</strong> mech<strong>an</strong>isms involved remain obscure. We identified a<br />
direct interaction between V0 c-subunit <strong>an</strong>d synaptobrevin which constitutes a molecular link to<br />
SNARE-mediated fusion. Acute perturbation of this interaction with V0 derived peptides
induced a decrease in release probability <strong>an</strong>d inhibited glutamatergic as well as cholinergic<br />
neurotr<strong>an</strong>smitter release in mammali<strong>an</strong> cortical slices <strong>an</strong>d cultured neurons <strong>from</strong> superior<br />
cervical g<strong>an</strong>glia respectively. Thus, V-ATPase V0 sector ensures two independent functions:<br />
proton tr<strong>an</strong>sport by a fully assembled V-ATPase <strong>an</strong>d a role in SNARE-dependent exocytosis.<br />
Disclosures: O. El far, None; J. Di giov<strong>an</strong>ni, None; S. Bodkkazi, None; S. Mochida,<br />
None; N. Samari, None; C. Leveque, None; F. Youssouf, None; A. Brechet, None; C. Iborra,<br />
None; Y. Maulet, None; D. Deb<strong>an</strong>ne, None; M.J. Seagar, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.14/F1<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: Progetto eccellenza CARIPARO: Physiopathology of <strong>the</strong> synapse: neurotr<strong>an</strong>smitters,<br />
neurotoxins <strong>an</strong>d novel <strong>the</strong>rapies.<br />
Post Doc University of Padova gr<strong>an</strong>t #CPDR078721/07<br />
Title: A rosette of SNARE complexes mediates synaptic vesicle exocytosis at <strong>the</strong><br />
D.mel<strong>an</strong>ogaster neuromuscular junction<br />
Authors: *A. MEGIGHIAN 1 , O. ROSSETTO 2 , M. SCORZETO 1 , S. PANTANO 4 , C.<br />
BENNA 3 , D. ZANINI 3 , M. A. ZORDAN 3 , C. MONTECUCCO 2 ;<br />
1 Hum<strong>an</strong> Anat. <strong>an</strong>d Physiol., 2 Biomed. Sci., 3 Biol., Univ. Padova, Padova, Italy; 4 Biomolecular<br />
Simulations Group, Inst. Pasteur of Montevideo, Montevideo, Uruguay<br />
Abstract: SNARE proteins are <strong>the</strong> main molecular constituents of <strong>the</strong> synaptic vesicle fusion<br />
machinery. The three SNARE proteins <strong>for</strong>m a four helix bundle which involves one cytoplasmic<br />
domain of <strong>the</strong> v-SNARE VAMP/synaptobrevin <strong>an</strong>d of <strong>the</strong> t-SNARE syntaxin , <strong>an</strong>d two<br />
cytoplasmic domains of <strong>the</strong> t-SNARE SNAP-25. The assembly of such SNARE complex plays a<br />
key role in <strong>the</strong> fusion process, <strong>an</strong>d it is currently viewed as <strong>the</strong> “minimal fusion machinery”<br />
(Weber et al., 1998; Sudhof <strong>an</strong>d Rothm<strong>an</strong>, <strong>2009</strong>).<br />
There are hints that a rosette of SNARE complexes have to assemble in order to allow <strong>the</strong><br />
<strong>for</strong>mation of a fusion pore during exocytosis. Studies using mutated SNARE proteins, or<br />
SNAREs cleaved with different BoNTs, suggest that <strong>the</strong> number of SNARE complexes placed<br />
around <strong>the</strong> fusion pore in a rosette-like pattern should be between 3 <strong>an</strong>d 10-15 depending on <strong>the</strong>
type of exocytotic vesicle <strong>an</strong>d cell (Hua et al., 2001; Montecucco et al., 2005; Lu et al., 2008).<br />
Cleavage of SNAP-25 by BoNT A occurs within a nine-aminoacid (aa) C-terminal region of<br />
SNAP-25. This truncated <strong>for</strong>m of SNAP-25 is still able to participate in <strong>the</strong> <strong>for</strong>mation of a stable<br />
SNARE complex, but it has a strong inhibitory effect on exocytosis, probably by preventing <strong>the</strong><br />
<strong>for</strong>mation of <strong>the</strong> SNARE rosette (Montecucco et al., 2005).<br />
Using computer graphics we modeled such a rosette of SNARE complexes <strong>an</strong>d have predicted<br />
which residues should be essential in <strong>the</strong> protein-protein contacts which mediate <strong>the</strong> rosette<br />
<strong>for</strong>mation in Drosophila mel<strong>an</strong>ogaster. Accordingly, we generated tr<strong>an</strong>sgenic lines carrying<br />
SNAP-25 mutated ei<strong>the</strong>r at R199 or R206 with al<strong>an</strong>ine. We expressed <strong>the</strong> tr<strong>an</strong>sgenes in a wild<br />
type background, based on <strong>the</strong> prediction of <strong>the</strong> rosette model that <strong>the</strong> mutated SNAP-25 iso<strong>for</strong>m<br />
would display a domin<strong>an</strong>t-negative effect on <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> rosette SNARE<br />
supercomplexes. We found a signific<strong>an</strong>t reduction of both evoked <strong>an</strong>d spont<strong>an</strong>eous<br />
neurotr<strong>an</strong>smitter release at <strong>the</strong> neuromuscular junction of <strong>the</strong> third instar larva with respect to<br />
controls, in <strong>the</strong> absence of overt NMJ morphological defects, providing indirect, but compelling<br />
evidence in favour of <strong>the</strong> involvement of a rosette of SNARE complexes in <strong>the</strong> neuroexocytosis<br />
of synaptic vesicles at <strong>the</strong> neuromuscular junction.<br />
Disclosures: A. Megighi<strong>an</strong>, None; O. Rossetto, None; M. Scorzeto, None; S. P<strong>an</strong>t<strong>an</strong>o,<br />
None; C. Benna, None; D. Z<strong>an</strong>ini, None; M.A. Zord<strong>an</strong>, None; C. Montecucco, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.15/F2<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NIH Gr<strong>an</strong>t GM081809<br />
NIH Gr<strong>an</strong>t NS060342<br />
FAER gr<strong>an</strong>t<br />
BRF gr<strong>an</strong>t<br />
Title: Isoflur<strong>an</strong>e affects neurotr<strong>an</strong>smitter release machinery by interacting with syntaxin 1A
Authors: *Z. XIE 1 , B. E. HERRING 2 , A. P. FOX 2 ;<br />
1 2<br />
Dept. of Anes<strong>the</strong>sia <strong>an</strong>d Critical care, Dept. of Neurobiology, Pharmacol. & Physiol., Univ.<br />
Chicago, Chicago, IL<br />
Abstract: General <strong>an</strong>es<strong>the</strong>tics are known to influence a number of receptors <strong>an</strong>d ion ch<strong>an</strong>nels.<br />
Some studies have investigated <strong>the</strong> effects of inhalational <strong>an</strong>es<strong>the</strong>tics on neurotr<strong>an</strong>smitter release.<br />
Low concentrations of <strong>an</strong>es<strong>the</strong>tic affected chemical tr<strong>an</strong>smission but nei<strong>the</strong>r impulse conduction<br />
nor cellular electrical properties were affected, raising <strong>the</strong> possibility of direct modulation of <strong>the</strong><br />
release machinery although effects on pre-synaptic ch<strong>an</strong>nels <strong>an</strong>d receptors have also been<br />
proposed as possible mech<strong>an</strong>isms. The large number of proteins targeted by general <strong>an</strong>es<strong>the</strong>tics<br />
has made it difficult to study <strong>the</strong>ir effects on <strong>the</strong> release machinery. In <strong>the</strong> present study we set<br />
out to determine if <strong>the</strong> volatile <strong>an</strong>es<strong>the</strong>tic, isoflur<strong>an</strong>e, directly alters <strong>the</strong> function of <strong>the</strong><br />
mammali<strong>an</strong> neurotr<strong>an</strong>smitter release machinery. To accomplish this, it was necessary to observe<br />
<strong>the</strong> effect of isoflur<strong>an</strong>e on evoked neurotr<strong>an</strong>smitter release independent of effects on ch<strong>an</strong>nels<br />
<strong>an</strong>d receptors. We used experimental paradigms that kept membr<strong>an</strong>e potential const<strong>an</strong>t, but<br />
which allowed [Ca 2+ ]i to be elevated by a known amount. PC12 cells were permeabilized by<br />
digitonin <strong>an</strong>d <strong>the</strong>n exposed to Ca 2+ , or alternatively cells were patch clamped in whole-cell<br />
configuration while being dialyzed with Ca 2+ . Amperometry was used to measure tr<strong>an</strong>smitter<br />
release. Additionally, optical measurement of ionomycin-evoked RH414 release was carried out<br />
in hippocampal neurons in <strong>the</strong> presence <strong>an</strong>d absence of isoflur<strong>an</strong>e. We found that clinically<br />
relev<strong>an</strong>t concentrations of isoflur<strong>an</strong>e inhibited neurotr<strong>an</strong>smitter release in PC12 cells <strong>an</strong>d<br />
hippocampal neurons in a dose-dependent m<strong>an</strong>ner. The EC50 of this inhibitory effect of<br />
isoflur<strong>an</strong>e was approximately 0.38 mM (1.3 MAC). Finally, experiments were per<strong>for</strong>med in<br />
PC12 cells which were tr<strong>an</strong>sfected with <strong>the</strong> syntaxin 1A mut<strong>an</strong>t, md130A. Overexpression of<br />
md130A in PC12 cells completely eliminated <strong>the</strong> reduction in neurotr<strong>an</strong>smitter release produced<br />
by isoflur<strong>an</strong>e, without affecting release itself. In summary, clinically relev<strong>an</strong>t concentrations of<br />
isoflur<strong>an</strong>e reduces neurotr<strong>an</strong>smitter release by <strong>an</strong> interaction with <strong>the</strong> release machinery.<br />
Syntaxin 1A, a SNARE protein, appears to be one of <strong>the</strong> primary targets <strong>for</strong> isoflur<strong>an</strong>e. It is<br />
likely that this inhibitory action of isoflur<strong>an</strong>e contributes to reduced neurotr<strong>an</strong>smitter release<br />
within <strong>the</strong> CNS, which may be relev<strong>an</strong>t to <strong>the</strong> <strong>an</strong>es<strong>the</strong>tized state.<br />
Disclosures: Z. Xie, None; B.E. Herring, None; A.P. Fox, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.16/F3<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion
Support: NIH Gr<strong>an</strong>t GM081809<br />
NIH Gr<strong>an</strong>t NS060342<br />
FAER Gr<strong>an</strong>t<br />
BRF Gr<strong>an</strong>t<br />
Title: Inhibition of <strong>the</strong> neurotr<strong>an</strong>smitter release machinery by <strong>the</strong> intravenous general <strong>an</strong>es<strong>the</strong>tics<br />
propofol <strong>an</strong>d etomidate<br />
Authors: B. E. HERRING 1 , Z. XIE 2 , K. MCMILLAN 1 , *A. B. HARKINS 3 , A. P. FOX 1 ;<br />
1 Dept. of Neurobiology, Pharmacol. <strong>an</strong>d Physiol., 2 Dept. of Anes<strong>the</strong>sia <strong>an</strong>d Critical care, Univ.<br />
of Chicago, Chicago, IL; 3 Dept. of Pharmacol. <strong>an</strong>d Pysiological Sci., St. Louis Univ., St. Louis,<br />
MO<br />
Abstract: The mech<strong>an</strong>ism of action of general <strong>an</strong>es<strong>the</strong>tics is only partially understood.<br />
Facilitation of inhibitory GABAA receptors plays <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> action of most<br />
<strong>an</strong>es<strong>the</strong>tics, but this mech<strong>an</strong>ism is thought to be especially relev<strong>an</strong>t in <strong>the</strong> case of intravenous<br />
<strong>an</strong>es<strong>the</strong>tics. More recent evidence suggests that <strong>an</strong>es<strong>the</strong>tics also inhibit excitatory synaptic<br />
tr<strong>an</strong>smission via a presynaptic mech<strong>an</strong>ism(s), but it has been difficult to determine whe<strong>the</strong>r <strong>the</strong>se<br />
agents act on <strong>the</strong> neurotr<strong>an</strong>smitter release machinery itself. We previously found that <strong>the</strong><br />
commonly used inhalational <strong>an</strong>es<strong>the</strong>tic, isoflur<strong>an</strong>e, dose-dependently inhibited <strong>the</strong><br />
neurotr<strong>an</strong>smitter release machinery in neurosecretory cells <strong>an</strong>d in cultured hippocampal neurons.<br />
We also found that <strong>the</strong> overexpression of a C-terminal truncated <strong>for</strong>m of syntaxin 1, named<br />
md130A, completely blocked <strong>the</strong> effects of isoflur<strong>an</strong>e on <strong>the</strong> neurotr<strong>an</strong>smitter release machinery;<br />
this result suggested that syntaxin 1A was a key mediator of <strong>the</strong> response to isoflur<strong>an</strong>e. In <strong>the</strong><br />
present study we sought to determine whe<strong>the</strong>r <strong>the</strong> intravenous <strong>an</strong>es<strong>the</strong>tics, propofol <strong>an</strong>d<br />
etomidate, also inhibit <strong>the</strong> neurotr<strong>an</strong>smitter release machinery. Using <strong>an</strong> experimental approach<br />
that elevates [Ca 2+ ]i at neurotr<strong>an</strong>smitter release sites, while bypassing <strong>an</strong>es<strong>the</strong>tic effects on<br />
ch<strong>an</strong>nels <strong>an</strong>d receptors, we show that clinically relev<strong>an</strong>t concentrations of propofol <strong>an</strong>d<br />
etomidate inhibit <strong>the</strong> neurotr<strong>an</strong>smitter release machinery in neurosecretory cells <strong>an</strong>d in cultured<br />
hippocampal neurons. Toge<strong>the</strong>r <strong>the</strong>se results suggest that a SNARE <strong>an</strong>d/or SNARE-associated<br />
protein represent import<strong>an</strong>t target(s) <strong>for</strong> both inhalational <strong>an</strong>d intravenous general <strong>an</strong>es<strong>the</strong>tics.<br />
Fur<strong>the</strong>rmore, overexpression of md130A completely eliminated <strong>the</strong> reduction in neurotr<strong>an</strong>smitter<br />
release produced by propofol, without affecting release itself, <strong>the</strong>reby establishing syntaxin 1A<br />
as a key intermediary in both isoflur<strong>an</strong>e <strong>an</strong>d propofol’s ability to inhibit <strong>the</strong> neurotr<strong>an</strong>smitter<br />
release machinery.<br />
Disclosures: B.E. Herring, None; Z. Xie, None; K. McMill<strong>an</strong>, None; A.B. Harkins,<br />
None; A.P. Fox, None.<br />
Poster
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.17/F4<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NARSAD Young Investigator Award to PGUR<br />
Title: Role of Stx 1 phosphorylation on SNARE complex <strong>for</strong>mation<br />
Authors: *P. G. ULERY-REYNOLDS 1 , M. A. CASTILLO 2 , S. GHOSE 3 , C. TAMMINGA 3 , J.<br />
RIZO-REY 4 ;<br />
1 2 3 4<br />
Neurol & Psychiatry, Neurol., Psychiatry, Biochem., Univ. Texas Southwestern Med. Ctr.,<br />
Dallas, TX<br />
Abstract: The pre-synaptic protein syntaxin 1 (Stx 1) is a critical component of <strong>the</strong><br />
neurotr<strong>an</strong>smitter release machinery. It is a member of <strong>the</strong> SNARE (soluble N-ethylmaleimidesensitive<br />
factor (NSF) attachment protein receptors) family, which are ubiquitous membr<strong>an</strong>e<br />
proteins necessary <strong>for</strong> membr<strong>an</strong>e fusion, <strong>an</strong>d thus involved in vesicle trafficking <strong>an</strong>d exocytosis.<br />
At <strong>the</strong> pre-synaptic active zone Stx 1 interacts with two o<strong>the</strong>r SNAREs, mainly SNAP-25<br />
(soluble NSF attachment protein of 25 kDa) also found on <strong>the</strong> axonal membr<strong>an</strong>e, <strong>an</strong>d<br />
synaptobrevin 2 (a.k.a. VAMP (vesicle-associated membr<strong>an</strong>e protein) 2) found on <strong>the</strong> membr<strong>an</strong>e<br />
of synaptic vesicles, to <strong>for</strong>m a heterotrimer known as <strong>the</strong> SNARE complex. Formation of this<br />
complex is necessary <strong>for</strong> neurotr<strong>an</strong>smitter release, as it mediates synaptic vesicle docking <strong>an</strong>d<br />
fusion with <strong>the</strong> axonal plasma membr<strong>an</strong>e. While <strong>the</strong> import<strong>an</strong>ce of Stx 1 in synaptic tr<strong>an</strong>smission<br />
is indisputable, very little is known about how its function is regulated. One of <strong>the</strong> most common<br />
ways of regulating <strong>the</strong> function of a protein is via phosphorylation, <strong>an</strong>d Stx 1 has been shown to<br />
be phosphorylated on Ser14 by <strong>the</strong> kinase CK2; however <strong>the</strong> role of phosphorylation in Stx 1<br />
function remains largely unknown. To address this question we have developed our own<br />
phospho-Stx 1-specific <strong>an</strong>tibody <strong>an</strong>d a series of phospho-Stx 1 mut<strong>an</strong>ts. Using a variety of<br />
biochemical techniques we have <strong>an</strong>alyzed <strong>the</strong> effect of phosphorylation on <strong>the</strong> ability of Stx 1 to<br />
bind to its pre-synaptic SNARE partners. Our findings suggest that phosphorylation of Stx 1 on<br />
Ser14 favors its interaction with SNAP25 <strong>an</strong>d <strong>for</strong>mation of <strong>the</strong> SNARE complex while does not<br />
seem to affect its interaction with Syb2.<br />
Disclosures: P.G. Ulery-Reynolds, None; M.A. Castillo, None; S. Ghose, None; C.<br />
Tamminga, None; J. Rizo-Rey, None.<br />
Poster
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.18/F5<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NSC-97-2311-B-002-001-MY2<br />
NSC-97-2321-B-002-042<br />
NSC-97-2311-B-002-007-MY3<br />
NHRI-EX97-9718NC<br />
Title: Fusion pore dynamics regulated by cyclic AMP<br />
Authors: *Y.-T. HSIAO 1 , C.-W. CHANG 1 , M.-H. HSU 2 , N. CHIANG 2 , C.-T. WANG 1,2 ;<br />
1 Inst. of Mol. <strong>an</strong>d Cell. Biol., 2 Dept. of Life Sci., Natl. Taiw<strong>an</strong> Univ., Taipei, Taiw<strong>an</strong><br />
Abstract: The gating <strong>an</strong>d conduct<strong>an</strong>ce of ion ch<strong>an</strong>nel are regulated by protein phosphorylation.<br />
Ca 2+ triggered exocytosis starts with a narrow connection of <strong>the</strong> vesicle lumen <strong>an</strong>d <strong>the</strong><br />
extracellular space, termed <strong>the</strong> fusion pore. Previous studies suggest that fusion pores are<br />
proteinaceous. However, it remains unknown if fusion pore dynamics c<strong>an</strong> be regulated by<br />
protein phosphorylation in a similar way to that <strong>for</strong> ion ch<strong>an</strong>nel. Several studies have shown that<br />
fusion pore dynamics are regulated by chronic stimulation of adenylate cyclase due to <strong>the</strong><br />
ch<strong>an</strong>ges in gene expression profile of synaptic proteins, such as synaptotagmin IV (Syt IV). In<br />
this study, we examine whe<strong>the</strong>r acute stimulation of adenylate cyclase could modulate fusion<br />
pore dynamics without ch<strong>an</strong>ging gene expression. We applied single-cell amperometry to detect<br />
<strong>the</strong> kinetic ch<strong>an</strong>ges of <strong>the</strong> fusion pores by directly applying <strong>for</strong>skolin (activator of adenylate<br />
cyclase) in PC12 cells <strong>for</strong> 20~200 sec. The foot signal preceding amperometric spikes (prespike<br />
foot, PSF) has been shown to represent <strong>the</strong> fusion pore dynamics. The PSF duration was<br />
signific<strong>an</strong>tly reduced by direct application of <strong>for</strong>skolin compared to control. In addition, <strong>the</strong><br />
spike frequency was decreased <strong>an</strong>d <strong>the</strong> latency of <strong>the</strong> first amperometric spike was increased by<br />
direct application of <strong>for</strong>skolin compared to control. By using real-time PCR, we found <strong>the</strong> level<br />
of tr<strong>an</strong>scripts was not ch<strong>an</strong>ged upon direct application of <strong>for</strong>skolin in terms of Syt IV, SNAP-<br />
25a, SNAP-25b <strong>an</strong>d SNAP-23. However, after chronic incubation of <strong>for</strong>kolin <strong>for</strong> 1~8 hours, <strong>the</strong><br />
level of tr<strong>an</strong>scripts was increased in terms of Syt IV, SNAP-25b <strong>an</strong>d SNAP-23. Thus <strong>the</strong> ch<strong>an</strong>ges<br />
in fusion pore dynamics upon direct application of <strong>for</strong>skolin c<strong>an</strong>not attribute to <strong>the</strong> ch<strong>an</strong>ges in<br />
gene expression. These results suggest protein phosphorylation through cAMP signaling may<br />
directly regulate fusion pore dynamics.
Disclosures: Y. Hsiao, None; C. Ch<strong>an</strong>g, None; M. Hsu, None; N. Chi<strong>an</strong>g, None; C. W<strong>an</strong>g,<br />
None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.19/F6<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: INComb FP7 HEALTH project no 223234<br />
Title: Distribution of <strong>the</strong> high affinity binding sites of Botulinum toxin type A in <strong>the</strong> intramural<br />
g<strong>an</strong>glia of <strong>the</strong> guinea-pig<br />
Authors: A. COELHO 1 , *A. A. SILVA 1 , F. CRUZ 2 , A. AVELINO 1 ;<br />
1 Fac Medicine, IBMC, Univ. of Porto, Portugal, Porto, Portugal; 2 Depart. of Urology, Hosp. S.<br />
João, Fac Medicine, IBMC, Univ. of Porto, Porto, Portugal<br />
Abstract: Botulinum neurotoxin type A (BoNT/A) administration has been successfully used in<br />
<strong>the</strong> treatment of symptoms associated with intractable overactive bladder. The specificity of<br />
BoNT/A is due to <strong>the</strong> presence of membr<strong>an</strong>e receptors (Synaptic Vesicle protein, SV2) that are<br />
exposed during neurotr<strong>an</strong>smitter exocytosis. Once internalized, BoNT/A undergoes a pHdependent<br />
con<strong>for</strong>mational ch<strong>an</strong>ge that causes <strong>the</strong> dissociation of <strong>the</strong> heavy <strong>an</strong>d light chains. The<br />
latter has endopeptidase activity that cleaves specific sites of <strong>the</strong> SNAP-25 protein preventing <strong>the</strong><br />
assembly of <strong>the</strong> synaptic fusion complex SNARE <strong>an</strong>d blocking neurotr<strong>an</strong>smitter release.<br />
BoNT/A effect lasts about 4 months in <strong>the</strong> skeletal muscle <strong>an</strong>d 9 months in <strong>the</strong> detrusor smooth<br />
muscle. However, <strong>the</strong> reasons <strong>for</strong> such differences were never fully elucidated. The guinea-pig<br />
bladder innervation is similar to that of hum<strong>an</strong>s, since it contains intramural parasympa<strong>the</strong>tic<br />
g<strong>an</strong>glia.<br />
In <strong>the</strong> present study we studied <strong>the</strong> distribution of SV2, uncleaved <strong>an</strong>d cleaved SNAP-25 in<br />
intramural g<strong>an</strong>glia of guinea-pigs after treatment with BoNT/A. The neurochemistry of BoNT/A<br />
sensitive structures was investigated using markers <strong>for</strong> parasympa<strong>the</strong>tic, sympa<strong>the</strong>tic <strong>an</strong>d sensory<br />
fibers.<br />
Guinea-pig bladders were injected with 10U of BoNT/A <strong>an</strong>d were collected 1,3 <strong>an</strong>d 8 days after.<br />
Cryostat sections were processed <strong>for</strong> dual immunofluorescent staining using <strong>an</strong>tibodies against<br />
SV2, SNAP-25 (cleaved <strong>an</strong>d uncleaved), vesicular acetylcholine tr<strong>an</strong>sporter (VAChT), tyrosine<br />
hydroxilase (TH) <strong>an</strong>d calcitonin-gene related peptide (CGRP).<br />
Guinea-pig parasympa<strong>the</strong>tic g<strong>an</strong>glia exhibit SV2 <strong>an</strong>d SNAP-25 immunoreactivity. Double
labeling showed extensive co-localization of <strong>the</strong> two proteins. Cleaved SNAP-25 was detected<br />
after BoNT/A administration. Both SV2 <strong>an</strong>d cleaved SNAP-25 immunoreactivity showed a<br />
higher degree of co-localization with cholinergic fibers, possibly preg<strong>an</strong>glionic, th<strong>an</strong> with<br />
adrenergic or peptidergic fibers.<br />
Our data show that: I) BoNT/A targets <strong>an</strong>d by-products of BoNT/A action are present in<br />
intramural g<strong>an</strong>glia of guinea pig. II) SNAP-25 cleavage occurs in intramural g<strong>an</strong>glia after<br />
BoNT/A administration. III) SNAP-25 is cleaved mainly in cholinergic, parasympa<strong>the</strong>tic fibers.<br />
IV) <strong>When</strong> injected intramurally BoNT/A action is exerted on pre <strong>an</strong>d post-g<strong>an</strong>glionic nerve<br />
terminals. These results may explain <strong>the</strong> differences in <strong>the</strong> duration of effect of BoNT/A between<br />
skeletal muscle <strong>an</strong>d urinary bladder smooth muscle.<br />
Disclosures: A. Coelho, None; A.A. Silva, None; F. Cruz, Allerg<strong>an</strong>, F. Consult<strong>an</strong>t/Advisory<br />
Board; A. Avelino, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.20/F7<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: Fondation pour la Recherche Médicale<br />
Fédération pour la Recherche sur le Cerveau<br />
Rotary International<br />
Agence Nationale de la Recherche<br />
Associaton Fr<strong>an</strong>çaise contre les Myopathies<br />
Max Pl<strong>an</strong>ck <strong>Society</strong><br />
Title: Role of Munc13 proteins in synaptic vesicle docking <strong>an</strong>alyzed using high-pressure<br />
freezing <strong>an</strong>d electron tomography<br />
Authors: *L. SIKSOU 1 , F. VAROQUEAUX 2 , O. PASCUAL 1 , A. TRILLER 1 , N. BROSE 2 , S.<br />
MARTY 1 ;<br />
1 INSERM U789, Paris, Fr<strong>an</strong>ce; 2 Max Pl<strong>an</strong>ck Inst. of Exptl. Med., Göttingen, Germ<strong>an</strong>y
Abstract: In <strong>the</strong> central nervous system, <strong>the</strong> release of neurotr<strong>an</strong>smitter <strong>from</strong> synaptic vesicles in<br />
<strong>the</strong> synaptic cleft mediates fast communication with <strong>the</strong> postsynaptic neuron. Synaptic vesicles<br />
are rendered release-competent at a Munc13-dependent priming step. Thereafter, <strong>the</strong> SNARE<br />
complex executes vesicular fusion with <strong>the</strong> plasma membr<strong>an</strong>e. Munc13s are thought to enable<br />
<strong>the</strong> assembly of <strong>the</strong> SNARE complex. Previous morphological studies using aldehyde-fixed cells<br />
suggested that vesicle docking to <strong>the</strong> membr<strong>an</strong>e is independent of Munc13, as vesicles were<br />
found to be docked even in <strong>the</strong> absence of Munc13. However, <strong>the</strong>se results might be misleading<br />
as aldehydes may modify <strong>the</strong> localization of synaptic vesicles in <strong>the</strong> axonal terminal.<br />
To reassess <strong>the</strong> role of Munc13 in vesicular docking we froze cultured mouse hippocampal slices<br />
under high-pressure. Indeed, this method permits to immobilize living cells/tissue almost<br />
inst<strong>an</strong>tly without employing aldehydes <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e circumvents <strong>the</strong> artifacts <strong>the</strong>y induce. Also,<br />
electron tomography was used to evaluate at high resolution <strong>the</strong> relationship between synaptic<br />
vesicles <strong>an</strong>d plasma membr<strong>an</strong>e.<br />
In control slices, we showed that synaptic vesicles are docked but not hemifused with <strong>the</strong><br />
membr<strong>an</strong>e. Ra<strong>the</strong>r, <strong>the</strong>y are apposed <strong>an</strong>d connected to it by fine lateral str<strong>an</strong>ds. In slices <strong>from</strong><br />
Munc13-deficient mice, synaptic vesicles are not docked to <strong>the</strong> membr<strong>an</strong>e. Instead, <strong>the</strong>y remain<br />
a few n<strong>an</strong>ometers away <strong>from</strong> <strong>the</strong> membr<strong>an</strong>e.<br />
These results suggest that hemifusion is not a stable intermediate. Moreover, <strong>the</strong>y indicate that<br />
Munc13 proteins are necessary <strong>for</strong> synaptic vesicle docking.<br />
Disclosures: L. Siksou, None; F. Varoqueaux, None; O. Pascual, None; A. Triller, None; N.<br />
Brose, None; S. Marty, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.21/F8<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Title: Characterization of C. eleg<strong>an</strong>s snapin mut<strong>an</strong>ts<br />
Authors: *J. E. RICHMOND, S. YU, A. A. MARTIN, A. O. BURDINA, H. PAREKH, S. M.<br />
KLOSTERMAN;<br />
Biolog Sci., Univ. Illinois Chicago, Chicago, IL<br />
Abstract: Synaptic vesicle priming is dependent on <strong>the</strong> assembly of SNARE complexes <strong>for</strong>med<br />
between synaptobrevin, SNAP-25 <strong>an</strong>d syntaxin. The subsequent calcium-dependent release of<br />
<strong>the</strong>se fusion-competent vesicles requires <strong>the</strong> recruitment of <strong>the</strong> calcium-sensor, synaptotagmin, a
protein which is also implicated in endocytosis. The interaction between <strong>the</strong> SNARE complex<br />
<strong>an</strong>d synaptotagmin is thought to be enh<strong>an</strong>ced by snapin, a protein first identified as a SNAP-25<br />
interacting molecule <strong>an</strong>d subsequently shown to localize with synaptotagmin to dense core<br />
vesicles. Consistent with <strong>the</strong> proposed function of snapin, chromaffin cells <strong>from</strong> snapin knockout<br />
mice exhibit a decrease in <strong>the</strong> size of <strong>the</strong> readily-releasable dense core vesicle pool,<br />
commensurate with reduced snapin/synaptotagmin-associated SNARE complex interactions.<br />
Whe<strong>the</strong>r snapin plays a similar role in synaptic vesicle release has yet to be fully explored. To<br />
address this question, we have begun to characterize a C. eleg<strong>an</strong>s snapin (SNPN-1) deletion<br />
mut<strong>an</strong>t, obtained <strong>from</strong> <strong>the</strong> C. eleg<strong>an</strong>s knockout consortium <strong>an</strong>d out-crossed in <strong>the</strong> lab.<br />
Preliminary pharmacological <strong>an</strong>d electrophysiological data show a modest reduction in<br />
cholinergic tr<strong>an</strong>smission in this snpn-1 mut<strong>an</strong>t, where as synaptotagmin (SNT-1) mut<strong>an</strong>ts exhibit<br />
a more severe defect in both assays. We are currently exploring whe<strong>the</strong>r expression of SNPN-1<br />
under <strong>the</strong> promoter <strong>for</strong> <strong>the</strong> vesicular ACh tr<strong>an</strong>sporter (Punc-17) rescues <strong>the</strong> snpn-1 mut<strong>an</strong>t<br />
cholinergic tr<strong>an</strong>smission defect. As a first step in assessing whe<strong>the</strong>r SNPN-1 is involved in SNT-<br />
1 recruitment to SNARE complexes following synaptic vesicle priming, we have generated snt-<br />
1;snpn-1 double mut<strong>an</strong>ts. Preliminary data suggest that <strong>the</strong> phenotype of <strong>the</strong> double mut<strong>an</strong>t is no<br />
more severe th<strong>an</strong> ei<strong>the</strong>r single mut<strong>an</strong>t suggesting <strong>the</strong>se two proteins may act in <strong>the</strong> same<br />
pathway. We are currently conducting electrophysiological experiments to explore <strong>the</strong> calciumdependence<br />
of <strong>the</strong> evoked phenotypes of <strong>the</strong>se single <strong>an</strong>d double mut<strong>an</strong>ts. In addition we are<br />
<strong>an</strong>alyzing mut<strong>an</strong>ts prepared by high-pressure freeze fixation <strong>for</strong> ultra-structural EM <strong>an</strong>alysis.<br />
Toge<strong>the</strong>r <strong>the</strong>se experiments should fur<strong>the</strong>r define <strong>the</strong> role of interactions between SNPN-1 <strong>an</strong>d<br />
SNT-1, <strong>an</strong>d elucidate snapin function with regard to synaptic tr<strong>an</strong>smission.<br />
Disclosures: J.E. Richmond, None; S. Yu, None; A.A. Martin, None; A.O. Burdina,<br />
None; H. Parekh, None; S.M. Klosterm<strong>an</strong>, None.<br />
Poster<br />
522. Neurtr<strong>an</strong>smitter Release: Docking <strong>an</strong>d Fusion<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 522.22/F9<br />
Topic: B.06.a. Docking <strong>an</strong>d fusion<br />
Support: NSF Gr<strong>an</strong>t 0544031<br />
Title: Sensitivity of Caenorhabditis eleg<strong>an</strong>s mut<strong>an</strong>ts to oxidative stress
Authors: L. GURENLIAN 1 , J. BODKIN 1 , M. CAFARCHIO 1 , C. MARTIN 1 , A. MINNITI 2 , N.<br />
INESTROSA 2 , *R. E. KOHN 1 ;<br />
1 Ursinus Col., Collegeville, PA; 2 Fac. de Cs. Biologicas P.U. Catolica de Chile, S<strong>an</strong>tiago, Chile<br />
Abstract: We are examining <strong>the</strong> effect of oxidative stress on development of Caenorhabditis<br />
eleg<strong>an</strong>s strains with defects in nervous system function. Oxidative stress c<strong>an</strong> alter activity <strong>an</strong>d<br />
expression of <strong>the</strong> enzyme, acetylcholinesterase (AChE), which breaks down <strong>the</strong><br />
neurotr<strong>an</strong>smitter, acetylcholine, in synapses. <strong>When</strong> AChE activity is inhibited, excess<br />
acetylcholine accumulates in synapses, causing paralysis. Abund<strong>an</strong>ce of acetylcholine in<br />
synapses is decreased in unc-13 mut<strong>an</strong>ts, as UNC-13 regulates release of neurotr<strong>an</strong>smitter into<br />
synapses, causing paralysis. We are determining whe<strong>the</strong>r oxidative stress alters <strong>the</strong> rate of<br />
development of C. eleg<strong>an</strong>s with increased abund<strong>an</strong>ce of neurotr<strong>an</strong>smitter in synapses, due to<br />
defects in AChE, <strong>an</strong>d decreased abund<strong>an</strong>ce of neurotr<strong>an</strong>smitter in synapses, due to defects in<br />
UNC-13. C. eleg<strong>an</strong>s embryos were grown in <strong>the</strong> presence of paraquat, which induces oxidative<br />
stress, <strong>an</strong>d <strong>the</strong> percentage of worms that developed to adulthood over five days was determined.<br />
In <strong>the</strong> presence of paraquat, rate of development decreased <strong>for</strong> all strains tested, except <strong>for</strong> <strong>the</strong><br />
unc-13 mut<strong>an</strong>t. unc-13 mut<strong>an</strong>t worms developed slowly <strong>an</strong>d did not reach adulthood during <strong>the</strong><br />
five days of <strong>the</strong> experiment in <strong>the</strong> presence or absence of paraquat. Worms with defects in genes<br />
that express AChE, ace-1 <strong>an</strong>d ace-2, developed similarly to wild type in <strong>the</strong> presence of<br />
paraquat. A strain with defects in ace-1, ace-2, <strong>an</strong>d unc-13 developed to adulthood signific<strong>an</strong>tly<br />
more slowly th<strong>an</strong> wild type in <strong>the</strong> presence of paraquat. We are acquiring data over a longer time<br />
period to determine whe<strong>the</strong>r development of unc-13 mut<strong>an</strong>ts to adulthood is altered by oxidative<br />
stress. These findings suggest that <strong>the</strong> slowed developmental rate of unc-13 mut<strong>an</strong>ts is reversed<br />
when more acetylcholine c<strong>an</strong> accumulate in synapses <strong>an</strong>d that this process is sensitive to<br />
oxidative stress.<br />
Disclosures: L. Gurenli<strong>an</strong>, None; J. Bodkin, None; M. Cafarchio, None; C. Martin,<br />
None; A. Minniti, None; N. Inestrosa, None; R.E. Kohn, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.1/F10<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: NINDS RO1-NS052529<br />
NINDS RO1-NS051505
NINDS UO1-NS058213<br />
NIDA P50DA005274<br />
Title: Prolonged exposure to CB1 c<strong>an</strong>nabinoid receptor agonist results in neuronal<br />
hyperexcitability as a consequence of decreased GABA-mediated synaptic function in primary<br />
hippocampal cultures<br />
Authors: *R. E. BLAIR 1 , L. S. DESHPANDE 2 , R. J. DELORENZO 2 ;<br />
2 Neurol., 1 Virginia Commonwealth Univ., Richmond, VA<br />
Abstract: Agonist-induced down-regulation of G-protein-coupled receptors (GPCRs) is <strong>an</strong><br />
import<strong>an</strong>t regulatory process throughout <strong>the</strong> CNS that mediates <strong>the</strong> control of both receptor<br />
function <strong>an</strong>d synaptic tr<strong>an</strong>smission. Similar use-dependent regulatory mech<strong>an</strong>isms have also<br />
been shown to occur <strong>for</strong> lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels. The presynaptic CB1 c<strong>an</strong>nabinoid receptor<br />
(CB1) is a GPCR that plays a pivotal role in mediating synaptic tr<strong>an</strong>smission via regulation of<br />
neurotr<strong>an</strong>smitter release <strong>an</strong>d thus, has become <strong>an</strong> attractive <strong>the</strong>rapeutic target in models of<br />
neuronal hyperexcitability <strong>an</strong>d excitotoxicity. Although acute exposure to CB1 agonists has been<br />
shown to infer suppression of neuronal hyperexcitability, recent findings have demonstrated that<br />
prolonged c<strong>an</strong>nabinoid exposure, with subsequent agonist-induced CB1 down-regulation,<br />
ultimately results in exacerbation of neuronal hyperexcitability. Such a dysregulation of<br />
presynaptic release mech<strong>an</strong>isms could result in prolonged saturating levels of neurotr<strong>an</strong>smitters<br />
which could affect <strong>the</strong> density <strong>an</strong>d/or sensitivity of postsynaptic receptors. This study set out to<br />
evaluate <strong>the</strong> effects of prolonged agonist-induced downregulation of <strong>the</strong> CB1 on postsynaptic<br />
GABAergic inhibitory function utilizing <strong>an</strong> in vitro preparation of primary hippocampal<br />
neuronal cultures (HNCs). HNCs were exposed <strong>for</strong> 24 h to <strong>the</strong> c<strong>an</strong>nabimimetic (+)-WIN55,212<br />
(+WIN; 1000 nM) <strong>an</strong>d evaluated <strong>for</strong> GABAA receptor function both electrophysiologically <strong>an</strong>d<br />
immunocytochemically. In addition, CB1 expression <strong>an</strong>d distribution was also evaluated<br />
immunocytochemically following prolonged agonist exposure. Upon removal of +WIN, currentclamp<br />
recording of neurons revealed pronounced hyperexcitability characterized by highfrequency<br />
spike discharges exceeding 3 Hz. Fur<strong>the</strong>rmore, voltage-clamp <strong>an</strong>alysis of mIPSCs<br />
revealed a 2.5 fold increase in frequency <strong>an</strong>d a 33% decrease in amplitude, indicating increased<br />
probability of presynaptic GABA release <strong>an</strong>d decrease in postsynaptic GABAA receptor<br />
function respectively. Prolonged +WIN exposure induced a dramatic down-regulation<br />
(98.2±1.1%) of membr<strong>an</strong>e CB1 at both inhibitory (VGAT positive) <strong>an</strong>d excitatory (VGLUT1<br />
positive) terminals. Fur<strong>the</strong>r immunocytochemical <strong>an</strong>alysis of neuronal processes revealed that<br />
prolonged +WIN exposure resulted in a signific<strong>an</strong>t decrease (34.9±7.2%) of membr<strong>an</strong>e GABAA<br />
receptor expression. The results <strong>from</strong> this study demonstrate that agonist-induced downregulation<br />
of <strong>the</strong> CB1 in primary HNCs produces neuronal hyperexcitability as a consequence of<br />
altered GABA-mediated synaptic function.<br />
Disclosures: R.E. Blair, None; L.S. Deshp<strong>an</strong>de, None; R.J. DeLorenzo, None.<br />
Poster
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.2/F11<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Title: Insulin-like growth factor-1 enh<strong>an</strong>ces synaptic tr<strong>an</strong>smission by a hypoxia-induced factor-<br />
1-mediated vascular endo<strong>the</strong>lial growth factor expression in hippocampal neuronal cultures<br />
Authors: Y.-F. HUANG 1 , C.-H. YANG 1 , C.-C. HUANG 1 , M.-H. TAI 2 , *K.-S. HSU 3 ;<br />
1 Dept. of Pharmacology, <strong>an</strong>d Inst. of Basic Med. Sci., National Cheng Kung University, Tain<strong>an</strong>,<br />
Taiw<strong>an</strong>; 2 Inst. of Biomed. Sci., National Sun Yat-Sen University, Kaohsiung, Taiw<strong>an</strong>; 3 Natl.<br />
Cheng Kung Univ., Tain<strong>an</strong> 701, Taiw<strong>an</strong><br />
Abstract: Hypoxia-induced factor-1 (HIF-1) is a tr<strong>an</strong>scriptional factor that coordinates <strong>the</strong><br />
adaptive responses to hypoxia in mammali<strong>an</strong> cells. It consists of a regulatory subunit HIF-1α,<br />
which accumulates under hypoxic condition, <strong>an</strong>d a constitutively expressed subunit HIF-1β. In<br />
addition to well characterized oxygen-dependent mode of action, various growth factors <strong>an</strong>d<br />
cytokines have recently been shown to stimulate HIF-1α expression <strong>an</strong>d <strong>the</strong>reby trigger <strong>the</strong><br />
tr<strong>an</strong>scription of numerous hypoxia-inducible genes in <strong>an</strong> oxygen-independent m<strong>an</strong>ner. In this<br />
study, we examined <strong>the</strong> functional relationship between insulin-like growth factor-1 (IGF-1)<br />
signaling, HIF-1α protein turnover, <strong>an</strong>d vascular endo<strong>the</strong>lial growth factor (VEGF) expression in<br />
hippocampal neuronal cultures. Our results show that IGF-1 induced a time- <strong>an</strong>d dose-dependent<br />
increase in HIF-1α protein expression, which was blocked by pretreatment with selective IGF-1<br />
receptor <strong>an</strong>tagonist, AG1024, tr<strong>an</strong>scriptional inhibitor, actinomycin D, or tr<strong>an</strong>slational inhibitors,<br />
cycloheximide <strong>an</strong>d <strong>an</strong>isomycin. In addition, pharmacological blockade of <strong>the</strong><br />
phosphatidylinositol 3-kinase/Akt/mammali<strong>an</strong> target of rapamycin signaling pathway but not <strong>the</strong><br />
extracellular signal-regulated kinase inhibited <strong>the</strong> IGF-1-induced HIF-1α expression. More<br />
import<strong>an</strong>tly, <strong>the</strong> increase in HIF-1α expression induced by IGF-1 was accomp<strong>an</strong>ied by increasing<br />
in <strong>the</strong> levels of VEGF mRNA <strong>an</strong>d protein <strong>an</strong>d enh<strong>an</strong>cing <strong>the</strong> excitatory synaptic tr<strong>an</strong>smission. In<br />
parallel, blockade of HIF-1α activity by echinomycin or lentiviral tr<strong>an</strong>sfection with domin<strong>an</strong>tnegative<br />
mut<strong>an</strong>t HIF-1α or shRNA targeting HIF-1α inhibited <strong>the</strong> increased expression of VEGF<br />
<strong>an</strong>d <strong>the</strong> enh<strong>an</strong>cement of synaptic tr<strong>an</strong>smission induced by IGF-1. Toge<strong>the</strong>r <strong>the</strong>se results suggest<br />
that IGF-1 may act through HIF-1-mediated VEGF expression to enh<strong>an</strong>ce excitatory synaptic<br />
tr<strong>an</strong>smission in hippocampal neurons <strong>an</strong>d reveal a novel role of HIF-1α in synaptic modulation.<br />
Disclosures: Y. Hu<strong>an</strong>g, None; C. Y<strong>an</strong>g, None; C. Hu<strong>an</strong>g, None; M. Tai, None; K. Hsu, None.<br />
Poster
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.3/F12<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Title: Estrogen acts through GPR-30 receptors to rapidly increase neurotr<strong>an</strong>smitter release <strong>from</strong><br />
hippocampal excitatory synapses<br />
Authors: *D. P. BALUCH, J. GEORGES, P. DEVICHE, W. J. TYLER;<br />
Sch. of Life Sci., Arizona State Univ., Tempe, AZ<br />
Abstract: Estrogen is thought to mediate behavior <strong>an</strong>d cognitive function by acting on <strong>the</strong><br />
estrogen receptors α <strong>an</strong>d β (ERα <strong>an</strong>d ERβ). The majority of evidence supporting <strong>the</strong>se actions<br />
stem <strong>from</strong> postsynaptic observations made in hippocampus <strong>an</strong>d cortex that estrogen induces<br />
dendritic spine <strong>for</strong>mation, mediates dendritic remodeling, <strong>an</strong>d regulates neurotr<strong>an</strong>smitter receptor<br />
turnover. Little is known however with regard to <strong>the</strong> influence of estrogen on presynaptic<br />
function although <strong>an</strong>y such actions would also likely contribute to <strong>the</strong> mech<strong>an</strong>isms underlying<br />
estrogen-mediated behavioral <strong>an</strong>d cognitive plasticity. Fur<strong>the</strong>rmore, <strong>the</strong> signaling affects of<br />
estrogen described to date are generally slow (hours to days), dependent on ERα activation <strong>an</strong>d<br />
require gene tr<strong>an</strong>scription. In both neuronal <strong>an</strong>d non-neuronal cell types, it has recently been<br />
shown that estrogen c<strong>an</strong> rapidly (minutes) increase internal calcium concentrations by activating<br />
<strong>the</strong> membr<strong>an</strong>e bound estrogen receptor GPR-30. There<strong>for</strong>e we investigated <strong>the</strong> influence of<br />
estrogen on neurotr<strong>an</strong>smitter release kinetics at hippocampal excitatory synapses using <strong>the</strong> pHdependent<br />
reporter of synaptic vesicle exo- <strong>an</strong>d endocytosis synaptopHluorin. Using dissociated<br />
cultures prepared <strong>from</strong> thy1-synaptopHluorin mice, we per<strong>for</strong>med optical imaging <strong>an</strong>d wholecell<br />
voltage-clamp studies. In <strong>the</strong>se studies, we first observed that GPR-30 receptors are indeed<br />
localized to hippocampal excitatory presynaptic terminals. Functionally, we observed <strong>an</strong> increase<br />
in excitatory neurotr<strong>an</strong>smitter release <strong>from</strong> hippocampal synapses within 30 minutes of estrogen<br />
treatment. Our preliminary data indicate estrogenic effects in <strong>the</strong> hippocampus are mediated in<br />
part through presynaptic GPR-30 receptors <strong>an</strong>d non-nuclear estrogen receptor signaling<br />
pathways. We are conducting fur<strong>the</strong>r studies to examine <strong>the</strong> signaling cascades underlying <strong>the</strong>se<br />
effects, as well as to fur<strong>the</strong>r delineate <strong>the</strong> rapid actions of estrogen on presynaptic function in <strong>the</strong><br />
hippocampus.<br />
Disclosures: D.P. Baluch, None; J. Georges, None; P. Deviche, None; W.J. Tyler, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.4/F13<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: gr<strong>an</strong>t <strong>from</strong> NIH (NS24970)<br />
gr<strong>an</strong>t <strong>from</strong> NIH (NS063192)<br />
Title: Ionic conditions modulate stimulus-induced capacit<strong>an</strong>ce ch<strong>an</strong>ges in isolated<br />
Neurohypophysial terminals<br />
Authors: H. G. MARRERO, *J. R. LEMOS;<br />
Dept Physiol, U.Mass.Med.Sch, Worcester, MA<br />
Abstract: Petidergic nerve terminals of <strong>the</strong> neurohypophysis (NH) secrete both oxytocin <strong>an</strong>d<br />
vasopressin upon stimulation by peptide-specific bursts of action potentials <strong>from</strong> magnocellular<br />
neurons. These bursts vary in both frequency <strong>an</strong>d action potential duration <strong>an</strong>d also induce in situ<br />
ionic ch<strong>an</strong>ges both inside <strong>an</strong>d outside <strong>the</strong> terminals in <strong>the</strong> NH. These temporary effects include<br />
<strong>the</strong> increase of external potassium <strong>an</strong>d decrease of external calcium, as well as increases in<br />
internal sodium <strong>an</strong>d chloride concentrations. In order to examine <strong>the</strong> effects that <strong>the</strong>se ionic<br />
ch<strong>an</strong>ges have on secretion, stimulus-induced capacit<strong>an</strong>ce recordings were per<strong>for</strong>med on isolated<br />
terminals of <strong>the</strong> neurohypophysis using action potential burst patterns of varying frequency <strong>an</strong>d<br />
action potential width. The results indicate that in NH terminals: 1) increased internal chloride<br />
concentration improves <strong>the</strong> efficacy of action potential-induced capacit<strong>an</strong>ce ch<strong>an</strong>ges, 2)<br />
increasing external potassium increases stimulus-induced capacit<strong>an</strong>ce ch<strong>an</strong>ges, 3) decreasing<br />
external calcium decreases <strong>the</strong> capacit<strong>an</strong>ce induced by low frequency broadened action<br />
potentials, while no difference in capacit<strong>an</strong>ce ch<strong>an</strong>ge is observed with high frequency unbroadened<br />
action potentials, 4) increasing internal sodium increases <strong>the</strong> capacit<strong>an</strong>ce ch<strong>an</strong>ge<br />
induced by low frequency bursts of broadened action potentials, more th<strong>an</strong> <strong>for</strong> high frequency<br />
bursts of narrow action potentials. These results are consistent with previous models of stimulusinduced<br />
secretion, where optimal secretory efficacy is determined by particular characteristics of<br />
action potentials within a burst. Our results suggest that positive effects of increased internal<br />
sodium <strong>an</strong>d external potassium during a burst may serve as a compensatory mech<strong>an</strong>ism <strong>for</strong><br />
secretion, counterbal<strong>an</strong>cing <strong>the</strong> negative effects of reduced external calcium. In this view, high<br />
frequency un-broadened action potentials (initial burst phase) would condition <strong>the</strong> terminals by<br />
increasing internal sodium <strong>for</strong> optimal secretion by <strong>the</strong> physiologically later phase of broadened<br />
action potentials. Thus, ionic ch<strong>an</strong>ges occurring during a burst may help to make such<br />
stimulation more efficient at inducing secretion. Fur<strong>the</strong>rmore, <strong>the</strong>se effects are thought to occur<br />
within <strong>the</strong> initial few seconds of incoming burst activity at both oxytocin <strong>an</strong>d vasopressin types<br />
of NH terminals.<br />
Disclosures: H.G. Marrero, None; J.R. Lemos , None.
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.5/F14<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: UDALL Center <strong>for</strong> Parkinson's Disease Research, NSO38367<br />
Americ<strong>an</strong> Parkinson's Disease Association<br />
Title: Mech<strong>an</strong>isms mediating ubiquitin-dependent synaptic physiology<br />
Authors: *G. V. RINETTI 1 , G. DAVID 3 , F. E. SCHWEIZER 2 ;<br />
1 Neurobio., 2 UCLA, Los Angeles, CA; 3 Baylor Col. of Med., Houston, TX<br />
Abstract: Introduction. There is subst<strong>an</strong>tial evidence supporting <strong>the</strong> notion that protein<br />
degradation, via <strong>the</strong> Ubiquitin Proteasome System (UPS), <strong>an</strong>d protein syn<strong>the</strong>sis, mediate<br />
postsynaptic plasticity e.g. LTP. By contrast, <strong>the</strong>re is less but increasing evidence that <strong>the</strong> UPS<br />
modulates synaptic physiology presynaptically. We have previously reported a fast (within<br />
minutes) <strong>an</strong>d strong (3-4 fold) increase in mEPSC <strong>an</strong>d mIPSC <strong>an</strong>d spont<strong>an</strong>eous postsynaptic<br />
currents in response to blocking <strong>the</strong> UPS pathway at <strong>the</strong> level of <strong>the</strong> proteasome or <strong>the</strong> E1<br />
activating enzyme. Although presynaptic ch<strong>an</strong>ges have been consistently confirmed after UPS<br />
inhibition in different species (<strong>from</strong> Drosophila to mammals), <strong>the</strong> suggested mech<strong>an</strong>isms <strong>an</strong>d/or<br />
c<strong>an</strong>didate proteins vary signific<strong>an</strong>tly <strong>from</strong> study to study.<br />
Methods. To find a mech<strong>an</strong>ism that explains our finding of <strong>an</strong> increase in minis <strong>an</strong>d spont<strong>an</strong>eous<br />
currents, we explored mech<strong>an</strong>isms that have been previously linked <strong>the</strong> UPS as a regulator of<br />
synaptic physiology using westerblotting <strong>an</strong>d electrophysiology. We incubated hippocampal rat<br />
cultures with proteasome or E1 blockers <strong>for</strong> ei<strong>the</strong>r 10 minutes or 2 hrs, <strong>an</strong>d per<strong>for</strong>med Westernblot<br />
<strong>an</strong>alysis with <strong>an</strong>tibodies against Munc13, Synaptotagmin, Rim1 <strong>an</strong>d P<strong>an</strong>-Ubiquitin.<br />
Additionally, we tested <strong>the</strong> effect UPS blockers on minis while stimulating cAMP-PKA<br />
pathways (<strong>for</strong>skolin), while increasing or decreasing calcium levels (BAPTA-AM or high<br />
calcium (10mM)) or while blocking protein syn<strong>the</strong>sis (cyclohexamide).<br />
Results. Preincubation of hippocampal neurons with ei<strong>the</strong>r proteasome or E1 blockers showed no<br />
protein level ch<strong>an</strong>ges of Munc13, Synaptotagmin <strong>an</strong>d Rim1. Additionally, minis increased under<br />
proteasome or E1 inhibition even after application of <strong>for</strong>skolin, BAPTA-AM, high calcium <strong>an</strong>d<br />
protein syn<strong>the</strong>sis inhibition. Interestingly, ubiquitinated proteins only increased after 2 hours of<br />
preincubation with proteasome blockers, which suggests that <strong>the</strong> rapid increase in mini<br />
frequency that we observe may not depend on aggregation of poly-ubiquitinated proteins, but
possibly a posttr<strong>an</strong>slational modification that could lead to ch<strong>an</strong>ges in <strong>the</strong> activity state of certain<br />
c<strong>an</strong>didate proteins.<br />
Conclusions. We conclude that although a specific mech<strong>an</strong>ism remains unknown, <strong>the</strong> increase in<br />
mini frequency we observe could be mediated by ch<strong>an</strong>ges in <strong>the</strong> ubiquitinated state of protein(s)<br />
that ch<strong>an</strong>ge protein function ra<strong>the</strong>r th<strong>an</strong> protein half-life.<br />
Disclosures: G.V. Rinetti, None; G. David, None; F.E. Schweizer, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.6/F15<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Ministry of Education, Culture, Sports, Science, <strong>an</strong>d Technology of Jap<strong>an</strong><br />
Jap<strong>an</strong> <strong>Society</strong> <strong>for</strong> <strong>the</strong> Promotion of Science <strong>for</strong> Young Scientists<br />
Title: Protease-activated receptor 1 induces suppression of inhibitory synaptic tr<strong>an</strong>smission in<br />
<strong>the</strong> hippocampus<br />
Authors: *Y. HASHIMOTODANI 1 , T. OHNO-SHOSAKU 2 , M. KANO 1 ;<br />
1 2<br />
Dept. of Neurophysiology, Univ. of Tokyo, Tokyo, Jap<strong>an</strong>; Dept. of Impairment Study,<br />
K<strong>an</strong>azawa Univ., K<strong>an</strong>azawa, Jap<strong>an</strong><br />
Abstract: Protease-activated receptors (PARs) constitute a family of seven-tr<strong>an</strong>smembr<strong>an</strong>e<br />
domain G protein-coupled receptors that are activated by proteolytic cleavage of a specific site<br />
within <strong>the</strong>ir extracellular N-terminus domains. This cleavage reveals a new N terminus, which<br />
acts as a te<strong>the</strong>red lig<strong>an</strong>d <strong>an</strong>d <strong>the</strong>n binds <strong>an</strong>d activates <strong>the</strong> receptor. PAR1, one of <strong>the</strong> four<br />
subtypes of PARs (PAR1-PAR4), is widely expressed in <strong>the</strong> brain, <strong>an</strong>d has been suggested to<br />
play a variety of roles in pathological <strong>an</strong>d physiological conditions. However, <strong>the</strong> roles of PAR1<br />
in modulation of synaptic tr<strong>an</strong>smission have not been well investigated. In this study, we<br />
examined effects of PAR1 activation on inhibitory postsynaptic currents (IPSCs) in cultured<br />
hippocampal neurons. We conducted paired whole-cell recordings <strong>from</strong> cultured rat hippocampal<br />
neurons <strong>an</strong>d recorded unitary IPSCs. Application of <strong>the</strong> PAR1 activator thrombin or specific<br />
agonist TFLLR induced a tr<strong>an</strong>sient suppression of IPSCs, which was blocked by <strong>the</strong> PAR1<br />
<strong>an</strong>tagonist SCH79797. The second application of thrombin or TFLLR failed to suppress IPSCs,<br />
indicating severe desensitization. <strong>When</strong> TFLLR <strong>an</strong>d thrombin were sequentially applied, <strong>the</strong>
effect of <strong>the</strong> secondly applied drug was diminished irrespective of <strong>the</strong> order of drug application,<br />
confirming <strong>the</strong> involvement of <strong>the</strong> same receptor. The PAR1-mediated IPSC suppression was<br />
accomp<strong>an</strong>ied by <strong>an</strong> increase in <strong>the</strong> paired-pulse ratio, suggesting that <strong>the</strong> IPSC suppression is of<br />
presynaptic origin. PAR1 is known to be expressed in both neurons <strong>an</strong>d glia, <strong>an</strong>d activation of<br />
glial PAR1 has been shown to induce calcium mobilization <strong>from</strong> internal calcium stores, which<br />
triggers release of modulators including glutamate <strong>an</strong>d ATP. There<strong>for</strong>e, it is possible that such<br />
modulators suppress IPSCs through presynaptic receptors. We observed that application of<br />
TFLLR induced intracellular calcium rise in glia that was completely blocked by pretreatment of<br />
thapsigargin. Import<strong>an</strong>tly, thapsigargin did not block <strong>the</strong> suppression of IPSCs by TFLLR,<br />
suggesting that modulators released <strong>from</strong> glia by PAR1 activation are not involved in <strong>the</strong><br />
TFLLR-induced IPSC suppression. We are now investigating whe<strong>the</strong>r neuronal PAR1 is<br />
necessary <strong>an</strong>d how IPSCs are suppressed by PAR1 activation.<br />
Disclosures: Y. Hashimotod<strong>an</strong>i, None; T. Ohno-Shosaku, None; M. K<strong>an</strong>o, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.7/F16<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Supported by <strong>the</strong> Deutsche Forschungsgemeinschaft (CMPB)<br />
Title: Chronic stress enh<strong>an</strong>ces K + fluxes during severe hypoxia<br />
Authors: *G. A. FLUEGGE 1,2 , B. HILDEBRANDT 3 , E. FUCHS 1,2 , M. MÜLLER 3,2 ;<br />
1 Germ<strong>an</strong> Primate Ctr., Goettingen, Germ<strong>an</strong>y; 2 DFG Res. Ctr. Mol. Physiol. of <strong>the</strong> Brain<br />
(CMPB), University of Göttingen, Germ<strong>an</strong>y; 3 Zentrum Physiologie und Pathophysiologie, Univ.<br />
of Göttingen, Göttingen, Germ<strong>an</strong>y<br />
Abstract: Chronic stress may impair <strong>the</strong> function of complex neuronal networks by inducing<br />
dendritic retraction, affecting neuronal excitability as well as synaptic plasticity, <strong>an</strong>d modulating<br />
glial cells. To elucidate <strong>the</strong> consequences of chronic restraint stress <strong>for</strong> <strong>the</strong> hippocampal<br />
<strong>for</strong>mation, we submitted adult male Sprague Dawley rats to daily restraint stress (6 hr/day) <strong>for</strong><br />
three weeks according to <strong>an</strong> established protocol. In acute hippocampal tissue slices of <strong>the</strong><br />
stressed rats, Schaffer collateral stimulation did not reveal obvious ch<strong>an</strong>ges in basal synaptic<br />
function or synaptic short term plasticity (paired pulse stimulation) within <strong>the</strong> CA1 subfield. Yet<br />
long term potentiation (100 Hz, 1 s) was markedly impaired after stress, <strong>an</strong>d this effect could be
mimicked by chemical poisoning of glial cells with fluoroacetate (5 mM, >1.5 h). In acute<br />
hippocampal slices oxygen withdrawal triggered, within 2-3 min, characteristic hypoxia-induced<br />
spreading depression-like events (HSD) that were identified by a sudden negative shift of <strong>the</strong><br />
extracellular DC potential by 15-20 mV. Comparing <strong>the</strong> key parameters of HSD showed no<br />
signific<strong>an</strong>t differences in <strong>the</strong> amplitudes of <strong>the</strong> extracellular DC potential shift or <strong>the</strong> time to<br />
HSD onset among control <strong>an</strong>d stressed rats. However, <strong>the</strong> duration of HSD - measured at <strong>the</strong> half<br />
amplitude level of <strong>the</strong> DC shift - was shortened by ~30% in stressed rats, suggesting accelerated<br />
recovery of membr<strong>an</strong>e potentials <strong>an</strong>d ionic distribution upon reoxygenation. Monitoring <strong>the</strong><br />
interstitial K + concentration indicated that in slices <strong>from</strong> stressed rats interstitial K + rose faster<br />
during <strong>the</strong> initial phase of hypoxia <strong>an</strong>d reached a ~70% higher level be<strong>for</strong>e HSD was ignited.<br />
The peak K + level reached at <strong>the</strong> height of HSD, or <strong>the</strong> undershoot of <strong>the</strong> prehypoxic K + baseline<br />
upon reoxygenation were, however, unch<strong>an</strong>ged. Fluoroacetate pretreatment failed to mimic <strong>the</strong><br />
enh<strong>an</strong>ced interstitial K + accumulation during early hypoxia. Analyzing <strong>the</strong> intrinsic optical signal<br />
of HSD revealed identical light scattering increases as well as similar HSD propagation<br />
velocities <strong>an</strong>d tissue invasion of <strong>the</strong> hippocampal <strong>for</strong>mation. These data suggest that restraint<br />
stress primarily targets K + fluxes during hypoxia. Unveiling <strong>the</strong> molecular mech<strong>an</strong>isms<br />
underlying <strong>the</strong> enh<strong>an</strong>ced interstitial K + accumulation in stressed rats as well as <strong>the</strong> shortening of<br />
HSD episodes requires fur<strong>the</strong>r detailed <strong>an</strong>alyses on <strong>the</strong> cellular level of hippocampal neurons <strong>an</strong>d<br />
glial cells. However, it appears that impaired glial K + buffering - as mimicked by glial cell<br />
poisoning with fluoroacetate - is not <strong>the</strong> primary cause <strong>for</strong> <strong>the</strong> elevated K + levels early during<br />
hypoxia.<br />
Disclosures: G.A. Fluegge, None; B. Hildebr<strong>an</strong>dt, None; E. Fuchs, None; M. Müller, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.8/F17<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: NIH Gr<strong>an</strong>t R01NS051561<br />
Title: Ischemia induces Ca 2+ -independent glutamatergic vesicle release through actin filament<br />
depolymerization in area CA1 of <strong>the</strong> hippocampus but not in cerebellar Purkinje cells<br />
Authors: *A. ANDRADE, D. J. ROSSI;<br />
Behavioral Neurosci., Oregon Hlth. & Sci. Univ., Beaverton, OR
Abstract: Tr<strong>an</strong>sient, non-catastrophic brain ischemia c<strong>an</strong> induce ei<strong>the</strong>r ischemic preconditioning<br />
or delayed, selective neuronal death in ischemia sensitive neurons. Altered glutamatergic<br />
signaling <strong>an</strong>d Ca 2+ homeostasis have been implicated in both processes. Here we use patchclamp<br />
recording <strong>an</strong>d Ca 2+ imaging to monitor early ch<strong>an</strong>ges in glutamate release <strong>an</strong>d<br />
cytoplasmic [Ca 2+ ] ([Ca 2+ ]c) in two slice models of brain ischemia. In rat hippocampal slices<br />
loaded with <strong>the</strong> Ca 2+ -sensitive dye Fura-2, ischemia causes <strong>an</strong> early increase in [Ca 2+ ]c that<br />
precedes <strong>the</strong> severe ischemic depolarization, <strong>an</strong>d is paralleled by <strong>an</strong> increase in <strong>the</strong> frequency of<br />
miniature excitatory post synaptic currents (mEPSC frequency in ischemia= 1.2±0.2Hz, p0.05 compared to control ischemia). Thus, <strong>the</strong><br />
increase in [Ca 2+ ]c <strong>an</strong>d <strong>the</strong> increase in mEPSCs are mutually independent. Stabilizing actin<br />
filaments with Jaspamide (20µM) prevented vesicle release induced by ischemia (mEPSC<br />
frequency in ischemia was 0.13±0.03Hz, <strong>for</strong> Jaspamide soaked slices <strong>an</strong>d 1.1±0.32Hz, <strong>for</strong><br />
interleaved control slices, p
Support: NIH<br />
FRAXA<br />
NIH/HD07333<br />
Title: Dopaminergic modulation of inhibitory currents in mouse medial prefrontal cortex<br />
Authors: *K. PAUL 1 , C. L. COX 2 ;<br />
1 Beckm<strong>an</strong> Inst., Univ. Illinois, Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a, IL; 2 Beckm<strong>an</strong> Institute, Departments<br />
of Mol. <strong>an</strong>d Integrative Physiol. <strong>an</strong>d Pharmacol., Univ. of Illinois at Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a,<br />
IL<br />
Abstract: The rodent prefrontal cortex receives subst<strong>an</strong>tial innervation <strong>from</strong> midbrain dopamine<br />
(DA)-containing neurons that target both pyramidal neurons <strong>an</strong>d inhibitory interneurons. In this<br />
study, we investigated whe<strong>the</strong>r DA altered <strong>the</strong> activity of miniature inhibitory postsynaptic<br />
currents (mIPSC) recorded <strong>from</strong> layer II/III pyramidal neurons in <strong>the</strong> medial prefrontal cortex<br />
(mPFC) of mice. Whole cell recordings were obtained <strong>from</strong> layer II/III pyramidal neurons using<br />
brain slices <strong>from</strong> P30-P48 postnatal age mice. IPSC recordings were optimized by using a Cs+containing<br />
pipette solution, <strong>an</strong>d a holding potential of 0 mV. Miniature IPSCs were recorded in<br />
<strong>the</strong> presence of TTX (1 µM) to block action potentials as well as CPP (10 µM) <strong>an</strong>d DNQX (20<br />
µM) to block NMDA <strong>an</strong>d nonNMDA glutamate currents, respectively. We found that DA (100<br />
µM) signific<strong>an</strong>tly increased <strong>the</strong> frequency <strong>an</strong>d amplitude of mIPSCs in 11 of 13 cells. The<br />
average frequency of mIPSCs was increased by 25% (p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.10/F19<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Parksinson's Disease Foundation Postdoctoral Fellowship<br />
NIH Gr<strong>an</strong>t NS046579<br />
Title: Dopaminergic regulation of calcium signaling in dendritic spines of striatal medium spiny<br />
neurons<br />
Authors: *M. J. HIGLEY, B. L. SABATINI;<br />
Harvard Med. Sch., Boston, MA<br />
Abstract: Perturbation of dopamine signaling in <strong>the</strong> basal g<strong>an</strong>glia is a hallmark feature of<br />
neurological disorders such as Parkinson’s disease, although <strong>the</strong> cellular mech<strong>an</strong>isms underlying<br />
<strong>the</strong> actions of this neuromodulator are poorly understood. Within <strong>the</strong> striatum, glutamatergic <strong>an</strong>d<br />
dopaminergic afferents converge onto individual dendritic spines of medium spiny projection<br />
neurons (MSNs). Postsynaptic activation of D2-type DA receptors (D2Rs) has been linked to<br />
modulation of synaptic tr<strong>an</strong>smission as well as long-term plasticity of synaptic connections.<br />
However, <strong>the</strong> specific synaptic targets regulated by D2Rs remain unclear. Here, we made wholecell<br />
recordings <strong>from</strong> fluorescently identified D2R-positive MSNs in brain slices taken <strong>from</strong> D2R-<br />
BAC-GFP mice. We paired 2-photon imaging <strong>an</strong>d local uncaging of glutamate to study<br />
synaptically-evoked calcium (Ca) signals within individual dendritic spines. Pharmacological<br />
m<strong>an</strong>ipulations revealed that NMDA-type glutamate receptors as well as L- <strong>an</strong>d N/P/Q-type<br />
voltage-gated Ca ch<strong>an</strong>nels (VGCCs) comprise <strong>the</strong> domin<strong>an</strong>t synaptic Ca sources in spines of<br />
<strong>the</strong>se cells. In addition, as in <strong>the</strong> hippocampus, R-type VGCCs appear to participate in a negative<br />
feedback loop limiting <strong>the</strong> magnitude of excitatory postsynaptic potentials (EPSPs). In contrast<br />
to synaptic activation, spine Ca influx evoked by dendritic invasion of back-propagating action<br />
potentials (bAPs) was mediated predomin<strong>an</strong>tly by L- <strong>an</strong>d R-type VGCCs with little contribution<br />
<strong>from</strong> N/P/Q-type ch<strong>an</strong>nels. Application of <strong>the</strong> selective D2R agonist quinpirole did not alter <strong>the</strong><br />
magnitude or kinetics of somatically-recorded EPSPs evoked by ei<strong>the</strong>r electrical stimulation of<br />
striatal afferents or direct glutamate uncaging on single spines. However, quinpirole signific<strong>an</strong>tly<br />
reduced <strong>the</strong> magnitude of <strong>the</strong> uncaging-evoked Ca signal. Quinpirole similarly reduced <strong>the</strong><br />
magnitude of spine Ca influx produced by bAPs. Blocking L-type VGCCs occluded <strong>the</strong> actions<br />
of quinpirole on both <strong>for</strong>ms of Ca signals, indicating that <strong>the</strong>se ch<strong>an</strong>nels are a principal synaptic<br />
target of D2Rs. Our results demonstrate that dopaminergic modulation of synaptic tr<strong>an</strong>smission<br />
in striatopallidal neurons acts primarily through alterations in local Ca influx, with limited effects<br />
on global voltage signaling.<br />
Disclosures: M.J. Higley, None; B.L. Sabatini, None.
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.11/F20<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Milton Fund<br />
HHMI<br />
NINDS<br />
Title: Experience-dependent modulation of striatal synaptic tr<strong>an</strong>smission by dopamine type 1<br />
receptor signals<br />
Authors: *Y. KOZOROVITSKIY, B. SABATINI;<br />
Neurobio., Harvard Med. School/HHMI, Boston, MA<br />
Abstract: The basal g<strong>an</strong>glia are involved in reward-based behavior through poorly understood<br />
neural mech<strong>an</strong>isms. Our study focuses on <strong>the</strong> development of striatum during <strong>the</strong> we<strong>an</strong>ing period<br />
in mice, when <strong>the</strong>y tr<strong>an</strong>sition <strong>from</strong> nursing to <strong>for</strong>aging <strong>for</strong> solid food. This period marks a time<br />
of signific<strong>an</strong>t reorg<strong>an</strong>ization of motivated behaviors. We have found a ch<strong>an</strong>ge in <strong>the</strong> synaptic<br />
response to dopamine in <strong>the</strong> striatum across <strong>the</strong> we<strong>an</strong>ing period <strong>an</strong>d showed that early life<br />
experience c<strong>an</strong> influence this process.<br />
Studies were done in acute brain slice of D1R BAC GFP mice using electrophysiology as well as<br />
combined MNI-glutamate uncaging <strong>an</strong>d 2-photon laser sc<strong>an</strong>ning microscopy. We found that<br />
dopaminergic stimulation of D1 receptors on medium spiny striatal neurons temporarily <strong>an</strong>d<br />
selectively downregulates NMDA-receptor dependent synaptic tr<strong>an</strong>smission during <strong>the</strong> periwe<strong>an</strong>ing<br />
period. The decrement in synaptic tr<strong>an</strong>smission has a post synaptic mech<strong>an</strong>ism <strong>an</strong>d is<br />
associated with reduced calcium influx at <strong>the</strong> level of single dendritic spines. Moreover,<br />
experiences which promote <strong>for</strong>aging <strong>for</strong> food <strong>an</strong>d activate <strong>the</strong> striatum be<strong>for</strong>e we<strong>an</strong>ing (e.g.,<br />
early we<strong>an</strong>ing or daily exploration of a complex environment), prevent <strong>the</strong> dopaminergic<br />
downregulation of synaptic tr<strong>an</strong>smission. We are currently exploring <strong>the</strong> mech<strong>an</strong>isms <strong>an</strong>d<br />
functional consequences of this phenomenon.<br />
Disclosures: Y. Kozorovitskiy, None; B. Sabatini, None.<br />
Poster
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.12/F21<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: HSFS<br />
SHRF<br />
CFI<br />
Title: Postsynaptic adenosine-induced persistent synaptic depression in hippocampus: Distinct<br />
regulation of GluR1 <strong>an</strong>d GluR2 receptors<br />
Authors: *F. S. CAYABYAB, Z. CHEN, C. PANCYR, N. L. LONGMUIR, M. NIAZI;<br />
Dept. of Physiol., Univ. of Saskatchew<strong>an</strong>, Saskatoon, SK, C<strong>an</strong>ada<br />
Abstract: Hypoxia triggers a massive accumulation of extracellular adenosine, which is widely<br />
accepted to cause presynaptic inhibition <strong>an</strong>d neuroprotection. However, <strong>the</strong> postsynaptic actions<br />
of adenosine are less well defined. Previously we reported that stimulation of adenosine A1<br />
receptors (A1Rs) activated p38 MAPK <strong>an</strong>d JNK which led to profound synaptic depression in<br />
<strong>the</strong> CA3-CA1 hippocampal synapses. Here, we demonstrate a novel mech<strong>an</strong>ism in which<br />
prolonged stimulation of A1Rs with N 6 -cyclopentyladenosine (CPA, 500 nM, 45 min) caused<br />
adenosine-induced persistent synaptic depression (APSD), which involved functional <strong>an</strong>d<br />
physical interactions between postsynaptic A1Rs <strong>an</strong>d GluR2-containing AMPA receptors. Field<br />
potential recordings <strong>from</strong> <strong>the</strong> hippocampal CA1 region showed that APSDs remained suppressed<br />
despite recovery of paired pulse facilitation to baseline levels during prolonged CPA washout.<br />
The levels of APSDs were attenuated by incubating slices with 2µM Tat-GluR2-3Y peptide, <strong>an</strong><br />
inhibitor of GluR2 endocytosis. We <strong>the</strong>n determined using biotinylation that prolonged CPA<br />
incubation signific<strong>an</strong>tly decreased GluR2 surface expression, <strong>an</strong>d this effect was reduced by <strong>the</strong><br />
Tat-GluR2-3Y peptide (but not by a scrambled version). We also confirmed that prolonged<br />
incubation of slices with CPA or hypoxia (20 min) reduced both GluR2 <strong>an</strong>d GluR1 proteins in<br />
hippocampal membr<strong>an</strong>e fractions. This CPA-mediated decrease in GluR2 surface expression was<br />
diminished by selective inhibitors of p38 MAPK (20µM SB203580, or 10µM SB239063), <strong>an</strong>d<br />
this inhibitory response was potentiated when ei<strong>the</strong>r p38 MAPK inhibitor was combined with a<br />
selective JNK inhibitor (5µM SP600125). In contrast to GluR2, <strong>the</strong> CPA-mediated GluR1<br />
internalization was insensitive to p38MAPK or JNK inhibitors. Finally, we found that a<br />
functional coupling of A1Rs <strong>an</strong>d GluR2-containing AMPA receptors could be facilitated by <strong>the</strong><br />
physical interactions of <strong>the</strong>se proteins as shown by co-immunoprecipitation studies. Toge<strong>the</strong>r,<br />
<strong>the</strong>se results suggest 1) that prolonged A1R stimulation induces internalization of GluR2containing<br />
AMPA receptors, which contributes to APSDs at a post-synaptic locus, <strong>an</strong>d 2) that in
contrast to GluR1 surface redistribution, <strong>the</strong> A1R-mediated internalization of GluR2 depends on<br />
p38 MAPK <strong>an</strong>d JNK activation. This differential regulation of GluR2 <strong>an</strong>d GluR1 receptors by<br />
A1R stimulation suggests that GluR2-deficient AMPA receptors may contribute to hypoxiainduced<br />
neuronal damage.<br />
Disclosures: F.S. Cayabyab, None; Z. Chen, None; C. P<strong>an</strong>cyr, None; N.L. Longmuir,<br />
None; M. Niazi, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.13/F22<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Germ<strong>an</strong> Research Council Gr986/9-1<br />
Title: Function of GABA tr<strong>an</strong>sporters GAT-1 <strong>an</strong>d GAT-3 in tonic <strong>an</strong>d phasic inhibition of<br />
striatal output neurons<br />
Authors: *R. GRANTYN, K. KIRMSE, M. SEMTNER, A. DVORZHAK, S. KIRISCHUK;<br />
Univ. Med. Charité, Berlin, Germ<strong>an</strong>y<br />
Abstract: GABAergic striatal output neurons (SONs) undergo cell death when mutation of<br />
huntingtin results in excessive accumulation of polyglutamine-containing proteins (Huntington’s<br />
disease). Deficits in <strong>the</strong> regulation of ambient GABA levels <strong>an</strong>d <strong>the</strong> resulting ch<strong>an</strong>ges in tonic<br />
<strong>an</strong>d/or phasic inhibition may contribute to excitotoxic cell death, but <strong>the</strong> role of GABA<br />
tr<strong>an</strong>sporters (GATs) is not well elucidated in <strong>the</strong> striatum. The present study aimed at clarifying<br />
<strong>the</strong> differential function of GAT-1 versus GAT-3, as <strong>the</strong>y were reported to display preferential<br />
expression in neurons <strong>an</strong>d astrocytes. The relative strength of <strong>the</strong> two tr<strong>an</strong>sporters was assessed<br />
by using <strong>the</strong> respective blockers, NO711 (10 µM) <strong>an</strong>d SNAP4114 (40 µM). Based on patchclamp<br />
recordings <strong>from</strong> SONs between postnatal days (P) 7 <strong>an</strong>d 34, we demonstrate that <strong>the</strong><br />
GABA(A) receptor-mediated tonic current (I(GABA)tonic) is weak or absent during <strong>the</strong> first 2<br />
postnatal weeks but <strong>the</strong>n signific<strong>an</strong>tly increases towards adulthood. At P28-34 block of GAT-1<br />
produced <strong>an</strong> about 10fold increase of I(GABA)tonic, whereas SNAP4114 had little effect on it.<br />
The dependency of GABA(A) receptor-mediated phasic synaptic responses on ambient GABA<br />
concentration ch<strong>an</strong>ges was evaluated on <strong>the</strong> basis of GABA(B) receptor-mediated ch<strong>an</strong>ges of<br />
stimulus-induced GABA release <strong>an</strong>d ch<strong>an</strong>ges in <strong>the</strong> amplitude/kinetics of qu<strong>an</strong>tal postsynaptic<br />
responses. It was found that in <strong>the</strong> adult striatum block of GAT-1 affected pre- <strong>an</strong>d postsynaptic
sites, as it decreased <strong>the</strong> amplitudes of evoked IPSCs (eIPSCs) <strong>an</strong>d miniature IPSCs (mIPSCs),<br />
prolonged eIPSCs <strong>an</strong>d increased <strong>the</strong>ir paired-pulse ratio (PPR). Block of GAT-3 produced a<br />
similar GABA(B) receptor-dependent effect on GABA release, but to reveal this effect required<br />
<strong>the</strong> presence of NO711, i.e. a block of GAT-1 activity. In contrast to I(GABA)tonic, <strong>an</strong> agedependency<br />
of GAT- or GAT-3 action on phasic GABAergic synaptic tr<strong>an</strong>smission has not been<br />
revealed. We conclude that in <strong>the</strong> murine striatum GAT-1, <strong>an</strong>d possibly GAT-3, operate in <strong>the</strong><br />
uptake mode <strong>an</strong>d <strong>the</strong>reby keep extracellular GABA at a level excluding persistent activation of<br />
presynaptic GABA(B) <strong>an</strong>d postsynaptic GABA(A) receptors. The astrocytic GABA tr<strong>an</strong>sporter<br />
GAT-3 may provide <strong>an</strong> additional uptake capacity when neuronal GAT-1 is insufficient, but this<br />
would affect phasic ra<strong>the</strong>r th<strong>an</strong> tonic inhibition of SONs.<br />
Disclosures: R. Gr<strong>an</strong>tyn, None; K. Kirmse, None; M. Semtner, None; A. Dvorzhak,<br />
None; S. Kirischuk, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.14/F23<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: Gr<strong>an</strong>t Support: MH78823<br />
Title: Potentiation of latent synapses by synaptic modulators<br />
Authors: *C. CHANG 1 , X. JIANG 2 , S. MENNERICK 3 ;<br />
2 Psychiatry, 3 Psychiatry; Anat. <strong>an</strong>d Neurobio., 1 Washington Univ, St Louis, St. Louis, MO<br />
Abstract: Release probability (pr) of hippocampal synapses varies across terminals. In addition,<br />
<strong>the</strong>re appears to be a population of very low pr or latent presynaptic terminals that are not<br />
effectively recruited by normal presynaptic activity. Whe<strong>the</strong>r <strong>the</strong>se synapses are affected by<br />
synaptic modulators is not clear. To address this question, we used MK801 in combination with<br />
low frequency stimulation to eliminate NMDA receptor mediated EPSCs (INMDAR) at high pr<br />
terminals. We evaluated <strong>the</strong> response <strong>from</strong> <strong>the</strong> remaining synapses to <strong>the</strong> synaptic modulators<br />
phorbol ester (PDBu, PKC activator <strong>an</strong>d C1-domain binding modulator) or <strong>for</strong>skolin (FSK,<br />
adenylic cyclase activator) by measuring <strong>the</strong> recovery of INMDAR. Acute application of PDBu, but<br />
not FSK, promoted <strong>the</strong> re-emergence of INMDAR after MK801 elimination of synaptic INMDAR. In<br />
<strong>the</strong> absence of MK801, PDBu <strong>an</strong>d FSK potentiated IAMPAR <strong>an</strong>d INMDAR to <strong>the</strong> same degree,<br />
indicating that nei<strong>the</strong>r PDBu nor FSK preferentially affects <strong>the</strong> level of synaptic APMAR or
NMDAR, <strong>an</strong>d also suggesting a presynaptic locus of modulation. In addition, <strong>the</strong> potentiation of<br />
INMDAR by PDBu was not seen after whole cell blockade of NMDAR, indicating that <strong>the</strong> insertion<br />
of NMDAR into postsynaptic sites does not contribute to PDBu-dependent potentiation of<br />
INMDAR. Pre-exposure to <strong>the</strong> PKC inhibitor Gö6983, which prevents <strong>the</strong> phosphorylation of<br />
NDMAR by PKC activation, did not abolish PDBu-dependent potentiation of INMDA, suggesting<br />
potentiation of INMDA by PDBu is not caused by <strong>the</strong> movement of unblocked NMDAR into<br />
postsynaptic sites. Blocking INMDAR at both high <strong>an</strong>d low pr synapses by MK801 in high<br />
extracellular calcium or with higher stimulation frequency did not completely prevent<br />
potentiation of INMDAR by PDBu, suggesting that PDBu potentiates synaptic output at <strong>the</strong><br />
synapses that have extremely low pr. Lastly, we used imaging to test whe<strong>the</strong>r PDBu recruits<br />
tr<strong>an</strong>smitter release <strong>from</strong> presynaptically silent synapses, which are defined as <strong>the</strong> synapses that<br />
are V-GluT1 immunoreactive but devoid of FM-1-43 uptake. Similar to electrophysiology<br />
results, acute exposure to PDBu, but not to FSK, reduced <strong>the</strong> percentage of presynaptically silent<br />
glutamatergic synapses. These results suggest that acute phorbol ester application, but not cAMP<br />
increases, unmasks latent presynaptic terminals. The results may also imply that basal status of<br />
synaptic activity might determine <strong>the</strong> efficacy of different synaptic modulators.<br />
Gr<strong>an</strong>t Support: MH78823<br />
Disclosures: C. Ch<strong>an</strong>g, None; X. Ji<strong>an</strong>g, None; S. Mennerick, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.15/F24<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Support: research fund of Drexel University College of Medicine(W-J Gao).<br />
NARSAD young investigator awards(W-J Gao).<br />
NIH R21 gr<strong>an</strong>t MH232307 (W-J Gao).<br />
Title: MK-801 induces distinct ch<strong>an</strong>ges of AMPA <strong>an</strong>d NMDA receptors in <strong>the</strong> fast spiking<br />
interneurons <strong>an</strong>d pyramidal cells in adolescent rat prefrontal cortex<br />
Authors: *H. WANG, W.-J. GAO;<br />
Dept Neurobio/Anat, Drexel Univ. Coll Med., Philadelphia, PA
Abstract: NMDA receptors are critical not only <strong>for</strong> normal prefrontal functions but also <strong>for</strong> <strong>the</strong><br />
pathological processes of schizophrenia. We recently reported that fast-spiking (FS) interneurons<br />
gradually lost NMDA receptors during cortical development <strong>an</strong>d most (~75%) of <strong>the</strong>se cells<br />
express calcium-permeable AMPA receptors (CP-AMPAR). The FS interneurons also exhibit<br />
m<strong>an</strong>y distinct physiological properties in <strong>the</strong> rat prefrontal cortex (PFC), including low NMDA<br />
receptors, prominent frequency-dependent short-term facilitation, <strong>an</strong>d characteristic ch<strong>an</strong>ges<br />
during adolescent period. We hypo<strong>the</strong>size that <strong>the</strong>se characteristic properties may make <strong>the</strong>se<br />
cells particularly vulnerable to disruptive influences in <strong>the</strong> PFC, thus contributing to <strong>the</strong> onset of<br />
m<strong>an</strong>y psychiatric disorders, including schizophrenia. Here we test this hypo<strong>the</strong>sis by<br />
investigating how low-dose administration of MK-801, <strong>an</strong> uncompetitive NMDA receptor<br />
<strong>an</strong>tagonist often used in schizophrenia <strong>an</strong>imal model, affects <strong>the</strong> functions of AMPA <strong>an</strong>d NMDA<br />
receptors in <strong>the</strong> FS interneurons compared with those in pyramidal cells in <strong>the</strong> adolescent rat<br />
PFC. The rats (PD30-45) were treated with MK 801 (0.1 mg/kg, i.p.) <strong>for</strong> five consecutive days<br />
<strong>an</strong>d sacrificed <strong>for</strong> in vitro patch-clamp recordings in PFC slices 24h after <strong>the</strong> last treatment. We<br />
found that <strong>the</strong> threshold of FS interneurons was increased <strong>an</strong>d spike number decreased<br />
signific<strong>an</strong>tly in MK-801-treated rats. Although <strong>the</strong> NMDA/AMPA ratios were similarly <strong>an</strong>d<br />
signific<strong>an</strong>tly decreased in both FS interneurons <strong>an</strong>d pyramidal cells, MK-801 induced several<br />
characteristic alterations in FS interneurons, including signific<strong>an</strong>tly increased ratio of AMPA-<br />
EPSC/stimulus intensity, reduced inwardly rectifying current, shifted reversal potential, <strong>an</strong>d<br />
decreased amplitude of NMDAR-mediated mEPSCs. In contrast, <strong>the</strong> frequency of NMDARmediated<br />
mEPSCs in pyramidal neurons was dramatically increased by administration of MK-<br />
801 <strong>an</strong>d <strong>the</strong> mEPSCs were effectively blocked by bath-application of APV, whereas nei<strong>the</strong>r<br />
amplitude nor kinetics of NMDAR-mediated mEPSCs in <strong>the</strong>se cells was affected by MK-801<br />
treatment, indicating a signific<strong>an</strong>t increase of presynaptic NMDA receptors. These results<br />
suggest a likely target-cell specific regulation of AMPA <strong>an</strong>d NMDA receptor by MK-801<br />
treatment in <strong>the</strong> adolescent rat PFC.<br />
Disclosures: H. W<strong>an</strong>g, None; W. Gao, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.16/F25<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action<br />
Title: Interneuronal ErbB4 receptor ablation alters GABAergic inhibition in pyramidal cells
Authors: *V. ZSIROS 1 , J. E. BELFORTE 2 , K. C. K. LLOYD 3 , K. NAKAZAWA 1 ;<br />
1 UGCB, NIMH, Be<strong>the</strong>sda, MD; 2 Facultad de Medicina, Univ. de Buenos Aires, Ciudad de<br />
Buenos Aires, Argentina; 3 Dept. of Anatomy, Physiol. <strong>an</strong>d Cell Biol., Univ. of Cali<strong>for</strong>nia, Davis,<br />
CA<br />
Abstract: Variation in ErbB4-neuroregulin signaling was associated with <strong>an</strong> increased<br />
susceptibility to schizophrenia both in genome-wide association <strong>an</strong>d postmortem brain studies.<br />
ErbB4 is a member of <strong>the</strong> receptor tyrosine kinases <strong>an</strong>d its lig<strong>an</strong>d, neuregulin1, was shown to<br />
modulate glutamatergic signaling by its interaction with NMDA receptors. However, <strong>the</strong> ErbB4<br />
receptor is preferentially expressed in interneurons in <strong>the</strong> adult neocortex. One c<strong>an</strong> hypo<strong>the</strong>size<br />
that ErbB4 signaling may directly alter GABAergic input to pyramidal cells by a presynaptic<br />
mech<strong>an</strong>ism, which may result in perturbation of pyramidal cell firing through cortical<br />
disinhibition. Alternatively, NMDA-receptor hypofunction in interneurons could be elicited upon<br />
deletion of postsynaptic ErbB4 in <strong>the</strong>se neurons. The aim of this study is to determine how<br />
neuregulin-ErbB4 signaling affects network activity in <strong>the</strong> prefrontal cortex in mice. Using a<br />
floxed-ErbB4 mouse line crossed to ei<strong>the</strong>r a cortical GABAergic cell-Cre line (Cre#4127) or a<br />
<strong>for</strong>ebrain excitatory cell-Cre line (G35-3), we conditionally inactivated ErbB4 in interneurons or<br />
in excitatory cells in <strong>the</strong> neocortex, respectively. Using whole cell patch-clamp recordings in <strong>the</strong><br />
adult mice prefrontal cortex we found that baseline presynaptic GABAergic tr<strong>an</strong>smission is<br />
altered in <strong>the</strong> interneuron-specific ErbB4 knockout line. In <strong>the</strong> presence of glutaminergic<br />
synaptic blockers, miniature IPSC frequency was lower <strong>an</strong>d both rise <strong>an</strong>d decay time const<strong>an</strong>ts<br />
were longer in mut<strong>an</strong>ts. In addition to synaptic tr<strong>an</strong>smission, cortical pyramidal cells are also<br />
known to express tonic inhibition through extrasynaptic or perisynaptic GABAergic receptors.<br />
<strong>When</strong> >100uM gabazine was added to block all <strong>the</strong> GABAergic current, however, we found no<br />
ch<strong>an</strong>ge in <strong>the</strong> overall membr<strong>an</strong>e current in <strong>the</strong> interneuron-specific ErbB4 knockout mice, unlike<br />
in <strong>the</strong> cells <strong>from</strong> control littermates. Based on <strong>the</strong>se results we suggest that ErbB4 ablation in<br />
interneurons results in alteration of GABAergic signaling in pyramidal cells <strong>an</strong>d we predict that<br />
presynaptic ablation of ErbB4 receptors result in a somewhat decreased inhibition of <strong>the</strong><br />
postsynaptic excitatory cells.<br />
Disclosures: V. Zsiros, None; J.E. Bel<strong>for</strong>te, None; K.C.K. Lloyd, None; K. Nakazawa, None.<br />
Poster<br />
523. Modulation: Mech<strong>an</strong>isms of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 523.17/F26<br />
Topic: B.07.g. Modulation: Mech<strong>an</strong>isms of action
Support: AHAF<br />
NIH Gr<strong>an</strong>t NS055804<br />
Title: Presenilin 1 modulates spont<strong>an</strong>eous but not evoked release <strong>from</strong> hippocampal neurons<br />
Authors: K. G. PRATT 1 , H. WATARI 2,1 , D. G. COOK 3,5 , *J. M. SULLIVAN 4,1,2 ;<br />
1 Physiol. & Biophysics Dept., 2 Grad. Program in Neurobio. & Behavior, 3 Dept. of Med., 4 Univ.<br />
Washington Sch. Med., Seattle, WA; 5 VA Med. Ctr. GRECC, Seattle, WA<br />
Abstract: Mutations in <strong>the</strong> genes encoding presenilins are <strong>the</strong> most common cause of familial<br />
Alzheimer’s disease (AD), but <strong>the</strong> specific role that presenilins play in <strong>the</strong> development of AD<br />
remains to be determined. An emerging hypo<strong>the</strong>sis proposes that synaptic dysfunction is<br />
responsible <strong>for</strong> <strong>the</strong> early stages of AD. To help adv<strong>an</strong>ce our underst<strong>an</strong>ding of <strong>the</strong><br />
neuropathological effects of mut<strong>an</strong>t presenilins, it will be useful to identify <strong>the</strong> role presenilin 1<br />
(PS1) normally plays in non-pathological synaptic function. To achieve this goal, we per<strong>for</strong>med<br />
<strong>an</strong> in-depth characterization of evoked <strong>an</strong>d spont<strong>an</strong>eous excitatory neurotr<strong>an</strong>smission in autaptic<br />
hippocampal cultures derived <strong>from</strong> PS1 null mouse embryos. Evoked tr<strong>an</strong>smission was unaltered<br />
in PS1 null neurons: excitatory postsynaptic current (EPSC) size, NMDA receptor-mediated<br />
current amplitude, paired-pulse ratio, depression during a train of action potentials <strong>an</strong>d recovery<br />
after delivery of <strong>the</strong> train were not signific<strong>an</strong>tly different <strong>from</strong> wild-type womb-mate controls.<br />
As reported previously <strong>for</strong> conventional cortical cultures of PS1 null neurons (Parent et al., J.<br />
Neurosci. 2005), however, <strong>the</strong> rate of spont<strong>an</strong>eous miniature EPSCs (mEPSCs) was dramatically<br />
increased. This increase in spont<strong>an</strong>eous mEPSC rate was reversed by acutely buffering<br />
intracellular calcium with EGTA, or by chronic Lenti virus-mediated expression of wild-type<br />
PS1. Experiments are currently underway to identify <strong>the</strong> source(s) of <strong>the</strong> elevated calcium<br />
responsible <strong>for</strong> <strong>the</strong> increase in spont<strong>an</strong>eous mEPSC rate in PS1 null neurons. Toge<strong>the</strong>r, our<br />
findings suggest that functional synapse number, postsynaptic receptor composition <strong>an</strong>d action<br />
potential-dependent release probability are not directly affected by <strong>the</strong> absence of PS1. Instead,<br />
<strong>an</strong> increase in spont<strong>an</strong>eous, action potential-independent release probability appears to be a<br />
primary consequence of eliminating PS1 in neurons, likely due to altered calcium signaling.<br />
Disclosures: K.G. Pratt, None; H. Watari, None; D.G. Cook, None; J.M. Sulliv<strong>an</strong> , None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.1/F27
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: P20 RR015583<br />
NS 33270<br />
Title: Excitatory amino acid tr<strong>an</strong>sporter 3 (EAAT3) alters synaptic plasticity <strong>an</strong>d spatial learning<br />
Authors: K. M. HOFFMAN 1 , A. N. ANGELL 1 , D. P. BONISLAWSKI 1 , D. J. POULSEN 1 , M.<br />
P. BABCOCK 2 , *M. P. KAVANAUGH 1 ;<br />
1 2<br />
NIH COBRE Neurosci., Univ. of Mont<strong>an</strong>a, Missoula, MT; Psychology, Mont<strong>an</strong>a State Univ.,<br />
Bozem<strong>an</strong>, MT<br />
Abstract: Mainten<strong>an</strong>ce of <strong>the</strong> extracellular glutamate concentration, largely by glutamate<br />
tr<strong>an</strong>sporters, is crucial to <strong>the</strong> selectivity <strong>an</strong>d specificity of neurotr<strong>an</strong>smission. Block of <strong>the</strong><br />
neuronal glutamate tr<strong>an</strong>sporter EAAT3 has been shown to cause glutamate spillover onto<br />
extrasynaptic targets <strong>an</strong>d enh<strong>an</strong>ce NMDA receptor activation (Diamond, 2001). EAAT3 is found<br />
in <strong>the</strong> hippocampus, <strong>an</strong>d its surface expression is greatly increased in CA1 following LTP<br />
(Levenson et al. 2002). In this study we found that block of EAAT3, through <strong>the</strong> use of ei<strong>the</strong>r<br />
viral or pharmacological techniques, reduced LTP <strong>an</strong>d impaired spatial learning <strong>an</strong>d memory in<br />
<strong>the</strong> Morris water maze in 5 month old Sprague-Dawley rats. We used recombin<strong>an</strong>t adenoassociated<br />
viral vectors encoding control GFP or EAAT3-<strong>an</strong>tisense, injected into <strong>the</strong> hippocampi<br />
of 8 week old rats to knock down EAAT3 expression in hippocampus. EAAT3 knockdown was<br />
confirmed by immunohistochemistry in EAAT3-<strong>an</strong>tisense injected rats. We found that <strong>the</strong>ta<br />
stimulation resulted in decreased LTP in EAAT3 <strong>an</strong>ti-sense injected rats compared to nullinjected<br />
rats (16.3 ± 3.0% n=32, 32.6 ± 5.5% n=25 respectively, p=0.01). Next, we utilized <strong>the</strong><br />
EAAT3 selective <strong>an</strong>tagonist benzyl-aspartate (BA; Esslinger et al. 2005), to fur<strong>the</strong>r confirm <strong>the</strong><br />
role of EAAT3. LTP following <strong>the</strong>ta stimulation in <strong>the</strong> presence of 30µM BA was signific<strong>an</strong>tly<br />
decreased compared to control conditions (16.0 ± 5.7% n=9, 31.5 ± 4.2% n=10 respectively, p<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.2/F28<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Title: D-aspartate exerts <strong>an</strong> opposing role upon age-dependent NMDAR-related decay of<br />
synaptic plasticity <strong>an</strong>d memory in mice<br />
Authors: A. USIELLO 1,2 , R. NISTICÒ 3,4 , F. NAPOLITANO 1 , *N. B. MERCURI 3,5 , F.<br />
ERRICO 1 ;<br />
1 Lab. of Behavioural Neurosci., CEINGE Biotecnologie Av<strong>an</strong>zate, Naples, Italy; 2 Dept. of Hlth.<br />
Sci., Univ. of Molise, Campobasso, Italy; 3 IRCCS -SANTA LUCIA FOUNDATION, Roma,<br />
Italy; 4 Dept. of Pharmacobiology, Univ. of Calabria, Arca Vacata di Rende, Italy; 5 Clinica<br />
Neurologica, Univ. Tor Vergata, Roma, Italy<br />
Abstract: Within <strong>the</strong> pool of endogenous amino acids, serine <strong>an</strong>d aspartate are <strong>the</strong> only two<br />
residues occurring at signific<strong>an</strong>t concentrations in free D-<strong>for</strong>m in <strong>the</strong> mammali<strong>an</strong> brain.<br />
Compelling evidence demonstrates that D-serine plays a crucial role in controlling neuronal<br />
functions in mammals. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>the</strong> biological signific<strong>an</strong>ce of D-Asp in <strong>the</strong> brain is<br />
still obscure. D-Asp appears with a peculiar temporal pattern of localization, being abund<strong>an</strong>t<br />
during brain embryo development <strong>an</strong>d strongly decreasing after birth. This phenomenon is <strong>the</strong><br />
result of <strong>the</strong> postnatal onset of D-Aspartate Oxidase (DDO) expression, <strong>the</strong> only known enzyme<br />
that strictly controls <strong>the</strong> endogenous levels of D-Asp. In our work, we highlighted <strong>the</strong> role of D-<br />
Asp on mouse hippocampal NMDAR-dependent functions, like synaptic plasticity <strong>an</strong>d memory,<br />
<strong>an</strong>d how this molecule <strong>an</strong>d its regulating enzyme DDO may influence <strong>the</strong> age-related decay of<br />
such processes. In this regard, we used two different experimental strategies to produce <strong>an</strong><br />
abnormal increase of endogenous D-Asp levels in <strong>the</strong> mouse: a genetic approach based on <strong>the</strong><br />
targeted deletion of Ddo gene <strong>an</strong>d a pharmacological approach based on <strong>the</strong> oral administration<br />
of D-Asp. We demonstrated that D-Asp acts as a potent in vitro <strong>an</strong>d in vivo neuromodulatory<br />
molecule upon central NMDAR tr<strong>an</strong>smission. Accordingly, we showed that this D-amino acid is<br />
able to strongly influence hippocampus-related functions at adulthood. Indeed, while upregulated<br />
levels of D-Asp increase NMDAR-dependent LTP <strong>an</strong>d spatial memory in young mice,<br />
its prolonged deregulation in mature <strong>an</strong>imals induces a dramatic acceleration of <strong>the</strong> agedependent<br />
decay of hippocampal functions. Moreover, we highlighted <strong>the</strong> ability of D-Asp to<br />
enh<strong>an</strong>ce NMDAR-dependent synaptic plasticity through <strong>an</strong> inducible “turn-on/turn-off-like<br />
mech<strong>an</strong>ism”. Strikingly, we also showed that administration of D-Asp at older stages of life<br />
strongly rescues hippocampal synaptic plasticity deterioration <strong>an</strong>d attenuates <strong>the</strong> natural<br />
cognitive decline of aged <strong>an</strong>imals.<br />
In conclusion, our data suggest that <strong>the</strong> in-embryo-occurring D-amino acid D-Asp, when<br />
“<strong>for</strong>ced” at late stages of life, discloses plasticity windows in <strong>the</strong> aging brain aimed to counteract<br />
<strong>the</strong> physiological reduction of NMDAR signaling.
Disclosures: A. Usiello, None; R. Nisticò, None; F. Napolit<strong>an</strong>o, None; N.B. Mercuri,<br />
None; F. Errico, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.3/F29<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: NIH (GM 60655)<br />
Title: Low-frequency potentiation (LFP) at <strong>the</strong> per<strong>for</strong><strong>an</strong>t path-dentate gyrus synapse in vivo is<br />
observed in ureth<strong>an</strong>e-, but not pentobarbital-<strong>an</strong>es<strong>the</strong>tized rats<br />
Authors: J. GONZALEZ 1 , S. R. HARRIS 1 , K. ANGUIANO 3 , *B. E. DERRICK 2,4 ;<br />
1 Biol., Univ. Texas at S<strong>an</strong> Antonio, The Neurosciences Institute, S<strong>an</strong> Antonio, TX; 2 Univ. Texas<br />
at S<strong>an</strong> Antonio, S<strong>an</strong> Antonio, TX; 3 Dept. of Biol. <strong>an</strong>d The Neurosciences Inst., The Univ. of<br />
Texas at S<strong>an</strong> Antonio, S<strong>an</strong> Antonio, TX; 4 Biol., The Neurosciences Inst., S<strong>an</strong> Antonio, TX<br />
Abstract: Several recent studies have focused on synaptic potentiation induced by low<br />
frequency stimulation <strong>an</strong>d its possible role in memory. Previously we reported low frequency<br />
stimulation (25-900 pulses, 0.5-1 Hz) of ei<strong>the</strong>r medial or lateral per<strong>for</strong><strong>an</strong>t path projections to <strong>the</strong><br />
dentate gyrus in awake, behaving <strong>an</strong>imals produces <strong>an</strong> immediate <strong>an</strong>d robust (100%) potentiation<br />
of per<strong>for</strong><strong>an</strong>t path dentate responses. This low-frequency potentiation (LFP) shows no decrement<br />
over 4 hrs, suggesting expression of <strong>the</strong> later, protein syn<strong>the</strong>sis-dependent phase of LTP.<br />
However, unlike late <strong>for</strong>ms of LTP that persists <strong>from</strong> days to weeks, LFP is relatively short-lived<br />
(< 24 hrs) <strong>an</strong>d NMDAr-independent, indicating LFP is distinct <strong>from</strong> LTP seen at <strong>the</strong>se same<br />
synapses when induced by <strong>the</strong>ta burst stimulation. We report here that, in <strong>an</strong>imals <strong>an</strong>es<strong>the</strong>tized<br />
with pentobarbital sodium, stimulation that normally induces LFP in behaving <strong>an</strong>imals (1 Hz,<br />
900 pulses) fails to induce LFP. LFS at 1 Hz stimulation at maximal current intensities similarly<br />
was ineffective in inducing LFP. LFP was observed with moderate stimulation intensities under<br />
ureth<strong>an</strong>e <strong>an</strong>es<strong>the</strong>sia, <strong>an</strong> <strong>an</strong>es<strong>the</strong>tic thought to have moderate effects on GABAergic inhibition,<br />
however <strong>the</strong> magnitude of LFP was smaller th<strong>an</strong> that observed in behaving <strong>an</strong>imals. We propose<br />
factors present during waking states (such as acetylcholine or neuromodulators related to<br />
arousal) are necessary <strong>for</strong> LFP induction, possibly by allowing sustained depolarization at lower<br />
stimulation frequencies. Toge<strong>the</strong>r, <strong>the</strong>se data indicate a distinct <strong>an</strong>d robust (but tr<strong>an</strong>sient) <strong>for</strong>m of<br />
cortico-dentate LTP in vivo is induced without bursting, <strong>an</strong>d supports <strong>the</strong> view that multiple<br />
<strong>for</strong>ms of LTP are found in <strong>the</strong> hippocampal <strong>for</strong>mation, even within a single synaptic population.
Moreover, <strong>the</strong> expression of some <strong>for</strong>ms of LTP is dependent on behavioral state. These distinct<br />
<strong>for</strong>ms of potentiation may have functionally distinct roles in learning <strong>an</strong>d memory.<br />
Disclosures: J. Gonzalez, None; S.R. Harris, None; K. Angui<strong>an</strong>o, None; B.E. Derrick, GM<br />
60655, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts<br />
as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.4/F30<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: NIH gr<strong>an</strong>t AA14294<br />
Title: A role <strong>for</strong> GABA-B autoreceptors in LTP induced by primed burst stimulation, but not<br />
repeated burst stimulation<br />
Authors: J. D. COOKE 1 , *L. M. GROVER 1 , W. R. HOLMES 2 ;<br />
1 2<br />
Pharmacology, Physiol. & Toxicology, Marshall Univ. Sch. of Med., Huntington, WV; Biol.<br />
Sci., Ohio Univ., A<strong>the</strong>ns, OH<br />
Abstract: Repeated burst stimulation at <strong>the</strong>ta-delta frequencies is highly effective <strong>for</strong> LTP<br />
induction (Grover et al., Learn Mem, <strong>2009</strong>). One reason <strong>for</strong> <strong>the</strong> effectiveness of repeated burst<br />
stimulation is <strong>the</strong> strong suppression of GABAergic inhibition which develops when bursts are<br />
repeated at intervals of 200 ms to 1 s. Because GABA-B autoreceptor-mediated disinhibition is<br />
maximal at <strong>an</strong> interval around 200 ms (<strong>the</strong>ta frequency), GABA-B receptors might be critical <strong>for</strong><br />
disinhibition during repeated burst stimulation, especially at <strong>the</strong>ta frequency. To test this<br />
possibility, we recorded GABA receptor-mediated IPSCs during repeated burst stimulation at<br />
intervals of 200 ms, 500 ms, <strong>an</strong>d 1 s, <strong>an</strong>d examined effects of <strong>the</strong> GABA-B receptor <strong>an</strong>tagonist,<br />
CGP-55845. CGP-55845 had little or no effect on IPSCs except during <strong>the</strong> second burst given at<br />
<strong>the</strong> 200 ms interval, where CGP-55845 greatly attenuated disinhibition (Grover <strong>an</strong>d Holmes, Soc<br />
Neurosci Abstr, 2008). Based on <strong>the</strong>se findings, we predicted that blocking GABA-B receptors<br />
would not affect LTP induced by repeated burst stimulation, but would inhibit LTP produced by<br />
primed burst stimulation at <strong>the</strong> 200 ms interval. To test <strong>the</strong>se predictions, we examined four<br />
stimulation conditions: primed burst stimulation consisting of two stimulus bursts (each burst<br />
containing 4 stimuli at 100 Hz) at a 200 ms inter-burst interval, <strong>an</strong>d repeated burst stimulation<br />
(20 bursts with each burst containing 4 stimuli at 100 Hz) given at inter-burst intervals of 200
ms, 500 ms, <strong>an</strong>d 1 s. In agreement with our predictions, primed burst stimulation induced<br />
signific<strong>an</strong>tly greater LTP under control conditions (me<strong>an</strong> increase in field EPSP of 13.4 ±6.4% at<br />
30 min post) compared to primed burst stimulation in 2 µM CGP-55845 (me<strong>an</strong> ch<strong>an</strong>ge in field<br />
EPSP of -4.5±4.9%, p0.80), at 500 ms intervals LTP averaged 21.7±4.4% (control)<br />
<strong>an</strong>d 31.0±5.9% (CGP-55845, p>0.20), <strong>an</strong>d at 1 s intervals LTP averaged 20.2±9.7% (control)<br />
<strong>an</strong>d 20.0±9.5% (CGP-55845, p>0.90). These findings support a role <strong>for</strong> GABA-B autoreceptormediated<br />
disinhibition only <strong>for</strong> LTP induced by primed burst stimulation.<br />
Disclosures: J.D. Cooke, None; L.M. Grover, None; W.R. Holmes, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.5/F31<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: FONDECYT Gr<strong>an</strong>t 1070028<br />
Title: Beta-adrenoceptor blockade reduces long-term synaptic potentiation <strong>an</strong>d BDNF<br />
expression in <strong>the</strong> rat occipital cortex<br />
Authors: *O. A. FLORES, H. NUÑEZ, H. PEREZ, C. MORGAN, L. VALLADARES, R.<br />
SOTO-MOYANO, A. HERNANDEZ;<br />
Inst. of Nutr. <strong>an</strong>d Food Technol. (INTA), S<strong>an</strong>tiago, Chile<br />
Abstract: Beta-adrenergic receptor stimulation c<strong>an</strong> signific<strong>an</strong>tly facilitate synaptic potentiation<br />
(LTP) in <strong>the</strong> hippocampus <strong>an</strong>d enh<strong>an</strong>ce memory processes, but its effect on neocortical plastic<br />
mech<strong>an</strong>isms is less conclusive. We studied in <strong>an</strong>es<strong>the</strong>tized rats <strong>the</strong> effect of <strong>the</strong> betaadrenoceptor<br />
<strong>an</strong>tagonist propr<strong>an</strong>olol on LTP induced in vivo by tet<strong>an</strong>izing stimulation applied to<br />
<strong>the</strong> occipital cortex. Since activity-dependent tr<strong>an</strong>scription of brain derived neurotrophic factor<br />
(BDNF) is required <strong>for</strong> tr<strong>an</strong>slating LTP associated to sensory experience into some types of<br />
learning, <strong>the</strong> effect of long-lasting propr<strong>an</strong>olol administration upon occipital cortex BDNF<br />
expression was also studied. Occipital cortex LTP was elicited by potentiating tr<strong>an</strong>scallosal<br />
evoked responses with a tet<strong>an</strong>izing train, 15 min after administration of a single dose of<br />
propr<strong>an</strong>olol (1.25, 2.5 or 5 mg/kg i.p.). Occipital cortex BDNF protein concentration was<br />
measured in additional rats receiving subcut<strong>an</strong>eous chronic administration of propr<strong>an</strong>olol (3
mg/kg/day) or saline during 3 days by me<strong>an</strong>s of a mini-osmotic pump. Results showed that<br />
propr<strong>an</strong>olol dose-dependently inhibited <strong>the</strong> ability of <strong>the</strong> occipital cortex to develop LTP, while<br />
chronic propr<strong>an</strong>olol administration decreased BDNF expression in this cortical region. These<br />
effects could be related to <strong>the</strong> reported disruptive actions induced on short- <strong>an</strong>d long-term<br />
memory <strong>for</strong>mation by pretreatment with beta-adrenoceptor <strong>an</strong>tagonists, <strong>an</strong>d probably involves<br />
activity ch<strong>an</strong>ges of kinases modulating NMDA <strong>an</strong>d/or AMPA receptors as well as<br />
downregulation of <strong>the</strong> cAMP-CREB-BDNF intracellular tr<strong>an</strong>sduction cascade, respectively.<br />
Gr<strong>an</strong>t fron FONDECYT: 1070028<br />
Disclosures: O.A. Flores, None; H. Nuñez, None; H. Perez, None; C. Morg<strong>an</strong>, None; L.<br />
Valladares, None; R. Soto-Moy<strong>an</strong>o, None; A. Hern<strong>an</strong>dez, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.6/F32<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: NINDS Gr<strong>an</strong>t PO1 NS-39546<br />
Title: Glutamatergic signaling at synapses undergoing morphallactic plasticity<br />
Authors: *Z. R. LYBRAND, M. J. ZORAN;<br />
Texas A&M Univ., College Station, TX<br />
Abstract: Lumbriculus variegatus undergoes a <strong>for</strong>m of synaptic plasticity during a regenerative<br />
process called neural morphallaxis. Discrete synaptic ch<strong>an</strong>ges in escape neural networks underlie<br />
<strong>the</strong> emergence of newly org<strong>an</strong>ized sensory fields as body segments ch<strong>an</strong>ge positional identity in<br />
response to injury. Sensory fields within L. variegatus are mediated by two gi<strong>an</strong>t fiber systems,<br />
<strong>the</strong> medial (MGF), which receives sensory inputs <strong>from</strong> <strong>the</strong> first third of <strong>the</strong> body segments, <strong>an</strong>d<br />
two lateral (LGF), which are activated by sensory inputs <strong>from</strong> <strong>the</strong> posterior two-thirds of <strong>the</strong><br />
body. During neural morphallaxis induced by tr<strong>an</strong>section of <strong>the</strong> body, posterior segments<br />
possessing only LGF sensory fields tr<strong>an</strong>s<strong>for</strong>m such that sensory inputs onto <strong>the</strong> MGF pathway<br />
emerge as new head segments are regenerated.<br />
The tr<strong>an</strong>s<strong>for</strong>mation of gi<strong>an</strong>t fiber sensory fields is incredibly rapid, suggesting that plasticity<br />
within <strong>the</strong>se neural circuits involves <strong>the</strong> unsilencing of nonfunctional synapses ra<strong>the</strong>r th<strong>an</strong><br />
synapse <strong>for</strong>mation. We hypo<strong>the</strong>size that <strong>the</strong>se silent synapses become unsilenced during <strong>an</strong><br />
activity-dependent plasticity similar to that involved in long term potentiation (LTP) reported <strong>for</strong>
o<strong>the</strong>r systems. Since some classes of sensory neurons in <strong>an</strong>nelids are glutamatergic, we have<br />
investigated whe<strong>the</strong>r ch<strong>an</strong>ges in glutamatergic tr<strong>an</strong>smission underlie sensory-to-interneuronal<br />
plasticity.<br />
Intact worms were immobilized pharmacologically with nicotine <strong>an</strong>d dissected to expose <strong>the</strong><br />
ventral nerve cord (VNC). A suction electrode placed adjacent to <strong>the</strong> setal bundle was used to<br />
activate sensory inputs while EPSPs were recorded in <strong>the</strong> MGF axon. Stimulation of sensory<br />
neurons produces a multicomponent EPSP. An initial depolarizing component was<br />
approximately 10mV. A second EPSP component was activated at higher voltages, resulting in a<br />
second larger wave<strong>for</strong>m of approximately 20mV. We hypo<strong>the</strong>size that one or both of <strong>the</strong>se EPSP<br />
components were glutamatergic. There<strong>for</strong>e, preparations were ba<strong>the</strong>d in a competitive AMPA<br />
receptor <strong>an</strong>tagonist, CNQX, <strong>an</strong>d <strong>the</strong> sensory inputs were activated. The application of CNQX (1<br />
<strong>an</strong>d 25µM) completely abolished stimulus-evoked, multicomponent EPSPs. Thus, at least <strong>the</strong><br />
initial depolarization of <strong>the</strong> MGF by sensory inputs is due to glutamate release. This appears<br />
similar to mech<strong>an</strong>isms of LTP, in that upon <strong>the</strong> activation of AMPARs, <strong>the</strong>re is sufficient<br />
depolarization of <strong>the</strong> postsynaptic cell to activate o<strong>the</strong>r voltage-dependent glutamate receptors.<br />
Ongoing studies are examining whe<strong>the</strong>r or not NMDAR <strong>an</strong>tagonists might also escape neural<br />
circuits <strong>an</strong>d plasticity associated with neural morphallaxis.<br />
Disclosures: Z.R. Lybr<strong>an</strong>d, None; M.J. Zor<strong>an</strong>, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.7/G1<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Title: Secretory vesicle-related gene CAPS2 knock-out mice exhibit <strong>the</strong> reduced number of<br />
hippocampal GABAergic interneuron <strong>an</strong>d <strong>the</strong> impairments in synaptic plasticity <strong>an</strong>d behavior<br />
Authors: *Y. SHINODA 1,2 , E. KINAMERI 1 , A. FURUYA 1 , T. SADAKATA 1,2 , T.<br />
FURUICHI 1,2 ;<br />
1 RIKEN BSI, Wako, Jap<strong>an</strong>; 2 JST/CREST, Kawaguchi, Jap<strong>an</strong><br />
Abstract: Calcium-dependent activator protein <strong>for</strong> secretion 2 (CAPS2) is a secretory vesicle<br />
associated protein <strong>an</strong>d is suggested to be associated with autism susceptibility. In this study, we<br />
<strong>an</strong>alyzed decreased number of interneuron <strong>an</strong>d its effect on synaptic plasticity <strong>an</strong>d behavioral<br />
phenotypes in KO mice. Theta-burst induced long-term potentiation (LTP) at CA3-CA1<br />
synapses was signific<strong>an</strong>tly reduced in KO mice. However, <strong>the</strong> reduction was recovered to <strong>the</strong>
control levels in <strong>the</strong> presence of picrotoxin. These results suggest that impaired GABAergic<br />
circuits might cause <strong>the</strong> reduction of <strong>the</strong>ta-burst induced LTP in KO mice. In spite of <strong>the</strong> LTP<br />
impairment, <strong>the</strong>re were no signific<strong>an</strong>t abnormalities in KO mice in hippocampus-dependent<br />
memory. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, KO mice showed <strong>an</strong>xiety-like behaviors in most of <strong>the</strong> tests. In<br />
conclusion, our study suggests that <strong>the</strong> impairment in GABAergic circuits of KO mice causes <strong>the</strong><br />
reduced hippocampal LTP <strong>an</strong>d may be associated with <strong>the</strong>ir <strong>an</strong>xiety-like behaviors.<br />
Disclosures: Y. Shinoda, None; E. Kinameri, None; A. Furuya, None; T. Sadakata, None; T.<br />
Furuichi, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.8/G2<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: NIH Gr<strong>an</strong>t MH60608<br />
HHMI Gr<strong>an</strong>t 52005120<br />
Title: Effects of <strong>the</strong> interlesion interval on <strong>the</strong> synaptic efficacy of <strong>the</strong> crossed temporodentate<br />
pathway after progressive entorhinal cortex lesions in rats<br />
Authors: M. ROBINSON, J. J. TAYLOR, P. J. SANCHEZ, S. RHODES, K. C. LANG, C.<br />
CRON, A. HERZOG, M. CUSHMAN, *J. J. RAMIREZ;<br />
Dept Psychol, Neurosci Prog, Davidson Col., Davidson, NC<br />
Abstract: The entorhinal cortex (EC) serves as a major source of input to <strong>the</strong> dentate gyrus (DG)<br />
of <strong>the</strong> hippocampal <strong>for</strong>mation in rats. Following a unilateral one-stage lesion of <strong>the</strong> entorhinal<br />
area, rats lose <strong>the</strong>ir ability to per<strong>for</strong>m a spatial working memory task, but <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce<br />
improves between 8 <strong>an</strong>d 12 days postlesion. This recovery period corresponds to <strong>the</strong> time at<br />
which <strong>the</strong> crossed temporodentate pathway (CTD), which originates in <strong>the</strong> intact contralateral<br />
EC, proliferates <strong>an</strong>d <strong>for</strong>ms new connections with <strong>the</strong> denervated DG. We have previously<br />
reported that per<strong>for</strong>ming <strong>the</strong> EC lesion in two stages (i.e. a progressive lesion) separated by a 6<br />
day interlesion interval (ILI) accelerates axonal sprouting, enh<strong>an</strong>ces synaptic efficacy of <strong>the</strong><br />
CTD, <strong>an</strong>d spares spatial memory in rats. The length of <strong>the</strong> ILI has previously been shown to<br />
affect <strong>the</strong> sprouting response of <strong>the</strong> commissural/associational fiber plexus after progressive EC<br />
lesions, with maximal exp<strong>an</strong>sion of <strong>the</strong> plexus being elicited with ILIs of 4 to 13 days. The
purpose of this experiment was to determine whe<strong>the</strong>r a 12-day ILI would enh<strong>an</strong>ce <strong>the</strong> synaptic<br />
efficacy of <strong>the</strong> CTD more effectively th<strong>an</strong> a 6-day ILI. Here, we explored <strong>the</strong> extracellular field<br />
excitatory postsynaptic potentials of <strong>the</strong> DG resulting <strong>from</strong> CTD stimulation as well as <strong>the</strong><br />
capacity of <strong>the</strong> CTD to support long-term potentiation (LTP). The rats were given a 15-day<br />
survival period after progressive or one-stage lesions. The DG responses to CTD stimulation<br />
were recorded be<strong>for</strong>e <strong>an</strong>d after a high-frequency tet<strong>an</strong>y protocol (8 trains of 8 pulses at 400Hz)<br />
typically used to induce LTP in this model preparation. The amplitude <strong>an</strong>d slope of <strong>the</strong> evoked<br />
responses <strong>for</strong> <strong>the</strong> 12-day ILI progressive group were signific<strong>an</strong>tly greater th<strong>an</strong> <strong>the</strong> control group;<br />
no o<strong>the</strong>r intergroup differences were found. LTP was not consistently observed in <strong>an</strong>y of <strong>the</strong><br />
groups we studied, though it was occasionally seen in <strong>the</strong> lesion groups. Thus, extending <strong>the</strong> ILI<br />
<strong>from</strong> 6 to 12 days enh<strong>an</strong>ced <strong>the</strong> evoked responses in <strong>the</strong> DG produced by CTD stimulation, but<br />
not <strong>the</strong> CTD’s capacity to support LTP.<br />
Disclosures: M. Robinson, None; J.J. Taylor, None; P.J. S<strong>an</strong>chez, None; S. Rhodes,<br />
None; K.C. L<strong>an</strong>g, None; C. Cron, None; A. Herzog, None; M. Cushm<strong>an</strong>, None; J.J. Ramirez,<br />
None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.9/G3<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: NIA Sleep Gr<strong>an</strong>t SP01AG017628-07<br />
Title: Blocking glia-derived ATP release prevents sleep loss-induced hippocampal synaptic<br />
plasticity deficit <strong>an</strong>d memory impairment in mice<br />
Authors: *C. FLORIAN 1 , C. G. VECSEY 1 , M. M. HALASSA 2 , P. G. HAYDON 2 , T. ABEL 1 ;<br />
1 2<br />
Dept. of Biol., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA; Dept. of Neurosci., Tufts Univ.,<br />
Boston, MA<br />
Abstract: Sleep disturb<strong>an</strong>ces contribute to cognitive deficits, suggesting that sleep is critical <strong>for</strong><br />
memory storage <strong>an</strong>d consolidation (Stickgold, 2005). However, <strong>the</strong> mech<strong>an</strong>isms by which sleep<br />
modulates memory remain unknown. Using a molecular genetic approach to disrupt SNAREdependent<br />
exocytosis specifically in astrocytes (dnSNARE mice), Pascual <strong>an</strong>d collaborators<br />
(2005) found that blocking gliotr<strong>an</strong>smission prevented <strong>the</strong> extracellular accumulation of<br />
adenosine, which is <strong>for</strong>med <strong>from</strong> ATP released by astrocytes. In a recent study of memory using
<strong>the</strong> novel object recognition task, dnSNARE mice were protected against <strong>the</strong> memory-degrading<br />
effects of sleep loss. The same protection against sleep deprivation was observed when wild-type<br />
mice were treated with <strong>the</strong> adenosine A1 receptor <strong>an</strong>tagonist 8-cyclopentyl-1,3-dimethylx<strong>an</strong>thine<br />
(CPT) (Halassa et al., <strong>2009</strong>). These findings suggest that astrocyte-derived adenosine may be a<br />
c<strong>an</strong>didate molecule responsible <strong>for</strong> cognitive deficits following sleep disturb<strong>an</strong>ces. The present<br />
study aimed to extend <strong>the</strong>se findings by specifically probing <strong>the</strong> effects of sleep deprivation on<br />
hippocampal function in dnSNARE or CPT-treated mice. In a first approach, we examined<br />
whe<strong>the</strong>r blockade of gliotr<strong>an</strong>smission or blockade of A1 receptor signaling could prevent <strong>the</strong><br />
effects of 6 hours of sleep deprivation on <strong>the</strong> hippocampal late phase of long-term potentiation<br />
(L-LTP), a <strong>for</strong>m of synaptic plasticity that is a cellular model of memory storage. We found that<br />
sleep deprivation impaired <strong>the</strong> spaced 4-train L-LTP in wild-type mice but not in <strong>the</strong> dnSNARE<br />
mice. Similarly this deficit in L-LTP resulting <strong>from</strong> sleep loss was rescued by <strong>the</strong> treatment of<br />
wild-type mice with chronic infusion of CPT. Because different classes of memory rely on<br />
different brain regions (novel object recognition depends on extra-hippocampal regions, whereas<br />
spatial memory is highly dependent on <strong>the</strong> hippocampus proper, e.g.), in a second approach we<br />
investigated whe<strong>the</strong>r memory in a spatial version of <strong>the</strong> object recognition task is also affected<br />
by sleep deprivation. We found that hippocampus-dependent memory deficits mediated by sleep<br />
deprivation were rescued in dnSNARE mice <strong>an</strong>d CPT-treated mice. Toge<strong>the</strong>r, <strong>the</strong>se data provide<br />
<strong>the</strong> first evidence that glia-released ATP is responsible <strong>for</strong> <strong>the</strong> effects of sleep loss on<br />
hippocampal synaptic plasticity <strong>an</strong>d hippocampus-dependent memory. This study suggests a new<br />
<strong>the</strong>rapeutic target to reverse <strong>the</strong> cognitive dysfunction induced by sleep disturb<strong>an</strong>ces.<br />
Disclosures: C. Flori<strong>an</strong>, None; C.G. Vecsey, None; M.M. Halassa, None; P.G. Haydon,<br />
None; T. Abel, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.10/G4<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: DFG STR 699/1-2<br />
DFG 436 POL 17/3/05<br />
DFG GZ 46RUS<br />
EMBO WPF2004/18
Title: Hippocampal LTP in stressed mice with <strong>an</strong>d without hedonic deficit: Effects of chronic<br />
treatment with citalopram<br />
Authors: K. TOKARSKI 1,2 , *A. DRAGUHN 1 , N. GORENKOVA 3,4 , O. DOLGOV 3,5 , E.<br />
SCHUNK 3,6 , M. KUNCHULIA 1,7 , H. W. STEINBUSCH 8 , T. STREKALOVA 3,8 ;<br />
1 Inst. Physiol & Pathophysiol, Unvi Heidelberg, 69120 Heidelberg, Germ<strong>an</strong>y; 2 Dept. of Physiol.,<br />
Inst. of Pharmacology, PAS, Kraków, Pol<strong>an</strong>d; 3 Central Inst. of Mental Hlth., M<strong>an</strong>nheim,<br />
Germ<strong>an</strong>y; 4 Inst. of Psychiatry, Ctr. <strong>for</strong> <strong>the</strong> Cell. Basis of Behaviour, King’s Col. London,<br />
London, United Kingdom; 5 Anokhin Inst. of Normal Physiology, RAS, Moscow, Russi<strong>an</strong><br />
Federation; 6 Dept. of Pharmacol., Innsbruck Med. Univ., Innsbruck, Austria; 7 I. Beritashvili Inst.<br />
of Physiol., Tbilisi, Georgia; 8 Sch. <strong>for</strong> Mental Hlth. <strong>an</strong>d Neurosci., Maastricht Univ., Maastricht,<br />
Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Depression is a mental illness, <strong>the</strong> core feature of which is <strong>an</strong>hedonia, a decreased<br />
ability to experience pleasures. Cognitive deficit is <strong>an</strong>o<strong>the</strong>r import<strong>an</strong>t characteristic of<br />
depression. Even though <strong>the</strong> biological basis of depression remains unclear, <strong>the</strong> available data<br />
point to <strong>an</strong> import<strong>an</strong>ce of <strong>the</strong> hippocampus in a pathogenesis of <strong>the</strong>se <strong>an</strong>d o<strong>the</strong>r depressive<br />
symptoms. Stress is a major pathogenetic factor of depression <strong>an</strong>d <strong>an</strong>hedonia, which was shown<br />
to affect <strong>the</strong> hippocampal <strong>for</strong>mation during depression, but also with o<strong>the</strong>r pathologies. So far<br />
employed experimental approaches did not allow <strong>for</strong> a selective <strong>an</strong>alysis of <strong>the</strong> relationship<br />
between hipocampal functions, experience of stress <strong>an</strong>d occurence of depressive symptoms. The<br />
aim of our study was to investigate <strong>the</strong> hippocampal plasticity in a mouse model of stressinduced<br />
<strong>an</strong>hedonia with internal control <strong>for</strong> chronic stress, in which tet<strong>an</strong>ically induced LTP in<br />
<strong>the</strong> CA1 area of <strong>the</strong> hippocampus was tested ex vivo in <strong>the</strong> hippocampal slices, <strong>an</strong>d relay <strong>the</strong>se<br />
data to parameters of sucrose test, a putative test <strong>for</strong> a hedonic state in rodents. In employed<br />
chronic stress model, <strong>an</strong>hedonic state is defined by a decrease in sucrose preference over water<br />
below 65%. After 4-week stress, approximately 60% of C57BL/6N mice are classified as<br />
<strong>an</strong>hedonic; <strong>the</strong> remaining <strong>an</strong>imals do not show a hedonic deficit <strong>an</strong>d o<strong>the</strong>r depressive-like<br />
behaviors <strong>an</strong>d used as a control <strong>for</strong> <strong>the</strong> effects of stress, which are not associated with <strong>an</strong>hedonia.<br />
Anhedonic mice demonstrated disrupted LTP in <strong>the</strong> CA1 area of <strong>the</strong> hippocampus, while no such<br />
ch<strong>an</strong>ges were observed in <strong>the</strong> non-<strong>an</strong>hedonic <strong>an</strong>imals. Citalopram, delivered 1 week be<strong>for</strong>e <strong>an</strong>d<br />
in a course of a 4-week stress procedure, reduced percentage of <strong>an</strong>hedonic <strong>an</strong>imals <strong>an</strong>d prevented<br />
<strong>an</strong> impairment in <strong>the</strong> hippocampal LTP. Thus, our data evidence disrupted hippocampal<br />
plasticity being specifically associated with a state of hedonic deficit, but not stress alone, in a<br />
mouse model of stress-induced <strong>an</strong>hedonia with inetrenal control <strong>for</strong> stress. Presented results<br />
fur<strong>the</strong>r define a relationship between hippocampal LTP, parameters of <strong>the</strong> sucrose test, <strong>an</strong>d<br />
effects of chronic <strong>an</strong>tidepress<strong>an</strong>t treatment in chronically stressed <strong>an</strong>imals with <strong>an</strong>d without<br />
hedonic deficit.<br />
Disclosures: K. Tokarski, None; A. Draguhn, None; N. Gorenkova, None; O. Dolgov,<br />
None; E. Schunk, None; M. Kunchulia, None; H.W. Steinbusch, None; T. Strekalova, None.<br />
Poster
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.11/G5<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: CIHR<br />
NSERC<br />
CFI<br />
Title: Electrophyisiological ch<strong>an</strong>ges in <strong>the</strong> ventral subicular-nucleus accumbens pathway<br />
following heroin self-administration <strong>an</strong>d acute food deprivation-induced reinstatement<br />
Authors: *S. TOBIN, S. D. GLASGOW, C. A. CHAPMAN, U. SHALEV;<br />
Concordia Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Long-term chronic exposure to opiates leads to dependency, a condition believed to<br />
involve maladaptive ch<strong>an</strong>ges in neuronal pathways. In particular, drugs of abuse have been<br />
shown to exert a profound effect on <strong>the</strong> mesolimbic dopamine pathway, including <strong>the</strong> nucleus<br />
accumbens (NAc) <strong>an</strong>d ventral tegmental area. Moreover, in vivo studies have demonstrated a<br />
signific<strong>an</strong>t reduction in LTP in <strong>the</strong> hippocampal CA1 region following opiate withdrawal. The<br />
ventral subiculum (vSub) is a primary output region <strong>for</strong> <strong>the</strong> hippocampal <strong>for</strong>mation <strong>an</strong>d has<br />
excitatory glutamatergic projections to <strong>the</strong> NAc. This pathway has been implicated in morphine<br />
withdrawal but has not been investigated with regard to heroin self-administration (SA) or acute<br />
food deprivation (FD)-induced reinstatement. Here, male Long Ev<strong>an</strong>s rats were impl<strong>an</strong>ted with a<br />
stimulating electrode into <strong>the</strong> vSub <strong>an</strong>d a recording electrode into <strong>the</strong> NAc <strong>an</strong>d trained to selfadminister<br />
heroin (.05 mg/kg/inf) <strong>for</strong> 13 days. Following training, rats received a minimum 4<br />
days of extinction at which time heroin was unavailable. Under extinction conditions rats were<br />
subjected to 48h FD <strong>an</strong>d one 2h reinstatement test. Evoked excitatory postsynaptic potentials<br />
(EPSPs) were recorded immediately following SA training, extinction, <strong>an</strong>d reinstatement,<br />
sessions. Moreover, following reinstatement, LTP was induced using high frequency stimulation<br />
(200 Hz). No differences were observed, relative to a drug-naïve control group, over <strong>the</strong> course<br />
of SA training or extinction. However, LTP demonstrated a signific<strong>an</strong>t effect of time <strong>an</strong>d a<br />
signific<strong>an</strong>t time X group interaction, with heroin trained rats showing a greater potentiation of<br />
EPSPs th<strong>an</strong> FD non-heroin trained <strong>an</strong>imals. Thus, acute FD may alter <strong>the</strong> propensity <strong>for</strong> <strong>the</strong><br />
induction of synaptic plasticity in <strong>the</strong> vSub-NAc pathway in a m<strong>an</strong>ner that is dependent upon<br />
prior experience with heroin.<br />
Disclosures: S. Tobin, None; S.D. Glasgow, None; C.A. Chapm<strong>an</strong>, None; U. Shalev, None.
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.12/G6<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: DFG FR 1034-7-1<br />
DFG SFB 779 B4<br />
Title: Electrical stimulation of <strong>the</strong> ventral tegmental area <strong>an</strong>d its influence on synaptic plasticity<br />
in hippocampal CA1 of freely moving rats<br />
Authors: S. FREY 1 , *J. FREY 2 ;<br />
1 Dept Neurophysiol., 2 Leibniz Inst. Neurobiol, Magdeburg, Germ<strong>an</strong>y<br />
Abstract: The prolonged mainten<strong>an</strong>ce of hippocampal long-term potentiation (LTP), i.e. <strong>the</strong><br />
protein syn<strong>the</strong>sis-dependent phase beyond 4-6 h (late-LTP) requires heterosynaptic events during<br />
LTP induction. We had previously shown in hippocampal slices in vitro, that late-LTP requires<br />
dopaminergic D1/D5-receptor activation in <strong>an</strong> associative, synergistic interaction with NMDAreceptor<br />
activation within a distinct time window (Frey at al. 1990, 1991, 1993; Frey <strong>an</strong>d Morris,<br />
1998). Our recent studies in <strong>the</strong> intact, freely behaving rat confirmed <strong>the</strong>se results: early-LTP,<br />
i.e. <strong>the</strong> protein syn<strong>the</strong>sis-independent tr<strong>an</strong>sient phase (with a duration of less th<strong>an</strong> 4-6 h) in <strong>the</strong><br />
CA1 region could be rein<strong>for</strong>ced into late-LTP by high-frequency stimulation of <strong>the</strong> ventral<br />
tegmental area (VTA) 15 min after LTP induction in <strong>the</strong> CA1 region of freely moving rats. The<br />
VTA, a modulatory input to <strong>the</strong> CA1 region is a heterogeneous group of dopaminergic cells <strong>an</strong>d<br />
a major component of <strong>the</strong> mesolimbic dopamine system. This rein<strong>for</strong>cement of early-LTP in<br />
CA1 was dependent on dopaminergic receptor activation <strong>an</strong>d on protein syn<strong>the</strong>sis <strong>an</strong>d followes<br />
rules of 'synaptic tagging' (Frey <strong>an</strong>d Morris, 1998).<br />
Now, we have extended our studies <strong>an</strong>d could show that high-frequency stimulation of <strong>the</strong> VTA<br />
alone, i.e. without LTP-induction in CA1, caused a delayed-onset potentiation <strong>for</strong> <strong>the</strong> recorded<br />
field-EPSP <strong>an</strong>d population spike (POP) in <strong>the</strong> CA1 region in response to test stimulation of <strong>the</strong><br />
contralateral CA3. This delayed-onset potentiation was dependent on <strong>the</strong> synergistic activation of<br />
both <strong>the</strong> glutamatergic <strong>an</strong>d <strong>the</strong> dopaminergic receptor activation, because paused glutamatergic<br />
test stimulation abolished this potentiation. These results again support our findings in<br />
hippocampal slices in vitro (Navakkode et al. 2007).<br />
For all experiments in vivo we used our recently implemented method of simult<strong>an</strong>eous recording<br />
of both, <strong>the</strong> field-EPSP <strong>an</strong>d <strong>the</strong> POP in <strong>the</strong> hippocampal CA1 in freely moving rats by
stimulation of <strong>the</strong> contralateral CA3.<br />
Acknowledgements: This work was supported by gr<strong>an</strong>ts of <strong>the</strong> DFG to JUF <strong>an</strong>d SF SFB 779 TP<br />
B4 <strong>an</strong>d FR1034-7-1 to JUF.<br />
References: Frey et al., Brain Res. 522 (1990), Frey et al. Neurosci. Lett. 129 (1991), Frey et al.<br />
Science 260 (1993), Frey & Morris TINS 21(1998), Navakkode et al. Neuropharmacol. 52<br />
(2007).<br />
Disclosures: S. Frey, None; J. Frey, None.<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.13/G7<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: MIUR<br />
Compagnia S<strong>an</strong> Paolo<br />
Regione Piemonte<br />
Title: Basolateral amygdala inactivation impairs learning-induced long-term potentiation in <strong>the</strong><br />
cerebellar cortex<br />
Authors: L. ZHU 1 , T. SACCO 1 , P. STRATA 1,2 , *B. SACCHETTI 1 ;<br />
1 Univ. Turin, Turin, Italy; 2 EBRI-S<strong>an</strong>ta Lucia Fndn. (IRCCS), Rome, Italy<br />
Abstract: Learning to fear d<strong>an</strong>gerous situations requires <strong>the</strong> participation of basolateral<br />
amygdala (BLA). In <strong>the</strong> present study, we provide evidence that BLA is import<strong>an</strong>t <strong>for</strong> <strong>the</strong><br />
synaptic streng<strong>the</strong>ning underlying in vivo memory <strong>for</strong>mation in <strong>the</strong> cerebellum. In <strong>the</strong> cerebellar<br />
vermis <strong>the</strong> parallel fibers (PF) to Purkinje cells (PC) synapse is potentiated one day following<br />
fear learning. Pretraining BLA inactivation abolished such a learning-induced LTP. Similarly,<br />
cerebellar LTP is impaired when BLA is blocked shortly, but not 6 h, after training. The last<br />
result shows that <strong>the</strong> effects of BLA inactivation on cerebellar plasticity, when present, are<br />
specifically related to memory processes <strong>an</strong>d not due to <strong>an</strong> interference with sensory or motor<br />
functions. These data suggest that, while <strong>the</strong> electrically-induced LTP is commonly obtained by<br />
repetitive stimulation of a specific synaptic pathway, learning-induced plasticity is a<br />
heterosynaptic phenomenon that requires inputs <strong>from</strong> o<strong>the</strong>r regions. Studies employing <strong>the</strong>
electrically-induced LTP in order to clarify <strong>the</strong> cellular mech<strong>an</strong>isms of memory should <strong>the</strong>re<strong>for</strong>e<br />
take into account <strong>the</strong> inputs arriving <strong>from</strong> o<strong>the</strong>r brain sites, considering <strong>the</strong>m as integrative units.<br />
In addition, our results indicate that BLA regulates activity-dependent processes occurring in<br />
cerebellar cortex during memory processes. Based on previous <strong>an</strong>d <strong>the</strong> present findings, we<br />
proposed that BLA evaluates <strong>the</strong> emotional content of sensory stimuli <strong>an</strong>d thus enables learningrelated<br />
plasticity to be <strong>for</strong>med in <strong>the</strong> cerebellum in order to respond appropriately to new stimuli<br />
or situations.<br />
Disclosures: L. Zhu, Compagnia di S<strong>an</strong> Paolo, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); T. Sacco,<br />
Regione Piemonte, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); P. Strata, MIUR, Regione Piemonte,<br />
Compagnia S<strong>an</strong> Paolo, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); B. Sacchetti, MIUR, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received).<br />
Poster<br />
524. LTP: Physiology <strong>an</strong>d Behavior I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 524.14/G8<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: Trinity College Faculty Research Gr<strong>an</strong>t<br />
Title: Role of early life stress in corticolimbic plasticity<br />
Authors: *J. H. BLAISE 1 , R. M. CLARK 2 , K. E. HAINES 3 ;<br />
1 Engin., 2 Interdisciplinary Sci. Program, 3 Biol., Trinity Col., Hart<strong>for</strong>d, CT<br />
Abstract: Numerous studies suggest that adverse experiences early in life induce neurochemical,<br />
morphological, <strong>an</strong>d functional ch<strong>an</strong>ges in <strong>the</strong> mammali<strong>an</strong> brain--ch<strong>an</strong>ges that endure into<br />
adulthood <strong>an</strong>d are closely linked with <strong>an</strong> increased risk of stress-related psychiatric disorders,<br />
such as major depressive disorder (MDD) <strong>an</strong>d post-traumatic stress disorder (PTSD). Neonatal<br />
isolation is a chronic early life stressor, shown to trigger a response of <strong>the</strong> hypothalamicpituitary-adrenocortical<br />
(HPA) axis which causes elevated levels of glucocorticoids within <strong>the</strong><br />
brain. The medial prefrontal cortex (mPFC) <strong>an</strong>d <strong>the</strong> basolateral amygdala (BLA) toge<strong>the</strong>r <strong>for</strong>m a<br />
corticolimbic system which is involved in mediating emotional arousal <strong>an</strong>d stress response
effects on cognitive functions, particularly executive function, fear extinction, <strong>an</strong>d working<br />
memory. A control group of Sprague-Dawley rat pups were subjected to neonatal isolation (ISO)<br />
<strong>for</strong> <strong>an</strong> hour a day <strong>from</strong> postnatal days PN2-9. Ano<strong>the</strong>r group of similar age <strong>an</strong>d species which<br />
were not h<strong>an</strong>dled (NH) served as controls. To assess <strong>the</strong> effects of neonatal isolation on synaptic<br />
plasticity of <strong>the</strong> mPFC-BLA synapse, ch<strong>an</strong>ges in long-term potentiation (LTP, a crucial<br />
component of synaptic plasticity believed to be <strong>the</strong> mech<strong>an</strong>ism <strong>for</strong> learning <strong>an</strong>d memory in <strong>the</strong><br />
brain) were measured <strong>an</strong>d compared between <strong>the</strong> two groups. Preliminary results indicate that<br />
LTP is reduced in ISO <strong>an</strong>imals compared to NH controls. Although preliminary, <strong>the</strong>se results<br />
suggest a reduction in mPFC output to BLA neurons which is likely caused by stress-induced<br />
decreases in mPFC dendritic arborization <strong>an</strong>d spine density_both of which are reflected in <strong>the</strong><br />
reduction of LTP in ISO rats compared to controls. Fur<strong>the</strong>r data collection is necessary to<br />
determine <strong>the</strong> exact mech<strong>an</strong>isms underlying <strong>the</strong>se effects induced by early life stress in this<br />
corticolimbic circuit.<br />
Disclosures: J.H. Blaise, None; R.M. Clark, None; K.E. Haines, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.1/G9<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Title: Sensitivity to higher-order statistical properties of excitation <strong>an</strong>d inhibition in rat visual<br />
cortical neurons<br />
Authors: *M. P. SCENIAK 1 , S. SABO 2 ;<br />
1 2<br />
Case Western Reserve, University Heights, OH; Pharmacol., Case Western Reserve Univ.,<br />
Clevel<strong>an</strong>d, OH<br />
Abstract: The gain of neuronal spiking responses in excitatory pyramidal neurons c<strong>an</strong> be<br />
modulated by varying <strong>the</strong> level of ‘background’ synaptic input (Ch<strong>an</strong>ce, F. et al., 2002). The<br />
dependence of response gain on <strong>the</strong> statistical properties of <strong>the</strong> excitatory to inhibitory bal<strong>an</strong>ce<br />
(EI) in cortical pyramidal neurons is not known. To investigate this, we simulated synaptic<br />
background inputs in vitro using a stochastic Orenstein-Uhlenbeck (OU) model (Fellous, J.M.,<br />
2003). Excitatory glutamatergic <strong>an</strong>d inhibitory GABAergic post-synaptic conduct<strong>an</strong>ces (PSCs)<br />
were simulated as two OU processes <strong>an</strong>d imposed on <strong>the</strong> cell through <strong>the</strong> real-time dynamicclamp<br />
system (RTXI). Fellous <strong>an</strong>d colleagues (2003) have shown that <strong>the</strong> OU model c<strong>an</strong> be used<br />
to independently define <strong>the</strong> me<strong>an</strong> <strong>an</strong>d st<strong>an</strong>dard deviation of <strong>the</strong> excitatory <strong>an</strong>d inhibitory PSCs.
We investigated <strong>the</strong> spiking response properties of neurons in vitro in rat (P14-P21) primary<br />
visual cortical brain slices. Current-clamp membr<strong>an</strong>e potential recordings were made <strong>from</strong><br />
regular spiking pyramidal excitatory neurons. We found that <strong>the</strong>re is a signific<strong>an</strong>t modulation of<br />
response gain by <strong>the</strong> statistical dependence of excitation <strong>an</strong>d inhibition in <strong>the</strong> background<br />
synaptic input. The statistical dependence of excitation <strong>an</strong>d inhibition were determined by<br />
varying ei<strong>the</strong>r <strong>the</strong> covari<strong>an</strong>ce of excitatory <strong>an</strong>d inhibitory stochastic processes or by defining a<br />
delay between <strong>the</strong> two processes <strong>for</strong> a given covari<strong>an</strong>ce. The dependence of neuronal firing on<br />
<strong>the</strong> higher-order statistics of <strong>the</strong> EI bal<strong>an</strong>ce, suggests that neurons are capable of encoding<br />
separate inhibitory pathways onto excitatory pyramidal neurons.<br />
Disclosures: M.P. Sceniak, None; S. Sabo, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.2/G10<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: McKnight Foundation<br />
Title: Emergence of intrinsic gain rescaling in <strong>the</strong> mammali<strong>an</strong> neocortex<br />
Authors: R. MEASE, *A. L. FAIRHALL, W. MOODY;<br />
Dept Physiol & Biophys, Univ. Washington, Seattle, WA<br />
Abstract: Gain rescaling is a fundamental <strong>for</strong>m of adaptation that allows a neuron or neural<br />
system to maximize in<strong>for</strong>mation encoding efficiency by ch<strong>an</strong>ging <strong>the</strong> mapping between inputs<br />
<strong>an</strong>d outputs to better sp<strong>an</strong> <strong>the</strong> r<strong>an</strong>ge of incoming environmental stimuli (Wark et al., 2007). Here,<br />
we examine intrinsic gain rescaling in synaptically-isolated single neurons in <strong>the</strong> developing<br />
mouse sensorimotor cortex during two developmental periods: post-natal day 0 (P0, <strong>the</strong> day of<br />
birth) <strong>an</strong>d post-natal day 7 (P7). We constructed a Linear-Nonlinear (LN) model <strong>for</strong> each neuron<br />
using in vitro whole-cell stimulation with gaussi<strong>an</strong> noise current scaled by a r<strong>an</strong>ge of amplitudes.<br />
By comparing <strong>the</strong> sampled nonlinear firing threshold functions, we qu<strong>an</strong>tified ch<strong>an</strong>ges in single<br />
neuron encoding <strong>for</strong> different stimulus distributions <strong>an</strong>d development stages.<br />
We found that gain rescaling ability is limited in spont<strong>an</strong>eously active P0 neurons but emerges<br />
with more mature firing properties by P7. Regardless of gain, <strong>the</strong> threshold shape of P7 neurons<br />
was surprisingly consistent across <strong>the</strong> population <strong>an</strong>d unaffected by tetrodotoxin block of<br />
spont<strong>an</strong>eous activity during earlier developmental stages. Fu<strong>the</strong>rmore, both development of gain
escaling <strong>an</strong>d convergence to a common population threshold occur as neurons develop a stable<br />
ratio of voltage-gated sodium <strong>an</strong>d potassium conduct<strong>an</strong>ces (G_Na/G_K). Increasing subthreshold<br />
G_Na/G_K by application of 4-aminopyridine improved gain rescaling in single P7 neurons <strong>an</strong>d<br />
increased <strong>the</strong> similarity of thresholds between neurons, suggesting that both observations may<br />
stem <strong>from</strong> a common biophysical mech<strong>an</strong>ism. Using a mammali<strong>an</strong> version of <strong>the</strong> Hodgkin-<br />
Huxley model neuron (Mainen et al., 1995), we found that both gain rescaling <strong>an</strong>d <strong>the</strong> shape of<br />
<strong>the</strong> rescaled threshold are entirely predicted by G_Na/G_K ratio across a wide r<strong>an</strong>ge of<br />
parameter values <strong>an</strong>d stimulus gains.<br />
Our findings suggest that <strong>the</strong> interaction of basic voltage-gated ch<strong>an</strong>nels which give rise to<br />
membr<strong>an</strong>e excitability may provide a simple mech<strong>an</strong>ism <strong>for</strong> gain rescaling by single neurons,<br />
even in <strong>the</strong> absence of slow or spike-triggered adaptation currents. Within a developmental<br />
context, <strong>the</strong> emergence of gain rescaling may promote stable patterns of network excitability<br />
prior to <strong>the</strong> onset of sensory experience.<br />
Wark, B, Lundstrom BN, <strong>an</strong>d Fairhall, AL (2007). Sensory adaptation. Current Opinion in<br />
Neurobiology. 17 (4), 423-9.<br />
Mainen ZF, Joerges J, Huguenard JR <strong>an</strong>d Sejnowski TJ (1995). A model of spike initiation in<br />
neocortical pyramidal neurons. Neuron 15, 1427-1439.<br />
Disclosures: R. Mease, None; A.L. Fairhall, None; W. Moody, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.3/G11<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: CIHR<br />
AZRDM-Al<strong>an</strong> Edwards Centre <strong>for</strong> Research on Pain postdoctoral fellowship<br />
Title: Properties of persistent firing in <strong>an</strong>terior cingulate cortex<br />
Authors: *Z. ZHANG, P. SEGUELA;<br />
McGill Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Recent studies using lesions, electrophysiology <strong>an</strong>d functional imaging revealed that<br />
<strong>the</strong> <strong>an</strong>terior cingulate cortex (ACC) participates in pain modulation <strong>an</strong>d is involved in <strong>the</strong><br />
emotional aspect of pain. ACC receives a dense cholinergic innervation <strong>an</strong>d is associated with
aversive avoid<strong>an</strong>ce behavior <strong>an</strong>d pain-related learning <strong>an</strong>d memory. It has been shown that ACC<br />
infusion of oxotremorine, a muscarinic receptor agonist, enh<strong>an</strong>ced nociceptive memory<br />
retention, while <strong>the</strong> muscarinic <strong>an</strong>tagonist scopolamine disrupted <strong>the</strong> acquisition of nociceptive<br />
memory in rats (Ortega-Legaspi et al., 2003; Malin et al., 2007). Here we report, using wholecell<br />
patch clamp recording of layer II/III principal neurons of rat ACC in acute slices, a longlasting<br />
non-synaptic cholinergic plateau potential (PP) <strong>an</strong>d persistent firing (PF) induced by<br />
carbachol (10 µM). Both PP <strong>an</strong>d PF were abolished by <strong>the</strong> muscarinic <strong>an</strong>tagonist atropine (1 µM)<br />
<strong>an</strong>d by <strong>the</strong> blockade of G-protein activation with GDPβS. In <strong>the</strong> presence of TTX (1 µM),<br />
current clamp recordings demonstrated that PP <strong>an</strong>d PF are mediated by a decrease in membr<strong>an</strong>e<br />
input resist<strong>an</strong>ce. Application of Ba 2+ , which blocks m<strong>an</strong>y K + ch<strong>an</strong>nels, enh<strong>an</strong>ced carbacholinduced<br />
PP <strong>an</strong>d PF but did not evoke <strong>the</strong>m. Intra-pipette BAPTA (10 mM) or Ca 2+ -free<br />
extracellular ACSF (0 mM, EGTA 1 mM) abolished PP <strong>an</strong>d PF, showing that <strong>the</strong>y are Ca 2+ -<br />
dependent. Bath perfusion of <strong>the</strong> L-type Ca 2+ ch<strong>an</strong>nel blocker nifedipine (50 µM) blocked PP<br />
<strong>an</strong>d PF. Inhibiting non-selective cation ch<strong>an</strong>nels with FFA (100 µM), 2-APB (100 µM) or SKF<br />
96365 (100 µM) also suppressed PP <strong>an</strong>d PF. Voltage clamp recording revealed a CCh sensitive,<br />
cation non-selective current which was inhibited by SKF96365. Interestingly, <strong>the</strong> group I mGluR<br />
agonist DHPG (20 µM) also induced similar PP <strong>an</strong>d PF in ACC <strong>an</strong>d this effect was occluded by<br />
saturating amount of CCh. Altoge<strong>the</strong>r, our data demonstrate, in layer II/III principal neurons of<br />
ACC, Ca 2+ -dependent PP <strong>an</strong>d PF mediated by a non-selective cation conduct<strong>an</strong>ce. PP <strong>an</strong>d PF,<br />
evoked by activation of muscarinic receptors <strong>an</strong>d group I mGluRs via a common signaling<br />
pathway, may play a mnemonic role in ACC, allowing storage of in<strong>for</strong>mation during pain signal<br />
processing.<br />
Disclosures: Z. Zh<strong>an</strong>g, None; P. Seguela, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.4/G12<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: FRM postdoctoral fellowship<br />
Title: Single interneurons in <strong>the</strong> molecular layer of <strong>the</strong> cerebellar cortex tr<strong>an</strong>sition Purkinje cells<br />
between “up” <strong>an</strong>d "down" states<br />
Authors: *C. OLDFIELD 1 , A. MARTY 1,2 , B. STELL 1,2 ;<br />
1 Univ. Paris Descartes, Paris, Fr<strong>an</strong>ce; 2 CNRS, Paris, Fr<strong>an</strong>ce
Abstract: We demonstrate here <strong>for</strong> <strong>the</strong> first time that interneurons in <strong>the</strong> molecular layer of <strong>the</strong><br />
cerebellum (MLIs) c<strong>an</strong> tr<strong>an</strong>sition <strong>the</strong> state of <strong>the</strong> Purkinje cells <strong>from</strong> "up" to "down" as well as<br />
<strong>from</strong> "down" to "up". Consistent with previous studies we show that Purkinje cells in slices of rat<br />
(PN 13 - 16) cerebellar cortex are bistable, alternating between <strong>an</strong> “up” state in which <strong>the</strong> cell<br />
fires spont<strong>an</strong>eous action potentials <strong>an</strong>d a silent “down” state. The bistability occurs at room<br />
temperature as well as at elevated temperatures <strong>an</strong>d when <strong>the</strong> cell is recorded under whole-cell<br />
patch-clamp conditions as well as when <strong>the</strong> cell is left unperturbed <strong>an</strong>d action potentials are<br />
recorded in <strong>an</strong> extracellular loose cell-attached configuration. Tr<strong>an</strong>sitions between states c<strong>an</strong> be<br />
elicited when MLIs are activated 10 times at 50 Hz with extracellular stimulation as well as<br />
when a single interneuron is activated with a similar pattern during paired interneuron-Purkinje<br />
cell recordings. The MLI-elicited “up” to “down” tr<strong>an</strong>sitions are highly reliable <strong>an</strong>d are normally<br />
followed by a deep hyperpolarization of <strong>the</strong> resting membr<strong>an</strong>e potential, suggestive of <strong>an</strong><br />
increased K+ conduct<strong>an</strong>ce. The MLI-elicited “down” to “up” tr<strong>an</strong>sitions require <strong>the</strong> activation of<br />
<strong>the</strong> hyperpolarization-activated cation ch<strong>an</strong>nels (H ch<strong>an</strong>nels) <strong>an</strong>d are blocked by <strong>the</strong> H ch<strong>an</strong>nel<br />
<strong>an</strong>tagonist ZD7288 (10 µM). Application of small hyperpolarizing steps to <strong>the</strong> Purkinje cells (50<br />
pA <strong>for</strong> 200 ms) also tr<strong>an</strong>sition <strong>the</strong> cells between “down” <strong>an</strong>d “up” states <strong>an</strong>d ZD7288 blocks <strong>the</strong><br />
ability of <strong>the</strong> hyperpolarizing step to tr<strong>an</strong>sition <strong>from</strong> “down” to “up” but not <strong>from</strong> “up” to<br />
“down”. There<strong>for</strong>e, we conclude that because of <strong>the</strong> endogenous H ch<strong>an</strong>nels in Purkinje cells,<br />
single MLIs are enough to tr<strong>an</strong>sition Purkinje cells between both states.<br />
Disclosures: C. Oldfield, None; A. Marty, None; B. Stell, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.5/G13<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: CIHR<br />
CFI<br />
CRC<br />
Title: Mech<strong>an</strong>isms <strong>for</strong> different modes of sensory proccesing in <strong>the</strong> central neurons of weakly<br />
electric fish
Authors: *T. DEEMYAD, M. J. CHACRON;<br />
McGill Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Most sensory systems (e.g., auditory <strong>an</strong>d somatosensory) need to discriminate<br />
between behaviorally relev<strong>an</strong>t signals that c<strong>an</strong> vary enormously in <strong>the</strong>ir spatiotemporal extent.<br />
Electorosensory neurons of weakly electric fish use <strong>the</strong> spatial extent of different stimulus<br />
categories in order to switch <strong>the</strong>ir frequency tuning <strong>from</strong> low to high frequencies (Chacron et al.<br />
2003). The electrosensory lateral line lobe (ELL) is <strong>the</strong> first <strong>an</strong>d only terminal of <strong>the</strong> peripheral<br />
sensory afferents. In <strong>the</strong> present study we aimed at finding mech<strong>an</strong>isms involving ch<strong>an</strong>ges in <strong>the</strong><br />
intrinsic properties of ELL neurons as well as <strong>the</strong>ir synaptic inputs that regulate <strong>the</strong>ir frequency<br />
tuning. In particular, we studied <strong>the</strong> role of KCNQ ch<strong>an</strong>nels, serotonin, <strong>an</strong>d acetylcholine in<br />
controlling <strong>the</strong> encoding of signals. KCNQ potassium ch<strong>an</strong>nels underlie <strong>the</strong> M current that is<br />
activated at subthreshold voltages, leading to a decrease in <strong>the</strong> afterdepolarization. As such, <strong>the</strong>se<br />
ch<strong>an</strong>nels c<strong>an</strong> regulate <strong>the</strong> firing pattern of neurons <strong>from</strong> tonic to bursting in m<strong>an</strong>y central <strong>an</strong>d<br />
peripheral areas including sympa<strong>the</strong>tic, hippocampal pyramidal <strong>an</strong>d striatal neurons. There is<br />
also evidence that serotonin induces burst firing in m<strong>an</strong>y area in CNS <strong>an</strong>d capable of altering <strong>the</strong><br />
neural processing of sensory in<strong>for</strong>mation. We used sharp electrode intracellular recordings in<br />
slice preparations of ELL to study <strong>the</strong> contribution of KCNQ ch<strong>an</strong>nels towards regulating<br />
frequency tuning. We fur<strong>the</strong>r investigated <strong>the</strong> effect of blocking KCNQ ch<strong>an</strong>nels by XE-991, a<br />
potent KCNQ ch<strong>an</strong>nel blocker, on AHPs <strong>an</strong>d its fundamental role in shaping burst firing in vitro.<br />
We show that blocking M current results in a reduction in <strong>the</strong> medium component of AHP.<br />
Fur<strong>the</strong>rmore, we observed a decrease in <strong>the</strong> response to high frequencies of neurons following<br />
blocking of M currents. As suggested by a previous <strong>the</strong>oretical study (Prescott <strong>an</strong>d Sejnowski<br />
2008), our finding show that KCNQ ch<strong>an</strong>nels improve <strong>the</strong> coding of high-frequency signals.<br />
Fur<strong>the</strong>rmore, we show that serotonergic <strong>an</strong>d cholinergic systems influence <strong>the</strong> activity of <strong>the</strong>se<br />
neurons in a similar m<strong>an</strong>ner to that observed by de-activating M currents (i.e., increased burst<br />
activity <strong>an</strong>d increased high-pass properties. These findings show how different modes of sensory<br />
processing <strong>an</strong>d in<strong>for</strong>mation tr<strong>an</strong>smission (e.g., low-pass vs. high-pass or burst vs. non-burst) c<strong>an</strong><br />
be modified by <strong>an</strong> interaction between different neurotr<strong>an</strong>smitters <strong>an</strong>d intrinsic properties of<br />
neurons.<br />
References:<br />
1.Chacron MJ, Doiron B, Maler L, Longtin A, Basti<strong>an</strong> J. Nature.2003,1;423(6935):77-81<br />
2. Prescott SA, Sejnowski TJ. J Neurosci. 2008 Dec 10; 28(50):13649-61.<br />
Disclosures: T. Deemyad, CIHR, CFI, CRC, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); M.J. Chacron,<br />
None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.6/G14<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NIH<br />
CCTB<br />
Title: Intrinsic properties, gamma oscillations, <strong>an</strong>d control of response timing<br />
Authors: E. M. CALLAWAY 1 , S. L. OTTE 1 , *K. J. NIELSEN 3 , A. R. HASENSTAUB 2 ;<br />
2 Crick-Jacobs Ctr., 1 Salk Inst. <strong>for</strong> Biol. Studies, La Jolla, CA; 3 Salk Inst., La Jolla, CA<br />
Abstract: Synchronization of neuronal activity, particularly at gamma (30-80Hz) frequencies, is<br />
related to cognitive functions such as attention <strong>an</strong>d memory; disruptions of this synchrony are<br />
associated with cognitive <strong>an</strong>d attentional deficits. The synchronized activity of inhibitory<br />
interneurons play a prominent role in <strong>the</strong> generation of gamma-b<strong>an</strong>d synchronization by<br />
en<strong>for</strong>cing temporal precision <strong>an</strong>d coordinating <strong>the</strong> activity of <strong>the</strong>ir post-synaptic targets.<br />
However, <strong>the</strong>re are m<strong>an</strong>y types of cortical neurons, each having specific neurochemical<br />
properties, patterns of connectivity, laminar distribution, <strong>an</strong>d electrophysiological characteristics.<br />
Thus, different types of neurons may vary in <strong>the</strong> amplitude <strong>an</strong>d timing of <strong>the</strong> synchronized<br />
inhibition <strong>the</strong>y receive, as well as in <strong>the</strong> integration of a given pattern of inhibitory inputs.<br />
Here we use a combination of electrophysiology, dynamic clamp, <strong>an</strong>d modeling to characterize<br />
<strong>the</strong> interactions between a neuron's intrinsic properties, <strong>the</strong> degree of gamma-b<strong>an</strong>d synchrony<br />
among its inhibitory inputs, <strong>an</strong>d its spike timing. We found that neurons varied in <strong>the</strong>ir spike<br />
timing relative to <strong>the</strong> peak of gamma frequency input, <strong>an</strong>d <strong>the</strong> degree to which <strong>the</strong>ir spike time<br />
depended on ch<strong>an</strong>ges in inhibitory synchrony. Observations <strong>from</strong> a single compartment model<br />
<strong>an</strong>d electrophysiological measurements of real neurons, suggest that <strong>the</strong> time const<strong>an</strong>t (Tm) of<br />
<strong>the</strong> neuron correlates with <strong>the</strong> spike phase <strong>an</strong>d phase precession; neurons with a short Tm fired<br />
closer to <strong>the</strong> peak of gamma inhibitory input <strong>an</strong>d exhibited phase precession, while neurons with<br />
a long Tm fired later <strong>an</strong>d were less affected by ch<strong>an</strong>ges in inhibitory synchrony. We confirmed<br />
<strong>the</strong>se findings by using dynamic clamp to artificially ch<strong>an</strong>ge <strong>the</strong> Tm of real neurons; decreasing<br />
<strong>the</strong> neuron’s Tm by adding leak conduct<strong>an</strong>ce resulted in phase precession.<br />
We conclude a neuron's intrinsic physiology subst<strong>an</strong>tially affects its response to gammasynchronized<br />
inhibitory inputs. These results suggest that <strong>the</strong> characteristic phase relationship of<br />
<strong>the</strong> discharges of neurons during gamma activity may be explained by differences in intrinsic<br />
properties ra<strong>the</strong>r th<strong>an</strong> differences in connectivity. Fur<strong>the</strong>r, we note that <strong>the</strong> relev<strong>an</strong>t physiology is<br />
not static, but may be altered by contextual or neuromodulatory factors; <strong>for</strong> inst<strong>an</strong>ce, a neuron's<br />
Tm decreases during intense synaptic activity, while acetylcholine c<strong>an</strong> alter <strong>the</strong> Tm const<strong>an</strong>t. We<br />
<strong>the</strong>re<strong>for</strong>e suggest that <strong>the</strong> effect of gamma oscillations on responsiveness is not fixed, but may be<br />
dynamically shaped (by factors such as neuromodulation or local background activity) to suit <strong>the</strong><br />
cortex's computational requirements during attention or cognition.
Disclosures: E.M. Callaway, None; S.L. Otte, None; K.J. Nielsen, None; A.R. Hasenstaub,<br />
None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.7/G15<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NIH Gr<strong>an</strong>t MH48432<br />
Title: ¬Heterogeneity in subthreshold membr<strong>an</strong>e properties of layer V pyramidal neurons in <strong>the</strong><br />
medial prefrontal cortex depends on <strong>the</strong>ir long-r<strong>an</strong>ge targets<br />
Authors: *N. C. DEMBROW, R. A. CHITWOOD, J. J. SIEGEL, B. E. KALMBACH, M. D.<br />
MAUK, D. JOHNSTON;<br />
Ctr. For Learning <strong>an</strong>d Memory, Univ. Texas, Austin, Austin, TX<br />
Abstract: Mnemonic persistent activity in <strong>the</strong> prefrontal cortex is a well-identified substrate <strong>for</strong><br />
m<strong>an</strong>y <strong>for</strong>ms of working memory. The inhibition of hyperpolarization-activated<br />
nonspecific-cation (h) currents in prefrontal cortex has been shown to streng<strong>the</strong>n persistent<br />
activity <strong>an</strong>d enh<strong>an</strong>ce working memory in both monkeys <strong>an</strong>d rodents. In order to underst<strong>an</strong>d how<br />
h-current modulation alters working memory, a crucial first step is to identify <strong>an</strong>d characterize<br />
intrinsic properties in <strong>the</strong> layer V pyramidal neurons that communicate this mnemonic activity to<br />
o<strong>the</strong>r regions. In this study we per<strong>for</strong>med somatic whole-cell patch clamp recordings of layer V<br />
pyramidal neurons in <strong>the</strong> prelimbic region of medial prefrontal cortex (mPFC) of 6-10 week old<br />
male Sprague Dawley rats. Our results suggest layer V pyramidal neurons within <strong>the</strong> mPFC may<br />
be segregated into two populations based on <strong>the</strong>ir subthreshold membr<strong>an</strong>e properties, particularly<br />
in several parameters dependent on <strong>the</strong> density <strong>an</strong>d kinetics of <strong>the</strong> h-current (input resist<strong>an</strong>ce,<br />
reson<strong>an</strong>ce frequency, sag ratio). Anatomical characterization using infusions of retrogradely<br />
tr<strong>an</strong>sported Lumafluor beads into different projection targets of mPFC pyramidal neurons<br />
revealed a correlation between intrinsic membr<strong>an</strong>e properties <strong>an</strong>d <strong>the</strong>ir projection targets.<br />
Specifically, infusions of Lumafluor beads into <strong>the</strong> pons labeled a subpopulation of layer V<br />
mPFC neurons (n = 26) that were characterized by lower input resist<strong>an</strong>ce (RN = 52.22 ± 2.39<br />
MΩ), higher reson<strong>an</strong>ce frequency (fR = 3.97 ± 0.15 Hz) <strong>an</strong>d a more pronounced sag ratio (SR:<br />
steady-state RN / max RN = 0.825 ± .006), in comparison to <strong>the</strong>ir adjacent (
input resist<strong>an</strong>ce (RN = 108.24 ± 20.77), lower reson<strong>an</strong>ce frequency (RF = 1.15 ± 0.49 Hz) <strong>an</strong>d<br />
little or no sag (SR = 0.96 ± .03). These results demonstrate a heterogeneity of h-current<br />
properties depending on whe<strong>the</strong>r pyramidal V mPFC neurons project to contralateral cortex or to<br />
subcortical structures. We conclude that <strong>the</strong> modulation of h-current associated with <strong>the</strong><br />
enh<strong>an</strong>cement of working memory might be more complex th<strong>an</strong> previously appreciated. The<br />
effects of h-current modulation might tr<strong>an</strong>sfer differently depending on <strong>the</strong> downstream target<br />
area of <strong>the</strong> layer V pyramidal neurons <strong>an</strong>d thus vary <strong>the</strong> way mnemonic persistent activity is<br />
tr<strong>an</strong>sferred across neuronal networks.<br />
Disclosures: N.C. Dembrow, None; R.A. Chitwood, None; J.J. Siegel, None; B.E.<br />
Kalmbach, None; M.D. Mauk, None; D. Johnston, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.8/G16<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NSF gr<strong>an</strong>t 0817969<br />
Title: Voltage-imaging <strong>from</strong> axons <strong>an</strong>d collaterals of cortical pyramidal neurons in brain slices<br />
Authors: M. POPOVIC 1 , A. FOUST 2 , D. MCCORMICK 2 , *D. ZECEVIC 1 ;<br />
1 Dept Cell & Mol. Physiol, 2 Dept Neurobio., Yale Univ. Sch. Med., New Haven, CT<br />
Abstract: Experimental evidence indicates that <strong>the</strong> functional capabilities of axons are much<br />
more diverse th<strong>an</strong> traditionally thought. Consequently, <strong>the</strong> field of axonal physiology is rapidly<br />
exp<strong>an</strong>ding. At <strong>the</strong> same time, m<strong>an</strong>y basic electrophysiological properties of cortical axons <strong>an</strong>d<br />
presynaptic terminals have largely been unexplored, owing to <strong>the</strong>ir small size <strong>an</strong>d inaccessibility<br />
to traditional recording techniques. Patch-electrode recordings have no spatial resolution (limited<br />
to recordings <strong>from</strong> a single site) <strong>an</strong>d do not allow <strong>for</strong> recording <strong>from</strong> axon collaterals or axons of<br />
interneurons. Thus, <strong>an</strong> import<strong>an</strong>t challenge in cellular neurobiology, both conceptually <strong>an</strong>d<br />
technically, is to develop <strong>an</strong> approach <strong>for</strong> recording membr<strong>an</strong>e potential (Vm) signals <strong>from</strong><br />
individual axons <strong>an</strong>d axon collaterals at adequate spatio-temporal resolution. The laser-excitation<br />
wide-field epi-fluorescence Vm-imaging has <strong>the</strong> potential to achieve <strong>the</strong> required sensitivity. We<br />
used <strong>the</strong> most sensitive voltage dye (in terms of S/N) <strong>for</strong> intracellular application (JPW3028) <strong>an</strong>d<br />
optimized both <strong>the</strong> resting florescence light intensity <strong>an</strong>d <strong>the</strong> relative fluorescence ch<strong>an</strong>ge in<br />
response to Vm ch<strong>an</strong>ge by utilizing a single wavelength laser excitation at 532 nm. The result
was a dramatic increase in <strong>the</strong> sensitivity allowing us to monitor AP signals <strong>from</strong> <strong>the</strong> axonal<br />
arbor in single trial recordings. Using this approach, we monitored AP initiation <strong>an</strong>d propagation<br />
in <strong>the</strong> axonal arbor of cortical layer 5 pyramidal neurons in slices at <strong>the</strong> full CCD frame rates of<br />
5 <strong>an</strong>d 10 KHz. The data show directly <strong>the</strong> axonal site of AP initiation as well as spike<br />
propagation characteristics at axonal br<strong>an</strong>ch-points. As <strong>the</strong> relationship between <strong>the</strong> dye signal<br />
<strong>an</strong>d <strong>the</strong> membr<strong>an</strong>e potential is strictly linear in <strong>the</strong> entire physiological r<strong>an</strong>ge, <strong>the</strong> wave<strong>for</strong>m of<br />
axonal APs is derived directly <strong>from</strong> optical recordings. The comparison of AP wave<strong>for</strong>ms <strong>from</strong><br />
<strong>the</strong> main axon, axon collaterals <strong>an</strong>d neighboring basal dendrites revealed dramatic differences.<br />
This type of in<strong>for</strong>mation, linked to a multi-compartmental numerical simulation <strong>an</strong>d to data on<br />
voltage-gated ch<strong>an</strong>nel distribution <strong>from</strong> immuno-cytochemical <strong>an</strong>alysis, should facilitate<br />
elucidating normal axonal physiology as well as abnormal ch<strong>an</strong>ges underlying a variety of<br />
neurological symptoms. In conclusion, our results show that a methodological watershed in<br />
axonal physiology has been passed by <strong>an</strong> adv<strong>an</strong>ce in sensitivity of Vm-imaging demonstrating<br />
<strong>the</strong> immense power of being able to directly record <strong>the</strong> events about which we could only<br />
<strong>the</strong>orize in <strong>the</strong> past on <strong>the</strong> basis of indirect evidence.<br />
Disclosures: M. Popovic, None; A. Foust, None; D. McCormick, None; D. Zecevic, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.9/G17<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NIH Gr<strong>an</strong>t R56-DK-068566<br />
NIH Gr<strong>an</strong>t R01-DK-068566<br />
NIH Gr<strong>an</strong>t R01-NS-051671<br />
Title: Analysis of axonal excitability modulated by high-frequency (KHz) biphasic electrical<br />
current<br />
Authors: *C. TAI 1 , H. LIU 1 , J. R. ROPPOLO 2 , W. C. DE GROAT 2 ;<br />
1 Dept. of Urology, 2 Dept. of Pharmacol. & Chem Biol, Univ. Pittsburgh, Pittsburgh, PA<br />
Abstract: Underst<strong>an</strong>ding how axonal excitability is modulated by high-frequency biphasic<br />
(HFB) electrical current could help to fur<strong>the</strong>r reveal <strong>the</strong> possible mech<strong>an</strong>isms underlying <strong>the</strong>
nerve conduction block phenomenon, promote its clinical application, <strong>an</strong>d design new<br />
stimulation method to selectively activate small nerve fibers. Currently, <strong>the</strong>re is no study that has<br />
investigated <strong>the</strong> modulation of axonal excitability induced by HFB electrical current. The goal of<br />
this study is to use computer simulation to <strong>an</strong>alyze this modulation process. A lumped circuit<br />
model of a myelinated axon based on Schwarz-Reid-Bostock (SRB) equations derived <strong>from</strong><br />
hum<strong>an</strong> axons is used. A monopolar point electrode is placed at <strong>the</strong> middle of a 6 cm long axon<br />
with a 1 mm dist<strong>an</strong>ce to <strong>the</strong> axon. The HFB electrical current is delivered via <strong>the</strong> point electrode<br />
to modulate <strong>the</strong> axonal excitability. After 30 ms, a single uniphasic test pulse is also applied via<br />
<strong>the</strong> same electrode to determine <strong>the</strong> axonal excitability during <strong>the</strong> HFB modulation. The results<br />
show that axonal excitability could be ei<strong>the</strong>r increased or decreased by HFB current depending<br />
on <strong>the</strong> current intensity. The increase of axonal excitability is due to <strong>the</strong> high level of sodium<br />
ch<strong>an</strong>nel activation, whereas <strong>the</strong> activation of both fast <strong>an</strong>d slow potassium ch<strong>an</strong>nels plays <strong>an</strong><br />
import<strong>an</strong>t role in decreasing axonal excitability. As <strong>the</strong> HFB current intensity increases, <strong>the</strong><br />
location determining <strong>the</strong> axonal excitability ch<strong>an</strong>ges <strong>from</strong> <strong>the</strong> nodes under <strong>the</strong> electrode within<br />
<strong>the</strong> “main lobe” region of <strong>the</strong> activating function to <strong>the</strong> nodes away <strong>from</strong> <strong>the</strong> electrode in <strong>the</strong><br />
“side lobe” region of <strong>the</strong> activating function. This simulation study also shows that <strong>the</strong><br />
modulation of axonal excitability by HFB electrical current could be potentially useful to<br />
selectively activate <strong>the</strong> small nerve fibers in a compound nerve trunk without activating <strong>the</strong> large<br />
fibers, indicating <strong>an</strong><strong>the</strong>r possible clinical application of <strong>the</strong> HFB stimulation method.<br />
Disclosures: C. Tai, None; H. Liu, None; J.R. Roppolo, None; W.C. de Groat, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.10/G18<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NSERC<br />
AHFMR<br />
Title: Interaction of motor neuron type <strong>an</strong>d location of dendritic ch<strong>an</strong>nels on bistable firing<br />
properties<br />
Authors: H. KIM 1 , *K. E. JONES 2,1 ;<br />
1 2<br />
Biomed. Engin., Univ. of Alberta, Edmonton, AB, C<strong>an</strong>ada; Fac Physical Educ. & Rec., Univ.<br />
Alberta, Edmonton, AB, C<strong>an</strong>ada
Abstract: Objective<br />
- This computational study investigated <strong>the</strong> interaction between parameters that distinguish<br />
different types of motor neurons (e.g. input resist<strong>an</strong>ce) <strong>an</strong>d <strong>the</strong> dendritic distribution of persistent<br />
voltage-gated calcium ch<strong>an</strong>nels (VGCC) on nonlinear bistable firing patterns. Previous<br />
computational studies hypo<strong>the</strong>sized that VGCCs must be located fur<strong>the</strong>r <strong>from</strong> <strong>the</strong> soma in large,<br />
low input resist<strong>an</strong>ce motor neurons to generate bistable firing (Gr<strong>an</strong>de et al. J Neurophysiol<br />
97:4023-35, 2007).<br />
Methods<br />
- We used a recently published method <strong>for</strong> generating reduced two-compartment models <strong>from</strong><br />
<strong>an</strong>atomically reconstructed motor neurons. Slow (S), fast fatigue-resist<strong>an</strong>t (FR) <strong>an</strong>d fast fatiguing<br />
(FF) motor neuron models were constructed. Each of <strong>the</strong>se three models was simulated with<br />
VGCCs located at three dist<strong>an</strong>ces <strong>from</strong> <strong>the</strong> soma: 200, 600, & 1000 um. The types <strong>an</strong>d<br />
distribution of voltage-gated ion ch<strong>an</strong>nels were <strong>the</strong> same <strong>for</strong> all reduced models. A tri<strong>an</strong>gular<br />
current was applied to <strong>the</strong> somatic compartment of all models superimposed on a bias current<br />
that normalized <strong>for</strong> differences in rheobase.<br />
- The onset time of <strong>the</strong> dendritic plateau potential <strong>an</strong>d hysteresis of frequency-current (f-I)<br />
relationship in <strong>the</strong> models were used as criteria to evaluate bistability.<br />
Results<br />
- Results show that <strong>the</strong> models with <strong>the</strong> higher input resist<strong>an</strong>ce (S-type motor neuron) have <strong>an</strong><br />
earlier time of onset <strong>for</strong> <strong>the</strong> plateau potential <strong>for</strong> all dist<strong>an</strong>ces. To generate bistable firing patterns<br />
in FF-type models <strong>the</strong> VGCCs needed to be located more distally.<br />
This study’s conclusions indicate that <strong>the</strong>re are strong interactions between <strong>the</strong> biophysical<br />
parameters that distinguish motor neurons of different types <strong>an</strong>d <strong>the</strong> presence of nonlinear<br />
bistable firing patterns. The previous hypo<strong>the</strong>sis by Gr<strong>an</strong>de et al. appears to be true: to generate<br />
bistable firing in FF-type, VGCCs need to be located more distal to <strong>the</strong> soma compared to S-type<br />
motor neurons.<br />
Disclosures: H. Kim, None; K.E. Jones, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.11/G19<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: St. Olaf College Summer Gr<strong>an</strong>t <strong>for</strong> Scholarly <strong>an</strong>d Artistic Activity<br />
Title: A computational model of a compound impulse in a leech neuron implicated in learning
Authors: *K. M. CRISP;<br />
St Olaf Col., Northfield, MN<br />
Abstract: Neuronal geometry has recently been cited to explain variable responses at electrical<br />
synapses, but with little <strong>the</strong>oretical basis. In <strong>the</strong> leech, sensitization of defensive shortening has<br />
been linked to a 3-neuron positive feedback loop that includes <strong>the</strong> electrically-coupled S- <strong>an</strong>d Cinterneurons.<br />
Individual action potentials in <strong>the</strong> S-interneuron in saline containing elevated<br />
divalent cation concentrations evoke impulses of various amplitudes in <strong>the</strong> soma of <strong>the</strong> Cinterneuron.<br />
Because <strong>the</strong> C-interneuron is electrically unexcitable near <strong>the</strong> soma <strong>an</strong>d makes<br />
m<strong>an</strong>y electrical junctions with <strong>the</strong> S-interneuron, <strong>the</strong> impulses recorded <strong>from</strong> <strong>the</strong> C-interneuron’s<br />
soma may be <strong>the</strong> summation of action potentials occurring at multiple, independent impulse<br />
initiation zones far <strong>from</strong> its soma <strong>an</strong>d close to <strong>the</strong> junctions with <strong>the</strong> S-interneuron. To explore<br />
this possibility, a multi-compartmental model of <strong>the</strong> C-interneuron was developed <strong>from</strong><br />
morphological <strong>an</strong>d physiological data using <strong>the</strong> modeling environment SNNAP. Simulations<br />
indicated that reducing membr<strong>an</strong>e excitability (to mimic <strong>the</strong> effects of saline containing elevated<br />
divalent ion concentrations) resulted in variable impulse amplitudes in <strong>the</strong> C-interneuron’s soma<br />
<strong>an</strong>d a failure to activate its chemical synapses onto <strong>the</strong> Retzius cell, as has been observed<br />
physiologically. These simulations support <strong>the</strong> conclusion that a widely used test <strong>for</strong><br />
monosynaptic connections using elevated divalent cation concentrations is able to confirm that a<br />
synaptic connection in <strong>the</strong> leech is polysynaptic, even though <strong>the</strong> first synapse in this disynaptic<br />
connection is <strong>an</strong> electrical junction.<br />
Disclosures: K.M. Crisp, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.12/G20<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: DMS 0703502<br />
DMS 081714<br />
Title: Divisive gain modulation with dynamic stimuli in integrate-<strong>an</strong>d-fire neurons
Authors: *C. LY 1 , B. DOIRON 1,2 ;<br />
1 2<br />
Ma<strong>the</strong>matics, Univ. of Pittsburgh, Pittsburgh, PA; Ctr. <strong>for</strong> <strong>the</strong> Neural Basis of Cognition,<br />
Pittsburgh, PA<br />
Abstract: The modulation of <strong>the</strong> sensitivity, or gain, of neural responses to input is <strong>an</strong> import<strong>an</strong>t<br />
component of neural computation. It has been shown that divisive gain modulation of neural<br />
responses c<strong>an</strong> result <strong>from</strong> a stochastic shunting <strong>from</strong> bal<strong>an</strong>ced (mixed excitation <strong>an</strong>d inhibition)<br />
background activity [Ch<strong>an</strong>ce, Abbott, Reyes 2002]. This gain control scheme was developed <strong>an</strong>d<br />
explored with static inputs, where <strong>the</strong> membr<strong>an</strong>e <strong>an</strong>d spike train statistics were stationary in time.<br />
However, input statistics, such as <strong>the</strong> firing rates of pre-synaptic neurons, are often dynamic,<br />
varying on time scales comparable to typical membr<strong>an</strong>e time const<strong>an</strong>ts. Using a population<br />
density approach <strong>for</strong> integrate-<strong>an</strong>d-fire neurons with dynamic <strong>an</strong>d temporally rich inputs, we find<br />
<strong>the</strong> same fluctuation-induced divisive gain modulation is operative <strong>for</strong> dynamic inputs driving<br />
nonequilibrium responses. Moreover, <strong>the</strong> degree of divisive scaling of <strong>the</strong> dynamic response is<br />
qu<strong>an</strong>titatively <strong>the</strong> same as <strong>the</strong> steady-state responses -- thus, gain modulation via bal<strong>an</strong>ced<br />
conduct<strong>an</strong>ce fluctuations generalizes in a straight-<strong>for</strong>ward way to a dynamic setting.<br />
Disclosures: C. Ly, None; B. Doiron, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.13/G21<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: NINDS 1 RO1 NS054911-01A1 (A.A.P.)<br />
NSF IGERT Fellowship (A.E.H.)<br />
Title: Conduct<strong>an</strong>ce relationships c<strong>an</strong> be used to constrain features of cellular identity<br />
Authors: *A. E. HUDSON 1,2 , A. A. PRINZ 1 ;<br />
1 Biology, Emory Univ., Atl<strong>an</strong>ta, GA; 2 Bioengineering, Georgia Inst. of Technol., Atl<strong>an</strong>ta, GA<br />
Abstract: Recent experimental evidence suggests that coordinated expression of ion ch<strong>an</strong>nels<br />
plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of<br />
<strong>the</strong> crustace<strong>an</strong> stomatogastric g<strong>an</strong>glion exhibits a unique set of positive linear correlations<br />
between conduct<strong>an</strong>ces. This data implies a causal relationship between expressed conduct<strong>an</strong>ce
correlations <strong>an</strong>d defined electrical activity type. To test this idea, we used <strong>an</strong> existing database of<br />
conduct<strong>an</strong>ce-based model neurons that sp<strong>an</strong> a wide r<strong>an</strong>ge of activity types. We partitioned this<br />
database based on various measures of intrinsic activity, to approximate distinctions between<br />
biological cell types. We <strong>the</strong>n tested individual conduct<strong>an</strong>ce pairs <strong>for</strong> linear dependence to<br />
identify correlations. Contrary to experimental evidence in which all conduct<strong>an</strong>ce correlations<br />
are positive, 30% of those seen in this database were negative relationships. Similarly, calcium<br />
correlations have not yet been published due to experimental constraints, however within <strong>the</strong><br />
database 73% of correlations involved at least one calcium conduct<strong>an</strong>ce. Only <strong>the</strong><br />
hyperpolarization-activated conduct<strong>an</strong>ce was not correlated with o<strong>the</strong>r conduct<strong>an</strong>ce parameters<br />
in <strong>an</strong>y activity type. Similar to experimental results, each activity type investigated had a unique<br />
combination of correlated conduct<strong>an</strong>ces, excluding those without correlations. In general, we<br />
found that <strong>the</strong> presence of identified correlations became more likely as <strong>the</strong> definition of activity<br />
type became more restrictive. Finally, it was seen that populations of models that con<strong>for</strong>m to a<br />
specific conduct<strong>an</strong>ce correlation have a higher likelihood of exhibiting <strong>the</strong> activity type where<br />
that correlation was observed. We conclude that regulating conduct<strong>an</strong>ce ratios c<strong>an</strong> support <strong>the</strong><br />
mainten<strong>an</strong>ce of a wide r<strong>an</strong>ge of cellular activity types, <strong>an</strong>d predict that previously unseen<br />
negative correlations <strong>an</strong>d correlations involving calcium conduct<strong>an</strong>ces are biologically plausible.<br />
Disclosures: A.E. Hudson, None; A.A. Prinz, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.14/G22<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Title: Voltage-clamp <strong>an</strong>alysis of ionic currents in dopaminergic neurons of <strong>the</strong> dorsal raphe<br />
nucleus<br />
Authors: *A. G. DOUGALIS, M. LI, M. A. UNGLESS;<br />
MRC Clin. Sci. Ctr., Imperial Col. London, London, United Kingdom<br />
Abstract: The dorsal raphe nucleus (DR) contains a small population of dopaminergic neurons<br />
scattered amongst <strong>the</strong> principal serotonergic cells (Hokfelt et al., 1984).<br />
We have previously reported <strong>the</strong> electrophysiological <strong>an</strong>d pharmacological properties of<br />
dopamine neurons of <strong>the</strong> DR in vitro (Dougalis et al., 2008) in tr<strong>an</strong>sgenic mice expressing green<br />
fluorescent protein (GFP) under <strong>the</strong> promoter region of <strong>the</strong> pitx3 gene, a selective marker of<br />
midbrain dopaminergic neurons (Zhao et al., 2004). Here, to complement our previous work, we
have used whole-cell electrophysiology in brain slices <strong>from</strong> pitx3-GFP mice (male, 2-6 months<br />
old) <strong>an</strong>d exploited a combination of voltage-clamp protocols <strong>an</strong>d pharmacological blockade to<br />
isolate <strong>an</strong>d <strong>an</strong>alyse <strong>the</strong> major intrinsic membr<strong>an</strong>e conduct<strong>an</strong>ces underlying <strong>the</strong>ir electrical<br />
behaviour. All breeding <strong>an</strong>d experimental procedures were conducted in accord<strong>an</strong>ce with <strong>the</strong><br />
Animals (Scientific Procedures) Act of 1986 (UK).<br />
Voltage-clamped dopamine neurons of <strong>the</strong> DR stepped to hyperpolarised holding potentials<br />
(more negative th<strong>an</strong> -85 mV) activated <strong>an</strong> inwardly rectifying, current reminiscent of <strong>the</strong><br />
hyperpolarisation-activated depolarising current (<strong>the</strong> IH current, ZD7228 [4-Ethylphenylamino-<br />
1,2-dimethyl-6-methylaminopyrimidinine]-sensitive). Voltage steps to depolarising holding<br />
potentials (more positive th<strong>an</strong> -45 mV) activated three outward potassium currents, including <strong>the</strong><br />
tr<strong>an</strong>sient A-type (IA current, 4-aminopyridine [4-AP]-sensitive), <strong>the</strong> slow delayed rectifier (IK<br />
current, tetraethylammonium [TEA]-sensitive) <strong>an</strong>d <strong>the</strong> dubbed “muscarinic” (IM current, XE991<br />
[10,10-bis(4-Pyridinylmethyl)-9(10H)-<strong>an</strong>thracenone]-sensitive). An apamin-sensitive, calciumactivated<br />
outward potassium current (IK(Ca)) was also activated by depolarisation <strong>an</strong>d<br />
contributed to <strong>the</strong> afterhyperpolarisation current (IAHP) recorded at -60 mV, while cadmium<br />
(Cd+2)-sensitive, low voltage-activated (LVA, T-type) <strong>an</strong>d high voltage-activated (HVA, Ltype,<br />
nimodipine-sensitive) calcium currents were also resolved.<br />
Our results show that dopamine neurons of <strong>the</strong> DR exhibit autorhythmicity <strong>an</strong>d may control <strong>the</strong>ir<br />
pacemaking activity through <strong>an</strong> interplay of intrinsic IK(Ca), IM <strong>an</strong>d IA conduct<strong>an</strong>ces <strong>an</strong>d <strong>the</strong>ir<br />
on-dem<strong>an</strong>d modification by synaptic activity.<br />
Disclosures: A.G. Dougalis, None; M. Li, None; M.A. Ungless, None.<br />
Poster<br />
525. Firing Properties of Neurons<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 525.15/G23<br />
Topic: B.10.b. Modulation of neuronal firing properties<br />
Support: FAPESP<br />
CNPq<br />
Proex<br />
NIH-Fogarty International Center<br />
Title: Rapid effects of 17-β−estradiol on central auditory neurons of <strong>the</strong> zebra finch
Authors: T. F. BECKHAUSER 1 , C. V. MELLO 2 , *R. M. LEAO 3 ;<br />
1 Physiol., Univ. of São Paulo, Ribeirão Preto, Brazil; 2 Behavioral Neurosci., Oregon Hlth. <strong>an</strong>d<br />
Sci. Univ., Portl<strong>an</strong>d, OR; 3 Dept Fisiol, Univ. Sao Paulo, Ribeirao Preto, Brazil<br />
Abstract: Besides <strong>the</strong>ir well-known actions as hormones, sexual steroids have been shown to act<br />
as neuromodulators of <strong>the</strong> activity of m<strong>an</strong>y central. They c<strong>an</strong> act ei<strong>the</strong>r through <strong>the</strong> well known<br />
nuclear receptors to affect gene tr<strong>an</strong>scription resulting in long-term ch<strong>an</strong>ges in neuronal<br />
physiology or via nontraditional, rapid (seconds to minutes) actions that certainly involve<br />
membr<strong>an</strong>e receptors. This fast route of steroids action in <strong>the</strong> brain results in regulation of<br />
membr<strong>an</strong>e potential <strong>an</strong>d neurotr<strong>an</strong>smission by regulating ion ch<strong>an</strong>nels <strong>an</strong>d neurotr<strong>an</strong>smitter<br />
receptors. In accord<strong>an</strong>ce steroids c<strong>an</strong> influence <strong>an</strong>imal behavior in seconds to minutes. The<br />
Nidopalium Caudomediallis (NCM) is a central auditory nucleus of <strong>the</strong> oscines (songbirds) rich<br />
in <strong>the</strong> estradiol-converting enzyme aromatase, <strong>an</strong>d syn<strong>the</strong>sizes estradiol <strong>from</strong> testosterone when<br />
<strong>the</strong> bird sings, <strong>an</strong>d this estradiol c<strong>an</strong> shape <strong>the</strong> activity of NCM neurons in vivo. In ths work we<br />
aimed to investigate <strong>the</strong> mech<strong>an</strong>isms of action of estradiol on NCM neurons.<br />
Recordings were made on parasagital brain slices of both male <strong>an</strong>d female zebrafinches<br />
(Taeniopygia guttata) containing <strong>the</strong> NCM <strong>an</strong>d adjacent areas. Estradiol signific<strong>an</strong>tly (p
Support: NIH/NCCAM R01 AT001928<br />
Title: Neuronal Aβ expression impairs serotonin signaling in a tr<strong>an</strong>sgenic C. eleg<strong>an</strong>s<br />
Authors: *M. K. BROWN 1 , L. E. DOSANJH 1 , Y. LUO 2 , C. LINK 3 ;<br />
2 Dept. of Pharmaceut. Sci., 1 Univ. of Maryl<strong>an</strong>d-Baltimore, Baltimore, MD; 3 Inst. <strong>for</strong> Behavioral<br />
Genet., Univ. of Colorado at Boulder, Boulder, CO<br />
Abstract: Alzheimer’s disease (AD) is <strong>the</strong> leading cause of dementia in <strong>the</strong> elderly. AD<br />
currently affects 5 million people in <strong>the</strong> United States <strong>an</strong>d is predicted to affect 14 million by <strong>the</strong><br />
year 2050. Though <strong>the</strong> underlying pathology of AD is still under scrutiny, genetic evidence has<br />
implicated amyloid beta (Aβ) as a determin<strong>an</strong>t factor in pathogenesis <strong>an</strong>d has led to widespread<br />
support of <strong>the</strong> amyloid cascade hypo<strong>the</strong>sis. This hypo<strong>the</strong>sis states that <strong>an</strong> increase in Aβ, whe<strong>the</strong>r<br />
by increased production or decreased clear<strong>an</strong>ce, leads to downstream effects including synaptic<br />
dysfunction <strong>an</strong>d cell death. Previously, <strong>the</strong> nematode C. eleg<strong>an</strong>s has been engineered to express<br />
intramuscular hum<strong>an</strong> Aβ <strong>an</strong>d this tr<strong>an</strong>sgenic model has been used to explore Aβ-induced<br />
toxicity, protein tr<strong>an</strong>sport <strong>an</strong>d to evaluate toxic species of <strong>the</strong> protein. However, <strong>the</strong> musclespecific<br />
models present a limited application to AD pathogenesis, <strong>an</strong>d a model that expresses Aβ<br />
in neurons was developed out of a necessity to study Aβ toxicity in relev<strong>an</strong>t cell types. Using a<br />
tr<strong>an</strong>sgenic C. eleg<strong>an</strong>s with p<strong>an</strong> neuronal expression of hum<strong>an</strong> Aβ, we evaluated early<br />
dysfunction caused by Aβ expression specific to neurons. We found neuronal expression of Aβ<br />
altered several behaviors we evaluated in this study. Neuronal expression of Aβ caused a<br />
signific<strong>an</strong>t decline in serotonin stimulated egg-laying <strong>an</strong>d progeny viability. Fur<strong>the</strong>r, <strong>the</strong>se<br />
tr<strong>an</strong>sgenic worms also displayed a shorter me<strong>an</strong> <strong>an</strong>d maximum lifesp<strong>an</strong>. Two <strong>for</strong>ms of learning<br />
behaviors, associative learning <strong>an</strong>d enh<strong>an</strong>ced slowing response (ESR), were both impaired by<br />
neuronal expression of Aβ. However, we did find that basal slowing response (BSR), <strong>the</strong><br />
dopamine-mediated counterpart to ESR, remained intact. Because differential responses were<br />
observed between dopamine <strong>an</strong>d serotonin mediated behaviors, <strong>the</strong>se results indicate that<br />
neuronal expression of Aβ selectively impairs serotonergic tr<strong>an</strong>smission in <strong>the</strong> C. eleg<strong>an</strong>s. These<br />
phenotypes of <strong>the</strong> neuronal Aβ expressing C. eleg<strong>an</strong>s mut<strong>an</strong>t provide a valid model <strong>for</strong> studying<br />
Aβ-induced synaptic dysfunction.<br />
Disclosures: M.K. Brown, None; L.E. Dos<strong>an</strong>jh, None; Y. Luo, None; C. Link, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.2/G25
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t AG029421<br />
Title: Novel age-dependent learning deficits in a mouse model of Alzheimer’s disease:<br />
Implications <strong>for</strong> tr<strong>an</strong>slational research<br />
Authors: *K. S. MONTGOMERY 1 , G. EDWARDS, III 1 , R. K. SIMMONS 1 , M. M.<br />
NICOLLE 2 , M. A. GLUCK 3 , C. E. MYERS 3 , J. L. BIZON 1 ;<br />
1 2<br />
Texas A&M Univ., College Station, TX; Wake Forest Univ. Sch. of Med., Winston-Salem,<br />
NC; 3 Rutgers Univ., Newark, NJ<br />
Abstract: Computational modeling predicts that <strong>the</strong> hippocampus plays <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong><br />
ability to apply previously learned in<strong>for</strong>mation to novel problems <strong>an</strong>d situations (referred to as<br />
<strong>the</strong> ability to generalize in<strong>for</strong>mation or simply as ‘tr<strong>an</strong>sfer learning’). These predictions have<br />
been tested in hum<strong>an</strong>s using a computer-based task on which individuals with hippocampal<br />
damage are able to learn a series of complex discriminations with two stimulus features (shape<br />
<strong>an</strong>d color), but are impaired in <strong>the</strong>ir ability to tr<strong>an</strong>sfer this in<strong>for</strong>mation to newly configured<br />
problems in which one of <strong>the</strong> features is altered. This deficit occurs despite <strong>the</strong> fact that <strong>the</strong><br />
feature predictive of <strong>the</strong> reward (<strong>the</strong> relev<strong>an</strong>t in<strong>for</strong>mation) is not ch<strong>an</strong>ged. The goal of <strong>the</strong> current<br />
study was to develop a mouse <strong>an</strong>alog of tr<strong>an</strong>sfer learning <strong>an</strong>d to determine if this new task was<br />
sensitive to pathological ch<strong>an</strong>ges in a mouse model of AD. We describe a task in which mice<br />
were able to learn a series of concurrent discriminations that contained two stimulus features<br />
(odor <strong>an</strong>d digging medium) <strong>an</strong>d could tr<strong>an</strong>sfer this learned in<strong>for</strong>mation to new problems in which<br />
<strong>the</strong> irrelev<strong>an</strong>t feature in each discrimination pair was altered. Moreover, we report age-dependent<br />
deficits specific to tr<strong>an</strong>sfer learning in <strong>the</strong> APP+PS1 mice relative to nontr<strong>an</strong>sgenic littermates.<br />
The robust impairment in tr<strong>an</strong>sfer learning may be more sensitive to AD-like pathology th<strong>an</strong><br />
traditional cognitive assessments in that no deficits were observed in <strong>the</strong> APP+PS1 mice on <strong>the</strong><br />
widely used Morris water maze task. These data describe a novel <strong>an</strong>d sensitive paradigm to<br />
evaluate mnemonic decline in AD mouse models that has unique tr<strong>an</strong>slational adv<strong>an</strong>tages over<br />
st<strong>an</strong>dard species-specific cognitive assessments (e.g. water maze <strong>for</strong> rodent <strong>an</strong>d delayed<br />
paragraph recall <strong>for</strong> hum<strong>an</strong>s).<br />
Disclosures: K.S. Montgomery, None; G. Edwards, None; R.K. Simmons, None; M.M.<br />
Nicolle, None; M.A. Gluck, None; C.E. Myers, None; J.L. Bizon, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 526.3/G26<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Adeno-associated viral tr<strong>an</strong>sduction of abeta <strong>an</strong>d tau genes enh<strong>an</strong>ces pathology <strong>an</strong>d<br />
cognitive deficits in mouse model of AD<br />
Authors: N. BARREZUETA, *A. E. EASTON, R. LIDGE, C. BOURIN, S. KEENAN, A.<br />
HENDRICSON, R. KNOX, C. ALBRIGHT, L. BRISTOW, A. M. CACACE;<br />
Bristol Myers Squibb, Walling<strong>for</strong>d, CT<br />
Abstract: Adeno-associated virus serotype-1 (AAV-1) is commonly used as a gene delivery<br />
vector when developing rodent models of neurodegeneration, (Wolfe, et. al.;Golde, et al). We<br />
developed this technique with <strong>the</strong> goal of producing <strong>an</strong>imal models <strong>for</strong> neurological disease.<br />
Initially, AAV-1 encoding green fluorescent protein (GFP) was injected into new born mice to<br />
validate <strong>the</strong> technology <strong>an</strong>d verify reproducibility of <strong>the</strong> injections. Within 3 weeks postinjection,<br />
mice showed GFP expression that increased with age in cortex, hippocampus, <strong>an</strong>d<br />
cerebellum. We used this method to develop a novel <strong>an</strong>imal model of Alzheimer’s disease, in<br />
which we tr<strong>an</strong>sduced a bicistronic cDNA encoding British Abeta 1-42 <strong>an</strong>d <strong>the</strong> hum<strong>an</strong> Tau gene<br />
containing <strong>the</strong> P301L mutation. AAV-1 (Bri-Abeta-42+P301Ltau) was injected into <strong>the</strong><br />
ventricles (1 ul/side) of newborn triple tr<strong>an</strong>sgenic mice*. Somatodendritic tau was detected in <strong>the</strong><br />
hippocampus by 3 weeks of age <strong>an</strong>d became robust by 6 months of age, compared to triple<br />
tr<strong>an</strong>sgenic mice injected with <strong>an</strong> empty vector. In addition, AAV-1 (Bri-Abeta-42+P301Ltau)<br />
mice display robust deficits in acquisition <strong>an</strong>d probe trials in <strong>the</strong> MWM task as well as a<br />
reduction in object recognition compared to empty vector <strong>an</strong>d WT controls. Cognitive deficits<br />
were reliably reproduced across cohorts of <strong>an</strong>imals. Similarly, deficits in LTP were observed in<br />
<strong>the</strong> AAV-1 (Bri-Abeta-42+P301Ltau) mice compared to empty vector controls. These data show<br />
that we have successfully used <strong>the</strong> adeno-associated viral method of gene tr<strong>an</strong>sduction to<br />
accelerate pathology <strong>an</strong>d behavioral deficits observed in triple tr<strong>an</strong>sgenic mice to create a robust<br />
<strong>an</strong>imal model of Alzheimer’s disease.<br />
*triple tr<strong>an</strong>sgenic mice carried mut<strong>an</strong>t <strong>for</strong>ms of APP, presenilin1, P301Ltau. Founders obtained<br />
<strong>from</strong> Fr<strong>an</strong>k LaFerla’s lab.<br />
Disclosures: N. Barrezueta, None; A.E. Easton, None; R. Lidge, None; C. Bourin, None; S.<br />
Keen<strong>an</strong>, None; A. Hendricson, None; R. Knox, None; C. Albright, None; L. Bristow,<br />
None; A.M. Cacace, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 526.4/G27<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t AG025824<br />
Title: Isolation stress exacerbates memory-related behavioral deficits in SAMP8 mice<br />
Authors: *H. DONG 1 , C. M. YUEDE 2 , K. M. MURPHY 1 , Q. CHEN 1 , S. A. FARR 3,4 , W. A.<br />
BANKS 3,4 , J. E. MORLEY 3,4 , J. G. CSERNANSKY 1 ;<br />
1 Dept Psychiatry <strong>an</strong>d Behavioral Sci., Northwestern Univ. Feinberg Sch. of Med., Chicago, IL;<br />
2 Psychiatry, Washington Univ., St. Louis, MO; 3 St. Louis VA Med. Ctr., St. Louis, MO; 4 Div. of<br />
Geriatric Research, Dept. of Intrnl. Med., St. Louis Univ. Sch. of Med., St. Louis, MO<br />
Abstract: Previously, we found that exposure to chronic isolation stress signific<strong>an</strong>tly increased<br />
tissue amyloid-β (Aβ) levels, amyloid plaque deposition, <strong>an</strong>d impaired memory function in<br />
Tg2576 mice, a tr<strong>an</strong>sgenic mouse model of Alzheimer’s disease (AD) (Dong et al. 2004, 2008).<br />
Evidence that stress accelerates Aβ- levels <strong>an</strong>d impairs memory function has also been reported<br />
in o<strong>the</strong>r tr<strong>an</strong>sgenic mouse models of AD (Joel et al. 2006; Green et al. 2006). However, <strong>the</strong><br />
effects of stress in non-tr<strong>an</strong>sgenic mouse models of AD have not been addressed. Senescenceaccelerated<br />
prone mice (SAMP) are serial models of accelerated senescence established through<br />
phenotypic selection <strong>from</strong> a common genetic pool of <strong>the</strong> AKR/J strain of mice. SAMP8, one of<br />
<strong>the</strong> SAMP substrains, has been proposed as a model <strong>for</strong> AD, as <strong>the</strong>se <strong>an</strong>imals exhibit premature<br />
memory loss <strong>an</strong>d increased amyloid precursor protein (APP) expression. In this study, SAMP8<br />
<strong>an</strong>d control mice (total n= 48, 24 each) were r<strong>an</strong>domly assigned to undergo ei<strong>the</strong>r isolation or<br />
group housing. The 12 SAMP8 <strong>an</strong>d 12 control mice in <strong>the</strong> isolation stress group were<br />
individually housed in a cage 1/3 <strong>the</strong> size of a st<strong>an</strong>dard mouse cage <strong>from</strong> we<strong>an</strong>ing <strong>for</strong> 6 months.<br />
Non-isolated mice were housed in groups of 3 in a st<strong>an</strong>dard-sized cage. After 3 <strong>an</strong>d 6 months of<br />
isolation stress, tests of fear conditioning <strong>an</strong>d spatial reversal learning were per<strong>for</strong>med.<br />
Following completion of behavioral testing, <strong>the</strong> <strong>an</strong>imals were sacrificed to assess tissue Aβ<br />
levels, amyloid plaque deposition, protein carbonyl levels <strong>an</strong>d hippocampal volumes. There was<br />
a signific<strong>an</strong>t effect of housing condition on <strong>the</strong> capacity <strong>for</strong> contextual (F(1,32)=7.98; p=0.008)<br />
<strong>an</strong>d cued memory (F(1,32)=11.99; p=0.0015) after 3 months of isolation stress, <strong>an</strong>d on spatial<br />
reversal learning after 6 months of isolation stress (F(1,16)=7.79; p=0.013). The hippocampal<br />
volumes of <strong>the</strong> isolated <strong>an</strong>imals were signific<strong>an</strong>tly smaller th<strong>an</strong> those of <strong>the</strong> non-isolated <strong>an</strong>imals<br />
(F(1,20)= 96.34; p
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.5/G28<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Gr<strong>an</strong>t PI-2006/15<br />
Gr<strong>an</strong>t PCC08-0064<br />
Title: Somatostatin correlation with amyloid-β peptide in <strong>the</strong> olfactory system of <strong>the</strong><br />
Alzheimer’s double (APPswe, PSEN1dE9) tr<strong>an</strong>sgenic mice model<br />
Authors: *D. SAIZ 1 , C. DE LA ROSA-PRIETO 1 , I. UBEDA-BAÑON 1 , L. ARGANDOÑA-<br />
PALACIOS 2 , S. GARCIA-MUÑOZGUREN 2 , R. INSAUSTI 1 , A. MARTINEZ-MARCOS 1 ;<br />
1 Univ. de Castilla-La M<strong>an</strong>, Albacete (Albacete), Spain; 2 Complejo Hospitalario Universitario de<br />
Albacete, Servicio de Neurología., Albacete, Spain<br />
Abstract: Alzheimer disease is <strong>the</strong> most prevalent neurodegenerative syndrome in developed<br />
countries. Olfactory deficits are frequent in Alzheimer’s disease patients. Such dysfunctions<br />
might be due to early affectation of <strong>the</strong> olfactory system by amyloid-β (Aβ) peptide present in<br />
senile plaques. It has been recently described that somatostatin reduction in aging <strong>an</strong>d/or<br />
Alzheimer disease appears to unleash <strong>the</strong> down-regulation of neprylisin enzyme, which provokes<br />
<strong>the</strong> up-regulation in Aβ deposition. The olfactory system of double tr<strong>an</strong>sgenic mice<br />
(APPswe/PSEN1dE9, The Jackson Laboratory) has been investigated in order to <strong>an</strong>alyze <strong>the</strong><br />
correlation in <strong>the</strong> distribution of somatostatin <strong>an</strong>d Aβ peptide. The olfactory bulb, <strong>an</strong>terior<br />
olfactory nucleus, olfactory tubercle, piri<strong>for</strong>m, amygdaloid <strong>an</strong>d entorhinal cortices have been<br />
<strong>an</strong>alyzed in homozygote mice at 2, 4, 6 <strong>an</strong>d 8 months of age <strong>an</strong>d compared with <strong>the</strong><br />
corresponding controls. Experimental procedures were carried out according to <strong>the</strong> guidelines of<br />
<strong>the</strong> Europe<strong>an</strong> Community on Welfare of Research Animals (Directive 86/69/EEC) <strong>an</strong>d <strong>the</strong><br />
Ethical Committee of Animal Research of <strong>the</strong> University of Castilla-La M<strong>an</strong>cha (gr<strong>an</strong>t PI-<br />
2006/15). Immunofluorescence procedures were carried out: primary <strong>an</strong>tibodies (rabbit <strong>an</strong>ti-Aβ,<br />
1:250, Cell Signaling <strong>an</strong>d goat <strong>an</strong>ti-somatostatin D-20, 1:1000, S<strong>an</strong>ta Cruz). Secondary<br />
<strong>an</strong>tibodies were ei<strong>the</strong>r biotinylated (1:200, Vector) or fluorescent (1:200, Alexas 488 donkey<br />
<strong>an</strong>ti-rabbit <strong>an</strong>d 568 donkey <strong>an</strong>ti-goat, respectively; Molecular Probes). Somatostatin positivecells<br />
were charted with <strong>an</strong> X-Y recording system (AccuStage, Minnesota Datametrics, MN,<br />
USA). Double immunofluorescence sections were visualized under a LSM 710 Zeiss confocal<br />
microscope (Carl Zeiss MicroImaging, Barcelona, Spain). Intensities of each fluorochrome were<br />
<strong>an</strong>alyzed using <strong>the</strong> profile tool of <strong>the</strong> ZEN software (Zeiss) to qu<strong>an</strong>titatively estimate doublelabeled<br />
cells. Aβ deposits are distributed in olfactory structures in early stages <strong>an</strong>d <strong>the</strong> expression<br />
is increased with age. The expression of somatostatin is compared in control <strong>an</strong>d homozygote<br />
<strong>an</strong>imals. In <strong>the</strong> latter, <strong>the</strong> expression of somatostatin is correlated with that of Aβ deposits at
different ages. Authors th<strong>an</strong>k Dr. Calvo <strong>for</strong> helping in genotyping <strong>an</strong>imals. Supported by <strong>the</strong><br />
Autonomous Government of Castilla-La M<strong>an</strong>cha-FEDER (Gr<strong>an</strong>ts PI-2006/15 <strong>an</strong>d PCC08-0064).<br />
Disclosures: D. Saiz, None; C. de la Rosa-Prieto, None; I. Ubeda-Bañon, None; L.<br />
Arg<strong>an</strong>doña-Palacios, None; S. Garcia-Muñozguren, None; R. Insausti, None; A. Martinez-<br />
Marcos, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.6/G29<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: JSPS Gr<strong>an</strong>t 20591469<br />
Title: Qu<strong>an</strong>titative evaluation of 11 C-PIB binding in amyloid precursor protein tr<strong>an</strong>sgenic mouse<br />
brain<br />
Authors: *C. SEKI, M. TOKUNAGA, S. HATTORI, M. SHIDAHARA, R. NAKAO, T.<br />
OKAUCHI, J. MAEDA, M. HIGUCHI, Y. KIMURA, T. SUHARA;<br />
Mol. Imaging Ctr., Natl. Inst. of Radiological Scienses, Chiba, Jap<strong>an</strong><br />
Abstract: Introduction 11 C-PIB is a widely-used PET lig<strong>an</strong>d to visualize amyloid β (Aβ)<br />
plaques in vivo. Although <strong>the</strong> nature of its binding properties is yet to be clarified, detailed<br />
qu<strong>an</strong>titative PET assays of living <strong>an</strong>imal models may provide critical insights into <strong>the</strong> molecular<br />
basis of <strong>the</strong> radiolig<strong>an</strong>d kinetics. Total volume of distribution (VT) derived <strong>from</strong> time-activity<br />
curves in <strong>the</strong> plasma (pTAC) <strong>an</strong>d brain (tTAC) is <strong>an</strong> indicator of <strong>the</strong> affinity of <strong>the</strong> lig<strong>an</strong>d <strong>for</strong> <strong>the</strong><br />
total binding sites in tissues. In this study, we compared regional VT values among aged wildtype<br />
(WT) <strong>an</strong>d amyloid precursor protein (APP) tr<strong>an</strong>sgenic (Tg) <strong>an</strong>d young WT mice to examine<br />
alterations of <strong>the</strong> lig<strong>an</strong>d kinetics in physiological aging <strong>an</strong>d pathological amyloidogenesis.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> applicability of reference tissue models that use only tTACs was assessed as<br />
more practical <strong>an</strong>alysis.<br />
Methods Female aged APP Tg (23-26 mo.; 20-29g; n=4) <strong>an</strong>d WT (25 mo.; 26g; n=1) <strong>an</strong>d male<br />
young control (YC) WT (9.7±1.8 wk; 24±2g; n=6) mice were studied. Simult<strong>an</strong>eously with <strong>the</strong><br />
intravenous 11 C-PIB (0.4-1.8 mCi) injection, 90-min PET data acquisition <strong>an</strong>d serial arterial<br />
blood sampling were started. For <strong>the</strong> blood samples, radioactivity per unit volume <strong>an</strong>d parent<br />
radiolig<strong>an</strong>d fraction in <strong>the</strong> plasma were measured to determine pTAC. Regional brain tTACs<br />
were obtained <strong>from</strong> dynamic PET data with <strong>the</strong> <strong>an</strong>atomical assist<strong>an</strong>ce of MRI. Regional VT was
estimated with compartment model <strong>an</strong>alysis (CMA) <strong>an</strong>d Log<strong>an</strong> <strong>an</strong>alysis (LA) 1 . For <strong>the</strong><br />
evaluation of <strong>the</strong> specific 11 C-PIB binding to Aβ plaques, VT ratio to <strong>the</strong> cerebellum (DVR) was<br />
also calculated using VT obtained with CMA <strong>an</strong>d LA. DVRs were also calculated by simplified<br />
reference tissue model (SRTM) 2 , <strong>an</strong>d were compared with those with CMA <strong>an</strong>d LA.<br />
Results <strong>an</strong>d Conclusion The 2-tissue model fitted tTACs in all regions including <strong>the</strong> cerebellum<br />
of Tg <strong>an</strong>d aged WT mice better th<strong>an</strong> did <strong>the</strong> 1-tissue model, whereas <strong>the</strong> 1-tissue model resulted<br />
in a better fit to <strong>the</strong> most tTACs of YC mice. Cerebellar VT values of Tg <strong>an</strong>d aged WT mice were<br />
higher th<strong>an</strong> those in YC mice. VT values in <strong>the</strong> neocortex <strong>an</strong>d hippocampus of Tg mice were<br />
elevated in consistency with <strong>the</strong> Aβ deposition. DVRs estimated with SRTM were well<br />
correlated with those determined with LA (r 2 =0.89) <strong>an</strong>d CMA (r 2 =0.88).<br />
The present data support <strong>the</strong> ability of qu<strong>an</strong>titative PET measurements in mice to distinctly<br />
demonstrate ch<strong>an</strong>ges in non-specific <strong>an</strong>d specific 11 C-PIB retentions related to aging <strong>an</strong>d amyloid<br />
pathology, respectively. The use of SRTM is also validated as a feasible, non-invasive <strong>an</strong>alysis<br />
to qu<strong>an</strong>titatively evaluate <strong>the</strong> interaction between 11 C-PIB <strong>an</strong>d Aβ plaques.<br />
1) Log<strong>an</strong> J, et al. J Cereb Blood Flow Metab, 1990.<br />
2) Lammertsma AA, et al., Neuroimage, 1996.<br />
Disclosures: C. Seki, None; M. Tokunaga, None; S. Hattori, None; M. Shidahara, None; R.<br />
Nakao, None; T. Okauchi, None; J. Maeda, None; M. Higuchi, None; Y. Kimura, None; T.<br />
Suhara, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.7/G30<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Intramural Research Program of <strong>the</strong> NIH, National Institute on Aging<br />
Title: Developing a non-hum<strong>an</strong> primate model of Alzheimer's disease using <strong>the</strong> noradrenergic<br />
neurotoxin DSP-4<br />
Authors: *K. B. DUFFY 1,2 , K. HAGELIN 2 , G. P. TINKLER 3 , D. K. INGRAM 4 , J. A.<br />
MATTISON 2 , M. OTTINGER 1 ;<br />
1 Dept. of Animal Science, Univ. of Maryl<strong>an</strong>d, College Park, MD; 2 Lab. of Exptl. Gerontology,<br />
NIA/NIH, Poolesville, MD; 3 Dept. of Psychology, Univ. of Iowa, Iowa City, IA; 4 Pennington<br />
Biomed. Res. Ctr., Louisi<strong>an</strong>a State Univ., Baton Rouge, LA
Abstract: The cholinergic neurotr<strong>an</strong>smitter system has been implicated as a key component of<br />
<strong>the</strong> neural degeneration in Alzheimer’s disease; however <strong>the</strong> involvement of catecholaminergic<br />
systems is less clear. Deregulation of this system may influence <strong>the</strong> development <strong>an</strong>d progression<br />
of Alzheimer’s disease (AD), specifically in patients who do not carry <strong>the</strong> gene mutations<br />
involved in familial AD. The locus coeruleus (LC) is <strong>the</strong> main site <strong>for</strong> norepinephrine (NE)<br />
syn<strong>the</strong>sis; degeneration results in decreased NE along with possible alterations in <strong>the</strong> o<strong>the</strong>r<br />
catecholamines. LC degeneration is a well-documented feature of AD; however its role remains<br />
unclear. Currently no <strong>an</strong>imal model exists <strong>for</strong> idiopathic AD, which accounts <strong>for</strong> <strong>the</strong> majority of<br />
all hum<strong>an</strong> cases. Common models of AD include tr<strong>an</strong>sgenic mice expressing select atypical<br />
hum<strong>an</strong> genes resulting in production of AD-like pathology <strong>an</strong>d pharmacological m<strong>an</strong>ipulations<br />
targeting specific neurotr<strong>an</strong>smitter systems thought to be involved in <strong>the</strong> development of AD.<br />
The purpose of this study was to ascertain <strong>the</strong> effects of <strong>the</strong> selective noradrenergic neurotoxin,<br />
N (2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) in squirrel monkeys. In rodents, DSP-4<br />
triggers biochemical <strong>an</strong>d <strong>an</strong>atomical ch<strong>an</strong>ges in brain regions innervated by <strong>the</strong> LC; however<br />
chronic peripherally-induced NE depletion in non-hum<strong>an</strong> primates is not well established. Each<br />
monkey received <strong>an</strong> injection of 50 mg/kg DSP-4 or vehicle <strong>an</strong>d subsequent injections 3 months<br />
<strong>an</strong>d 6 months later (10 mg/kg DSP-4 or vehicle) to maintain NE depletion. Three months after<br />
<strong>the</strong> last injection, <strong>the</strong> monkeys were necropsied. Catecholamine content was evaluated by ELISA<br />
in several brain regions to assess NE depletion <strong>an</strong>d alterations in dopamine. Pilot data indicated<br />
>65% NE depletion in cortical <strong>an</strong>d limbic brain regions 30 days following injection of<br />
neurotoxin, while data <strong>from</strong> <strong>the</strong> long term study showed comparatively less depletion in adult<br />
<strong>an</strong>d aged <strong>an</strong>imals. More depletion was observed in aged <strong>an</strong>imals following exposure to DSP-4<br />
<strong>an</strong>d <strong>an</strong> age related decrease was noted in <strong>the</strong> LC of vehicle treated <strong>an</strong>imals. This study examines<br />
a mech<strong>an</strong>ism <strong>for</strong> influencing AD pathology development in squirrel monkeys which has been<br />
shown effective in o<strong>the</strong>r species. Non-hum<strong>an</strong> primates are <strong>an</strong> ideal c<strong>an</strong>didate <strong>for</strong> developing a<br />
model <strong>for</strong> <strong>the</strong> study of idiopathic AD because amyloid pathology occurs naturally late in life.<br />
Additionally, no current <strong>an</strong>imal model of AD exists with close similarity to hum<strong>an</strong>s across a<br />
wide array of physiological responses.<br />
Disclosures: K.B. Duffy, None; K. Hagelin, None; G.P. Tinkler, None; D.K. Ingram,<br />
None; J.A. Mattison, None; M. Ottinger, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.8/G31<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models
Support: CIHR Gr<strong>an</strong>t<br />
Title: Characterization of a novel tr<strong>an</strong>sgenic mouse model overexpressing mut<strong>an</strong>t hum<strong>an</strong> APP in<br />
<strong>the</strong> absence of <strong>the</strong> cholesterol trafficking Npc1 protein<br />
Authors: *M. MAULIK 1 , J. YANG 2 , J. KIM 2 , K. PEAKE 3 , J. E. VANCE 3 , D. WESTAWAY 2 ,<br />
S. KAR 2 ;<br />
1 Ctr. <strong>for</strong> Neurosci., 2 Ctr. <strong>for</strong> Prion <strong>an</strong>d Protein Folding Dis., 3 Group on Mol. <strong>an</strong>d Cell Biol. of<br />
Lipids, Dept. of Med., Univ. of Alberta, Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is<br />
characterized neuropathologically by <strong>the</strong> presence of extracellular β-amyloid (Aβ) containing<br />
neuritic plaques, intracellular phosphotau-positive neurofibrillary t<strong>an</strong>gles, <strong>an</strong>d loss of neurons in<br />
selected brain regions. The pathophysiology of AD is thought to result <strong>from</strong> <strong>the</strong> accumulation of<br />
Aβ peptides derived <strong>from</strong> <strong>the</strong> proteolytic cleavage of β-amyloid precursor protein (APP).<br />
Various in vitro as well as in vivo studies suggest that cholesterol plays a critical role in APP<br />
processing <strong>an</strong>d Aβ production. However, <strong>the</strong> precise mech<strong>an</strong>isms by which cholesterol<br />
modulates Aβ production/accumulation <strong>an</strong>d contributes to AD pathogenesis still remain unclear.<br />
To address this issue, we developed a new line of tr<strong>an</strong>sgenic (tg) mice by crossing mut<strong>an</strong>t hum<strong>an</strong><br />
APP tg (APP +/- ) mice, that recapitulate m<strong>an</strong>y features of AD with Niem<strong>an</strong>n Pick type C1<br />
knockout (Npc1 -/- ) mice, which show intracellular cholesterol accumulation. APP +/- ,Npc1 -/-<br />
double mut<strong>an</strong>ts (ANPC-tg) exhibit a slower growth rate, higher brain-to-body ratio <strong>an</strong>d mortality<br />
th<strong>an</strong> ei<strong>the</strong>r APP +/- or Npc1 -/- single mut<strong>an</strong>ts. They also display cognitive <strong>an</strong>d motor deficits as<br />
evident <strong>from</strong> <strong>the</strong>ir poor per<strong>for</strong>m<strong>an</strong>ce in object recognition memory <strong>an</strong>d rotarod tests,<br />
respectively. At all major brain regions studied, <strong>the</strong> ANPC-tg mice exhibit intra-neuronal<br />
cholesterol accumulation like <strong>the</strong> Npc1 -/- mice <strong>an</strong>d also show enh<strong>an</strong>ced neuropathological<br />
ch<strong>an</strong>ges including Aβ plaques, neuronal loss, reactive gliosis <strong>an</strong>d myelin degradation.<br />
Collectively, <strong>the</strong>se results indicate that overexpression of mut<strong>an</strong>t hum<strong>an</strong> APP in <strong>the</strong> absence of<br />
<strong>the</strong> cholesterol trafficking protein Npc1 exacerbates AD pathology. The ANPC-tg mice<br />
constitute a novel <strong>an</strong>imal model to study <strong>the</strong> impact of aberr<strong>an</strong>t intra-neuronal cholesterol<br />
distribution on APP biology. [Supported by C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research]<br />
Disclosures: M. Maulik, None; J. Y<strong>an</strong>g, None; J. Kim, None; K. Peake, None; J.E. V<strong>an</strong>ce,<br />
None; D. Westaway, None; S. Kar, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.9/G32
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t 1 RO1 NS55118<br />
Title: A behavioral characterization of 5XFAD mouse model of Alzheimer’s disease<br />
Authors: *W. J. XU, M. E. ANDERSON, A. MANSOURI, W. E. VAN NOSTRAND, J. K.<br />
ROBINSON;<br />
Stony Brook Univ., Stony Brook, NY<br />
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by <strong>the</strong><br />
presence of amyloid plaques, neurofibrillary t<strong>an</strong>gles, <strong>an</strong>d subsequent loss of neurons in <strong>the</strong> brain.<br />
It affects a large percentage of <strong>the</strong> elderly population <strong>an</strong>d currently <strong>the</strong>re is no cure. A number of<br />
tr<strong>an</strong>sgenic mouse models of <strong>the</strong> disease have been developed addressing different aspects of <strong>the</strong><br />
pathology. One of <strong>the</strong> newer strains of <strong>the</strong>se mice, <strong>the</strong> 5XFAD, co-expresses mut<strong>an</strong>t <strong>for</strong>ms of<br />
both hum<strong>an</strong> APP <strong>an</strong>d PS1. A growing literature exists on <strong>the</strong> histological m<strong>an</strong>ifestations of <strong>the</strong>se<br />
genes in <strong>the</strong> 5XFAD. However, detailed behavioral characterizations of this strain had been<br />
lacking. The present study sought to address this issue by examining <strong>the</strong> 5XFAD mouse in a<br />
battery of behavioral tasks at early <strong>an</strong>d late adult time points. Spatial memory impairment was<br />
evident in <strong>the</strong> 5XFAD when compared to age-matched wild-type <strong>an</strong>imals in <strong>the</strong> absence of<br />
pronounced motor or exploratory deficits. These data suggest that <strong>the</strong> 5XFAD strain not only<br />
exhibits <strong>the</strong> amyloid pathology of AD but also <strong>the</strong> behavioral m<strong>an</strong>ifestations of <strong>the</strong> disorder.<br />
Disclosures: W.J. Xu, None; M.E. Anderson, None; A. M<strong>an</strong>souri, None; W.E. V<strong>an</strong><br />
Nostr<strong>an</strong>d, None; J.K. Robinson, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.10/G33<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIA<br />
Title: APP/Aβ expression in cortical input areas elicits molecular alterations within gr<strong>an</strong>ule cells<br />
of <strong>the</strong> dentate gyrus <strong>an</strong>d behavioral abnormalities
Authors: *J. A. HARRIS 1,2 , N. DEVIDZE 1 , M. T. THWIN 1 , T. HAMTO 1 , D. KIM 1 , K. HO 1 ,<br />
G.-Q. YU 1 , L. MUCKE 1,2 ;<br />
1 Gladstone Inst. Neurol Dis, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 2 Neurol., UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: In Alzheimer’s disease (AD), gr<strong>an</strong>ule cells of <strong>the</strong> dentate gyrus are vulnerable to<br />
APP/Aβ-induced molecular <strong>an</strong>d functional impairments. Aβ may exert <strong>the</strong>se effects within<br />
gr<strong>an</strong>ule cells, extracellularly after being released <strong>from</strong> gr<strong>an</strong>ule cells or synaptic contacts, or by<br />
affecting neurons in dist<strong>an</strong>t regions that provide afferent input to gr<strong>an</strong>ule cells. Our goal is to<br />
identify where APP/Aβ first acts within <strong>the</strong> vulnerable entorhinal-hippocampal network to<br />
ultimately elicit network dysfunction <strong>an</strong>d molecular <strong>an</strong>d behavioral deficits. To dissect <strong>the</strong> role<br />
of APP/Aβ expression in different, but interconnected, brain regions, we used tr<strong>an</strong>sgenic mice<br />
expressing APP primarily in layer II/III pyramidal cells of <strong>the</strong> medial entorhinal cortex (EC),<br />
which provides <strong>the</strong> primary excitatory afferent input to gr<strong>an</strong>ule cells of <strong>the</strong> dentate gyrus. No<br />
APP expression was detected in dentate gr<strong>an</strong>ule cells of <strong>the</strong>se mice. At 4 <strong>an</strong>d 9 months of age,<br />
tr<strong>an</strong>sgenic mice showed signific<strong>an</strong>t alterations in several behavioral assays characteristic of o<strong>the</strong>r<br />
mouse models of AD, including hyperactivity in <strong>the</strong> open field, disinhibition in <strong>the</strong> elevated plus<br />
maze, <strong>an</strong>d spatial memory deficits in <strong>the</strong> Morris water maze. At 6 months of age, tr<strong>an</strong>sgenic mice<br />
had molecular alterations in gr<strong>an</strong>ule cells that were previously shown to correlate with<br />
behavioral deficits in tr<strong>an</strong>sgenic mice with p<strong>an</strong>neuronal expression of APP, including reductions<br />
in calbindin <strong>an</strong>d fos levels. In <strong>the</strong> tr<strong>an</strong>sgenic mice with restricted APP expression, amyloid<br />
plaque deposition was already extensive at 6 months of age in cortical regions that expressed<br />
APP, but very little to no Aβ was deposited within <strong>the</strong> dentate gyrus at this age. These results<br />
suggest that molecular <strong>an</strong>d functional deficits within gr<strong>an</strong>ule cells of <strong>the</strong> hippocampus are due to<br />
a tr<strong>an</strong>s-synaptic effect of Aβ. Whe<strong>the</strong>r <strong>the</strong>se deficits are due to a soluble, synaptically released<br />
species of Aβ acting directly on gr<strong>an</strong>ule cells, or to ch<strong>an</strong>ges in afferent input induced by Aβ in<br />
<strong>the</strong> entorhinal cortex remains to be determined. Underst<strong>an</strong>ding where Aβ first triggers network<br />
dysfunction may enable <strong>the</strong> design of new strategies to better protect <strong>the</strong> most vulnerable brain<br />
regions in AD patients.<br />
Disclosures: J.A. Harris , None; N. Devidze, None; M.T. Thwin, None; T. Hamto, None; D.<br />
Kim, None; K. Ho, None; G. Yu, None; L. Mucke, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.11/G34<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models
Support: Alzheimer's Association<br />
APA Diversity Program in Neuroscience<br />
Title: Memory <strong>for</strong>mation, <strong>for</strong>ebrain neuronal number <strong>an</strong>d amyloid burden in Tg2576 mice<br />
lacking alpha7 nicotinic acetylcholine receptors<br />
Authors: *C. M. HERNANDEZ, W. SONG, K. T. DINELEY;<br />
Neurol., Univ. of Texas Med. Br., Galveston, TX<br />
Abstract: Alpha7 nicotinic acetylcholine receptors (nAChRs) are expressed within <strong>the</strong> septohippocampal<br />
<strong>an</strong>d baso-cortical cholinergic system. These long projections <strong>an</strong>d <strong>the</strong>ir respective<br />
targets are particularly vulnerable in Alzheimer’s disease (AD). AD is a neurodegenerative<br />
disease marked by amyloid plaques, presynaptic cholinergic dysfunction, loss of neuropil, <strong>an</strong>d a<br />
decrease in nAChR density in <strong>the</strong> cortex <strong>an</strong>d hippocampus. We tested <strong>the</strong> role that alpha7<br />
nAChRs per<strong>for</strong>m in <strong>the</strong> etiology of AD by genetically deleting <strong>the</strong> alpha7 nAChR subunit <strong>from</strong><br />
<strong>the</strong> Tg2576 mouse model <strong>for</strong> AD. Thus, Tg2576 mice tr<strong>an</strong>sgenic <strong>for</strong> mut<strong>an</strong>t hum<strong>an</strong> amyloid<br />
precursor protein (APP) were crossed with alpha7 nAChR null mice (A7KO) in order to render<br />
<strong>an</strong> <strong>an</strong>imal with elevated amyloid-beta in <strong>the</strong> absence of alpha7 nAChRs (A7KO-APP). Studies in<br />
our lab have demonstrated that <strong>an</strong> early onset fear conditioning learning deficit observed in <strong>the</strong><br />
Tg2576 mice is more severe in <strong>the</strong> A7KO-APP <strong>an</strong>imals; we are currently evaluating <strong>the</strong> behavior<br />
of <strong>the</strong>se mice in additional learning <strong>an</strong>d memory tasks. Fur<strong>the</strong>rmore, biochemical experiments in<br />
<strong>the</strong>se mice demonstrate that <strong>the</strong> A7KO-APP mouse has half <strong>the</strong> hippocampal amyloid-beta load<br />
as Tg2576 mice by 5 months of age. As such, experiments are in progress to evaluate <strong>an</strong>d<br />
compare <strong>the</strong> plaque pathology (percent load, number of plaques <strong>an</strong>d size of individual plaques)<br />
in <strong>for</strong>ebrain regions of aged (12+ months) <strong>an</strong>imals. Preliminary data suggest that amyloid burden<br />
“hotspots” <strong>for</strong> both groups of mice are within subregions of <strong>the</strong> hippocampus <strong>an</strong>d temporal<br />
cortex. In addition, comparisons of neuron counts are being completed <strong>an</strong>d suggest that A7KO-<br />
APP mice exhibit a signific<strong>an</strong>t hippocampal neuron loss with age versus APP mice. Thus, in a<br />
milieu of elevated amyloid-beta, loss of alpha7 nAChRs triggers <strong>the</strong> types of neurodegenerative<br />
ch<strong>an</strong>ges that are a hallmark of AD. Similar to AD, <strong>the</strong>se pathologic ch<strong>an</strong>ges likely contribute to<br />
fur<strong>the</strong>r decline in cognitive function.<br />
Disclosures: C.M. Hern<strong>an</strong>dez, None; W. Song, None; K.T. Dineley, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.12/G35
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: A pilot study of amyloid beta in rhesus monkey brain, CSF <strong>an</strong>d plasma<br />
Authors: Q. ZHAO 1 , Q. CAI 2 , L. ZHU 1 , J. LU 1 , S. LI 1 , *S. JIAO 3,1 , J. WANG 1 ;<br />
1 2 3<br />
Lab. Animal Sci., E/M Lab., Capital Med. Univ., Beijing, China; Tarcine BioMed Inc.,<br />
Beijing, China<br />
Abstract: Accumulation of <strong>the</strong> amyloid beta (Aβ) peptide in <strong>the</strong> brain plays <strong>an</strong> import<strong>an</strong>t role in<br />
<strong>the</strong> pathogenesis of Alzheimer’s disease (AD). Aβ40 <strong>an</strong>d Aβ42 levels were evaluated in brain,<br />
cerebral spinal fluid (CSF) <strong>an</strong>d plasma <strong>from</strong> 4 aged (19-23 yr) <strong>an</strong>d 6 young (5-10 yr) rhesus<br />
monkeys. CSF <strong>an</strong>d plasma of each <strong>an</strong>imal were assessed independently using a s<strong>an</strong>dwich<br />
ELISA. Concentrations of Aβ40 <strong>an</strong>d Aβ42 were calculated using assay st<strong>an</strong>dards on <strong>the</strong> same<br />
plate as a qu<strong>an</strong>tifiable technique. As <strong>an</strong>imals age, <strong>the</strong> amount of Aβ40/42 was signific<strong>an</strong>tly<br />
increased beginning at 19 years old. The immunohistochemical study of monkey brain was<br />
conducted to determine localization of Aβ deposits in <strong>the</strong> various regions including frontal,<br />
parietal, temporal cortex <strong>an</strong>d hippocampus. Senile plaques (SPs) were immunostained with<br />
<strong>an</strong>tibodies against Aβ40 <strong>an</strong>d Aβ42. A small number of Aβ40 stained scattered patches of<br />
gr<strong>an</strong>ular <strong>an</strong>d intracellular Aβ40 particles localized within cortical neurons <strong>an</strong>d glial cells<br />
regardless of <strong>the</strong> age of <strong>the</strong> monkey. Similarly, Aβ42 also immunostained cortical neurons <strong>an</strong>d<br />
large neurites, but mostly in aged monkeys. In <strong>the</strong> brains of aged monkeys, extracellular Aβ42<br />
plaques were mostly presented in <strong>the</strong> frontal lobe <strong>an</strong>d also intracellular gr<strong>an</strong>ular deposition of<br />
Aβ42 in <strong>the</strong> cortical neurons <strong>an</strong>d axons. The immuno-electron microscopy <strong>an</strong>alyses of <strong>the</strong> brain<br />
<strong>from</strong> a 23-year-old monkey fur<strong>the</strong>r revealed localization of <strong>the</strong> ultrastructure of Aβ deposits. Aβ<br />
fibrils were stained with <strong>an</strong>ti-rat IgG conjugated to 5 nm colloidal gold. Within brain sections<br />
labeled with Aβ40, <strong>the</strong> majority of colloidal gold particles existed in <strong>the</strong> cytoplasm <strong>an</strong>d <strong>the</strong><br />
nucleus of neurons <strong>an</strong>d glial cells, mostly located in <strong>the</strong> frontal lobe. In <strong>the</strong> sections labeled with<br />
Aβ42, most of colloidal gold were found in neurons <strong>an</strong>d glial cells in <strong>the</strong> frontal cortex <strong>an</strong>d<br />
hippocampus. The amount of Aβ42 tagged on <strong>the</strong> gold particles was higher th<strong>an</strong> that of Aβ40. A<br />
number of gold particles were engulfed by liposomes, which is related to <strong>the</strong> function of<br />
"dissidents" identification. In <strong>the</strong> cerebral cortex of all aged rhesus brains, Aβ deposits were also<br />
found in leptomeningial <strong>an</strong>d penetrating cortical vessels in <strong>the</strong> <strong>for</strong>m of amyloid <strong>an</strong>giopathy.<br />
These results suggest that intensive investigation of age-related ch<strong>an</strong>ges in Aβ deposits <strong>an</strong>d SPs<br />
spont<strong>an</strong>eously <strong>for</strong>m in <strong>the</strong> monkey brain will contribute to a better underst<strong>an</strong>ding of pathogenesis<br />
of Alzheimer’s disease.<br />
Disclosures: Q. Zhao, None; Q. Cai, None; L. Zhu, None; J. Lu, None; S. Li, None; S. Jiao,<br />
None; J. W<strong>an</strong>g, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.13/G36<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Characterization of Tg2576 mice on a 129S6 genetic background: Consistent behavioral<br />
deficits <strong>an</strong>d pathological markers of Alzheimer’s disease<br />
Authors: *N. R. RUSTAY 1 , E. A. CRONIN 2 , P. CURZON 2 , S. MARKOSYAN 2 , R. S.<br />
BITNER 2 , T. A. ELLIS 1 , J. F. WARING 1 , M. W. DECKER 2 , L. E. RUETER 2 , K. E.<br />
BROWMAN 2 ;<br />
1 Exptl. Imaging <strong>an</strong>d Biomarkers, 2 Neurosci., Abbott Labs., Abbott Park, IL<br />
Abstract: Tg2576 mice, which possess <strong>the</strong> hum<strong>an</strong> “Swedish” APP mutation, demonstrate both<br />
memory deficits in behavioral tasks as well as Aβ plaque pathology. These mice were originally<br />
constructed <strong>an</strong>d are routinely maintained on a mixed C57BL/6 X SJL genetic background. This<br />
heterogeneic background exhibits a high frequency of a retinal degeneration allele <strong>an</strong>d is prone<br />
to high variability in behavioral assays. The same APP mutation is also available maintained on a<br />
129S6 genetic background, providing more genetic homogeneity. However, little data are<br />
published regarding <strong>the</strong> effects of high levels of APP expression on behavior <strong>an</strong>d pathology in<br />
this background. We sought to determine whe<strong>the</strong>r tr<strong>an</strong>sgenic mice expressing <strong>the</strong> Swedish<br />
mutation on <strong>the</strong> 129S6 background show similar behavioral deficits as those on <strong>the</strong> mixed<br />
background. Separate groups of mice on <strong>the</strong> 129S6 background were tested at 3 different ages<br />
(6-7, 11-12, or 18-19 months) in 3 different behavioral assays (locomotor activity, Y-maze<br />
spont<strong>an</strong>eous alternation, <strong>an</strong>d contextual fear conditioning). Differences were detected between<br />
WT <strong>an</strong>d Tg mice in locomotor activity at 6-7 <strong>an</strong>d 18-19 months, Y-maze spont<strong>an</strong>eous alternation<br />
at 6-7 <strong>an</strong>d 11-12 months, <strong>an</strong>d contextual fear conditioning at 6-7, 11-12, <strong>an</strong>d 18-19 months. For<br />
comparison, Tg mice on <strong>the</strong> mixed B6/SJL background tested at 6-7 months only demonstrated<br />
signific<strong>an</strong>t impairment in <strong>the</strong> Y-maze in one of 2 cohorts tested, <strong>an</strong>d in <strong>the</strong> contextual fear<br />
conditioning assay. Tr<strong>an</strong>sgenic mice on both backgrounds were also compared <strong>for</strong> Aβ pathology.<br />
Despite <strong>the</strong> behavioral differences observed, similar Aβ pathology was observed between Tg<br />
mice on <strong>the</strong> two genetic backgrounds. These results indicate that mice on <strong>the</strong> 129S6 genetic<br />
background may generate more consistent <strong>an</strong>d robust behavioral differences, providing a useful<br />
model <strong>for</strong> testing novel <strong>the</strong>rapeutic agents <strong>for</strong> Alzheimer’s disease.<br />
Disclosures: N.R. Rustay, Abbott Laboratories, A. Employment (full or part-time); Abbott<br />
Laboratories, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
E.A. Cronin, Abbott Laboratories, A. Employment (full or part-time); P. Curzon, Abbott<br />
Laboratories, A. Employment (full or part-time); S. Markosy<strong>an</strong>, Abbott Laboratories, A.<br />
Employment (full or part-time); R.S. Bitner, Abbott Laboratories, A. Employment (full or parttime);<br />
T.A. Ellis, Abbott Laboratories, A. Employment (full or part-time); J.F. Waring, Abbott<br />
Laboratories, A. Employment (full or part-time); M.W. Decker, Abbott Laboratories, A.<br />
Employment (full or part-time); L.E. Rueter, Abbott Laboratories, A. Employment (full or parttime);<br />
K.E. Browm<strong>an</strong>, Abbott Laboratories, A. Employment (full or part-time).
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.14/G37<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: R01 AG-021982<br />
1F31NS063650-01A1<br />
Title: Tr<strong>an</strong>sient cerebral hypoperfusion divergently alters Aβ <strong>an</strong>d tau pathology<br />
Authors: *M. KOIKE, K. N. GREEN, M. BLURTON-JONES, F. M. LAFERLA;<br />
UCI, Irvine, CA<br />
Abstract: Alzheimer’s Disease (AD) is a progressive, neurodegenerative disorder <strong>an</strong>d <strong>the</strong> most<br />
common cause of dementia among <strong>the</strong> elderly [1]. M<strong>an</strong>y successful <strong>an</strong>imal models have been<br />
developed to study AD. Although <strong>the</strong>se models have led to import<strong>an</strong>t insights, it is rare that<br />
patients develop AD alone. Over half of AD patients suffer <strong>from</strong> 3 or more co-morbid diseases<br />
[2]; hence, it is imperative to develop models investigating <strong>the</strong> impact of co-morbidities on AD.<br />
A number of clinical studies have found that cerebral hypoperfusion injuries are associated with<br />
higher levels of amyloid-beta (A) <strong>an</strong>d decreased tau, both pathological proteins associated with<br />
AD [3-5]. However, <strong>the</strong> relationship between <strong>the</strong>se co-morbidities <strong>an</strong>d AD development is<br />
largely unknown. We developed <strong>an</strong> oligemic model of cerebral hypoperfusion in <strong>the</strong> 3xTg-AD<br />
Alzheimer’s disease mouse model, by bilaterally occluding <strong>the</strong> common carotid artery <strong>for</strong> 4<br />
minutes <strong>the</strong>n allowing <strong>the</strong> blood flow to return. This injury did not result in cerebral cell death at<br />
<strong>an</strong>y of <strong>the</strong> 3 time points we assessed, 48-hours, 3-weeks <strong>an</strong>d 3-months post-injury, 48-hours, 3weeks<br />
or 3-months post insult. We found that this mild, tr<strong>an</strong>sient, cerebral-hypoperfusion insult<br />
acutely increases A as measured by enzyme-linked immunosorbent assay. However, this same<br />
oligemic insult causes a dramatic decrease in total tau by immunohistochemistry <strong>an</strong>d western<br />
blot <strong>an</strong>alysis, accurately modeling <strong>the</strong> hum<strong>an</strong> clinical condition. Fur<strong>the</strong>rmore, we found that <strong>the</strong><br />
decrease in total tau levels were coincident with elevated autophagy markers, indicating that <strong>the</strong><br />
autophagolysosomal pathway may be involved in <strong>the</strong> tau clear<strong>an</strong>ce we observed. This pattern of<br />
altered tau <strong>an</strong>d A was maintained <strong>for</strong> 3-weeks but reverted to baseline <strong>for</strong> both proteins by 3months<br />
post-oligemia. The long lasting, divergent response of Aß <strong>an</strong>d tau pathology in our mice<br />
mimics <strong>the</strong> hum<strong>an</strong> clinical data, indicating <strong>the</strong> possibility that cerebral hypoperfusion may<br />
exacerbate <strong>the</strong> ch<strong>an</strong>ges in AD-related pathology in patients.<br />
1. Breteler, M.M. Neurobiol Aging, 2000. 21(2): p. 153-60.
2. Doraiswamy, P.M., et al. J Gerontol A Biol Sci Med Sci, 2002. 57(3): p. M173-7.<br />
3. Snowdon, D.A., et al. Jama, 1997. 277(10): p. 813-7.<br />
4. Goulding, J.M., et al. J Neurol Neurosurg Psychiatry, 1999. 67(5): p. 654-7.<br />
5. Tesco, G., et al. Neuron, 2007. 54(5): p. 721-37.<br />
Disclosures: M. Koike , None; K.N. Green, None; M. Blurton-Jones, None; F.M. LaFerla,<br />
None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.15/G38<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: EU FP6-037831<br />
EU HEALTH-F2-2007-201159<br />
Sigrid Juselius Foundation<br />
Academy of Finl<strong>an</strong>d<br />
Title: Overexpression of truncated TrkB brain derived neurotrophic factor receptor has a strong<br />
phenotype but does not interact with Alzheimer’s disease associated APP/PS1 tr<strong>an</strong>sgene in mice<br />
Authors: *H. J. TANILA 1 , S. KEMPPAINEN 1 , T. RANTAMAKI 2 , E. KARKKAINEN 1 , S.<br />
STAVEN 1 , E. CASTREN 2 ;<br />
1 Univ. Kuopio, Kuopio, Finl<strong>an</strong>d; 2 Neurosci. Ctr., Univ. of Helsinki, Helsinki, Finl<strong>an</strong>d<br />
Abstract: Background: Brain derived neurotrophic factor BDNF is one of <strong>the</strong> main regulators<br />
of neurogenesis <strong>an</strong>d neurite out growth <strong>an</strong>d plays also <strong>an</strong> import<strong>an</strong>t role in neuronal plasticity.<br />
Levels of BDNF <strong>an</strong>d its main receptor TrkB are reported to decrease in Alzheimer’s disease<br />
(AD). The present study modeled this situation by crossing APPswe/PS1dE9 tr<strong>an</strong>sgenic mice<br />
(AP) with a tr<strong>an</strong>sgenic line overexpressing <strong>the</strong> truncated <strong>for</strong>m of TrkB-receptor (T1).<br />
Methods: Mice were tested at <strong>the</strong> age of 12 months in Morris water maze, object recognition test<br />
as well as <strong>for</strong> <strong>the</strong>ir spont<strong>an</strong>eous exploratory activity in a new environment. At <strong>the</strong> end of <strong>the</strong><br />
study <strong>the</strong> mice were perfused <strong>an</strong>d brains were collected <strong>for</strong> ei<strong>the</strong>r immunohistochemistry or<br />
biochemical <strong>an</strong>alyses.
Results: AP genotype was strongly associated with impaired spatial learning <strong>an</strong>d memory but<br />
had no effect on object recognition. In contrast, T1 genotype had no signific<strong>an</strong>t effect on spatial<br />
memory but was associated with robust decrease in object exploration <strong>an</strong>d recognition memory.<br />
Both genotypes affected exploratory activity, such that AP genotype increase while T1 genotype<br />
decrased activity. No signific<strong>an</strong>t interactions were observed between <strong>the</strong>se two genetic factors.<br />
T1 genotype did not affect <strong>the</strong> extent of amyloid pathology in AP mice. In AP carrying mice,<br />
cortical BDNF levels were increased independent of <strong>the</strong>ir T1 status. Both AP <strong>an</strong>d T1 genotype<br />
associated with reduced neurogenesis in <strong>the</strong> dentate gyrus, which correlated with reduced<br />
exploratory activity in T1 carrying mice.<br />
Conclusions: Constitutive hypofunction of TrkB receptor results in a hypoactive phenotype,<br />
whereas APPswe/PS1dE9 mice show selective age-dependent memory loss.<br />
Disclosures: H.J. T<strong>an</strong>ila, None; S. Kemppainen, None; T. R<strong>an</strong>tamaki, None; E.<br />
Karkkainen, None; S. Staven, None; E. Castren, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.16/H1<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: KI Foundations: Loo <strong>an</strong>d H<strong>an</strong>s Osterm<strong>an</strong>s foundation<br />
The Swedish Brain Foundation<br />
The Gun <strong>an</strong>d Bertil Stohne´s foundation<br />
Title: Phosphorylation of tau <strong>an</strong>d related signal tr<strong>an</strong>sduction pathways in Tg2576 tr<strong>an</strong>sgenic<br />
mice<br />
Authors: *C. UNGER LITHNER, T. MUSTAFIZ, A. NORDBERG;<br />
Karolinska Institutet, Stockholm, Sweden<br />
Abstract: Although genetic <strong>an</strong>d biochemical studies have suggested a cardinal role <strong>for</strong> βamyloid<br />
(Aβ) in Alzheimer’s disease (AD), <strong>the</strong> underlying mech<strong>an</strong>ism(s) of how Aβ induces<br />
neurodegeneration is still unclear. In this study, Tg2576 mice were investigated at different time<br />
points, <strong>from</strong> 7 days to 15 months of age, to characterize <strong>the</strong> ch<strong>an</strong>ges in Aβ <strong>an</strong>d tau<br />
phosphorylation, as well as <strong>the</strong> signal tr<strong>an</strong>sduction pathways that might be involved following <strong>an</strong>
excess of Aβ in <strong>the</strong> brain. We have observed high Aβ levels in <strong>the</strong> cortex of Tg2576 mice at 7<br />
days of age, similar to <strong>the</strong> levels at 15 months. We also found <strong>an</strong> increase in tau phosphorylation<br />
at 7 <strong>an</strong>d 90 days of age followed by a decrease at 15 months of age in <strong>the</strong> Tg2576 mice<br />
compared to non-tr<strong>an</strong>sgenic controls. Our results indicates that GSK3β might be responsible <strong>for</strong><br />
<strong>the</strong> increased phosphorylation of tau, due to <strong>the</strong> reduced phosphorylation of GSK3β at Ser 9 . In<br />
addition, we continued to investigate <strong>the</strong> MAPK-pathways by characterizing <strong>the</strong> activity of p38<br />
<strong>an</strong>d JNK. Both p38 <strong>an</strong>d p-54 JNK activation were reduced at this early age compared to nontr<strong>an</strong>sgenic<br />
controls. The p38 activation was increased in <strong>an</strong> age-dependent m<strong>an</strong>ner in 7 to 90<br />
day-old Tg2576 mice followed by a reduction at 15 months of age, while <strong>the</strong>re was <strong>an</strong> increase<br />
in both p46 <strong>an</strong>d p54 JNK levels at 21day-old Tg2576 mice compared with 7day-old Tg2576<br />
mice which remained const<strong>an</strong>t even at 15 months of age. In conclusion, we show that Tg2576<br />
mice have early tau phosphorylation as well as ch<strong>an</strong>ges in GSK3β phosphorylation <strong>an</strong>d MAPK<br />
signal tr<strong>an</strong>sduction pathways. The results in this study might contribute to fur<strong>the</strong>r underst<strong>an</strong>ding<br />
of <strong>the</strong> complex interaction between Aβ <strong>an</strong>d tau, <strong>the</strong> two major neuropathological hallmarks of<br />
AD.<br />
Disclosures: C. Unger Lithner, None; T. Mustafiz, None; A. Nordberg, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.17/H2<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: PTDC/SAU-OSM/64093/2006<br />
SFRH/BPD/28853/2006<br />
Title: Tr<strong>an</strong>sthyretin <strong>an</strong>d amyloid beta peptide level regulation in Alzheimer’s disease: Evidence<br />
<strong>from</strong> a new tr<strong>an</strong>sgenic mouse model<br />
Authors: S. OLIVEIRA, *M. M. SARAIVA;<br />
Inst. Biol Molec Cell, Porto, Portugal<br />
Abstract: Several lines of evidence have been suggesting that tr<strong>an</strong>sthyretin (TTR) - a plasma<br />
<strong>an</strong>d cerebrospinal fluid (CSF)-circulating 55 kDa homotetrameric protein - has <strong>an</strong> import<strong>an</strong>t role<br />
in Alzheimer’s disease (AD). TTR <strong>an</strong>d o<strong>the</strong>r few CSF proteins (such as apolipoproteins E, J, <strong>an</strong>d<br />
A1) have been shown to bind soluble amyloid beta peptide (Aβ) in vitro <strong>an</strong>d to prevent Aβ <strong>from</strong>
<strong>for</strong>ming amyloid fibers. Fur<strong>the</strong>r in vitro studies have recently shown that TTR is able to cleave<br />
Aβ. Interestingly, decreased levels of TTR have been found in <strong>the</strong> CSF of elderly AD patients<br />
<strong>an</strong>d <strong>the</strong> levels of TTR in AD patients have been shown to be inversely correlated with amyloidplaque<br />
load.<br />
In order to elucidate <strong>the</strong> neuroprotective role of TTR in AD, we have developed a murine model<br />
of AD in a TTR deficient or null background - APPswe/PS1A246E/mTTR. Thioflavin S staining<br />
of brain sections showed that APPswe/PS1A246E/mTTR <strong>an</strong>imals bear brain amyloid deposits<br />
<strong>from</strong> as early as 5-6 months of age. Notably, at this time point, <strong>the</strong> levels of <strong>for</strong>mic acid-soluble<br />
Aβ1-42 are signific<strong>an</strong>tly increased in APPswe/PS1A246E/mTTR -/- <strong>an</strong>imals compared with both<br />
APPswe/PS1A246E/mTTR +/- <strong>an</strong>d APPswe/PS1A246E/mTTR +/+ littermate counterparts. Fur<strong>the</strong>r,<br />
<strong>the</strong> levels of <strong>for</strong>mic acid-soluble Aβ1-40 are also signific<strong>an</strong>tly increased in<br />
APPswe/PS1A246E/mTTR -/- mice compared with APPswe/PS1A246E/mTTR +/+ <strong>an</strong>imals.<br />
Determination of amyloid-plaque load by thioflavin S staining showed increased amyloid-plaque<br />
burden in APPswe/PS1A246E/mTTR -/- <strong>an</strong>imals. With regard to plasma levels of Aβ, we<br />
observed elevated levels of Aβ1-42 <strong>an</strong>d Aβ1-40 in APPswe/PS1A246E/mTTR -/- mice compared<br />
with both APPswe/PS1A246E/mTTR +/- <strong>an</strong>d APPswe/PS1A246E/mTTR +/+ . CSF <strong>an</strong>alyses also<br />
revealed that APPswe/PS1A246E/mTTR -/- mice present increased levels of Aβ1-42 <strong>an</strong>d Aβ1-40<br />
compared with APPswe/PS1A246E/mTTR +/- <strong>an</strong>imals.<br />
Our data clearly indicate elevated levels <strong>an</strong>d accelerated deposition of brain Aβ in <strong>the</strong> absence of<br />
TTR in this strain of mice, <strong>an</strong>d provide strong in vivo evidence <strong>for</strong> a role of TTR in <strong>the</strong><br />
regulation of brain Aβ levels.<br />
Disclosures: S. Oliveira, None; M.M. Saraiva, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.18/H3<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: CIHR to RQ<br />
CIHR to JM<br />
FRSQ-INSERM to RQ & SK<br />
Title: Age-dependent alterations of brain Apoptosis-Inducing Factor (AIF) expression in<br />
TgCRND8 tr<strong>an</strong>sgenic model of Alzheimer’s disease
Authors: *S. M. KRANTIC 1 , W. YU 2 , J.-G. CHABOT 2 , M. HILL 3 , J. MCLAURIN 3 , R.<br />
QUIRION 2 ;<br />
1 INSERM U29, Inst. Neurobiol Medit(INMED), Marseille, Fr<strong>an</strong>ce; 2 Dept. of Psychiatry, McGill<br />
Univ., Douglas Mental Hlth. Univ. Inst., Montreal, QC, C<strong>an</strong>ada; 3 Dept. of Med., Ctr. <strong>for</strong> Res. in<br />
Neurodegenerative Dis., Toronto, ON, C<strong>an</strong>ada<br />
Abstract: β-amyloid (Aβ) triggers neuronal death via Apotosis Inducing Factor (AIF) in vitro<br />
(Movsesy<strong>an</strong>, J Neurochem, 2004). This mitochondrial (62 kDa) protein is required <strong>for</strong> <strong>the</strong><br />
normal function of <strong>the</strong> respiratory chain: it is syn<strong>the</strong>sized as a 67 kDa precursor but triggers cell<br />
death when cleaved (57 kDa) <strong>an</strong>d tr<strong>an</strong>slocated to <strong>the</strong> nucleus (Kr<strong>an</strong>tic, Prog Neurobiol, 2007).<br />
We used tr<strong>an</strong>sgenic TgCRND8 mice (bearing Swedish KM670/671NL <strong>an</strong>d Indi<strong>an</strong>a V717F<br />
mutations in <strong>the</strong> Aβ-precursor protein gene) to assess in vivo whe<strong>the</strong>r Aβ burden <strong>an</strong>d plaque<br />
accumulation, characteristic <strong>for</strong> Alzheimer’s disease (AD), trigger cell death via AIF. TgCRND8<br />
mice display detectable neuronal death at 7 months (Bellucci, Neurobiol Dis, 2006) <strong>an</strong>d have<br />
plaque deposition pattern reflecting that seen in AD with cortex/hippocampus, striatum <strong>an</strong>d<br />
cerebellum being affected in 100% of <strong>an</strong>imals at 4, 6 <strong>an</strong>d 9 months of age, respectively (Christie<br />
et al, 2001). Our preliminary Western blot data indicate that <strong>the</strong> expression of AIF precursor <strong>an</strong>d<br />
57 kDa <strong>for</strong>m do not vary between age groups in <strong>the</strong> four brain regions studied. Fur<strong>the</strong>rmore, in<br />
<strong>the</strong> cortex <strong>an</strong>d cerebellum, <strong>the</strong>re were no differences in <strong>the</strong> expression of ei<strong>the</strong>r 62 or 57 kDa<br />
<strong>for</strong>ms. In contrast, levels of 62 kDa AIF in hippocampus <strong>an</strong>d striatum increase in 4- <strong>an</strong>d 6months<br />
aged TgCRND8 mice. Altoge<strong>the</strong>r, <strong>the</strong>se data suggest that AIF might contribute to <strong>the</strong> Aβ<br />
mediated-neuronal death by impinging on mitochondrial functions ra<strong>the</strong>r th<strong>an</strong> by nuclear<br />
tr<strong>an</strong>slocation of <strong>the</strong> 57 kDa <strong>for</strong>m.<br />
Disclosures: S.M. Kr<strong>an</strong>tic , None; W. Yu, None; J. Chabot, None; M. Hill, None; J.<br />
McLaurin, None; R. Quirion, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.19/H4<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t AG-05119<br />
NIH Gr<strong>an</strong>t NS-45093<br />
NIH Gr<strong>an</strong>t NS-33376
NIH Gr<strong>an</strong>t AG-12282<br />
Joseph Drown Foundation<br />
Stephen D. Bechtel Foundation<br />
Alzheimer’s Association<br />
Title: In vivo oxidative stress in brain of Alzheimer disease tr<strong>an</strong>sgenic mice: Requirement <strong>for</strong><br />
methionine 35 in amyloid β-peptide of hum<strong>an</strong> mut<strong>an</strong>t APP<br />
Authors: *M. LANGE 1,2 , R. SOWELL 1,2 , R. SULTANA 1,2 , D. BREDESEN 3 , D.<br />
BUTTERFIELD 1,2 ;<br />
1 2 3<br />
Chem., S<strong>an</strong>ders-Brown Ctr. on Aging, Univ. of Kentucky, Lexington, KY; Buck Inst. <strong>for</strong> Age<br />
Res., Novato, CA<br />
Abstract: Oxidative stress <strong>an</strong>d damage induced by Alzheimer disease (AD) pathology has been<br />
shown to disrupt protein <strong>an</strong>d lipid function, eventually leading to apoptosis. Of <strong>the</strong> 20 c<strong>an</strong>onical<br />
amino acids, methionine (Met) is one of <strong>the</strong> most easily oxidized. Specifically, Met35 of <strong>the</strong><br />
amyloid-β peptide (1-42) [Aβ(1-42)] (corresponding to hum<strong>an</strong> amyloid precursor protein (APP)<br />
residue 631) has been implicated in <strong>the</strong> oxidative <strong>an</strong>d neurotoxic properties of Aβ(1-42) in AD.<br />
As a small oligomer, Aβ(1-42) c<strong>an</strong> undergo a one-electron oxidation of <strong>the</strong> Met35 sulfur atom,<br />
leading to <strong>an</strong> S-centered free radical that gives rise to protein <strong>an</strong>d lipid oxidation in neurons. In<br />
vitro studies have demonstrated that substitution of <strong>the</strong> sulfur atom of Met35 with a CH2 group<br />
[norleucine (Nle)], abrogates <strong>the</strong> oxidative <strong>an</strong>d neurotoxic effects of <strong>the</strong> resulting Aβ(1-<br />
42)M35Nle peptide, while maintaining <strong>the</strong> same length <strong>an</strong>d hydrophobicity of Aβ(1-42).<br />
In <strong>the</strong> current study, 9 month-old tr<strong>an</strong>sgenic (Tg) PDAPP mice with <strong>the</strong> Swedish <strong>an</strong>d Indi<strong>an</strong>a<br />
mutations in hum<strong>an</strong> APP (APPSw,In) were used as in vivo models of AD. Additionally, 9<br />
month-old Tg mice matched <strong>for</strong> genetic background <strong>an</strong>d APPSw,In expression level, carrying <strong>an</strong><br />
additional M631L mutation substituting leucine (Leu) <strong>for</strong> Met at residue 35 of Aβ, were created<br />
to test <strong>the</strong> hypo<strong>the</strong>sis that Met35 is critical <strong>for</strong> oxidative stress in <strong>the</strong> PDAPP mouse model of<br />
AD. Because no codon <strong>for</strong> Nle exists, Leu (-CH(CH3)2) was used, <strong>for</strong> it has a naturally<br />
occurring codon <strong>an</strong>d exhibits similar length <strong>an</strong>d hydrophobicity to Met. Oxidative stress levels in<br />
brain of PDAPP, PDAPP(M631L), <strong>an</strong>d respective age-matched Non-Tg mice were measured as<br />
indexed by protein carbonyls, 3-nitrotyrosine, <strong>an</strong>d protein-bound 4-hydroxy-2-nonenal. Each<br />
parameter was signific<strong>an</strong>tly elevated in brain of PDAPP mice, indicating oxidative stress, when<br />
compared to brain <strong>from</strong> Non-Tg littermates of <strong>the</strong> same age. In contrast, <strong>an</strong>d consistent with <strong>the</strong><br />
in vitro studies, all indices of oxidative damage were completely prevented in PDAPP(M631L)<br />
mice compared to age-matched PDAPP mice. Moreover, no signific<strong>an</strong>t differences in <strong>the</strong> levels<br />
of APP, Aβ(1-42), Aβ(1-40), or <strong>the</strong> ratio of Aβ(1-42)/Aβ(1-40) were found between PDAPP <strong>an</strong>d<br />
PDAPP(M631L) mice, suggesting that loss of oxidative stress in vivo in brain of<br />
PDAPP(M631L) mice results solely <strong>from</strong> <strong>the</strong> Met35Leu mutation. This is <strong>the</strong> first in vivo study<br />
to demonstrate <strong>the</strong> requirement of <strong>the</strong> Aβ Met35 residue <strong>for</strong> oxidative stress in brain of a<br />
mammali<strong>an</strong> model of AD.
Disclosures: M. L<strong>an</strong>ge, None; R. Sowell, None; R. Sult<strong>an</strong>a, None; D. Bredesen, None; D.<br />
Butterfield, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.20/H5<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Study of <strong>the</strong> association between peripheral vascular disease <strong>an</strong>d Alzheimer's disease<br />
Authors: *Y. SHIH 1 , C.-W. LEE 2 , Y.-T. WU 3 , M.-J. JIANG 3 , Y.-M. KUO 3 ;<br />
1 Inst. of Cell Biol. <strong>an</strong>d Anatomy, Natl. Chung Kung Univ., Tain<strong>an</strong>, Taiw<strong>an</strong>; 2 Basic medical<br />
sciences, Natl. Cheng Kung Univ., Tain<strong>an</strong>, Taiw<strong>an</strong>; 3 Cell Biol. <strong>an</strong>d Anatomy, Natl. Chen Kung<br />
Univ., Tain<strong>an</strong>, Taiw<strong>an</strong><br />
Abstract: Alzheimer's disease (AD) is <strong>an</strong> age-related <strong>an</strong>d irreversible brain disorder. Two<br />
abnormal structures in <strong>the</strong> brain are <strong>the</strong> hallmarks of AD: amyloid plaques <strong>an</strong>d neurofibrillary<br />
t<strong>an</strong>gles. Plaques consist of largely insoluble deposits of beta-amyloid which snipped <strong>from</strong> a<br />
larger protein called amyloid precursor protein (APP). Plaques <strong>an</strong>d t<strong>an</strong>gles in <strong>the</strong> neuropil may<br />
affect neuronal function <strong>an</strong>d also contribute to <strong>the</strong> neuronal damage. Accumulated evidence<br />
suggests that cerebrovascular pathologies are early features of AD. Reduced blood flow, such as<br />
structural alterations, a<strong>the</strong>rosclerotic lesions, <strong>an</strong>d impaired hemodynamic responses has also been<br />
reported as one of <strong>the</strong> most consistent physiological deficits in AD.I hypo<strong>the</strong>size that peripheral<br />
artery disease will lead tocerebral vascular alteration <strong>an</strong>d cerebral amyloid <strong>an</strong>giopathy (CAA)<br />
<strong>for</strong>med. The aim of this study was to investigate association of peripheral artery disease,cerebral<br />
vascular alteration <strong>an</strong>d neuron degeneration in young L<strong>an</strong>yu Miniature Pig which under<br />
abdominal aortic <strong>an</strong>eurysm. Seven L<strong>an</strong>yu minipigs (age <strong>from</strong> 8 to 12 weeks) were divided into<br />
two groups. The experimental group received aortic coarctation with a Teflon-patch<br />
circumferentially wrapping around a segment of infrarenal abdominal aorta, approximately 2 cm<br />
away <strong>from</strong> <strong>the</strong> bifurcation of common iliac arteries. The sham operational control received aortic<br />
segment isolation only <strong>an</strong>d base line group didn’t receive surgical intervention. And observe <strong>the</strong><br />
morphology <strong>an</strong>d biochemistry ch<strong>an</strong>ges in each region of brain.It doesn’t demonstrate signific<strong>an</strong>t<br />
ch<strong>an</strong>ge in frontal cortex by H&E stain,pyridine enh<strong>an</strong>ce silver stain <strong>an</strong>d thioflavin-s stain<br />
between experimental group <strong>an</strong>d sham operational control in <strong>an</strong>terior inferior cortex.According<br />
to my result, it seems peripheral artery disease may not attend to cerebral vasculature alteration<br />
<strong>an</strong>d lead to CAA <strong>for</strong>med in a young individual. I suppose that aging might be <strong>the</strong> maintain risk<br />
factor to this process. And according to this result, I hypo<strong>the</strong>size that cerebral vascular alteration
caused by peripheral artery disease may not <strong>an</strong> beta-amyloid intermediate in early stage. In my<br />
future work, I will use aging <strong>an</strong>d peripheral artery disease mice to figure out this problem.<br />
Disclosures: Y. Shih, None; C. Lee, None; Y. Wu, None; M. Ji<strong>an</strong>g, None; Y. Kuo, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.21/H6<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Neurological impairment <strong>an</strong>d motor neuron degeneration in <strong>the</strong> spinal cord of Tg2576<br />
mouse model of Alzheimer disease<br />
Authors: *J.-S. SEO, Y.-H. LEEM, K.-W. LEE, P.-L. HAN;<br />
Div. N<strong>an</strong>o Sci., Ewha Wom<strong>an</strong>s Univ. Sch., Seoul, Republic of Korea<br />
Abstract: The tr<strong>an</strong>sgenic mice Tg2576 is widely used as a murine model of Alzheimer disease<br />
(AD). Tg2576 mice show AD-like pathophysiologic ch<strong>an</strong>ges, such as amyloid plaque deposits<br />
<strong>an</strong>d gliosis in <strong>the</strong> brain starting at 9-12 months of age <strong>an</strong>d <strong>the</strong>se ch<strong>an</strong>ges are accomp<strong>an</strong>ied with<br />
progressive memory impairments. In <strong>the</strong> present study, we demonstrate that Tg2576 mice<br />
display severe spinal cord deficits, which have not been yet described. Alzheimer disease (AD)<br />
mouse model Tg2576 mice at 10-16 months of age showed a severe defect in <strong>the</strong> hindlimb<br />
extension reflex <strong>an</strong>d displayed abnormal body trembling <strong>an</strong>d hindlimb tremors when suspended<br />
by <strong>the</strong> tail. The frequency <strong>an</strong>d severity of <strong>the</strong>se abnormalities overtly detected at 10 months of<br />
age, <strong>an</strong>d became gradually worsened. Histological <strong>an</strong>alyses showed that neuronal cells including<br />
cholinergic neurons in <strong>the</strong> lumbar cord of Tg2576 mice showed a severe reduction in number.<br />
Tg2576 mice showed high levels of Abeta <strong>an</strong>d gradual increase of oxidative stress in <strong>the</strong> spinal<br />
cord. Administration of curcumin, a natural compound with <strong>an</strong>ti-oxid<strong>an</strong>t property, notably<br />
reversed motor function deficits of Tg2576 mice. The neuronal loss in <strong>the</strong> lumbar cord was also<br />
rescued by curcumin. EM <strong>an</strong>alysis revealed that <strong>the</strong> numbers of <strong>the</strong> sciatic nerve fibers along<br />
with myelination were severely defective, which were partially rescued by curcumin. These<br />
results suggest that Tg2576 mice display severe degeneration of motor neurons in <strong>the</strong> spinal cord<br />
via high ROS accumulation <strong>an</strong>d produce associated motor function deficits.<br />
Key words: Motor neurons, spinal cord, ROS, Tg2576<br />
Disclosures: J. Seo, None; Y. Leem, None; K. Lee, None; P. H<strong>an</strong>, None.
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.22/H7<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: SNF310000-117806<br />
Title: Alzheimers disease-like neuropathology following in-utero infection in wild-type mice<br />
Authors: *I. KNUESEL, J. DOEHNER, A. MADHUSUDAN, K. BREU, J.-M. FRITSCHY;<br />
Univ. of Zurich, Zurich, Switzerl<strong>an</strong>d<br />
Abstract: M<strong>an</strong>y age-associated degenerative diseases are associated with abnormal<br />
proteinaceous aggregates, often in <strong>the</strong> <strong>for</strong>m of amyloid deposits. In Alzheimer’s disease (AD),<br />
beta-amyloid (Aβ) peptides are <strong>the</strong> major plaque components. The linkage of rare familial AD<br />
mutations, which all affect <strong>the</strong> proteolysis of amyloid precursor protein leading to increased<br />
production of aggregation-prone Aβ peptides, supports a central role of Aβ in AD<br />
pathophysiology. However, it is currently unknown which type of Aβ aggregate plays a primary<br />
causative part <strong>an</strong>d whe<strong>the</strong>r similar mech<strong>an</strong>isms also underlie late-onset, sporadic AD. There is<br />
growing experimental evidence that soluble Aβ species in <strong>the</strong> <strong>for</strong>m of pre-fibrillary oligomers<br />
may be import<strong>an</strong>t triggers of neuronal malfunction <strong>an</strong>d cell death. We have reported previously<br />
that normal aging in rodents <strong>an</strong>d primates is accomp<strong>an</strong>ied by loss of Reelin-expressing neurons<br />
<strong>an</strong>d accumulation of Reelin-enriched amyloid-like aggregates in <strong>the</strong> hippocampal <strong>for</strong>mation.<br />
These associated alterations correlate with age-related working memory impairments in wildtype<br />
mice. Moreover, a prenatal immune challenge resulted in plaque deposition at <strong>an</strong> earlier age<br />
compared to control mice, suggesting a role of early neurodevelopmental <strong>an</strong>d immunological<br />
abnormalities <strong>for</strong> aging-related neuropathology. Here, we extended <strong>the</strong>se investigations by<br />
examining 6 <strong>an</strong>d 15 months-old wild type mice that were exposed in-utero to <strong>the</strong> viral mimic <strong>an</strong>d<br />
cytokine releaser, polyinosinic:polycytidylic acid (PolyI:C), using biochemical <strong>an</strong>d<br />
immunohistochemical approaches, combined with immuno-electron microscopy to characterize<br />
<strong>the</strong> impact of a prenatal infection on Aβ <strong>an</strong>d neurofibrillary t<strong>an</strong>gle pathology. We report a<br />
signific<strong>an</strong>t reduction in Reelin expression accomp<strong>an</strong>ied by <strong>the</strong> <strong>for</strong>mation of Thioflavine Spositive<br />
<strong>an</strong>d Aβ-immunoreactive extracellular deposits selectively in <strong>the</strong> hippocampal <strong>for</strong>mation<br />
of PolyI:C-exposed mice. At 6 months of age, <strong>the</strong> area covered by Reelin- <strong>an</strong>d Aβ-positive<br />
deposits in <strong>the</strong> brain was signific<strong>an</strong>tly enlarged as compared to saline controls. In 15 months-old<br />
subjects, a fur<strong>the</strong>r aggravation of <strong>the</strong> amyloid pathology associated with abnormal levels of<br />
phoshorylated tau was observed following PolyI:C-exposure, suggesting that <strong>an</strong> in-utero
infection induces long-term immunological abnormalities favoring AD-like neuropathology in<br />
normal wild type mice.<br />
Disclosures: I. Knuesel, None; J. Doehner, None; A. Madhusud<strong>an</strong>, None; K. Breu, None; J.<br />
Fritschy, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.23/H8<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Behavioral <strong>an</strong>d histological characteristics of 192 IgG-saporin injected rats depending on<br />
injection site <strong>an</strong>d dose<br />
Authors: *D. JEONG 1 , Y. HWANG 2 , D. LEE 2 , J. CHANG 2 ;<br />
1 2<br />
Brain Korea 21 Project Med. Sci.&Yonsei Univ. Coll. of Med., Yonsei Univ. Coll. of Med.,<br />
Seoul, Republic of Korea<br />
Abstract: Cholinergic neuronal deficits are evident in both Alzheimer’s disease dementia (AD)<br />
<strong>an</strong>d vascular dementia (VaD). Forebrain Cholinergic neurons in <strong>the</strong> nucleus basalis<br />
magnocellularis (NBM) project primarily to <strong>the</strong> neocortex, <strong>an</strong>d those in <strong>the</strong> medial septum<br />
project to <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong>y make <strong>an</strong> import<strong>an</strong>t role in memory function. We used 192<br />
IgG-saporin to mimic deficits of cholinergic neurons at AD <strong>an</strong>d VaD. 192 IgG-saporin is<br />
composed with monoclonal <strong>an</strong>tibody had a low affinity to <strong>the</strong> rat nerve growth factor receptor<br />
p75 <strong>an</strong>d ribosomal inactivating protein, called saporin. <strong>When</strong> injected intracerebroventricularly<br />
or directly into <strong>the</strong> basal <strong>for</strong>ebrain cholinergic complex, 192 IgG-saporin selectively destroys<br />
cholinergic neurons. M<strong>an</strong>y experimenters had used 192 IgG-saporin to investigate cholinergic<br />
function but it had been used in different doses <strong>an</strong>d sites of lesion. This makes it difficult to<br />
compare <strong>the</strong> degrees of impairment produced by different lesions.<br />
Consequently, our aim is observation of behavioral <strong>an</strong>d histological ch<strong>an</strong>ges depending on<br />
injection site <strong>an</strong>d dose of 192 IgG-saporin. We injected 192 IgG-saporin (0.63ug/ul) in medial<br />
septum (dose: 0.05ul, 0.1ul, 0.2ul) or lateral ventricle (dose: 6ul, 8ul, 10ul). 192 IgG-saporin<br />
injected rats were compared with Dulbecco’s phosphate buffered saline injected rats.<br />
Neurological deficit <strong>an</strong>d functional outcome were determined by immuohistochemistry using<br />
<strong>an</strong>ti-cholineacetyltr<strong>an</strong>sferase <strong>an</strong>tibody <strong>an</strong>d behavioral test, called water maze.<br />
In immunohistological study, <strong>the</strong> extent of <strong>the</strong> cholinergic lesion was showed in <strong>the</strong> basal<br />
<strong>for</strong>ebrain complex region of 8ul <strong>an</strong>d 10ul of 192 IgG-saporin injected rats. In behavioral study,
sham <strong>an</strong>d lesion groups were able to learn <strong>the</strong> reference aspect of <strong>the</strong> water maze within 5day of<br />
training. In probe test, we observed signific<strong>an</strong>t decrease in time in target quadr<strong>an</strong>t, plat<strong>for</strong>m <strong>an</strong>d<br />
plat<strong>for</strong>m crossings, <strong>an</strong>d increase in latency to first crossing at 8ul <strong>an</strong>d 10ul of 192 IgG-sapoin<br />
injected rats (p
points (3, 6, 9, 12 <strong>an</strong>d 18 months). We also <strong>an</strong>alyzed <strong>the</strong> total number of 5-HT neurons within<br />
<strong>the</strong> raphe nuclei.<br />
3xTg-AD <strong>an</strong>imals showed a signific<strong>an</strong>t increase in SERT fibres within <strong>the</strong> hippocampus in a<br />
subfield, strata <strong>an</strong>d age specific comportment. This increase was restricted to hippocampal CA1<br />
stratum lacunosum moleculare when compared to control. No ch<strong>an</strong>ges were observed in <strong>an</strong>y of<br />
<strong>the</strong> o<strong>the</strong>r hippocampal subfields <strong>an</strong>d/or layers. This increase appear early in life (3 months,<br />
61%), partially recovering through midlife but strongly reappearing later in life (18 months)<br />
accounting <strong>for</strong> a 74% increase when compared to control <strong>an</strong>imals. Ultrastructurally, we observed<br />
that this increase in SERT fibre density was associated with <strong>an</strong> increase in <strong>the</strong> area density of<br />
SERT-positive axon terminals at both 3 <strong>an</strong>d 18 months of age (7 <strong>an</strong>d 4 fold increase,<br />
respectively); but without <strong>an</strong>y evident synaptic contact. In addition, no signific<strong>an</strong>t ch<strong>an</strong>ges were<br />
found in <strong>the</strong> total number of 5-HT neurons within raphe nuclei at <strong>an</strong>y age; suggesting ei<strong>the</strong>r a 5-<br />
HT fibre sprouting or a syn<strong>the</strong>sis increase. Our results suggest that <strong>an</strong> imbal<strong>an</strong>ce of 5-HT<br />
neurotr<strong>an</strong>smission might contribute to <strong>the</strong> cognitive deficits associated with AD.<br />
Disclosures: H. Norist<strong>an</strong>i, None; M. Olabarria, None; A. Verkhratsky, None; J.J.<br />
Rodriguez, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.25/H10<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: 5R01AG026330-03<br />
Univ of Washington ADRC P50AG05136<br />
Title: Microglia CD47 expression <strong>an</strong>d function are altered with age <strong>an</strong>d Alzheimer's disease<br />
Authors: *A. M. FLODEN, C. K. COMBS;<br />
Univ. North Dakota, Gr<strong>an</strong>d Forks, ND<br />
Abstract: Alzheimer’s disease (AD) is <strong>an</strong> age-associated disease characterized by increased<br />
accumulation of extracellular β-Amyloid (Aβ) plaques within <strong>the</strong> brain. Histological examination<br />
has also demonstrated robust, increased microglial activation in diseased brains versus controls<br />
often in association with <strong>the</strong>se fibrillar peptide aggregates. The paradoxical presence of<br />
increased, reactive microglia yet accumulating extracellular debris suggests that <strong>the</strong>se cells may
e phagocytically compromised during disease. Our previous data demonstrated decreased Aβ<br />
fibril uptake <strong>from</strong> adult murine microglia in spite of increased ability to adhere to Aβ fibrils<br />
when compared to postnatal microglia. The ability of postnatal cells was inhibited by<br />
pretreatment with <strong>the</strong> CD47 binding peptide, 4N1k, suggesting that CD47 function was involved<br />
in phagocytic uptake. The 4N1k peptide inhibited ability of adult microglia to adhere to Aβ<br />
fibrils again demonstrating a role <strong>for</strong> CD47 in microglia-Aβ interaction. These data led us to<br />
hypo<strong>the</strong>size that levels of expression, localization, or <strong>the</strong> specific signaling response of CD47<br />
would differ between young <strong>an</strong>d adult microglia <strong>an</strong>d control versus AD brains. To test this<br />
hypo<strong>the</strong>sis, acutely isolated murine microglia <strong>from</strong> postnatal day 0 or adult mice were isolated<br />
<strong>an</strong>d immunostained <strong>for</strong> CD47 immunoreactivity <strong>an</strong>d stimulated with or without <strong>the</strong> 4N1k peptide<br />
to assess ch<strong>an</strong>ges in protein phosphotyrosine levels by Western <strong>an</strong>alysis. Western blotting<br />
demonstrated no ch<strong>an</strong>ge in protein phosphotyrosine levels at ei<strong>the</strong>r age following 4N1k<br />
stimulation suggesting no apparent tyrosine kinase dependent signaling occurred subsequent to<br />
4N1k stimulation in <strong>the</strong>se cells. Immunostaining of <strong>the</strong> isolated cells demonstrated little<br />
immunoreactivity in <strong>the</strong> postnatal cells compared to those isolated <strong>from</strong> <strong>the</strong> adult <strong>an</strong>imals<br />
supporting <strong>the</strong> observation of increased CD47-dependent adhesiveness of adult versus postnatal<br />
microglia. Western blot <strong>an</strong>alysis of AD versus age-matched control brains revealed no<br />
signific<strong>an</strong>t decrease in total CD47 protein levels. Immunostaining demonstrated some robust<br />
microglial CD47 immunoreactivity in <strong>the</strong> AD brain versus control in support of <strong>the</strong> role of this<br />
protein in mediating microglia-Aβ interaction. Collectively, <strong>the</strong>se data suggest that microglia<br />
may increase CD47 protein levels with age or disease <strong>an</strong>d this protein is involved primarily with<br />
ability of <strong>the</strong> cells to bind to Aβ fibrils but not necessarily Aβ uptake with age.<br />
Disclosures: A.M. Floden, None; C.K. Combs, None.<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.26/H11<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Time course of neuronal tau, glial <strong>an</strong>d synaptic pathology in inducible tau tr<strong>an</strong>sgenic mice<br />
(rTg4510)<br />
Authors: *A. J. MILICI 1 , T. T. KAWABE 1 , T. M. BROWN 1 , D. CAOUETTE 1 , J. E.<br />
FINLEY 1 , S. M. O'NEILL 1 , K. E. G. RICHTER 1 , D. MORTON 2 , D. T. STEPHENSON 1 , C. D.<br />
HICKS 1 ;<br />
1 Neurosci. RU, 2 DSRD, Pfizer, Groton, CT
Abstract: rTg4510 mice conditionally expressing <strong>the</strong> P301L mutation of tau develop a rapid<br />
onset of hyperphosphorylated tau <strong>an</strong>d associated pathological ch<strong>an</strong>ges. In <strong>the</strong> present study, we<br />
describe regional <strong>an</strong>d temporal ch<strong>an</strong>ges in phospho-tau epitopes, synaptic <strong>an</strong>d glial markers in<br />
rTg4510 mice by immunohistochemistry (IHC). At 3.5 months, phosphorylated epitopes of tau<br />
(AT8, AT100 & pS262) were detected in <strong>the</strong> CA1 region of <strong>the</strong> hippocampus <strong>an</strong>d M1 (primary<br />
motor) region of cortex with a similar pattern of progression. Such markers increased at 6<br />
months <strong>an</strong>d by 8 months, both pTau epitopes were detected throughout <strong>the</strong> cortex <strong>an</strong>d<br />
hippocampus. In contrast, a different pTau epitope (AT180) was detected throughout <strong>the</strong> cortex<br />
<strong>an</strong>d hippocampus at 3.5 months <strong>an</strong>d staining increased over time. Markers <strong>for</strong> astrocytes <strong>an</strong>d<br />
microglia in <strong>the</strong> cortex <strong>an</strong>d hippocampus increased between 3.5 <strong>an</strong>d 8 months of age. Coinciding<br />
with <strong>the</strong> increased expression of glial <strong>an</strong>d pTau epitopes, we observed a loss of pyramidal<br />
neurons in <strong>the</strong> CA1 region by 6 months. Pyramidal neurons decreased <strong>from</strong> 4-6 layers thick at<br />
3.5 months to 2 cell layers by 6-8 months. By 10 months CA1 pyramidal neurons <strong>for</strong>med a<br />
discontinuous single cell layer. Evaluation of pre <strong>an</strong>d post synaptic markers has identified age<br />
related reduction of synapsin 2 in <strong>the</strong> CA3 mossy fiber region of <strong>the</strong> hippocampus <strong>an</strong>d reduced<br />
GluR1 staining in <strong>the</strong> CA1 region with no ch<strong>an</strong>ge in <strong>the</strong> expression of synaptophysin or drebrin.<br />
The progressive accumulation of pTau, neuronal loss, differential effect on synapses <strong>an</strong>d glial<br />
activation mimic m<strong>an</strong>y aspects of hum<strong>an</strong> AD neuropathology with a time course that is amenable<br />
to drug intervention.<br />
Disclosures: A.J. Milici, Pfizer, A. Employment (full or part-time); T.T. Kawabe, Pfizer, A.<br />
Employment (full or part-time); T.M. Brown, Pfizer, A. Employment (full or part-time); D.<br />
Caouette, Pfizer, A. Employment (full or part-time); J.E. Finley, Pfizer, A. Employment (full or<br />
part-time); S.M. O'Neill, Pfizer, A. Employment (full or part-time); K.E.G. Richter, Pfizer, A.<br />
Employment (full or part-time); D. Morton, Pfizer, A. Employment (full or part-time); D.T.<br />
Stephenson, Pfizer, A. Employment (full or part-time); C.D. Hicks, Pfizer, A. Employment (full<br />
or part-time).<br />
Poster<br />
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.27/H12<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t AG022101<br />
NIH Gr<strong>an</strong>t AG15490
Alzheimer's Association IIRG-02-3758<br />
Title: Disrupted expression <strong>an</strong>d extracelluar processing of brevic<strong>an</strong> in mice with amyloid<br />
deposits overexpressing hAPPsw<br />
Authors: *P. E. GOTTSCHALL 1 , J. M. AJMO 2 , K. R. PENNYPACKER 2 , D. MORGAN 3 , M.<br />
N. GORDON 3 ;<br />
1 Dept Pharmacol Toxicol, Univ. Ark<strong>an</strong>sas Med. Sci., Little Rock, AR; 2 Mol. Pharmacol. <strong>an</strong>d<br />
Physiol., Univ. of South Florida Col. of Med., Tampa, FL; 3 Byrd Alzheimer Inst., Univ. of South<br />
Florida, Tampa, FL<br />
Abstract: Aggregation <strong>an</strong>d deposition of amyloid-β (Aβ) in <strong>the</strong> <strong>for</strong>ebrain of Alzheimer’s disease<br />
subjects may disturb <strong>the</strong> molecular org<strong>an</strong>ization of <strong>the</strong> extracellular microenvironment that<br />
modulates neural <strong>an</strong>d synaptic plasticity. Proteoglyc<strong>an</strong>s are major components of this<br />
extracellular environment. Brevic<strong>an</strong> is one of a small family of aggregating, chondroitin sulfatebearing<br />
proteoglyc<strong>an</strong>s that is abund<strong>an</strong>tly expressed in <strong>the</strong> central nervous system <strong>an</strong>d it may<br />
affect neurite outgrowth <strong>an</strong>d stabilize synapses. Versic<strong>an</strong> is closely related to brevic<strong>an</strong> but it is<br />
deposited mostly in white matter. Turnover of brevic<strong>an</strong> <strong>an</strong>d versic<strong>an</strong>, after cleavage by <strong>the</strong><br />
matrix metalloproteinases (MMP) or a disintegrin <strong>an</strong>d metalloproteinase with thrombospondin<br />
motifs (ADAMTS), also influences neural plasticity. To test <strong>the</strong> hypo<strong>the</strong>sis that soluble or<br />
plaque-derived Aβ modifies <strong>the</strong> accumulation <strong>an</strong>d/or turnover of extracellular proteoglyc<strong>an</strong>s, we<br />
examined whe<strong>the</strong>r <strong>the</strong> expression <strong>an</strong>d processing of brevic<strong>an</strong> <strong>an</strong>d versic<strong>an</strong> were altered in <strong>the</strong><br />
brains of tr<strong>an</strong>sgenic mice overexpressing hum<strong>an</strong>, amyloid precursor protein with <strong>the</strong> Swedish<br />
mutation (APPsw) as a model of Alzheimer’s disease. The molecular weight of chondroitin<br />
sulfate-containing brevic<strong>an</strong> was consistently <strong>an</strong>d markedly lower in hippocampal tissue extract<br />
<strong>from</strong> 15-19 month old APPsw mice bearing Aβ deposits compared to non-tr<strong>an</strong>sgenic littermate<br />
mice. Pre-treatment of extracts with chondroitinase revealed a brevic<strong>an</strong> core protein with a<br />
molecular weight identical between APPsw <strong>an</strong>d non-tr<strong>an</strong>sgenic mice. In addition, <strong>the</strong> abund<strong>an</strong>ce<br />
of a major MMP-derived (but not ADAMTS-derived) proteolytic fragment of brevic<strong>an</strong> was<br />
strikingly diminished in several telencephalic regions of APPsw mice. Immunoreactive MMP-<br />
<strong>an</strong>d ADAMTS-derived proteolytic fragments of brevic<strong>an</strong> were localized to <strong>the</strong> periphery of core,<br />
amyloid plaques, a region with dystrophic neurites <strong>an</strong>d decreased synaptic marker expression.<br />
No signific<strong>an</strong>t ch<strong>an</strong>ges were observed in <strong>the</strong> abund<strong>an</strong>ce of <strong>the</strong> core protein or proteolytic<br />
fragment of versic<strong>an</strong>. These results suggest that Aβ or APP exert inhibitory effects selectively on<br />
proteolytic cleavage mech<strong>an</strong>isms responsible <strong>for</strong> processing of brevic<strong>an</strong>, <strong>an</strong>d not versic<strong>an</strong>. In<br />
addition, syn<strong>the</strong>tic mech<strong>an</strong>isms that add chondroitin sulfate to <strong>the</strong> core protein may be altered in<br />
<strong>the</strong>se mice. Since brevic<strong>an</strong> stabilizes synaptic structures <strong>an</strong>d inhibits synaptic plasticity, defective<br />
brevic<strong>an</strong> processing observed in amyloid-β-bearing mice <strong>an</strong>d potentially end-stage hum<strong>an</strong><br />
Alzheimer’s disease, may contribute to deficient neural plasticity.<br />
Disclosures: P.E. Gottschall, None; J.M. Ajmo, None; K.R. Pennypacker, None; D.<br />
Morg<strong>an</strong>, None; M.N. Gordon, None.<br />
Poster
526. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 526.28/H13<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Functional <strong>an</strong>alysis of <strong>the</strong> hippocampus in APP tr<strong>an</strong>sgenic mice - Deficits in synaptic <strong>an</strong>d<br />
cognitive functions<br />
Authors: *C. PEREZ CRUZ 1 , M. W. NOLTE 1 , N. R. RUSTAY 2 , G. GROSS 1 , U. EBERT 1 ;<br />
1 2<br />
Abbott, Neurosci. Research, GPRD, Ludwigshafen, Germ<strong>an</strong>y; Abbott, Neurosci. Research,<br />
GPRD, Abbott Park, IL<br />
Abstract: Alzheimer’s disease (AD) is <strong>the</strong> most common <strong>for</strong>m of late-life dementia, affecting 30<br />
million individuals worldwide. One major hallmark of AD is <strong>the</strong> massive loss of synapses that<br />
occurs at <strong>an</strong> early clinical stage of <strong>the</strong> disease. The aim of this study was to evaluate synaptic<br />
modifications by qu<strong>an</strong>tifying spine number in basal <strong>an</strong>d apical dendrites of CA1 pyramidal<br />
neurons, assessing cognitive deficits, <strong>an</strong>d evaluating protein levels of <strong>the</strong> synapse-associated<br />
immediate early gene, Arc, a well known marker <strong>for</strong> neuronal activity, <strong>an</strong>d of somatostatin in 5.5<br />
months-old Tg2576 mice (n= 5 - 6 / group). Arc mRNA is rapidly tr<strong>an</strong>sported to dendritic<br />
processes <strong>for</strong> local tr<strong>an</strong>slation induced upon synaptic activity (Tzingounis <strong>an</strong>d Nicoll, 2006).<br />
Somatostatin is a neuronal marker of a specific subpopulation of interneurons that has been<br />
reported to be affected in several <strong>an</strong>imal models of AD <strong>an</strong>d aging (Ramos et al., 2006, Vela et<br />
al., 2003). Our results show that Tg2576 mice presented a 34% reduction in <strong>the</strong> density of<br />
mushroom type spines in basal dendrites proximal to soma compared to wild type littermates<br />
(p
Disclosures: C. Perez Cruz, Abbott, A. Employment (full or part-time); M.W. Nolte, Abbott,<br />
A. Employment (full or part-time); N.R. Rustay, Abbott, A. Employment (full or part-time); G.<br />
Gross, Abbott, A. Employment (full or part-time); U. Ebert, Abbott, A. Employment (full or<br />
part-time).<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.1/H14<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: Alzheimer Forschungsgemeinschaft<br />
Title: Immunoreactivity <strong>for</strong> beta-secretase (BACE) increases in hum<strong>an</strong> brain with <strong>the</strong> severity of<br />
Alzheimer's disease<br />
Authors: *K. D. GEIGER 1 , L. RAJENDRAN 2,3 , C. U. EWALD 4 , L. M. DRACH 5 ;<br />
1 Univ. Dresden, Dresden, Germ<strong>an</strong>y; 2 Max Pl<strong>an</strong>ck Inst. of Mol. Cell Biol. <strong>an</strong>d Genet., Dresden,<br />
Germ<strong>an</strong>y; 3 Systems <strong>an</strong>d Cell Biol. of Neurodegeneration, Univ. of Zürich, Zürich, Switzerl<strong>an</strong>d;<br />
4 Dept. of Neurology, Ev<strong>an</strong>gelisches Kr<strong>an</strong>kenhaus, Unna, Germ<strong>an</strong>y; 5 Dept. of Gerontopsychiatry,<br />
Carl-Friedrich-Flemming-Hospital, Schwerin, Germ<strong>an</strong>y<br />
Abstract: Proteolytic processing of <strong>the</strong> amyloid precursor protein (APP) by beta <strong>an</strong>d gammasecretases<br />
resulting in cumulative amyloid-beta (Abeta) production has been indicated as a main<br />
etiological basis of Alzheimer’s disease (AD). Experimental evidence suggests that co-residence<br />
of APP <strong>an</strong>d BACE1 in <strong>the</strong> endosomal compartments promotes amyloidogenesis. Upregulation of<br />
BACE 1 is suggested as a cause <strong>for</strong> some <strong>for</strong>ms of late onset AD. Here, we investigated <strong>the</strong><br />
distribution of BACE on sections of hum<strong>an</strong> brains <strong>from</strong> Alzheimer patients.<br />
We studied paraffin-embedded tissues of 3-5 brain regions <strong>from</strong> 55 autopsy cases with<br />
Alzheimer’s disease of varying severity, including 20 cases with Lewy-body disease (DLB) in<br />
combination with Alzheimer’s disease <strong>an</strong>d 10 cases of vascular encephalopathy in comparison<br />
with 12 normal controls. Diagnoses were obtained by morphology, silver impregnation <strong>an</strong>d<br />
immunocytochemistry using <strong>an</strong> adapted version of <strong>the</strong> CERAD classification. The sections were<br />
evaluated semi-qu<strong>an</strong>titatively using H&E, modified Bielschowsky silver staining <strong>an</strong>d<br />
immunocytochemistry (IR) <strong>for</strong> Ezrin (3C12), BACE1, hyperphosphorylated Tau (AT8), GFAP,<br />
Ubiquitin <strong>an</strong>d HLA-DR (MHC II). Apoptosis was evaluated using <strong>the</strong> TUNEL method.<br />
We found expression of BACE1 mostly in neurons <strong>an</strong>d some astrocytes of Alzheimer brains.<br />
Only minimal BACE-IR was observed in neurons of vascular encephalopathy brains, none in
normal controls. BACE IR increased signific<strong>an</strong>tly with <strong>the</strong> severity of Alzheimer-associated<br />
pathology. The more severe cases showed additional BACE-IR in astrocytes, which <strong>the</strong>n showed<br />
a similar distribution as Ezrin-IR in astrocyts <strong>an</strong>d was associated with neuronal apoptosis.<br />
Our data demonstrate <strong>the</strong> overexpression of BACE in a large number of neurons in hum<strong>an</strong><br />
tissues of Alzheimer cases which is associated with increased amyloid depositis of progressive<br />
disease. The more severe BACE- overload in late stage Alzheimer’s disease may also affect<br />
activated astrocytes <strong>an</strong>d thus contribute to <strong>an</strong> enh<strong>an</strong>ced neuronal damage.<br />
Disclosures: K.D. Geiger, None; L. Rajendr<strong>an</strong>, None; C.U. Ewald, None; L.M. Drach,<br />
None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.2/H15<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: IDPH<br />
Title: Elevated BACE1 expression in tr<strong>an</strong>sgenic mouse models of Alzheimer's disease is<br />
associated with synaptic/axonal pathology <strong>an</strong>d amyloidogenesis<br />
Authors: X.-X. YAN 1 , *X.-M. ZHANG 4 , Y. CAI 1 , H. CAI 5 , K. XIONG 6 , R. G. STRUBLE 2 , P.<br />
C. WONG 7 , P. R. PATRYLO 3 , R. W. CLOUGH 1 ;<br />
1 Anat., Sou<strong>the</strong>rn Illinois Univ. Sch. of Med., Carbondale, IL; 2 Neurol., Sou<strong>the</strong>rn Illinois Univ.<br />
Sch. of Med., Springfield, IL; 3 Physiol., Sou<strong>the</strong>rn Illinois Univ. Sch. of Med., Carbondale, IL;<br />
4 Anat., Carbondale, IL; 5 Lab. of Neurogenetics, Natl. Inst. on Aging, Be<strong>the</strong>sda, MD; 6 Dept. of<br />
Anat. <strong>an</strong>d Neurobio., Central South Univ. Xi<strong>an</strong>gya Med. Sch., Ch<strong>an</strong>gsha, China; 7 Dept. of<br />
Pathology <strong>an</strong>d Neurosci., The Johns Hopkins Univ. Sch. of Med., Baltimore, MD<br />
Abstract: How amyloid plaques develop in <strong>the</strong> brain represents <strong>the</strong> central yet unsettled<br />
question in Alzheimer disease (AD) pathogenesis. Competing hypo<strong>the</strong>ses postulate that βamyloid<br />
peptide (Aβ) deposits are ei<strong>the</strong>r <strong>the</strong> cause or <strong>the</strong> consequence of vascular, glial <strong>an</strong>d<br />
neuronal ch<strong>an</strong>ges. Amyloidogenic proteins including β-amyloid precursor protein (APP), <strong>an</strong>d its<br />
cleavage enzymes, β-secretase (BACE1) <strong>an</strong>d γ-secretase, appear to be expressed largely in<br />
neurons, supporting a notion that Aβ deposits originate <strong>from</strong> neuronal components. Amyloid<br />
depositions may exist ei<strong>the</strong>r as neuritic or diffuse <strong>for</strong>ms, with <strong>the</strong> <strong>for</strong>mer being associated with<br />
dystrophic neurites. This phenomenon appears as if dystrophic neurites <strong>an</strong>d Aβ deposits do not
necessarily precede one to <strong>an</strong>o<strong>the</strong>r. Synapses <strong>an</strong>d axonal terminals have been considered <strong>the</strong><br />
origin of extracellular Aβ deposits. However, <strong>an</strong>atomic evidence <strong>for</strong> early synaptic/axonal<br />
alterations preceding or coevolving with plaque onset <strong>an</strong>d evolution is yet to be established. This<br />
study determined <strong>the</strong> localization of elevated BACE1 expression relative to Aβ accumulation <strong>an</strong>d<br />
synaptic/neuritic alterations in tr<strong>an</strong>sgenic (Tg) mice harboring familial AD (FAD) mutations<br />
(5XFAD, 2XFAD, 3xTg-AD mice). BACE1 immunoreactivity (ir) coexisted with compact<br />
plaques in <strong>the</strong> cortex <strong>an</strong>d hippocampus of all tr<strong>an</strong>sgenics, occurring in dystrophic axonal neurites<br />
of various neuronal origins (GABAergic, glutamatergic, cholinergic or catecholaminergic).<br />
Compact plaques within <strong>the</strong> cortical grey matter appeared to mainly derive <strong>from</strong> BACE/Aβ<br />
labeled presynaptic terminals surrounding pyramidal neurons with cytoplasmic Aβ-ir, <strong>an</strong>d some<br />
of <strong>the</strong>se terminals undergone sprouting to become overt dystrophic neurites. Primitive plaques<br />
appeared to evolve into cored plaques as extracellular Aβ accumulated within/around <strong>the</strong><br />
growing mass of sprouting dystrophic neurites. In old 3xTg-AD mice, <strong>the</strong> superficial cortical<br />
layers largely expressed diffuse plaques. Development of <strong>the</strong>se diffuse plaques with age was<br />
associated with increased BACE1/Aβ labeling at axonal terminals lacking apparent sprouting or<br />
dystrophy. Toge<strong>the</strong>r, BACE1 overexpression, generally in axonal terminals, coincided<br />
<strong>an</strong>atomically with <strong>the</strong> development of neuritic <strong>an</strong>d diffuse plaques in tr<strong>an</strong>sgenic models of AD.<br />
Specifically, sprouting/dystrophic axon terminals appear to play <strong>an</strong> active role in <strong>the</strong> <strong>for</strong>mation<br />
<strong>an</strong>d evolution of neuritic plaques potentially by producing <strong>an</strong>d releasing Aβ into surrounding<br />
extracellular space.<br />
Disclosures: X. Y<strong>an</strong>, None; X. Zh<strong>an</strong>g, None; Y. Cai, None; H. Cai, None; K. Xiong,<br />
None; R.G. Struble, None; P.C. Wong, None; P.R. Patrylo, None; R.W. Clough, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.3/H16<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: : Alzheimer’s Association <strong>an</strong>d McKnight Brain Disorders Award (IB)<br />
Europe<strong>an</strong> union <strong>an</strong>d Methusalem gr<strong>an</strong>t (BDS)<br />
Title: Presenilins function as ER Ca 2+ leak ch<strong>an</strong>nels in neurons: Implications <strong>for</strong> Alzheimer’s<br />
disease pathogenesis
Authors: *H. ZHANG 1 , S. SUN 1 , A. HERREMAN 2 , B. D. STROOPER 2 , I.<br />
BEZPROZVANNY 1 ;<br />
1 UTSouthwestern Med., dallas, TX; 2 K.U.Leuven, LEUVEN, Belgium<br />
Abstract: Alzheimer’s disease (AD) is a progressive <strong>an</strong>d irreversible neurodegenerative<br />
disorder. Mutations in presenilins 1 <strong>an</strong>d 2 (PS1 <strong>an</strong>d PS2) account <strong>for</strong> 40% of familial AD (FAD)<br />
cases. In <strong>the</strong> previous studies with mouse embryonic fibroblasts (MEF) cell lines <strong>from</strong> presenilin<br />
(PS) double-knockout (DKO) mice we demonstrated that presenilins control endoplasmic<br />
reticulum (ER) calcium (Ca 2+ ) levels by acting as passive ER Ca 2+ leak ch<strong>an</strong>nels <strong>an</strong>d that m<strong>an</strong>y<br />
familial Alzheimer's disease (FAD)-linked mutations in presenlins disrupt this function (Tu at al,<br />
2006; Nelson at al, 2007). To determine if presenilins function as ER Ca 2+ leak ch<strong>an</strong>nels in<br />
neurons, we per<strong>for</strong>med Ca 2+ imaging experiments with hippocampal neuronal cultures <strong>from</strong> PS<br />
conditional double knockout (cDKO) mice (PS1 floxed/floxed ; PS2 -/-) infected with Cre-encoding<br />
lentiviruses. We observed increased size of ionomycin (IO)-sensitive Ca 2+ pool <strong>an</strong>d increased<br />
caffeine response in PS-null neurons when compared with control cultures. In a series of rescue<br />
experiments we demonstrated that PS1 wild type rescue constructs, but not PS1-FAD reduce <strong>the</strong><br />
size of IO-sensitive Ca 2+ pool <strong>an</strong>d caffeine response. We also observed <strong>the</strong> increased size of<br />
ionomycin (IO)-sensitive Ca 2+ pool <strong>an</strong>d caffeine responses in hippocampal cultures <strong>from</strong> PS1-<br />
M146V KI mice. These results support <strong>the</strong> hypo<strong>the</strong>sis that presenilins function as ER Ca 2+ leak<br />
ch<strong>an</strong>nels in hippocampal neurons. In additional experiments we found that Ry<strong>an</strong>odine receptors<br />
compensate <strong>for</strong> loss of ER Ca 2+ leak function of presenilins in hippocampal neurons. Our results<br />
suggest that abnormal ER Ca 2+ homeostasis may play <strong>an</strong> import<strong>an</strong>t role in AD pathogenesis.<br />
Disclosures: H. Zh<strong>an</strong>g, None; S. sun, None; A. Herrem<strong>an</strong>, None; B.D. Strooper, None; I.<br />
bezprozv<strong>an</strong>ny, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.4/H17<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: Swiss National Science Foundation (A.I.M. <strong>an</strong>d P.C.F., gr<strong>an</strong>t 310000-116652/1)<br />
NCCR “Neural Plasticity <strong>an</strong>d Repair” (P.C.F)<br />
Scholarship Lem<strong>an</strong>ic Neuroscience Program 2007/2008
Title: γ-Secretase activity alterations affect Wnt pathway gene tr<strong>an</strong>scription in CHO cells -<br />
Implications <strong>for</strong> Alzheimer’s disease<br />
Authors: *A. I. MAGOLD 1 , M. CACQUEVEL 1 , K. HARSHMAN 2 , P. C. FRAERING 1 ;<br />
1 EPFL Swiss Inst. of Technol. / Brain Mind Inst., Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d; 2 CIG - Plate-<strong>for</strong>me<br />
DAF, UNIL, University of Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d<br />
Abstract: Introduction: γ-Secretase is <strong>an</strong> intra-membr<strong>an</strong>e-cleaving aspartyl protease implicated<br />
in <strong>the</strong> processing of a wide r<strong>an</strong>ge of type I membr<strong>an</strong>e proteins including <strong>the</strong> Notch receptor <strong>an</strong>d<br />
APP. It thus regulates diverse cellular <strong>an</strong>d biological processes including <strong>the</strong> differentiation of<br />
neuronal embryonic stem- or intestinal stem cells. Proteolysis of <strong>the</strong>se proteins by γ-secretase<br />
triggers signaling cascades by releasing intracellular domains (ICDs) which, following<br />
association with adaptor proteins <strong>an</strong>d nuclear tr<strong>an</strong>slocation, modulate <strong>the</strong> tr<strong>an</strong>scription of<br />
different genes by binding directly to <strong>the</strong>ir promoters. The pronounced proliferative <strong>an</strong>d<br />
regenerative effects of Notch signaling <strong>an</strong>d its implication in <strong>the</strong> generation of <strong>the</strong> Abetapeptides,<br />
makes γ-secretase a <strong>the</strong>rapeutic target <strong>for</strong> several types of c<strong>an</strong>cer <strong>an</strong>d <strong>for</strong> Alzheimer’s<br />
disease.<br />
Methodology <strong>an</strong>d Results:<br />
To investigate <strong>the</strong> broad effects of γ-secretase activity onto <strong>the</strong> cellular tr<strong>an</strong>scriptome, Chinese<br />
hamster ovary (CHO) cells with enh<strong>an</strong>ced γ-secretase were compared to cells with abolished γsecretase<br />
activity via a microarray designed <strong>for</strong> a genetically close species, mouse. By applying a<br />
cut-off based on <strong>the</strong> false discovery rate (FDR, i.e. <strong>the</strong> probability to wrongly accept a difference<br />
between <strong>the</strong> two conditions) with <strong>an</strong> adj.p value of 0.005, we found 2658 EST clones to be<br />
differentially expressed, with 1241 EST clones of increased <strong>an</strong>d 1417 EST clones of decreased<br />
tr<strong>an</strong>scription with enh<strong>an</strong>ced γ-secretase activity. Out of <strong>the</strong> 35 most prominently tr<strong>an</strong>scriptionally<br />
altered genes, evaluated by real time PCR, 22 c<strong>an</strong>didate genes were confirmed to be<br />
differentially expressed. First, functional clustering <strong>an</strong>d interactome <strong>an</strong>alysis of encoded proteins<br />
of top score differentially expressed genes via <strong>the</strong> string 8.0 datab<strong>an</strong>k revealed strong alterations<br />
of Wnt pathways gene tr<strong>an</strong>scription through several key players.<br />
Next, <strong>an</strong>d because γ-secretase is causative <strong>for</strong> most early onset <strong>an</strong>d directly connected with late<br />
onset Alzheimer’s disease (AD) risk, top score differentially expressed genes were investigated<br />
in hum<strong>an</strong> AD cortex tissue as well. Whereas m<strong>an</strong>y showed tendencies only, <strong>the</strong> gene TERA,<br />
which is implicated in brain development, neural stem cell niche mainten<strong>an</strong>ce <strong>an</strong>d Wnt<br />
<strong>an</strong>tagonism in neural differentiation, showed signific<strong>an</strong>tly altered tr<strong>an</strong>scription levels in hum<strong>an</strong><br />
AD cortex.<br />
Signific<strong>an</strong>ce:<br />
Our findings will potentially help to decipher <strong>the</strong> biology of γ-secretase, <strong>an</strong>d better underst<strong>an</strong>d<br />
<strong>the</strong> roles of this enzyme in gene tr<strong>an</strong>scription <strong>an</strong>d AD.<br />
Disclosures: A.I. Magold, Swiss National Science Foundation (A.I.M. <strong>an</strong>d P.C.F., gr<strong>an</strong>t<br />
310000-116652/1), B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Lem<strong>an</strong>ic Neuroscience Scholarship<br />
2007/2008, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind<br />
support); M. Cacquevel, None; K. Harshm<strong>an</strong>, DNA array facility of <strong>the</strong> Center <strong>for</strong> Integrative<br />
Genomics (CIG) of <strong>the</strong> Faculty of Biology <strong>an</strong>d Medicine of <strong>the</strong> University o Laus<strong>an</strong>ne, A.<br />
Employment (full or part-time); P.C. Fraering, Swiss National Science Foundation (A.I.M. <strong>an</strong>d
P.C.F., gr<strong>an</strong>t 310000-116652/1), B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); NCCR “Neural Plasticity <strong>an</strong>d<br />
Repair” (P.C.F), B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.5/H18<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Title: Gamma secretase modulators ch<strong>an</strong>ge <strong>the</strong> amyloid beta profile in brain, CSF, <strong>an</strong>d plasma in<br />
guinea pigs<br />
Authors: *K. M. WOOD 1 , K. BALES 1 , M. LACHYANKAR 1 , J. J. CONBOY 2 , K. F.<br />
GEOGHEGAN 2 , E. Q. WANG 3 , E. K. SYLVAIN 1 , M. MARCONI 1 , K. WALLACE 1 , J.<br />
HODGE 3 , E. L. HUDSON 3 , L. ZHANG 3 , B. TATE 1 , C. STIFF 4 , D. S. JOHNSON 4 ;<br />
1 Neurosci. Biol., 2 Exploratory Medicinal Sci., 3 Dept. of Pharmacokinetics, Dynamics <strong>an</strong>d<br />
Metabolism, 4 Neurosci. Chem., Pfizer, Inc, Groton, CT<br />
Abstract: A principle component of amyloid plaques, which accumulates in <strong>the</strong> brain of<br />
Alzheimer disease (AD) patients, is <strong>the</strong> Aβ42 peptide. Agents that selectively lower <strong>the</strong> level of<br />
Aβ42 may represent a disease-modifying approach <strong>for</strong> <strong>the</strong> treatment of AD. There<strong>for</strong>e, we<br />
compared <strong>the</strong> Aβ42 lowering efficacy <strong>an</strong>d exposure-response relationships of three gamma<br />
secretase modulators in guinea pig brain, CSF <strong>an</strong>d plasma. All methods were approved by <strong>the</strong><br />
IACUC. Guinea pigs were treated with gamma secretase modulators that had whole-cell IC50s<br />
against AβX-42 r<strong>an</strong>ging <strong>from</strong> 74 to 212 nM. Following acute oral doses of 0.32 to 100 mg/kg,<br />
<strong>an</strong>alyses of <strong>the</strong> effects on Aβ levels were conducted at time points r<strong>an</strong>ging <strong>from</strong> 1 to 72 h.<br />
Compound levels were measured in brain, CSF <strong>an</strong>d plasma by LC-MS. Aβ1-X, X-40, 1-38 <strong>an</strong>d<br />
X-42 were measured in brain, CSF <strong>an</strong>d plasma by Dissociation-Enh<strong>an</strong>ced L<strong>an</strong>th<strong>an</strong>ide<br />
Fluorescent Immunoassay (DELFIA). Free (unbound) levels of compound in brain r<strong>an</strong>ged <strong>from</strong><br />
0.42 to 90.3 nM. Each compound produced dose-responsive effects <strong>an</strong>d time-dependent ch<strong>an</strong>ges<br />
in AβX-42 <strong>an</strong>d Aβ1-38 in all compartments sampled. Elevations of Aβ1-38 <strong>an</strong>d corresponding<br />
decreases of AβX-42 were more pronounced in plasma <strong>an</strong>d CSF th<strong>an</strong> in brain. Moreover, <strong>the</strong><br />
signific<strong>an</strong>t ch<strong>an</strong>ges that we observed in brain AβX-42 <strong>an</strong>d Aβ1-38 occurred with little or no<br />
effect on <strong>the</strong> total qu<strong>an</strong>tity of Aβ produced. To fur<strong>the</strong>r elucidate ch<strong>an</strong>ges in specific Aβ iso<strong>for</strong>ms,<br />
brain samples <strong>from</strong> guinea pig were <strong>an</strong>alyzed by immunoprecipitation followed by MALDI-TOF<br />
mass spectrometry. Mass spectrometry showed that Aβ species with <strong>the</strong> C-terminus at residue 34
were elevated by treatment with <strong>the</strong> gamma secretase modulators. Additionally, we observed a<br />
robust increase in Aβ1-37 <strong>an</strong>d Aβ11-37 in brain. An extended time course study revealed a<br />
complex pharmacokinetic/pharmacodynamic relationship in which a long-lived reduction in<br />
brain AβX-42 was observed. Taken toge<strong>the</strong>r, our results demonstrate that gamma secretase<br />
modulators effectively reduce AβX-42 while differentially affecting various Aβ fragments in<br />
brain, plasma <strong>an</strong>d CSF of guinea pigs.<br />
Disclosures: K.M. Wood, Pfizer, Inc., A. Employment (full or part-time); K. Bales, Pfizer,<br />
Inc., A. Employment (full or part-time); M. Lachy<strong>an</strong>kar, Pfizer, Inc., A. Employment (full or<br />
part-time); J.J. Conboy, Pfizer, Inc., A. Employment (full or part-time); K.F. Geogheg<strong>an</strong>,<br />
Pfizer, Inc., A. Employment (full or part-time); E.Q. W<strong>an</strong>g, Pfizer, Inc., A. Employment (full or<br />
part-time); E.K. Sylvain, Pfizer, Inc., A. Employment (full or part-time); M. Marconi, Pfizer,<br />
Inc., A. Employment (full or part-time); K. Wallace, Pfizer, Inc., A. Employment (full or parttime);<br />
J. Hodge, Pfizer, Inc., A. Employment (full or part-time); E.L. Hudson, Pfizer, Inc., A.<br />
Employment (full or part-time); L. Zh<strong>an</strong>g, Pfizer, Inc., A. Employment (full or part-time); B.<br />
Tate, Pfizer, Inc., A. Employment (full or part-time); C. Stiff, Pfizer, Inc., A. Employment (full<br />
or part-time); D.S. Johnson, Pfizer, Inc., A. Employment (full or part-time).<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.6/H19<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: NIA- RO1AG033570<br />
Title: Presenilin-1 plays a role in neural stem cell self-renewal in <strong>the</strong> adult brain<br />
Authors: *A. GADADHAR 1 , R. A. MARR 2 , O. LAZAROV 1 ;<br />
1 Anat. <strong>an</strong>d Cell Biol., Univ. of Illinois At Chicago, Chicago, IL; 2 Neurosci., The Rosalind<br />
Fr<strong>an</strong>klin Univ. of Med. <strong>an</strong>d Sci., North Chicago, IL<br />
Abstract: Neural stem cells exist in <strong>the</strong> adult brain in at least two neurogenic niches: <strong>the</strong><br />
subventricular zone <strong>an</strong>d <strong>the</strong> subgr<strong>an</strong>ule layer of <strong>the</strong> dentate gyrus. There, <strong>the</strong>y self-renew,<br />
proliferate <strong>an</strong>d give rise to glia <strong>an</strong>d neuroblasts that migrate to <strong>the</strong> olfactory bulb <strong>an</strong>d to <strong>the</strong><br />
gr<strong>an</strong>ule layer of <strong>the</strong> dentate gyrus, respectively, <strong>an</strong>d integrate <strong>the</strong>re. Increasing evidence suggests<br />
that newly-<strong>for</strong>med neurons play a role in numerous aspects of learning <strong>an</strong>d memory, <strong>an</strong>d<br />
rein<strong>for</strong>ce various aspects of brain plasticity. Impairments in neurogenesis may compromise <strong>the</strong>se
processes, leading to deficits in learning <strong>an</strong>d memory <strong>an</strong>d reduced plasticity. Alzheimer’s disease<br />
(AD) is a neurodegenerative disease characterized by cognitive impairments <strong>an</strong>d progressive<br />
memory loss. Impairments in neurogenesis may ei<strong>the</strong>r underlie <strong>the</strong>se deficits or exacerbate <strong>the</strong>m.<br />
The familial <strong>for</strong>ms of <strong>the</strong> disease (FAD) are caused by mutations in amyloid precursor protein<br />
(APP), presenilin-1 (PS1) <strong>an</strong>d presenilin-2 (PS2). Increasing evidence suggests that neurogenesis<br />
is altered in <strong>the</strong> AD brain <strong>an</strong>d in tr<strong>an</strong>sgenic mice harboring mut<strong>an</strong>t PS1. However, <strong>the</strong> nature of<br />
<strong>the</strong>se alterations is not yet fully understood. To determine a role <strong>for</strong> PS1 in neurogenesis in <strong>the</strong><br />
adult brain we developed a lentiviral vector system to knock down PS1 expression in neurogenic<br />
areas in <strong>the</strong> adult brain in a spatially- <strong>an</strong>d temporally-specific m<strong>an</strong>ner. We show that silencing<br />
PS1 expression in neurogenic areas dramatically decreases extent of neural stem cell selfrenewal,<br />
<strong>an</strong>d <strong>the</strong>ir proliferation <strong>an</strong>d induces cell differentiation. This study determines a key role<br />
<strong>for</strong> PS1 in neurogenesis in <strong>the</strong> adult brain. Import<strong>an</strong>tly, this study suggests that dysfunction of<br />
PS1, such as in FAD, directly misregulates neurogenesis.<br />
Disclosures: A. Gadadhar, None; R.A. Marr, None; O. Lazarov, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.7/H20<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: NIH Gr<strong>an</strong>t P01 NS047308<br />
Research Fellowship Award, Alder Foundation (July 1, <strong>2009</strong> – June 30, 2010)<br />
Title: Modeling <strong>an</strong> <strong>an</strong>ti-amyloid combination <strong>the</strong>rapy <strong>for</strong> Alzheimer’s disease<br />
Authors: *V. W. CHOW 1 , A. V. SAVONENKO 2 , T. MELNIKOVA 1 , H. KIM 1 , D. L. PRICE 3 ,<br />
T. LI 1 , P. C. WONG 4 ;<br />
1 Pathology/Neuropathology, 2 Pathology/Neuropathology, Neurol., 3 Pathology/Neuropathology,<br />
Neurology, Neurosci., 4 Pathology/Neuropathology, Neurosci., Johns Hopkins Med.<br />
Institutions|910003650|0, Baltimore, MD<br />
Abstract: Although γ-secretase <strong>an</strong>d BACE1 are well-recognized <strong>the</strong>rapeutic targets <strong>for</strong><br />
Alzheimer’s Disease (AD), untoward side effects associated with strong inhibition or reduction<br />
of <strong>the</strong>se aspartyl proteases have raised concerns regarding <strong>the</strong>ir <strong>the</strong>rapeutic potential. We showed<br />
recently that moderate decrease of ei<strong>the</strong>r γ-secretase or BACE1 provides modest beneficial
effects in <strong>the</strong> brain without <strong>an</strong>y mech<strong>an</strong>ism-based toxicity. Because <strong>the</strong> processing of APP to<br />
generate Aβ requires both γ-secretase <strong>an</strong>d BACE1, it is possible that reductions of both enzymes<br />
would provide additive protection against Aβ amyloidosis. Here, we assess <strong>the</strong> value of this<br />
novel <strong>an</strong>ti-amyloid combination <strong>the</strong>rapy using a mouse model of amyloidosis lacking one allele<br />
each of Aph-1a <strong>an</strong>d BACE1 (APPswe/PS1∆E9;Aph-1a +/- ;BACE1 +/- mice). We show that <strong>the</strong><br />
genetic reductions of both Aph-1a <strong>an</strong>d BACE1 additively attenuate <strong>the</strong> amyloid burden <strong>an</strong>d<br />
ameliorate cognitive deficits occurring in APPswe/PS1∆E9 mice. Import<strong>an</strong>tly, Aph-1a +/- ;<br />
BACE1 +/- mice exhibit normal life-sp<strong>an</strong> as compared to littermate controls <strong>an</strong>d <strong>the</strong>y do not show<br />
<strong>an</strong>y overt pathological or behavioral abnormality. Taken toge<strong>the</strong>r, our results identify a novel<br />
<strong>an</strong>ti-amyloid combination <strong>the</strong>rapy of targeting both γ-secretase <strong>an</strong>d BACE1 as <strong>an</strong> effective <strong>an</strong>d<br />
safe <strong>the</strong>rapeutic strategy <strong>for</strong> AD.<br />
Disclosures: V.W. Chow, None; A.V. Savonenko, None; T. Melnikova, None; H. Kim,<br />
None; D.L. Price, None; T. Li, None; P.C. Wong, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.8/H21<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: Americ<strong>an</strong> Federation <strong>for</strong> Aging Research (XM)<br />
Alzheimer’s Association (GT)<br />
Title: Role of γ-secretase subunit palmitoylation on amyloid deposition in a tr<strong>an</strong>sgenic mouse<br />
model of Alzheimer’s disease<br />
Authors: *X. MECKLER 1 , J. ROSEMAN 1 , H. CHENG 1 , S. PEI 1 , M. KEAT 1 , B.<br />
KASSARJIAN 1 , A. PARENT 1 , G. THINAKARAN 2 ;<br />
1 Neurobio., 2 Neurobiology, Neurol. <strong>an</strong>d Pathology, The Univ. of Chicago, Chicago, IL<br />
Abstract: Alzheimer’s disease (AD) is <strong>the</strong> leading cause of dementia affecting <strong>the</strong> elderly.<br />
Amyloid peptide (Aβ) is <strong>the</strong> main constituent of senile plaques, a characteristic lesion of <strong>the</strong><br />
disease. Aβ is produced by <strong>the</strong> sequential cleavage of its precursor protein APP by β- <strong>an</strong>d γsecretase.<br />
Several studies have previously highlighted <strong>the</strong> import<strong>an</strong>ce of cholesterol-rich lipid<br />
raft membr<strong>an</strong>e microdomains in this processing of APP. In particular, β-secretase, components<br />
of <strong>the</strong> γ-secretase complex <strong>an</strong>d its APP substrates localize to lipid rafts. Inhibiting <strong>the</strong> association
of <strong>the</strong>se proteins with lipid raft domains could potentially down-regulate Aβ production <strong>an</strong>d<br />
should be considered as a potential <strong>the</strong>rapeutic <strong>for</strong> Alzheimer’s disease. Import<strong>an</strong>tly, o<strong>the</strong>r γsecretase<br />
substrates do not localize to lipid raft. There<strong>for</strong>e displacing γ-secretase <strong>from</strong> lipid rafts<br />
should affect only <strong>an</strong>d specifically APP processing. We have recently identified <strong>the</strong><br />
palmitoylation of nicastrin <strong>an</strong>d Aph1, two γ-secretase subunits, as a critical post-tr<strong>an</strong>slational<br />
modification <strong>for</strong> <strong>the</strong> association of <strong>the</strong>se proteins to lipid rafts. However in N2a neuroblastoma<br />
cells, palmitoylation-deficient γ-secretase is still able to reach lipid raft microdomains <strong>an</strong>d<br />
process APP, suggesting <strong>the</strong> involvement of additional signal. In addition to lipid raft targeting,<br />
palmitoylation may have import<strong>an</strong>t function in γ-secretase neuronal trafficking as it is <strong>the</strong> case<br />
<strong>for</strong> m<strong>an</strong>y o<strong>the</strong>r proteins. We hypo<strong>the</strong>sized that γ-secretase palmitoylation is required <strong>for</strong><br />
amyloidogenic processing of APP in neurons <strong>an</strong>d that its inhibition is able to reduce Aβ<br />
production <strong>an</strong>d deposition without affecting o<strong>the</strong>r γ-secretase substrates processing. To test this<br />
hypo<strong>the</strong>sis we have generated tr<strong>an</strong>sgenic mice overexpressing wild type or palmitoylationdefective<br />
mut<strong>an</strong>t nicastrin <strong>an</strong>d Aph1. We have crossed <strong>the</strong>se mice with <strong>the</strong> AD tr<strong>an</strong>sgenic mouse<br />
model 85Dbo that overexpresses mut<strong>an</strong>t hum<strong>an</strong> APP <strong>an</strong>d presenilin 1. Here, we characterized<br />
<strong>the</strong>se mice by <strong>an</strong>alyzing Aβ deposition in <strong>the</strong> brain to determine if γ-secretase subunit<br />
palmitoylation is critical <strong>for</strong> APP processing in vivo. Fur<strong>the</strong>rmore, we pl<strong>an</strong> to <strong>an</strong>alyze <strong>the</strong><br />
processing of o<strong>the</strong>r γ-secretase-substrates, such as Notch, N-Cadherin <strong>an</strong>d DCC in <strong>the</strong>se mice, to<br />
determine if <strong>the</strong>y are affected by γ-secretase palmitoylation deficiency.<br />
Supported by <strong>the</strong> Americ<strong>an</strong> Federation <strong>for</strong> Aging Research (XM), <strong>an</strong>d Alzheimer’s Association<br />
(GT).<br />
Disclosures: X. Meckler, None; J. Rosem<strong>an</strong>, None; H. Cheng, None; S. Pei, None; M. Keat,<br />
None; B. Kassarji<strong>an</strong>, None; A. Parent, None; G. Thinakar<strong>an</strong>, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.9/H22<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: NIH P01-NS047308<br />
Title: Mossy fiber LTP deficits in BACE1 knockouts c<strong>an</strong> be rescued by activation of alpha 7<br />
nicotinic acetylcholine receptors<br />
Authors: *H. WANG 1,2 , P. WONG 3 , H.-K. LEE 1,2 ;<br />
1 Biol., Univ. Maryl<strong>an</strong>d, College Park, MD; 2 Neurosci. <strong>an</strong>d Cognitive Sci. (NACS) Program,
Univ. of Maryl<strong>an</strong>d, College Park, MD; 3 Departments of Pathology <strong>an</strong>d Neurosci., Johns Hopkins<br />
Sch. of Med., Baltimore, MD<br />
Abstract: Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), <strong>the</strong> β-secretase<br />
required <strong>for</strong> <strong>the</strong> <strong>for</strong>mation of β-amyloid (Aβ) peptides, is thought to be one of <strong>the</strong> key <strong>the</strong>rapeutic<br />
targets that c<strong>an</strong> prevent <strong>the</strong> progression of Alzheimer’s disease (AD). Although complete<br />
ablation of BACE1 gene prevents Aβ <strong>for</strong>mation, our previous study showed that BACE1<br />
knockout mice display severe presynaptic deficits at mossy fiber to CA3 synapses in<br />
hippocampus, one of <strong>the</strong> major loci of BACE1 expression in <strong>the</strong> brain. We hypo<strong>the</strong>size that such<br />
deficits are likely due to abnormal presynaptic Ca 2+ regulation. Cholinergic system has been<br />
implicated in several neurodegenerative disorders including AD, in some cases via activation of<br />
nicotinic acetylcholine receptors (nAChRs), among which are Ca 2+ permeable α7-nAChRs. Here<br />
we present data that brief application of nicotine at mossy fiber to CA3 synapses c<strong>an</strong> restore<br />
presynaptic release (as measured by a signific<strong>an</strong>tly decrease in paired-pulse facilitation (PPF)<br />
ratio) <strong>an</strong>d rescue mossy fiber LTP in BACE1 knockouts. This effect was blocked by αbungarotoxin,<br />
<strong>an</strong> <strong>an</strong>tagonist of α7-nAChRs, <strong>an</strong>d mimicked by a specific agonist of α7-nAChRs<br />
PNU 282987. Our data suggest that nicotine c<strong>an</strong> recover <strong>the</strong> presynaptic deficits in BACE1<br />
knockouts at mossy fiber to CA3 synapses by activating α7-nAChRs.<br />
Disclosures: H. W<strong>an</strong>g, None; P. Wong, None; H. Lee, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.10/H23<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: CIHR<br />
Title: Regulation of BACE1 by zinc signaling<br />
Authors: *Y. DENG, W. ZHOU, W. SONG;<br />
Townsend Family Laboratories, Dept. of Psychiatry, Univ. of British Columbia, V<strong>an</strong>couver, BC,<br />
C<strong>an</strong>ada<br />
Abstract: One of <strong>the</strong> pathological features of Alzheimer’s disease (AD) is <strong>the</strong> extracellular<br />
amyloid plaques, which is <strong>for</strong>med by deposition of 40-42 residue amyloid β protein (Aβ). Aβ is<br />
derived <strong>from</strong> cleavage of β-amyloid precursor protein (APP) by β-secretase <strong>an</strong>d γ-secretase. Zinc
is <strong>an</strong> essential mineral <strong>an</strong>d is involved in m<strong>an</strong>y aspects of cell metabolism. Zinc plays <strong>an</strong><br />
import<strong>an</strong>t role in DNA <strong>an</strong>d protein syn<strong>the</strong>sis, immune function, <strong>an</strong>d cell division. Disturb<strong>an</strong>ce of<br />
Zinc homeostasis has been implicated in neurodegeneration. To determine whe<strong>the</strong>r zinc affects<br />
APP processing <strong>an</strong>d Alzheimer’s disease pathogenesis, cells have been treated with different<br />
dosage of zinc sulfate <strong>an</strong>d cadmium chloride. The expression level of BACE1 was measured by<br />
RT-PCR. We found that low dosage of zinc <strong>an</strong>d cadmium treatment altered <strong>the</strong> BACE1<br />
expression. To fur<strong>the</strong>r examine whe<strong>the</strong>r Zinc affects BACE1 gene expression via its effect on<br />
BACE1 gene tr<strong>an</strong>scription, BACE1 promoter reporter plasmids were tr<strong>an</strong>sfected into cells <strong>an</strong>d<br />
<strong>the</strong>n treated with Zinc sulfate <strong>an</strong>d cadmium chloride <strong>an</strong>d <strong>the</strong> promoter activity was measured by<br />
a dual luciferase assay. Our preliminary data showed that <strong>the</strong> promoter activity was modulated<br />
by Zinc treatment. These results indicate that heavy metal ions like zinc <strong>an</strong>d cadmium may play<br />
<strong>an</strong> import<strong>an</strong>t role in regulating BACE1 gene expression.<br />
Disclosures: Y. Deng, None; W. Zhou, None; W. Song, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.11/H24<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: EU FP7 project LipiDiDiet, Gr<strong>an</strong>t Agreement N° 211696.<br />
Title: B-vitamin supplementation is necessary <strong>for</strong> <strong>the</strong> brain gamma-secretase activity reducing<br />
effects of docosahexaenoic acid <strong>an</strong>d uridine monophosphate<br />
Authors: N. VAN WIJK 1 , J. KUCHENBECKER 2 , A. A. M. KUIPERS 1 , M. C. DE WILDE 1 ,<br />
M. O. W. GRIMM 2 , T. HARTMANN 2 , P. J. KAMPHUIS 1 , *L. M. BROERSEN 1 ;<br />
1 D<strong>an</strong>one Res., Wageningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Saarl<strong>an</strong>d Univ., Homburg/Saar, Germ<strong>an</strong>y<br />
Abstract: Introduction Alzheimer’s disease (AD) is characterized by neurodegeneration <strong>an</strong>d<br />
<strong>the</strong> deposition of dense plaques in <strong>the</strong> brain containing beta-amyloid (abeta). Abeta peptides are<br />
believed to promote pro-inflammatory responses <strong>an</strong>d to activate neurotoxic pathways causing<br />
neuronal dysfunction characterized by a loss of spines <strong>an</strong>d neurites leading to neuronal death.<br />
Abeta is produced by <strong>the</strong> successive clipping of <strong>the</strong> amyloid precursor protein by beta- <strong>an</strong>d<br />
gamma-secretase, two membr<strong>an</strong>e-associated enzymes. Although <strong>the</strong> etiology of AD is not<br />
known it has been recognized that a clear link exists between AD <strong>an</strong>d nutrition. Previously, it<br />
was shown that nutrients may affect brain processes relev<strong>an</strong>t to AD, including neuronal
membr<strong>an</strong>e syn<strong>the</strong>sis. For inst<strong>an</strong>ce, docosahexaenoic acid (DHA) <strong>an</strong>d uridine monophosphate<br />
(UMP) were shown to increase <strong>the</strong> <strong>for</strong>mation of neuronal membr<strong>an</strong>es <strong>an</strong>d spines. Here we tested<br />
<strong>the</strong> influence of nutrients that have previously been shown to affect neuronal membr<strong>an</strong>e<br />
syn<strong>the</strong>sis or membr<strong>an</strong>e-bound enzymatic activity relev<strong>an</strong>t <strong>for</strong> AD. The current study assessed <strong>the</strong><br />
effects of DHA <strong>an</strong>d UMP on brain gamma-secretase activity as well as <strong>the</strong> role of B-vitamins in<br />
enabling <strong>the</strong>se actions.<br />
Methods Experiment 1: Gerbils were fed with UMP enriched diet <strong>an</strong>d gavaged daily with DHA<br />
<strong>from</strong> different sources. Experiment 2: Rats were fed DHA + UMP in combination with a Bvitamin<br />
deficient diet or a B-vitamin supplemented diet. After 4 weeks intervention <strong>the</strong> <strong>an</strong>imals<br />
were killed, <strong>the</strong>ir brains collected <strong>an</strong>d homogenized. Activity of gamma-secretase was measured<br />
with a fluorescent assay using a gamma-secretase specific peptide. The rate of fluorescent signal<br />
increase caused by peptide breakdown was taken as a measure of gamma-secretase activity.<br />
Plasma homocysteine levels were determined in experiment 2.<br />
Results Combined dietary supplementation of DHA <strong>an</strong>d UMP reduced brain gamma secretase<br />
activity only in <strong>the</strong> presence of sufficient B vitamins. Single nutrient interventions had no effect<br />
on gamma-secretase activity. Homocysteine levels were lowered in rats receiving B-vitamins in<br />
<strong>the</strong>ir diet.<br />
Conclusion These data show that a multi-nutrient approach is more successful in lowering<br />
gamma-secretase activity th<strong>an</strong> single nutrient intervention, suggesting that a multi-nutrient<br />
approach might offer <strong>an</strong> effective method in <strong>the</strong> m<strong>an</strong>agement of AD.<br />
Disclosures: N. v<strong>an</strong> Wijk, D<strong>an</strong>one Research, A. Employment (full or part-time); J.<br />
Kuchenbecker, None; A.A.M. Kuipers, D<strong>an</strong>one Research, A. Employment (full or part-time);<br />
M.C. de Wilde, D<strong>an</strong>one Research, A. Employment (full or part-time); M.O.W. Grimm,<br />
None; T. Hartm<strong>an</strong>n, None; P.J. Kamphuis, D<strong>an</strong>one Research, A. Employment (full or parttime);<br />
L.M. Broersen, D<strong>an</strong>one Research, A. Employment (full or part-time).<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.12/H25<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Title: Resveratrol- dependent SIRT1 activation modulates presenilin- 1 expression with<br />
implications <strong>for</strong> Alzheimer ’s disease<br />
Authors: *J. N. DILEO 1 , G. N. LATORRE 2 , S. M. ZAKHARY 2 , B. H. HALLAS 2 , L. K.<br />
FRIEDMAN 2 , G. TORRES 2 , J. R. LEHESTE 2 ;
1 2<br />
Neurosci, Nor<strong>the</strong>rn Boulevard, Old Westbury, NY; Neurosci., New York Col. of Osteo. Med.,<br />
Old Westbury, NY<br />
Abstract: Presenilin 1 (PSEN1) plays <strong>an</strong> essential catalytic role in <strong>the</strong> gamma-secretase/PS1<br />
complex which, if malfunctioning, is responsible <strong>for</strong> <strong>the</strong> accumulation of extracellular beta<br />
amyloid (BA) plaques associated with Alzheimer’s Disease (AD). The gamma secretase/PS1<br />
complex also modulates <strong>the</strong> proteolytic cleavage of <strong>the</strong> Notch tr<strong>an</strong>smembr<strong>an</strong>e receptor, <strong>the</strong>reby<br />
affecting proliferation <strong>an</strong>d differentiation of adult neural stem cells (NSC). Resveratrol (RES), a<br />
potent phytochemical, has unique <strong>an</strong>ti-aging <strong>an</strong>d neuroprotective qualities most of which are<br />
mediated by <strong>the</strong> NAD-dependent protein deacetylase Sirtuin 1 (SIRT1). RES-dependent SIRT1<br />
activation mimics <strong>the</strong> effects of a caloric restriction (CR) diet, which holds promise in slowing<br />
<strong>the</strong> signs <strong>an</strong>d symptoms of AD in cellular <strong>an</strong>d <strong>an</strong>imal models. Here we are presenting a novel<br />
link between RES-dependent SIRT1 activation <strong>an</strong>d PSEN1 expression. Our objective is to<br />
demonstrate <strong>the</strong> potential of this interaction <strong>for</strong> neural health in respect to AD. SIRT1 DNA<br />
targets were initially identified with a combined chromatin immunoprecipitation (ChIP)/DNA<br />
cloning strategy using hum<strong>an</strong> embryonic kidney cells (HEK-293). Here, SIRT1 was found to<br />
associate with <strong>an</strong> upstream non-coding DNA region implicated in <strong>the</strong> tr<strong>an</strong>scriptional regulation<br />
of psen1. Qu<strong>an</strong>titative PCR (QPCR) <strong>an</strong>alysis in HEK-293 cells after 48 hours of RES (50mcM)<br />
exposure revealed <strong>an</strong> almost three-fold increase in SIRT1 expression. At <strong>the</strong> same time psen1<br />
expression was increased two-fold in this cellular model. To validate our cell-based findings in<br />
vivo, we supplemented <strong>the</strong> diet of mature Sprague-Dawley rats with RES (1g/kg) <strong>for</strong> 28 days.<br />
Upon recovery of experimental <strong>an</strong>d control brains, we prepared one half <strong>for</strong><br />
immunohistochemical <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r <strong>for</strong> gene expressional <strong>an</strong>alysis. Whereas SIRT1 appeared<br />
unaffected, <strong>the</strong> expression of PSEN1 was increased four-fold in rat hippocampus <strong>an</strong>d two-fold in<br />
prefrontal cortex. A preliminary immunohistochemical <strong>an</strong>alysis suggests a positive correlation<br />
between RES treatment <strong>an</strong>d NSC proliferation in <strong>the</strong> subventricular zone of <strong>the</strong> lateral ventricles<br />
<strong>an</strong>d <strong>the</strong> subgr<strong>an</strong>ular zone of <strong>the</strong> dentate gyrus. We conclude that RES-dependent SIRT1<br />
activation results in a signific<strong>an</strong>t increase of PSEN1 expression in vitro <strong>an</strong>d in vivo. While<br />
mut<strong>an</strong>t PSEN1 is directly involved in <strong>the</strong> altered processing of BA, <strong>the</strong> knockout of PSEN1<br />
produces AD-like neurodegeneration in <strong>the</strong> absence of cerebral BA plaques. This suggests that<br />
gamma-secretase inhibitors might not be <strong>the</strong> best choice <strong>for</strong> <strong>the</strong> treatment of AD <strong>an</strong>d actually<br />
exacerbate neurodegeneration. Augmentation of PSEN1 via RES on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d may hold a<br />
unique <strong>the</strong>rapeutic potential <strong>for</strong> <strong>the</strong> treatment of AD.<br />
Disclosures: J.N. Dileo, None; G.N. LaTorre, None; S.M. Zakhary, None; B.H. Hallas,<br />
None; L.K. Friedm<strong>an</strong>, None; G. Torres, None; J.R. Leheste, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 527.13/H26<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Title: Elevation of BACE levels in a-beta rat model of Alzheimer's disease: Exacerbation by<br />
chronic stress <strong>an</strong>d prevention by nicotine - Fur<strong>the</strong>r validation of <strong>the</strong> model<br />
Authors: *M. ZAGAAR, M. SRIVAREERAT, T. TRAN, K. ALKADHI;<br />
Pharmacol. & Pharmaceut. Sci., Univ. of Houston, Houston, TX<br />
Abstract: In Alzheimer’s disease (AD), progressive accumulation of β-amyloid (Aβ)-peptides<br />
impairs nicotinic acetylcholine receptor (nAChR) function by <strong>an</strong> unknown mech<strong>an</strong>ism believed<br />
to involve α7 <strong>an</strong>d α4β2-nAChRs. Additionally, <strong>the</strong> beta-site amyloid precursor protein (APP)cleaving<br />
enzyme (BACE1), <strong>the</strong> rate limiting enzyme in pathogenic Aβ production, is expressed<br />
at high levels in hippocampal <strong>an</strong>d cortical regions of AD brains (e.g. Fukumoto et al., Arch<br />
Neurol 59:1381-1389, 2002). We measured hippocampal area CA1 protein levels of BACE1 <strong>an</strong>d<br />
α7- <strong>an</strong>d α4β2-nAChR subunits using <strong>an</strong> Aβ-infused (14 day osmotic pump icv infusion of 300<br />
pmol/day Aβ1-40 <strong>an</strong>d Aβ1-42) rat model of AD in <strong>the</strong> presence <strong>an</strong>d absence of chronic stress<br />
<strong>an</strong>d/or chronic nicotine treatment. There was a signific<strong>an</strong>t increase in <strong>the</strong> levels of BACE in Aβinfused<br />
rats, which was markedly intensified by chronic (4-6 weeks) stress, but normalized by<br />
chronic treatment with nicotine (1 mg/kg x2/day). The levels of <strong>the</strong> three subunits α7 <strong>an</strong>d α4 <strong>an</strong>d<br />
β2 were signific<strong>an</strong>tly decreased in Aβ-infused rats, but were also normalized by chronic nicotine<br />
treatment. Chronic stress in Aβ-infused rats had no signific<strong>an</strong>t effect on <strong>the</strong> levels of <strong>the</strong> nAChR<br />
subunits. The increased BACE levels <strong>an</strong>d decreased nAChR levels, which are established<br />
hallmarks of AD, provide additional support <strong>for</strong> <strong>the</strong> validity of <strong>the</strong> Aβ icv-infused rat as a model<br />
of AD.<br />
Disclosures: M. Zagaar, None; M. Srivareerat, None; T. Tr<strong>an</strong>, None; K. Alkadhi, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.14/H27<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: JSPS Research Fellow
Title: Tr<strong>an</strong>smembr<strong>an</strong>e domain 1 of Presenilin 1 contributes to <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> catalytic pore<br />
of γ-secretase<br />
Authors: *S. TAKAGI 1,3 , C. SATO 2,3 , T. TOMITA 1,3 , T. IWATSUBO 1,2,3 ;<br />
1 2<br />
Grad. Sch. of Pharmaceut. Sci., Grad. Sch. of Med., The Univ. of Tokyo, Tokyo, Jap<strong>an</strong>;<br />
3<br />
CREST, Tokyo, Jap<strong>an</strong><br />
Abstract: Generation <strong>an</strong>d deposition of amyloid-β peptide (Aβ) are implicated in <strong>the</strong><br />
pathogenesis of Alzheimer’s disease (AD). Thus, γ-secretase, that is responsible <strong>for</strong> <strong>the</strong> Aβ<br />
generation, is one of <strong>the</strong> plausible <strong>the</strong>rapeutic targets <strong>for</strong> AD. Enzymatic, pharmacological, <strong>an</strong>d<br />
chemical biological studies revealed that γ-secretase is <strong>an</strong> intramembr<strong>an</strong>e-cleaving aspartyl<br />
protease that endoproteolyzes a scissile bond within <strong>the</strong> tr<strong>an</strong>smembr<strong>an</strong>e domain (TMD) of<br />
various substrates. Presenilin (PS), a multisp<strong>an</strong>ning membr<strong>an</strong>e protein, harbors a pair of catalytic<br />
aspartates within its TMD6 <strong>an</strong>d 7 as <strong>the</strong> proteolytic subunit of γ-secretase. However, fine<br />
structural in<strong>for</strong>mation about this atypical protease is lacking, because γ-secretase is a high<br />
molecular weight membr<strong>an</strong>e protein complex that consists of PS, Nicastrin, Aph-1 <strong>an</strong>d Pen-2.<br />
We have <strong>an</strong>alyzed <strong>the</strong> structure of PS1 by substituted cysteine accessibility method (SCAM), a<br />
biochemical method by which structures of various membr<strong>an</strong>e proteins have been <strong>an</strong>alyzed in a<br />
functional state. Using SCAM, we found that PS1 harbors a hydrophilic “catalytic pore” <strong>for</strong>med<br />
by TMD6, 7, <strong>an</strong>d 9 within <strong>the</strong> membr<strong>an</strong>e (Sato et al., J Neurosci. 2006, 2008). The remaining<br />
question is how <strong>the</strong> N-terminal region of PS1 contributes to <strong>the</strong> γ-secretase activity. Here, we<br />
report that <strong>the</strong> structure <strong>an</strong>d functional role of PS1 TMD1, that has been shown to be<br />
indispensable <strong>for</strong> <strong>the</strong> catalytic activity (Wat<strong>an</strong>abe et al., J Biol. Chem. 2005). We found that a<br />
couple of amino acid residues in TMD1 face to <strong>the</strong> hydrophilic environment within <strong>the</strong> lipid<br />
bilayer. We <strong>the</strong>n <strong>an</strong>alyzed <strong>the</strong> relative position of TMD1 to <strong>the</strong> catalytic pore by crosslinking<br />
experiments of PS1 that harbors one substituted cysteine each within <strong>the</strong> amino- <strong>an</strong>d carboxylterminal<br />
regions. Cysteine mut<strong>an</strong>ts (i.e., M84C <strong>an</strong>d L89C) in TMD1 were crosslinked with<br />
L383C within <strong>the</strong> GxGD motif at TMD7 as well as with L435C in <strong>the</strong> PAL motif at TMD9.<br />
These data indicate that <strong>the</strong> cytosolic side of PS1 TMD1 locates in proximity to <strong>the</strong> catalytic<br />
pore. Finally, we found that <strong>the</strong> hydrophilicity of cytosolic side of TMD1 was affected by <strong>the</strong><br />
tr<strong>an</strong>sition state <strong>an</strong>alogue-type γ-secretase inhibitor. Collectively, <strong>the</strong>se data suggest that TMD1<br />
faces to <strong>the</strong> catalytic pore <strong>an</strong>d functions as a subsite in <strong>the</strong> γ-secretase-mediated intramembr<strong>an</strong>e<br />
cleavage.<br />
Disclosures: S. Takagi, None; C. Sato, None; T. Tomita, None; T. Iwatsubo, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 527.15/H28<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: Korea National Institute of Health Intramural Research Gr<strong>an</strong>t (091-4845-300-210-13)<br />
Title: Caspases-2 <strong>an</strong>d -8 are involved in <strong>the</strong> presenilin1/gamma-secretase-dependent cleavage of<br />
amyloid precursor protein after <strong>the</strong> induction of apoptosis<br />
Authors: *Y. KOH, S. CHAE, C. YOO, C. JO, S.-M. YUN, S. JO;<br />
Div. of Brain Dis., Natl. Inst. of Hlth., Seoul, Republic of Korea<br />
Abstract: The presenilin/γ-secretase protease cleaves m<strong>an</strong>y type-I membr<strong>an</strong>e proteins, including<br />
<strong>the</strong> amyloid β-protein (Aβ) precursor (APP). Previous studies have shown that apoptosis induces<br />
alterations in Aβ production in a caspase-dependent m<strong>an</strong>ner. Here, we report that staurosporine<br />
(STS)-induced apoptosis induces caspase-8 <strong>an</strong>d/or -2 dependent γ-secretase activation. STS<br />
increased γ-secretase-dependent cleavage of E-cadherin (E-cad/CTF2) in T47D cells. This<br />
cleavage of E-cadherin was blocked by caspase-2 <strong>an</strong>d -8 inhibitors. There was no inhibitory<br />
effect of o<strong>the</strong>r caspases (-1, -3, -5, 6, -9) on E-cad/CTF2 production. Caspase-2 <strong>an</strong>d -8 inhibitors<br />
also inhibited Aβ production by STS in H4 cells expressing <strong>the</strong> Swedish mut<strong>an</strong>t of APP (HSW)<br />
or APP-C99 (H4-C99). This finding was fur<strong>the</strong>r supported by evidence that siRNA tr<strong>an</strong>sfection,<br />
depleting caspase-2 or -8 levels, lowered Aβ production in HSW <strong>an</strong>d H4-C99 cells. These results<br />
suggest that caspase-2 <strong>an</strong>d/or -8 is involved in presenilin/γ- secretase activation <strong>an</strong>d Aβ<br />
production in apoptosis.<br />
Disclosures: Y. Koh, Korea National Institute of Health Intramural Research Gr<strong>an</strong>t (091-4845-<br />
300-210-13), B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); S. Chae, None; C. Yoo, None; C. Jo, None; S. Yun,<br />
None; S. Jo, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.16/H29<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: ADDF # 280601 (IB)
Title: Ry<strong>an</strong>odine receptors <strong>an</strong>d neuronal survival in Alzheimer’s disease<br />
Authors: S. SUN, H. ZHANG, *I. BEZPROZVANNY;<br />
Dept Physiol, ND12.502B, UT Southwestern Med. Ctr. Dallas, Dallas, TX<br />
Abstract: The abnormal calcium signaling in endoplasmic reticulum (ER) has been suggested to<br />
play <strong>an</strong> import<strong>an</strong>t role in familial Alzheimer’s disease (FAD) pathogenesis (Bezprozv<strong>an</strong>ny <strong>an</strong>d<br />
Mattson, 2008, Trends Neurosci, 31:454-463). Ry<strong>an</strong>odine receptors (Ry<strong>an</strong>R) are <strong>the</strong> major<br />
resident calcium release ch<strong>an</strong>nels in <strong>the</strong> ER. Three Ry<strong>an</strong>R subtypes are expressed in mammali<strong>an</strong><br />
brain. Here we investigated <strong>an</strong> expression <strong>an</strong>d function of 3 Ry<strong>an</strong>R subtypes in two different<br />
FAD mouse models - APPPS1 mice (Thy1-APPKM670/671NL; Thy1-PS1L166P) (Radde at al,<br />
2006, EMBO Rep, 7:940-946) <strong>an</strong>d 3xTG mice (PS1-M146V KI, Thy1-APP-Swe, Thy1-tau-<br />
P301L) (Oddo at al, 2003, Neuron, 39:409-421). By qPCR <strong>an</strong>alysis we determined that Ry<strong>an</strong>R2<br />
is <strong>the</strong> major subtype expressed in hippocampal <strong>an</strong>d cortical regions of <strong>the</strong>se mice between 2 <strong>an</strong>d<br />
9 months of age. We fur<strong>the</strong>r determined that expression levels of Ry<strong>an</strong>R1 <strong>an</strong>d Ry<strong>an</strong>R3 is<br />
increased <strong>an</strong>d expression levels of Ry<strong>an</strong>R2 are decreased in postnatal hippocampal neuronal<br />
cultures <strong>from</strong> 3XTG mice when compared to wild type mice. Specific knockdown of Ry<strong>an</strong>R2<br />
expression by Lenti-shRNAi protected 3xTG cultured hippocampal neurons <strong>from</strong> glutamateinduced<br />
toxicity in vitro. These results indicate that function of Ry<strong>an</strong>R is altered in FAD neurons<br />
<strong>an</strong>d that Ry<strong>an</strong>R2 constitute a potential target <strong>for</strong> treatment of AD.<br />
Disclosures: S. Sun, None; H. Zh<strong>an</strong>g, None; I. Bezprozv<strong>an</strong>ny, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.17/H30<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Title: The cortical distribution of beta secretase in Alzheimer disease <strong>an</strong>d o<strong>the</strong>r dementias<br />
Authors: *R. G. STRUBLE 1 , B. E. MOORE 2 , S. BECKMAN-RANDALL 3 , X. X. YAN 4 ;<br />
2 Pathology, 1 Sou<strong>the</strong>rn Illinois Univ., Springfield, IL; 3 Alzheimer Dis. Ctr., Sou<strong>the</strong>rn Illinois<br />
Univ., Springfield,, IL; 4 Anat., Sou<strong>the</strong>rn Illinois Univ., Carbondale,, IL<br />
Abstract: Beta-secretase (BACE) initiates <strong>the</strong> <strong>for</strong>mation of beta amyloid <strong>from</strong> precursor protein<br />
<strong>an</strong>d has been identified as a possible target <strong>for</strong> pharmacological intervention. Whe<strong>the</strong>r BACE<br />
expression is increased in Alzheimer’s disease is controversial. There<strong>for</strong>e, it is import<strong>an</strong>t to
clarify its distribution in hum<strong>an</strong> tissue in normal <strong>an</strong>d diseased samples. We used<br />
immunocytochemical techniques to characterize <strong>the</strong> distribution of BACE1-like<br />
immunoreactivity. METHODS: Tissue was obtained within 24 hours of death, fixed over night<br />
in 4% para<strong>for</strong>maldehyde, cryoprotected, <strong>an</strong>d frozen. A polyclonal <strong>an</strong>tisera directed against<br />
BACE1 (Chemicon) was used in <strong>an</strong> indirect immunoperoxidase technique with DAB as <strong>the</strong><br />
chromogen on 30um sections. We primarily focused on inferotemporal cortex but included<br />
samples of occipital cortex, hippocampus <strong>an</strong>d cerebellum. We also combined BACE<br />
immunoreactivity with immuno-labeling of beta amyloid, glial fibrillary acidic protein,<br />
parvalbumin <strong>an</strong>d calbindin. RESULTS: BACE1-like immunoreactivity was found in cortical<br />
tissues <strong>from</strong> AD, progressive supr<strong>an</strong>uclear palsy, lewy body disease, frontotemporal dementia<br />
(hippocampus) <strong>an</strong>d normal controls. BACE-immunoreactive processes, axons <strong>an</strong>d dendrites,<br />
were co-localized in amyloid plaques. Pericellular distribution, extending along <strong>the</strong> apical<br />
dendrite of pyramidal cells, was <strong>the</strong> most common pattern <strong>an</strong>d few neurons were completely<br />
filled with immunoreactive product. Thin sections (5-10um) emphasized a predomin<strong>an</strong>t<br />
pericellular pattern. More BACE immunoreactive neurons were found in supragr<strong>an</strong>ular th<strong>an</strong><br />
infragr<strong>an</strong>ular layers of neocortex. The gr<strong>an</strong>ule cell layer (IV) showed less immunoreactivity. The<br />
most intense immunoreactivity in preliminary studies, was found in striate cortex (Area 17) with<br />
less present in peristriate cortex. In cerebellum <strong>the</strong> most striking observation was BACE-like<br />
immunoreactivity in rare Golgi, basket <strong>an</strong>d stellate neurons but none in Purkinje cells. Rare<br />
BACE-immunoreactive baskets were also seen. Of note was that we saw no glia expressing<br />
BACE, even in regions devoid of neurons (fronto-temporal dementia). CONCLUSIONS: 1) 1)<br />
BACE1 is normally expressed in hum<strong>an</strong> cortical tissue, most likely in neuronal elements; 2) <strong>the</strong><br />
pericellular distribution of BACE1 suggests that it may be preferentially located in interneurons;<br />
3) BACE is normally expressed in brains <strong>from</strong> both controls <strong>an</strong>d those with neurological<br />
diseases; <strong>an</strong>d 4) we could not detect a clear alteration in BACE1-like immunoreactivity in AD<br />
cases compared to o<strong>the</strong>r dementing diseases or normal controls. BACE1 appears to have some<br />
role in normal function, particularly involving some, but not all interneurons.<br />
Disclosures: R.G. Struble, None; B.E. Moore, None; S. Beckm<strong>an</strong>-R<strong>an</strong>dall, None; X.X. Y<strong>an</strong>,<br />
None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.18/H31<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin
Support: <strong>the</strong> Korea Research Foundation Gr<strong>an</strong>t funded by <strong>the</strong> Kore<strong>an</strong> Government (MOEHRD,<br />
Basic Research Promotion Fund) (KRF-2006-331-E00287)<br />
<strong>the</strong> Korea Research Foundation Gr<strong>an</strong>t funded by <strong>the</strong> Kore<strong>an</strong> Government<br />
(MOEHRD)(E00151/R05-2004-000-12444-0)<br />
Title: Calsenilin regulates presenilin 1/γ-secretase-mediated N-cadherin cleavage <strong>an</strong>d β-catenin<br />
signaling<br />
Authors: C. JANG 1,2 , W. WASCO 3 , J. D. BUXBAUM 4 , Y.-S. KIM 2 , *E.-K. CHOI 1 ;<br />
1 Lab. of Cell. Aging & Neurodegeneration, Ilsong Inst. Life Sci, Hallym Univ., Any<strong>an</strong>g,<br />
Gyeonggi-Do, Republic of Korea; 2 Dept. of Microbiology, Sch. of Medicine, Hallym Univ.,<br />
Chuncheon, Republic of Korea; 3 MassGeneral Inst. <strong>for</strong> Neurodegenerative Dis., Massachusetts<br />
Gen. Hospital, Harvard Med. Sch., Charlestown, MA; 4 Mount Sinai Sch. of Med., New York,<br />
NY<br />
Abstract: Presenilin 1 (PS1) is a component of <strong>the</strong> γ-secretase complex that cleaves various<br />
type-I tr<strong>an</strong>smembr<strong>an</strong>e proteins including <strong>the</strong> amyloid precursor protein, Notch, CD44, Ncadherin,<br />
<strong>an</strong>d E-cadherin. N-cadherin is <strong>an</strong> essential adhesion molecule that <strong>for</strong>ms a complex<br />
with PS1/γ-secretase <strong>an</strong>d β-catenin in plasma membr<strong>an</strong>e <strong>an</strong>d that is sequentially cleaved by <strong>the</strong><br />
matrix metalloproteinase ADAM 10 <strong>an</strong>d PS1/γ-secretase producing a soluble cytoplasmic Ncadherin<br />
C-terminal fragment 2 (Ncad/CTF2). Here, we demonstrate that <strong>the</strong> expression of<br />
calsenilin, a presenilin-interacting protein that has been shown to have multiple functions in<br />
different subcellular compartments, leads to disturbing of PS1/γ-secretase-mediated N-cadherin<br />
ε-cleavage resulting in signific<strong>an</strong>t accumulation of N-cadherin C-terminal fragment 1<br />
(Ncad/CTF1) as well as reduction of cytoplasmic Ncad/CTF2 intracellular domain release along<br />
with deceleration of <strong>the</strong> cell surface delivery of PS1-CTF in SH-SY5Y neuroblastoma cells.<br />
Interestingly, we have found that <strong>the</strong> expression of calsenilin redistributed β-catenin <strong>from</strong> cell<br />
surface to cytoplasmic pool <strong>an</strong>d <strong>the</strong>reby negatively regulated target genes of T-cell factor/βcatenin<br />
nuclear signaling. Taken toge<strong>the</strong>r, our findings suggest that calsenilin is a novel negative<br />
regulator of <strong>the</strong> N-cadherin processing <strong>an</strong>d plays <strong>an</strong> import<strong>an</strong>t role in β-catenin signaling<br />
Disclosures: C. J<strong>an</strong>g, None; W. Wasco, None; J.D. Buxbaum, None; Y. Kim, None; E. Choi,<br />
None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.19/H32
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: NIH gr<strong>an</strong>ts AG15379<br />
NIH gr<strong>an</strong>ts AG26593<br />
Title: PS1 con<strong>for</strong>mation correlates with Aβ42/40 ratio <strong>an</strong>d is modulated by calcium<br />
Authors: *K. UEMURA, H. KOWA, C. M. LILL, X. LI, M. BANKS, L. HERL, J. A.<br />
PETERS, A. IVANOV, Z. FAN, P. JONES, B. T. HYMAN, O. BEREZOVSKA;<br />
Neurology/Alzheimer research unit, Massachusetts Gen. Hosp., Boston, MA<br />
Abstract: An increase in <strong>the</strong> Aβ42/40 ratio potentially leads to <strong>the</strong> pathogenesis of Alzheimer’s<br />
disease (AD) by accelerating <strong>the</strong> amyloid deposition in senile plaques. Mutations in PS1 or PS2,<br />
or o<strong>the</strong>r m<strong>an</strong>ipulations that lead to a structural ch<strong>an</strong>ge in <strong>the</strong> γ-secretase, lead to <strong>an</strong> alteration in<br />
<strong>the</strong> Aβ42/40 ratio <strong>an</strong>d, in <strong>the</strong> case of PS1 or PS2 mutations, to autosomal domin<strong>an</strong>t AD. Thus,<br />
monitoring structural alterations of PS1 in living cells in vitro <strong>an</strong>d in vivo could be a useful<br />
indicator of <strong>the</strong> γ-secretase con<strong>for</strong>mation, which could be exploited <strong>the</strong>rapeutically. Here, we<br />
develop a Forster reson<strong>an</strong>ce energy tr<strong>an</strong>sfer (FRET) probe, GFP-PS1-RFP (G-PS1-R), which<br />
consists of a donor (GFP) <strong>an</strong>d <strong>an</strong> acceptor (mRFP) fluorophores in <strong>the</strong> 1:1 ratio. The<br />
fluorophores are fused to <strong>the</strong> PS1 N-terminus (NT) <strong>an</strong>d TM6-7 loop domain, respectively,<br />
allowing monitoring of PS1 con<strong>for</strong>mational ch<strong>an</strong>ges by measuring ch<strong>an</strong>ges in <strong>the</strong> PS1 NT-loop<br />
proximity. We show that G-PS1-R c<strong>an</strong> be incorporated into <strong>the</strong> γ-secretase complex,<br />
reconstituting activity in PS1/2 deficient cells. Using Fluorescence lifetime imaging microscopy<br />
(FLIM) <strong>an</strong>d spectral FRET techniques, we show that this new probe c<strong>an</strong> be a read out of <strong>the</strong> PS1<br />
NT-loop proximity, which c<strong>an</strong> be used both in living neurons in vitro <strong>an</strong>d in mouse brain in vivo.<br />
Similarly to <strong>the</strong> untagged PS1, <strong>the</strong> G-PS1-R con<strong>for</strong>mation ch<strong>an</strong>ged after introduction of <strong>the</strong><br />
L166P FAD mutation. Fur<strong>the</strong>rmore, <strong>the</strong> probe reported rapid ch<strong>an</strong>ges in <strong>the</strong> PS1 con<strong>for</strong>mation in<br />
response to calcium influx in primary neurons in culture. This observation strongly supports <strong>the</strong><br />
hypo<strong>the</strong>sis that PS1/γ-secretase exists in a dynamic state, which c<strong>an</strong> be allosterically modulated.<br />
Thus, <strong>the</strong> new G-PS1-R probe we report here presents a useful tool <strong>for</strong> <strong>an</strong>alysis of <strong>the</strong><br />
physiological mech<strong>an</strong>ism underlying ch<strong>an</strong>ges in PS1 con<strong>for</strong>mation/function in living cells <strong>an</strong>d<br />
could be used <strong>for</strong> drug screening in AD <strong>the</strong>rapeutics.<br />
Disclosures: K. Uemura, None; H. Kowa, None; C.M. Lill, None; X. Li, None; M. B<strong>an</strong>ks,<br />
None; L. Herl, None; J.A. Peters, None; A. Iv<strong>an</strong>ov, None; Z. F<strong>an</strong>, None; P. Jones, None; B.T.<br />
Hym<strong>an</strong>, None; O. Berezovska, None.<br />
Poster<br />
527. Beta <strong>an</strong>d Gamma Secretase Function <strong>an</strong>d Metabolism I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 527.20/H33<br />
Topic: C.02.d. Beta <strong>an</strong>d gamma secretase, BACE <strong>an</strong>d presenilin<br />
Support: NIH/NIA<br />
Alzheimer’s Association<br />
Americ<strong>an</strong> Health Assist<strong>an</strong>ce Foundation<br />
Americ<strong>an</strong> Federation <strong>for</strong> Aging Research<br />
Title: Insights into elevated Aβ secretion by BACE-GPI processing of APP<br />
Authors: *K. S. VETRIVEL 1 , X. MECKLER 1 , Y. CHEN 1 , G. THINAKARAN 2 ;<br />
1 2<br />
Dept. Neurobio. <strong>an</strong>d Neurol., Dept. Neurobiology, Neurol. <strong>an</strong>d Pathology, Univ. Chicago,<br />
Chicago, IL<br />
Abstract: Alzheimer’s associated β-amyloid (Aβ) peptide is generated <strong>from</strong> amyloid precursor<br />
protein (APP) by <strong>the</strong> sequential proteolytic processing by BACE1 <strong>an</strong>d γ-secretase. Previous<br />
studies implicated cholesterol <strong>an</strong>d lipid rafts in Aβ production <strong>an</strong>d deposition. We examined raft<br />
association of BACE1 without m<strong>an</strong>ipulating cellular cholesterol levels in order to exclude<br />
pleiotropic effects associated with cholesterol depletion. We found that BACE1 is palmitoylated<br />
at 4 Cys residues at <strong>the</strong> junction of tr<strong>an</strong>smembr<strong>an</strong>e <strong>an</strong>d cytosolic domains <strong>an</strong>d Ala substitution of<br />
<strong>the</strong>se residues result in complete displacement of BACE1 <strong>from</strong> detergent-resist<strong>an</strong>t lipid raft<br />
membr<strong>an</strong>e domains. However, displacing BACE1 <strong>from</strong> lipid rafts did not have discernable<br />
influence on Aβ production. This finding is in contrast to <strong>an</strong> earlier study that reported elevated<br />
Aβ production by <strong>the</strong> expression of BACE-GPI, a vari<strong>an</strong>t of BACE1 that gets exclusively<br />
targeted to lipid rafts. In order to gain additional insights into BACE-GPI processing of APP, we<br />
characterized raft association, subcellular localization <strong>an</strong>d biochemical characterization of APP<br />
processing in N2a APPwt cells stably expressing wtBACE <strong>an</strong>d BACE-GPI. In agreement with<br />
previous study, addition of GPI to BACE1 results in increased raft association of BACE1<br />
compared to BACE1wt. Fur<strong>the</strong>r studies using cell surface biotinylation <strong>an</strong>d confocal microscopy<br />
revealed that BACE-GPI is mainly localized in cell surface, whereas wtBACE1 largely resides in<br />
endosomes. These results indicate that BACE-GPI cleaves APP mainly in cell surface lipid rafts,<br />
leading to <strong>the</strong> secretion of elevated levels of Aβ.<br />
Disclosures: K.S. Vetrivel, None; X. Meckler, None; Y. Chen, None; G. Thinakar<strong>an</strong>, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.1/H34<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Intracerebroventricular injections of mu-P-75 saporin c<strong>an</strong> produce memory deficits<br />
without impairing motor deficits in a mouse model of Alzheimer’s disease<br />
Authors: *J. J. MATCHYNSKI, S. LOWRANCE, J. ROSSIGNOL, N. PUCKETT, N.<br />
DERKORVER, J. RADWAN, K. TRAINOR, M. SANDSTROM, G. DUNBAR;<br />
Central Michig<strong>an</strong> Univ., Mount Pleas<strong>an</strong>t, MI<br />
Abstract: Intracerbroventricular injections of mu-P-75 saporin (Adv<strong>an</strong>ced Targeting Systems,<br />
S<strong>an</strong> Diego, CA) effectively <strong>an</strong>d efficiently destroys cholinergic neurons <strong>an</strong>d creates memory<br />
deficits in mice, mimicking some of <strong>the</strong> key symptoms of Alzheimer’s disease. Early attempts to<br />
use mu-P-75 saporin in mice required a relatively high me<strong>an</strong> effective dose (ED50) of 3.6 µg in<br />
order to create behavioral deficits (Berger-Sweeney et al., 2001, The Journal of Neuroscience,<br />
21: 8164-8173; Hunter et al, 2004, Europe<strong>an</strong> Journal of Neuroscience, 19: 3305-3316). Recent<br />
adv<strong>an</strong>ces in producing <strong>the</strong> saporin have lowered <strong>the</strong> ED50 to doses to 0.4 µg, although <strong>the</strong><br />
resulting memory deficits are tr<strong>an</strong>sient, <strong>an</strong>d doses above 0.8 µg c<strong>an</strong> cause motor deficits (Moreau<br />
et al., 2008, Hippocampus, 18: 610-622). In <strong>an</strong> ef<strong>for</strong>t to elucidate <strong>the</strong> behavioral effects of a<br />
higher (0.8 µg) dose, we gave bilateral intracerbroventricular injections of mu-P-75 saporin<br />
(n=6) or sterile phosphate buffered saline (n=3) into C57/BL6 mice <strong>an</strong>d assessed <strong>the</strong>ir cognitive<br />
abilities on both a Morris water maze (MWM) <strong>an</strong>d <strong>an</strong> object-recognition task, while monitoring<br />
<strong>the</strong>ir motor abilities using a rotarod task. Mice receiving <strong>the</strong> mu-P-75 saporin per<strong>for</strong>med<br />
signific<strong>an</strong>tly worse th<strong>an</strong> sham <strong>an</strong>imals on <strong>an</strong> object recognition task <strong>an</strong>d tended to have longer<br />
latencies <strong>an</strong>d swim paths during <strong>the</strong> seven days of MWM testing. Import<strong>an</strong>tly, no between-group<br />
differences were observed <strong>for</strong> latency to fall on <strong>the</strong> rotarod task. Collectively, <strong>the</strong>se results<br />
suggest that <strong>the</strong> 0.8 µg dose of saporin is both safe <strong>an</strong>d effective <strong>for</strong> mimicking AD-like memory<br />
deficits, without causing signific<strong>an</strong>t motor deficits.<br />
Disclosures: J.J. Matchynski, None; S. Lowr<strong>an</strong>ce, None; J. Rossignol, None; N. Puckett,<br />
None; N. Derkorver, None; J. Radw<strong>an</strong>, None; K. Trainor, None; M. S<strong>an</strong>dstrom, None; G.<br />
Dunbar, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.2/H35<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Arizona Alzheimer's Consortium<br />
Arizona Alzheimer's Disease Core Center (P30 AG19610) Pilot Gr<strong>an</strong>t Program<br />
Barrow Neurological Foundation<br />
HHMI-funded ASU SOLUR Program<br />
Title: Comprehensive polynutrient dietary supplementation improves cognition in 3xTG mice<br />
Authors: *J. E. VALLA 1,2 , B. B. BRADEN 3,2 , L. ENGLER 3,2 , A. GARCIA 3,2 , J. LIND 1,2,3 , N.<br />
L. YOUNG 1,2,3 , B. BOWMAN 3,2 , A. B. WOLF 1,2,3 , K. PAGE 1,2,3 , Y. KUSNE 1,2,3 , N. B.<br />
EMERSON LOMBARDO 4,5 , H. BIMONTE-NELSON 3,2 ;<br />
1 Barrow Neurolog. Institute, St. Joseph's Hosp. & Med. Ctr., Phoenix, AZ; 2 Arizona Alzheimer's<br />
Consortium, Phoenix, AZ; 3 Arizona State Univ., Tempe, AZ; 4 Boston Univ. Sch. of Med.,<br />
Boston, MA; 5 VAMC, Bed<strong>for</strong>d, MA<br />
Abstract: Animal model <strong>an</strong>d epidemiological studies suggest that some diets c<strong>an</strong> be<br />
neuroprotective <strong>an</strong>d slow cognitive decline in early stage AD. The beneficial properties of <strong>the</strong>se<br />
diets likely arise <strong>from</strong> increased omega-3’s, <strong>an</strong>ti-oxid<strong>an</strong>ts, <strong>an</strong>ti-inflammatory properties, B<br />
vitamins, as well as attenuation of insulin resist<strong>an</strong>ce <strong>an</strong>d reduced oxidation of LDL cholesterol.<br />
To maximize <strong>the</strong> additive <strong>an</strong>d likely synergistic effects of <strong>the</strong>se <strong>an</strong>d o<strong>the</strong>r nutrients, <strong>an</strong><br />
interdisciplinary team of clinici<strong>an</strong>s <strong>an</strong>d nutritionists designed <strong>the</strong> Memory Preservation Nutrition<br />
Supplement Program (MPNSP), designed to deliver diverse, broad-based, high-quality<br />
nutritional supplementation. In this study, we administered MPNSP supplements incorporated<br />
into st<strong>an</strong>dard mouse chow to triply-tr<strong>an</strong>sgenic mice known to develop features of AD with age.<br />
These supplements included 1) a phyto-nutrient powder comprised of 100% org<strong>an</strong>ic freeze-dried<br />
fruits <strong>an</strong>d vegetables, spices, grains <strong>an</strong>d probiotics; 2) <strong>an</strong> amalgam of herbs <strong>an</strong>d spices chosen <strong>for</strong><br />
<strong>the</strong>ir reported <strong>an</strong>ti-inflammatory properties (e.g., turmeric, resveratrol, ginger, Baikal Skullcap);<br />
<strong>an</strong>d 3) high-quality cod liver oil. Mice (N=56) were fed ei<strong>the</strong>r supplemented or st<strong>an</strong>dard chow<br />
beginning at <strong>an</strong> average age of 38 weeks (r<strong>an</strong>ge 29-46 weeks) in age- <strong>an</strong>d sex-matched cohorts in<br />
a full 2x2 design (genotype x diet). After approx 4 months of supplementation, mice were<br />
cognitively tested on a delayed match to position spatial working <strong>an</strong>d recent memory plus maze<br />
(DMS) task, as well as <strong>the</strong> spatial reference memory Morris maze. Preliminary <strong>an</strong>alyses indicate<br />
that triply-tr<strong>an</strong>sgenic mice on <strong>the</strong> nutrient-rich diet exhibited better cognitive per<strong>for</strong>m<strong>an</strong>ce th<strong>an</strong><br />
tr<strong>an</strong>sgenic mice that received <strong>the</strong> control diet; <strong>the</strong> diet improved learning on both <strong>the</strong> DMS task<br />
<strong>an</strong>d <strong>the</strong> Morris maze task. Preliminary <strong>an</strong>alyses also suggest that <strong>the</strong> diet had no signific<strong>an</strong>t effect<br />
in <strong>the</strong> wildtype mice. These results suggest that polynutrient supplementation c<strong>an</strong> be beneficial in<br />
countering <strong>the</strong> functional consequences of AD-related brain ch<strong>an</strong>ges. Studies are underway to<br />
deduce <strong>the</strong> effects of <strong>the</strong> supplementation on pathophysiological markers of AD in <strong>the</strong>se mice.
Collectively, <strong>the</strong>se findings, taken with prior research, indicate that diet could be a safe <strong>an</strong>d costeffective<br />
method of influencing AD-related functional ch<strong>an</strong>ge.<br />
Disclosures: J.E. Valla, None; B.B. Braden, None; L. Engler, None; A. Garcia, None; J.<br />
Lind, None; N.L. Young, None; B. Bowm<strong>an</strong>, None; A.B. Wolf, None; K. Page, None; Y.<br />
Kusne, None; N.B. Emerson Lombardo, None; H. Bimonte-Nelson, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.3/H36<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: AAV-mediated tau expression in rat hippocampus as a novel tauopathy model <strong>for</strong> <strong>the</strong><br />
development of <strong>the</strong>rapeutics<br />
Authors: *M. LI 1 , C. EHRENFELS 2 , P. SNODGRASS-BELT 3 , M. H. ARNOLD 2 , T.<br />
ENGBER 2 , T. BUSSIERE 2 , C. AMBROSE 2 ;<br />
1 2<br />
Dept Neuropharmacol, Biogenidec, Inc, Cambridge, MA; Biogenidec, Cambridge, MA;<br />
3<br />
bioenidec, Cambridge, MA<br />
Abstract: Abnormal accumulation of <strong>the</strong> microtubule-associated protein tau is a pathological<br />
hallmark of Alzheimer’s disease <strong>an</strong>d o<strong>the</strong>r neurodegenerative diseases. Most models used <strong>for</strong><br />
drug development are tr<strong>an</strong>sgenic models that have variable disease onsets <strong>an</strong>d disparate levels of<br />
pathologic protein. Viral vectors have been extensively used in recent years to express specific<br />
gene products in naive <strong>an</strong>imals to mimic pathological conditions of hum<strong>an</strong> diseases <strong>an</strong>d may<br />
offer <strong>an</strong> alternative to <strong>the</strong> more traditional tr<strong>an</strong>sgenic model. The aim of this study was to<br />
develop a model <strong>for</strong> testing potential tau-reducing <strong>the</strong>rapeutics by inducing expression of hum<strong>an</strong><br />
mut<strong>an</strong>t P301L tau in normal rats via AAV-mediated gene tr<strong>an</strong>sfer, which in turn would result in<br />
<strong>the</strong> development of tau pathology in <strong>the</strong> hippocampus.<br />
We constructed recombin<strong>an</strong>t AAV serotype 1 carrying <strong>the</strong> HA-tagged hum<strong>an</strong> P301L tau <strong>an</strong>d<br />
green fluorescent protein (GFP) <strong>an</strong>d stereotaxically injected into <strong>the</strong> dorsal hippocampus of 3month-old<br />
male Sprague-Dawley rats. Animals were euth<strong>an</strong>ized 2, 4 <strong>an</strong>d 6 weeks after injection.<br />
Hippocampal tissue was collected <strong>for</strong> Western blot <strong>an</strong>d histological <strong>an</strong>alysis. Western Blot<br />
<strong>an</strong>alysis using <strong>an</strong>ti-hum<strong>an</strong> tau <strong>an</strong>tibodies (TAUY9, Invitrogen, CA), <strong>an</strong>ti-HA <strong>an</strong>tibody (Roche,<br />
CT) <strong>an</strong>d <strong>an</strong>ti GFP <strong>an</strong>tibody (MBL, MA) showed robust expression of hum<strong>an</strong> mut<strong>an</strong>t tau <strong>an</strong>d GFP<br />
in <strong>the</strong> hippocampus as early as 2 weeks after injection. Immunohistochemistry, using <strong>an</strong>tibodies<br />
against phosphorylated tau (AT8, PIERCE) <strong>an</strong>d total tau (Tau5, Invitrogen, CA), <strong>an</strong>d <strong>an</strong>ti-GFP
<strong>an</strong>tibody demonstrated neuronal expression of GFP <strong>an</strong>d tau immunoreactivity throughout <strong>the</strong><br />
hippocampus.<br />
These data suggest that <strong>the</strong> localized gene tr<strong>an</strong>sfer of hum<strong>an</strong> mutated tau in naive rats could be a<br />
valuable model to study tau-related neurodegeneration <strong>an</strong>d to assess <strong>the</strong> in vivo efficacy of<br />
<strong>the</strong>rapeutics designed to reduce levels of pathological tau protein.<br />
Disclosures: M. Li, None; C. Ehrenfels, None; P. Snodgrass-Belt, None; M.H. Arnold,<br />
None; T. Engber, None; T. Bussiere, None; C. Ambrose, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.4/H37<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Stephen D. Bechtel Jr. Foundation <strong>an</strong>d <strong>the</strong> NIH.<br />
Title: Aβ-induced epilepti<strong>for</strong>m activity disrupts learning <strong>an</strong>d enh<strong>an</strong>ces <strong>for</strong>getting in tr<strong>an</strong>sgenic<br />
models of Alzheimer’s disease<br />
Authors: L. VERRET, M. THWIN, K. HO, L. MUCKE, *J. J. PALOP;<br />
Gladstone Instit <strong>an</strong>d UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Alzheimer’s disease (AD) is associated with high levels of amyloid-β peptide (Aβ),<br />
increased incidence of seizures, <strong>an</strong>d progressive cognitive decline. We previously demonstrated<br />
that high Aβ levels trigger generalized epileptic activity <strong>an</strong>d intense remodeling of hippocampal<br />
circuits in a mouse model of AD (hAPPJ20). However, causal relations between Aβ-induced<br />
epileptic activity, remodeling of hippocampal circuits, <strong>an</strong>d cognitive decline have not yet been<br />
established. To test whe<strong>the</strong>r epileptic activity critically contributes to hippocampal remodeling<br />
<strong>an</strong>d cognitive impairments in tr<strong>an</strong>sgenic mouse models of AD, we pharmacologically<br />
m<strong>an</strong>ipulated Aβ-induced epilepti<strong>for</strong>m activity in hAPPJ20 mice <strong>an</strong>d evaluated <strong>the</strong> effect of such<br />
m<strong>an</strong>ipulation on hippocampal remodeling <strong>an</strong>d cognitive decline. Chronic exacerbation of Aβinduced<br />
electroencephalographic (EEG) epileptic activity enh<strong>an</strong>ced hippocampal remodeling,<br />
including NPY/GABAergic sprouting in <strong>the</strong> molecular layer, ectopic expression of NPY in <strong>the</strong><br />
mossy fibers, <strong>an</strong>d depletions of calbindin in <strong>the</strong> gr<strong>an</strong>ule cells, suggesting that Aβ-induced<br />
remodeling of hippocampal circuits is indeed a downstream effect of aberr<strong>an</strong>t excitatory<br />
neuronal activity. Import<strong>an</strong>tly, increasing Aβ-induced epileptic activity disrupted acquisition of<br />
new memories (learning), which relies heavily on hippocampal functions, but also retrieval of
consolidated memories (remembering), which appears to involve neocortical functions. We<br />
conclude that aberr<strong>an</strong>t excitatory neuronal activity represents <strong>an</strong> early step in <strong>the</strong> pathogenic<br />
cascade leading <strong>from</strong> Aβ to cognitive deficits in tr<strong>an</strong>sgenic mouse models of AD <strong>an</strong>d possibly<br />
also in hum<strong>an</strong>s with AD.<br />
Supported by <strong>the</strong> Stephen D. Bechtel Jr. Foundation <strong>an</strong>d <strong>the</strong> NIH.<br />
Disclosures: L. Verret, None; M. Thwin, None; K. Ho, None; L. Mucke, None; J.J. Palop,<br />
None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.5/H38<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t 1 RO1 NS55118<br />
Title: Oper<strong>an</strong>t behavioral characterization of tr<strong>an</strong>sgenic murine models of Alzheimer’s disease<br />
Authors: *A. L. BLACKSHEAR, W. XU, M. ANDERSON, F. XU, W. E. VAN NOSTRAND,<br />
J. K. ROBINSON;<br />
SUNY Stony Brook, Stony Brook, NY<br />
Abstract: A common method of modeling aspects of Alzheimer’s disease in <strong>an</strong>imals is <strong>the</strong><br />
tr<strong>an</strong>sgenic mouse. M<strong>an</strong>y tr<strong>an</strong>sgenic models of Alzheimer’s disease have been created, r<strong>an</strong>ging<br />
<strong>from</strong> single tr<strong>an</strong>sgenic to quintuple tr<strong>an</strong>sgenic models. While <strong>the</strong> neuropathology of <strong>the</strong>se<br />
tr<strong>an</strong>sgenic strains has been well characterized, little systematic behavioral study has been<br />
conducted. One import<strong>an</strong>t way that <strong>the</strong>se <strong>an</strong>imals have not been studied is with coordinated,<br />
comprehensive behavioral evaluation among separate strains that would allow <strong>for</strong> direct intercomparisons<br />
between strains. The present study applied rigorous behavioral methodology while<br />
comparing three strains of tr<strong>an</strong>sgenic mice; <strong>the</strong> Tg-2576, one of <strong>the</strong> earliest <strong>an</strong>d best<br />
characterized single tr<strong>an</strong>sgenic Aβ models; <strong>the</strong> Tg-SwDI, a recent multiple Aβ tr<strong>an</strong>sgenic that<br />
recapitulates microvascular amyloid accumulation observed in Alzheimer’s disease <strong>an</strong>d related<br />
familial cerebral amyloid <strong>an</strong>giopathy disorders; <strong>an</strong>d <strong>the</strong> 3x-Tg, a recent <strong>an</strong>d increasingly studied<br />
triple tr<strong>an</strong>sgenic <strong>an</strong>imal that allows <strong>for</strong> <strong>the</strong> study of <strong>the</strong> interactions of three import<strong>an</strong>t<br />
pathologies in Alzheimer’s disease; tau, presenilin <strong>an</strong>d Aβ. All three strains were assessed on a<br />
thorough <strong>an</strong>d interlocking battery of oper<strong>an</strong>t tasks, r<strong>an</strong>ging <strong>from</strong> simple rule learning to delayed<br />
recall, as well as a number of oper<strong>an</strong>t tests of motor <strong>an</strong>d sensory ability. For comparison, <strong>the</strong>
mice were also assessed using o<strong>the</strong>r more commonly used tests, including <strong>the</strong> open field, Barnes<br />
maze, <strong>an</strong>d Morris water maze, which dem<strong>an</strong>ded more spatial orientation <strong>an</strong>d spatial memory<br />
abilities th<strong>an</strong> <strong>the</strong> oper<strong>an</strong>t tasks. After behavioral testing, post-mortem studies of <strong>the</strong> brains of<br />
each strain were conducted, to allow correlation between <strong>the</strong> specific neuropathology of each<br />
strain <strong>an</strong>d its corresponding behavioral deficits. The results of this study allow unique inferences<br />
to be made about <strong>the</strong> possible contributions of single genetic mutations, as well as combinations<br />
of mutations, to <strong>the</strong> neuropathology <strong>an</strong>d behavioral ch<strong>an</strong>ges in Alzheimer’s disease.<br />
Disclosures: A.L. Blackshear, None; W. Xu, None; M. Anderson, None; F. Xu, None; W.E.<br />
V<strong>an</strong> Nostr<strong>an</strong>d, None; J.K. Robinson, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.6/I1<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t 5T32AG00025512<br />
Title: Staging neuropathological ch<strong>an</strong>ges in a murine model of Alzheimer’s disease<br />
Authors: *D. E. HURTADO, L. MOLINA-PORCEL, A. K. ABOAGYE, J. Q.<br />
TROJANOWSKI, V. M.-Y. LEE;<br />
Dept Pathol & Lab. Med., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: OBJECTIVE: The pathology of Alzheimer’s Disease includes amyloid plaques<br />
comprised of β-amyloid (Aβ) peptides, <strong>an</strong>d neurofibrillary t<strong>an</strong>gles (NFT) consisting of<br />
hyperphosphorylated tau. M<strong>an</strong>y tr<strong>an</strong>sgenic models have been engineered to recapitulate <strong>the</strong>se<br />
pathologies, however few models exist which concurrently bear both pathologies. These models<br />
have adv<strong>an</strong>ced our underst<strong>an</strong>ding of <strong>the</strong> pathogenesis of Alzheimer’s Disease, however <strong>the</strong>re are<br />
few reports which comprehensively map regional <strong>an</strong>d temporal neuropathological ch<strong>an</strong>ges.<br />
Staging neuropathological ch<strong>an</strong>ges will allow investigators to better define a temporal <strong>an</strong>d spatial<br />
region to study disease pathogenesis. Our aim was to develop a novel bigenic plaque <strong>an</strong>d t<strong>an</strong>gle<br />
model <strong>an</strong>d comprehensively map <strong>the</strong> regional <strong>an</strong>d temporal distribution of pathology.<br />
METHOD: We developed a bigenic model by mating two previously reported tr<strong>an</strong>sgenic lines:<br />
PDAPP <strong>an</strong>d P301S. The PDAPP model was developed by Games et al. <strong>an</strong>d encodes a hum<strong>an</strong><br />
APP minigene carrying <strong>the</strong> V717F mutation, while <strong>the</strong> P301S model was developed by<br />
Yoshiyama et al. <strong>an</strong>d encodes <strong>the</strong> P301S mut<strong>an</strong>t hum<strong>an</strong> tau. Crossing <strong>the</strong>se lines generated a
plaque <strong>an</strong>d t<strong>an</strong>gle model (Bi-TAPP) which we used to immunohistochemically map <strong>the</strong> spatial<br />
<strong>an</strong>d temporal distribution of pathology. We <strong>an</strong>alyzed both sexes of <strong>the</strong> Bi-TAPP model <strong>an</strong>d<br />
compared bigenic models with <strong>the</strong> parental monogenic line. Our regional <strong>an</strong>d temporal<br />
distribution was limited to three bregma regions <strong>an</strong>d three time points. We <strong>an</strong>alyzed several<br />
phospho-tau sites indicative of NFT <strong>for</strong>mation, <strong>an</strong>d plaque <strong>for</strong>mation by utilizing APP/Aβ<br />
specific <strong>an</strong>tibodies.<br />
RESULTS: By mapping <strong>the</strong> temporal <strong>an</strong>d regional distribution of hyperphosphorylated tau <strong>an</strong>d<br />
plaque <strong>for</strong>mation, we observed distinct stages of phospho-tau progression reminiscent of Braak<br />
Stages I - VI. Briefly, we observed progression of AT8 positive tau staining initiated within <strong>the</strong><br />
entorhinal <strong>an</strong>d perirhinal region, <strong>an</strong>d along layers 2 of <strong>the</strong> neocortex. At later stages we observed<br />
<strong>the</strong> appear<strong>an</strong>ce of tau pathology within <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong> striatum. In late-end staging, we<br />
report development of tau pathology throughout deeper layers of <strong>the</strong> neocortex, a greater<br />
distribution within <strong>the</strong> hippocampus <strong>an</strong>d striatum, <strong>an</strong>d a more profound distribution within <strong>the</strong><br />
midbrain. Finally, we semi-qu<strong>an</strong>titatively evaluated variations in pathology between mice of<br />
similar ages. By staging <strong>the</strong> neuropathological ch<strong>an</strong>ges in our Bi-TAPP model, we have better<br />
defined temporal <strong>an</strong>d spatial points to study disease pathogenesis.<br />
Disclosures: D.E. Hurtado, None; L. Molina-Porcel, None; A.K. Aboagye, None; J.Q.<br />
Troj<strong>an</strong>owski, None; V.M. Lee, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.7/I2<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: FP-6 Integrated Project LSHG-CT-512018 (AMC) by <strong>the</strong> EC<br />
Fondazione Telethon<br />
Compagnia di S<strong>an</strong> Paolo<br />
Itali<strong>an</strong> Ministry of University <strong>an</strong>d Research (MUR)<br />
Title: Age-related variations of peroxisome proliferator activated receptor alpha in TG2576<br />
mouse brain
Authors: *S. MORENO 1 , F. FANELLI 1 , L. CRISTIANO 2 , E. BENEDETTI 2 , B. D'ANGELO 2 ,<br />
F. GIARDI 2 , G. SALESI 1 , M. D'AMELIO 3 , C. BERNARDI 4 , A. CIMINI 2 , M. CERÙ 2 ;<br />
1 Univ. Roma Tre, Rome, Italy; 2 Dept. of Basic <strong>an</strong>d Applied Biol., Univ. of L’Aquila, L’Aquila,<br />
Italy; 3 IRCCS Fondazione S<strong>an</strong>ta Lucia, Rome, Italy; 4 Unità Operativa Complessa di Anatomia<br />
Patologica, S. Filippo Neri Hosp., Rome, Italy<br />
Abstract: Alzheimer’s Disease (AD) is <strong>the</strong> most common <strong>for</strong>m of dementia <strong>an</strong>d it is<br />
characterized by progressive neurodegeneration. Histopathological ch<strong>an</strong>ges include β-amyloid<br />
(Aβ) plaques, neurofibrillary t<strong>an</strong>gles, inflammation <strong>an</strong>d neuronal loss. Growing evidence<br />
suggests that Aβ plays a critical <strong>an</strong>d early role in AD pathogenesis, involving production of<br />
reactive oxygen species (ROS), with consequent oxidative stress. Moreover, <strong>the</strong>re is increasing<br />
evidence that neurotoxicity in AD is mediated by neuroinflammatory processes.<br />
Peroxisome proliferator-activated receptors (PPARα, β, <strong>an</strong>d γ) are lig<strong>an</strong>d-activated tr<strong>an</strong>scription<br />
factors belonging to <strong>the</strong> nuclear receptors superfamily, accomplishing import<strong>an</strong>t functions<br />
related to lipid <strong>an</strong>d glucose metabolism <strong>an</strong>d to cell survival. PPARs are involved in <strong>the</strong><br />
physiological aging process, as decreased PPAR activity is associated with increased levels of<br />
ROS <strong>an</strong>d inflammatory mediators. Recent studies have revealed a protective role of PPARα in<br />
age-related inflammation, as a negative modulator of proinflammatory gene expression.<br />
The aim of this work was to investigate <strong>the</strong> possible involvement of PPARα in AD, as studied in<br />
a tr<strong>an</strong>sgenic mouse model (Tg2576). Previous morphological, electrophysiological <strong>an</strong>d<br />
behavioural studies demonstrated that neuronal deficits in Tg2576 mice are established in a timedependent<br />
m<strong>an</strong>ner.<br />
We examined PPARα expression <strong>an</strong>d localization during <strong>the</strong> disease progression (at ages<br />
comprised between 3 <strong>an</strong>d 18 months), by a combined biochemical <strong>an</strong>d morphological approach,<br />
focussing on <strong>the</strong> neocortex <strong>an</strong>d <strong>the</strong> hippocampus. The presence <strong>an</strong>d distribution of this<br />
tr<strong>an</strong>scription factor in Tg2576 <strong>an</strong>imals was <strong>the</strong>n compared with <strong>the</strong>ir Wt littermates. Data<br />
obtained by RT-PCR, Western Blotting, immunofluorescence, <strong>an</strong>d immunohistochemistry show<br />
age-related variations in <strong>the</strong> expression <strong>an</strong>d localization of PPARα. These ch<strong>an</strong>ges depend upon<br />
<strong>the</strong> brain area considered <strong>an</strong>d <strong>the</strong> genotype. Interestingly, <strong>the</strong> intracellular distribution of <strong>the</strong><br />
protein ch<strong>an</strong>ges with age, r<strong>an</strong>ging <strong>from</strong> <strong>an</strong> exclusively nuclear to a mainly cytoplasmic<br />
localization, depending on <strong>the</strong> age, genotype, brain area <strong>an</strong>d cell type. These variations strongly<br />
suggest differences in <strong>the</strong> nuclear receptor functionality <strong>an</strong>d/or nongenomic functions of PPARα<br />
protein.<br />
Taken toge<strong>the</strong>r, our data emphasize <strong>the</strong> role of PPARα in age-related inflammatory processes,<br />
ei<strong>the</strong>r physiological or pathological.<br />
Disclosures: S. Moreno, None; F. F<strong>an</strong>elli, None; L. Cristi<strong>an</strong>o, None; E. Benedetti, None; B.<br />
D'Angelo, None; F. Giardi, None; G. Salesi, None; M. D'Amelio, None; C. Bernardi,<br />
None; A. Cimini, None; M. Cerù, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.8/I3<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: EU 037831 MEMORIES<br />
Title: Effects of knocking out Vps10p-domain receptor family proteins in <strong>an</strong>ti-NGF tr<strong>an</strong>sgenic<br />
mice<br />
Authors: *S. CAPSONI 1 , G. AMATO 1 , D. VIGNONE 1 , A. NYKJAER 2 , T. E. WILLNOW 3 , A.<br />
CATTANEO 1,4 ;<br />
1 Europe<strong>an</strong> Brain Res. Inst., Rome, Italy; 2 NeuronIcon APS, Aarhus, Denmark; 3 Max Delbrueck<br />
Ctr. <strong>for</strong> Mol. Med., Berlin, Germ<strong>an</strong>y; 4 Scuola Normale Superiore, Pisa, Italy<br />
Abstract: The VPS10P-domain class of multifunctional type-1 receptors, expressed in neurons,<br />
target a r<strong>an</strong>ge of lig<strong>an</strong>ds, including neuropetides, neurotrophic factors <strong>an</strong>d tr<strong>an</strong>smembr<strong>an</strong>e<br />
proteins. Recent findings have revealed that VPS10P-domain receptors influence neuronal<br />
viability <strong>an</strong>d function through <strong>the</strong> regulation of protein tr<strong>an</strong>sport <strong>an</strong>d signal tr<strong>an</strong>sduction. In<br />
particular, two members of this receptor family, SorLa/LR11 <strong>an</strong>d sortilin, might be involved in<br />
<strong>the</strong> pathophysiology of several disorders, including Alzheimer’s disease.<br />
SorLa interacts with <strong>the</strong> amyloid precursor protein APP, acting as a sorting receptor that protects<br />
APP <strong>from</strong> being processed into beta amyloid (Andersen et al., PNAS 2005). In APP tr<strong>an</strong>sgenic<br />
mice, knocking out SorLa determines <strong>an</strong> increase in Aβ peptide production <strong>an</strong>d plaque <strong>for</strong>mation<br />
(Dodson et al., J. Neurosci. 2008). Interestingly, inherited or acquired ch<strong>an</strong>ges in SorLA<br />
expression or function have been associated to late-onset Alzheimer’s disease (Rogaeva et al.,<br />
Nat Genet. 2007). A second VPS10P-domain receptor, sortilin, is a co-receptor <strong>for</strong> proNGF, <strong>the</strong><br />
immature <strong>for</strong>m of NGF (Nykajer et al., Nature 2004; J<strong>an</strong>sen et al, Nature Neurosci. 2007), <strong>an</strong>d<br />
plays a signific<strong>an</strong>t part in <strong>the</strong> observed patterns of age-related proNGF-mediated neurotoxicity<br />
(Al-Shawi et al., Eur. J. Neurosci. 2008). An abnormal increase in proNGF : NGF signalling<br />
ratio, permissive of apoptotic <strong>an</strong>d neurodegenerative events, have been suggested to be one of<br />
<strong>the</strong> epigenetic factors triggering <strong>the</strong> onset of sporadic Alzheimer’s disease (Catt<strong>an</strong>eo et al., J.<br />
Alzheimer Dis. 2008). This proNGF/NGF unbal<strong>an</strong>ce is clearly reproduced in vivo in <strong>the</strong> AD11<br />
<strong>an</strong>ti-NGF mouse model (Capsoni et al., PNAS 2000), whereby <strong>the</strong> selective neutralization of <strong>the</strong><br />
mature <strong>for</strong>m of NGF, versus unprocessed proNGF, has been shown to determine <strong>the</strong> Alzheimerlike<br />
neurodegeneration (Capsoni et al. SfN Meeting 2005; Capsoni & Catt<strong>an</strong>eo Cell. Mol.<br />
Neurobiol. 2006).<br />
In this work, we have exploited mice in which <strong>the</strong> SorLa <strong>an</strong>d sortilin genes have been deleted by<br />
homologous recombination (Andersen et al., PNAS 2005; J<strong>an</strong>sen et al, Nature Neurosci. 2007) to<br />
verify <strong>the</strong>ir roles in <strong>the</strong> neurodegeneration in AD11 mice.<br />
To this purpose, AD11 mice were crossed with SorLa or sortilin knock-out mice <strong>an</strong>d <strong>the</strong> progeny<br />
<strong>an</strong>alysised at <strong>the</strong> behavioural <strong>an</strong>d histological level, at different ages (1, 3, <strong>an</strong>d 6 months).
We show that SorLa <strong>an</strong>d sortilin differently affect behaviour <strong>an</strong>d neuropathology in <strong>an</strong> agedependent<br />
m<strong>an</strong>ner.<br />
Disclosures: S. Capsoni, None; G. Amato, None; D. Vignone, None; A. Nykjaer, NeuronIcon<br />
APS, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); T.E.<br />
Willnow, None; A. Catt<strong>an</strong>eo, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.9/I4<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH gr<strong>an</strong>t P30 AG19610<br />
Arizona Alzheimer’s Consortium<br />
Barrow Neurological Foundation<br />
B<strong>an</strong>ner Alzheimer’s Institute<br />
Title: Regional cerebral glucose metabolism in a 3xTG mouse model of Alzheimer’s disease<br />
Authors: *R. M. CASPAR 1,2,3 , Y. KUSNE 1,2,3 , E. M. REIMAN 4,5,6,3 , J. E. VALLA 1,3 ;<br />
1 Barrow Neurolog. Institute, St. Joseph's Hosp. & Med. Ctr., Phoenix, AZ; 2 Arizona State Univ.,<br />
Tempe, AZ; 3 Arizona Alzheimer's Consortium, Phoenix, AZ; 4 B<strong>an</strong>ner Alzheimer's Inst.,<br />
Phoenix, AZ; 5 Univ. of Arizona, Tucson, AZ; 6 Tr<strong>an</strong>slational Genomics Res. Inst., Phoenix, AZ<br />
Abstract: We have previously used fluorodeoxyglucose (FDG) autoradiography to detect <strong>an</strong>d<br />
track <strong>the</strong> pattern of metabolic declines in two different tr<strong>an</strong>sgenic mouse models of fibrillar betaamyloid<br />
pathology, including <strong>the</strong> PDAPP mouse, which overexpresses a mut<strong>an</strong>t <strong>for</strong>m of hum<strong>an</strong><br />
APP mutation, <strong>an</strong>d <strong>the</strong> PSAPP mouse, which overexpresses mut<strong>an</strong>t <strong>for</strong>ms of <strong>the</strong> hum<strong>an</strong> APP <strong>an</strong>d<br />
PS1 genes, both known to cause early-onset Alzheimer’s disease (AD). In this study, we used <strong>the</strong><br />
same approach to study a triply-tr<strong>an</strong>sgenic (3xTG) model of AD, which overexpresses hum<strong>an</strong><br />
APP, PS1 <strong>an</strong>d tau mutations. Densitometric measurements <strong>from</strong> 55 brain regions were<br />
characterized <strong>an</strong>d compared in 2, 12, <strong>an</strong>d 18 month-old 3xTG <strong>an</strong>d wildtype control mice<br />
(n=12/group). As in <strong>the</strong> previously studied tr<strong>an</strong>sgenic mouse models, normalized regional FDG<br />
counts were preferentially altered in <strong>the</strong> 2- <strong>an</strong>d 12-month-old 3xTG mice, in a brain regional
pattern reminiscent of our previous <strong>an</strong>alyses in <strong>the</strong> o<strong>the</strong>r mouse models. This included particular<br />
regional vulnerability in a circuit known to underlie spatial <strong>an</strong>d discriminative learning <strong>an</strong>d to be<br />
preferentially involved in memory <strong>an</strong>d AD, prominently including <strong>the</strong> posterior<br />
cingulate/retrosplenial cortex. This effect did not appear to be age-dependent <strong>an</strong>d did not worsen<br />
appreciably between 2 <strong>an</strong>d 12 months of age. However, by 18 months of age, <strong>the</strong> 3xTG mice had<br />
signific<strong>an</strong>t reductions in absolute FDG counts in virtually every region of <strong>the</strong> brain, including<br />
cortical <strong>an</strong>d subcortical gray matter, cerebellar <strong>an</strong>d brainstem regions. Overall, our <strong>an</strong>alyses<br />
highlight consistencies in brain glucose uptake abnormalities across multiple mouse models of<br />
amyloid-associated pathophysiology. These mouse brain regional ch<strong>an</strong>ges are homologous to<br />
some alterations seen in PET sc<strong>an</strong>s <strong>from</strong> hum<strong>an</strong> AD patients <strong>an</strong>d could thus be useful biomarkers<br />
<strong>for</strong> early testing of novel interventions.<br />
Disclosures: R.M. Caspar, None; Y. Kusne, None; E.M. Reim<strong>an</strong>, None; J.E. Valla, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.10/I5<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: SAF2004-07802<br />
FIS PI 061582<br />
CIBERNED<br />
Title: Clinicopathological correlates of amyloid plaques recognized by <strong>an</strong> <strong>an</strong>tibody against a<br />
con<strong>for</strong>mational epitope specific to oligomeric Abeta in a doble APP/TAU tr<strong>an</strong>sgenic mouse<br />
model<br />
Authors: *B. DA ROCHA, JR 1,3 , M. COMA 2,3 , T. SCOTTON 2 , L. SERENO 3 , M.<br />
RODRIGUEZ 3 , J. ESPANA 4 , C. SAURA 4 , B. HYMAN 2 , T. GOMEZ-ISLA 2,3 ;<br />
1 Massachusetts Gen. Hosp., CHARLESTOWN, MA; 2 Massachusetts Gen. Hosp., Charlestown,<br />
MA; 3 Hosp. de la S<strong>an</strong>ta Creu i S<strong>an</strong>t Pau, Barcelona, Spain; 4 Univ. Autonoma de Barcelona,<br />
Barcelona, Spain<br />
Abstract: Background: It has been suggested that pre-fibrillar oligomeric assemblies of Aβ<br />
might be <strong>the</strong> proximate effectors of synaptic loss <strong>an</strong>d neuronal damage in Alzheimer’s disease
(AD). We have recently developed <strong>an</strong>d characterized a double tr<strong>an</strong>sgenic mouse model (line<br />
APP sw -tau vlw ) based on co-expression of hum<strong>an</strong> mut<strong>an</strong>t amyloid precursor protein <strong>an</strong>d tau. This<br />
model mimics some of <strong>the</strong> most salient features of hum<strong>an</strong> AD including amyloid deposition,<br />
abnormal tau phosphorylation /neurofibrillary t<strong>an</strong>gle (NFT) <strong>for</strong>mation, gliosis, neuronal loss in<br />
selectively vulnerable brain regions such as <strong>the</strong> entorhinal cortex (EC) <strong>an</strong>d <strong>the</strong> CA1 region of <strong>the</strong><br />
hippocampus, <strong>an</strong>d cognitive deficits. Interestingly, <strong>the</strong> neuronal cell loss in this model precedes<br />
robust amyloid deposition raising <strong>the</strong> possibility that soluble species of Aβ could be implicated<br />
in neuronal dysfunction <strong>an</strong>d death in this mouse model. Objectives: To study <strong>the</strong><br />
clinicopathological correlates of brain amyloid deposits labelled by NAB61 (kind gift of Virginia<br />
Lee), <strong>an</strong> <strong>an</strong>tibody that recognizes a con<strong>for</strong>mational epitope specific to oligomeric Aβ, in APP sw -<br />
tau vlw mice. Material <strong>an</strong>d Methods: Aβ burden was qu<strong>an</strong>tified on NAB61 immunostained<br />
sections by using a Bioqu<strong>an</strong>t image <strong>an</strong>alysis system in <strong>the</strong> EC, CA1 region of <strong>the</strong> hippocampus<br />
<strong>an</strong>d cingulate cortex. The correlations of this subset of plaques with o<strong>the</strong>r Aβ measurements,<br />
stereological counts of neurons <strong>an</strong>d astrocytes, <strong>an</strong>d levels of expression of CREB-targeted genes<br />
were investigated. Results: NAB61 positive plaque burden closely correlated with total amyloid<br />
burden, as labeled by 4G8 <strong>an</strong>tibody, <strong>an</strong>d ThioS positive fibrillar dense-core plaque load in <strong>the</strong><br />
brain of APP sw -tau vlw mice. Of note, <strong>the</strong> amount of oligomeric Aβ deposits, but not <strong>the</strong> total<br />
amount of plaques, signific<strong>an</strong>tly correlated with number of astrocytes, amount of neuronal cell<br />
death <strong>an</strong>d c-fos expression levels in <strong>the</strong> CA1 region of <strong>the</strong> hippocampus in <strong>the</strong>se mice.<br />
Conclusion: Unlike total brain amyloid burden, a subset of Aβ deposits recognized by a<br />
con<strong>for</strong>mational oligomer-specific <strong>an</strong>tibody strongly correlates with neuronal cell loss,<br />
inflammation <strong>an</strong>d memory-related gene expression in <strong>the</strong> hippocampus of APP sw -tau vlw mice.<br />
These results favor <strong>the</strong> idea that oligomeric Aβ might be implicated in neuronal damage in this<br />
double APP/tau tr<strong>an</strong>sgenic mouse model.<br />
Disclosures: B. Da rocha, None; M. Coma, None; T. Scotton, None; L. Sereno, None; M.<br />
Rodriguez, None; J. Esp<strong>an</strong>a, None; C. Saura, None; B. Hym<strong>an</strong>, None; T. Gomez-Isla, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.11/I6<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Alzheimer Assoc. II RG08-88<br />
Title: Neuroserpin inhibition of tissue plasminogen activator may be critical in <strong>the</strong> loss of<br />
cognitive ability <strong>an</strong>d amyloid-beta accumulation in Alzheimer disease
Authors: *N. W. SEEDS 1 , S. FABBRO 2 ;<br />
1 Neurosci. Prgm., 2 Hum<strong>an</strong> Med. Genet. Prgm., Univ. Colorado Sch. of Med., Aurora, CO<br />
Abstract: Alzheimer disease (AD) is characterized by <strong>the</strong> accumulation of amyloid-beta (Aβ)<br />
oligomers <strong>an</strong>d aggregated plaques. Plasmin, one of several enzymes that degrade Aβ, is low in<br />
AD brain due to dramatic inhibition of tissue plasminogen activator (tPA) activity by increased<br />
levels of neuroserpin (J.Neurochem 109:303-315, <strong>2009</strong>). An <strong>an</strong>imal model where Aβ1-42 peptide<br />
is injected into <strong>the</strong> frontal cortex of plasminogen activator (PA) system knockout mice shows<br />
that <strong>the</strong> absence of tPA leads to very slow clear<strong>an</strong>ce of <strong>the</strong> peptide compared to wildtype<br />
C57Bl/6 mice. In contrast, Aβ1-42 injection into brains of neuroserpin knockout mice leads to<br />
rapid clear<strong>an</strong>ce of Aβ peptide <strong>from</strong> <strong>the</strong> frontal cortex as compared to both wildtype <strong>an</strong>d PAI-1<br />
knockout mice. Although <strong>the</strong>se findings support <strong>the</strong> suggestion that neuroserpin inhibition of<br />
tPA promotes Aβ accumulation, this model does not mimic <strong>the</strong> AD brain where Aβ1-42<br />
aggregates over time into trimers or plaques that promote disease. There<strong>for</strong>e, we used <strong>the</strong> AD<br />
tr<strong>an</strong>sgenic mouse that develops plaques <strong>an</strong>d has cognitive deficits to breed AD+ mice that were<br />
lacking individual PA system genes. Interestingly, AD+/neuroserpin-/- mice show no detectable<br />
Aβ1-40 <strong>an</strong>d much less Aβ1-42 peptide by ELISA th<strong>an</strong> AD+ mice. These mice have fewer <strong>an</strong>d much<br />
smaller Aβ plaques as compared to <strong>the</strong> AD+ tr<strong>an</strong>sgenic mice, <strong>an</strong>d show more active tPA<br />
associated with <strong>the</strong>se smaller plaques. More import<strong>an</strong>tly, AD+/neuroserpin-/- mice show nearly<br />
normal spatial learning in <strong>the</strong> Morris water maze when compared to <strong>the</strong> poor per<strong>for</strong>m<strong>an</strong>ce seen<br />
in 9 to11 months’ old AD+ tr<strong>an</strong>sgenic mice. Thus, removal of <strong>the</strong> tPA inhibitor neuroserpin<br />
leads to reduced Aβ accumulation in brain <strong>an</strong>d restoration of near normal cognitive function.<br />
(Supported in part by <strong>the</strong> Alzheimer Association)<br />
Disclosures: N.W. Seeds, Alzheimer Association, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); S. Fabbro,<br />
None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.12/I7<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Shapiro Foundation<br />
NIH AG10688
Title: Cortical <strong>an</strong>d hippocampal ChAT activity <strong>an</strong>d NGF receptor levels in <strong>the</strong> 3xTg mouse<br />
model of Alzheimer’s disease<br />
Authors: *E. J. MUFSON 1 , S. E. PEREZ 2 , M. NADEEM 2 , S. E. COUNTS 2 , K.-J. OH 2 , C.<br />
OVERK 3 , M. D. IKONOMOVIC 4 ;<br />
1 Neurol Sci., 2 Dept. of Neurolog. Sci., 3 Dept. of Neurol Sci., Rush Univ. Med. Ctr., Chicago, IL;<br />
4 Departments of Neurol. <strong>an</strong>d Psychiatry, Univ. of Pittsburgh Sch. of Med., Pittsburgh, PA<br />
Abstract: Cholinergic basal <strong>for</strong>ebrain (CBF) neurons, which provide <strong>the</strong> primary source of<br />
cholinergic innervation to <strong>the</strong> cortex <strong>an</strong>d hippocampus require <strong>the</strong> neurotrophin NGF <strong>an</strong>d its<br />
cognate receptors (TrkA <strong>an</strong>d p75 NTR ) <strong>for</strong> <strong>the</strong>ir biological activity <strong>an</strong>d degenerate in Alzheimer’s<br />
disease (AD). It has been suggested that accumulation of Aβ within CBF projection sites initiates<br />
a cascade of events resulting in dysfunction of <strong>the</strong> cholinotrophic basal <strong>for</strong>ebrain system in AD.<br />
The triple tr<strong>an</strong>sgenic mouse model of AD (3xTg-AD), which displays Aβ plaques <strong>an</strong>d t<strong>an</strong>gle-like<br />
pathology, provides <strong>an</strong> excellent in vivo model to determine <strong>the</strong> effect of Aβ deposition upon<br />
choline acetyltr<strong>an</strong>sferase (ChAT) activity as well as TrkA <strong>an</strong>d p75 NTR protein levels in <strong>the</strong> cortex<br />
<strong>an</strong>d hippocampus. ChAT activity was determined by <strong>the</strong> Fonnum radioenzymatic assay <strong>an</strong>d<br />
TrkA <strong>an</strong>d p75 NTR protein levels were <strong>an</strong>alyzed by qu<strong>an</strong>titative immunoblotting applied to<br />
samples of cerebral cortex <strong>an</strong>d hippocampus harvested <strong>from</strong> young (2-3 months; n=12) <strong>an</strong>d<br />
middle-age (13-15 months; n=13) 3xTg-AD mice <strong>an</strong>d <strong>the</strong>ir respective non-tr<strong>an</strong>sgenic (ntg)<br />
littermates (n=22). In ntg mice, <strong>the</strong>re was a signific<strong>an</strong>t age-related increase in ChAT activity<br />
levels in <strong>the</strong> cortex (p=0.001) but not in <strong>the</strong> hippocampus. By contrast, in <strong>the</strong> 3xTg-AD mice<br />
cortical ChAT activity remained stable across age, whereas in <strong>the</strong> hippocampus it was<br />
signific<strong>an</strong>tly decreased with age (p=0.015). Cortical TrkA levels were increased across age in ntg<br />
mice (p = 0.027) but were unch<strong>an</strong>ged in 3xTg-AD mice. Hippocampal TrkA levels did not<br />
ch<strong>an</strong>ge with age in ei<strong>the</strong>r mouse genotype, although TrkA levels in <strong>the</strong> 3xTg-AD hippocampus<br />
were signific<strong>an</strong>tly higher th<strong>an</strong> ntg mice independent of age. With respect to p75 NTR levels, <strong>the</strong>re<br />
was no age-related ch<strong>an</strong>ge in <strong>the</strong> cortex or hippocampus of <strong>the</strong> ntg mice. However, 3xTg-AD<br />
mice displayed a signific<strong>an</strong>t reduction in p75 NTR levels with age (p=0.006) in <strong>the</strong> hippocampus<br />
but not in <strong>the</strong> cortex. Interestingly, Aβ plaques were present only in <strong>the</strong> hippocampus of middleage<br />
3xTg-AD mice. This suggests a role <strong>for</strong> Aβ deposition in <strong>the</strong> reduction of hippocampal<br />
ChAT activity <strong>an</strong>d p75 NTR levels but not TrkA receptor levels. Moreover, <strong>the</strong>se data indicate that<br />
ch<strong>an</strong>ges in cortical <strong>an</strong>d hippocampal ChAT activity <strong>an</strong>d NGF receptors levels seen in 3xTg-AD<br />
mice do not fully mimic those seen during <strong>the</strong> progression of AD (Counts <strong>an</strong>d Mufson, 2005).<br />
Disclosures: E.J. Mufson, Ceregene, Inc, E. Ownership Interest (stock, stock options, patent or<br />
o<strong>the</strong>r intellectual property); Ceregene, F. Consult<strong>an</strong>t/Advisory Board; S.E. Perez, None; M.<br />
Nadeem, None; S.E. Counts, None; K. Oh, None; C. Overk, None; M.D. Ikonomovic, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.13/I8<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Cellular mech<strong>an</strong>isms of aberr<strong>an</strong>t neuronal activity <strong>an</strong>d cognitive deficits in mouse models<br />
of Alzheimer’s disease<br />
Authors: *J. CHIN, H.-P. LING, S. C. LEISER, J. BROWN, R. NAGY, N. BREYSSE, D. YU,<br />
M. PANGALOS, A. RANDALL, A. WOOD, M. R. BOWLBY, P. REINHART;<br />
Wyeth Res., Princeton, NJ<br />
Abstract: The amyloid precursor protein (APP) <strong>an</strong>d amyloid β peptides (Aβ) cleaved <strong>from</strong> it<br />
contribute to cognitive impairments in Alzheimer’s disease (AD). Recent reports have<br />
demonstrated that APP/Aβ perturbs neuronal activity <strong>an</strong>d might also underlie <strong>the</strong> clinical<br />
observations that AD is associated with increased seizure incidence. Although it was previously<br />
unclear whe<strong>the</strong>r seizures are secondary to <strong>the</strong> disease or contribute to cognitive impairments,<br />
new results <strong>from</strong> tr<strong>an</strong>sgenic mouse models of AD suggest that seizure activity <strong>an</strong>d resulting<br />
compensatory responses of <strong>the</strong> brain play <strong>an</strong> import<strong>an</strong>t role in AD-related cognitive decline.<br />
Recent reports suggested that alterations in voltage-gated sodium ch<strong>an</strong>nels (VGSCs) might<br />
contribute to epilepti<strong>for</strong>m activity in AD <strong>an</strong>d related mouse models. Like APP, VGSCs are<br />
substrates <strong>for</strong> BACE1 <strong>an</strong>d γ-secretase, <strong>the</strong> two proteases responsible <strong>for</strong> production of neurotoxic<br />
Aβ. Alterations in VGSCs lead to aberr<strong>an</strong>t neuronal activity <strong>an</strong>d seizures, <strong>an</strong>d contribute to some<br />
<strong>for</strong>ms of epilepsy which when uncontrolled is associated with cognitive decline. Thus, <strong>the</strong><br />
increased levels/activities of BACE1 in AD patients <strong>an</strong>d mice may impact overall neuronal<br />
activity by altering VGSC levels.<br />
We used biochemical <strong>an</strong>d immunohistochemical methods to determine whe<strong>the</strong>r AD mouse<br />
models exhibit alterations in VGSCs that correspond to aberr<strong>an</strong>t brain activity <strong>an</strong>d cognitive<br />
deficits. We found that both Tg2576 <strong>an</strong>d PS1/APP mouse models of AD exhibit altered protein<br />
levels of VGSCs. These alterations were age-dependent <strong>an</strong>d were observed at ages when<br />
epilepti<strong>for</strong>m activity became evident in in vivo EEG recordings. Moreover, <strong>the</strong> magnitude of<br />
VGSC alterations qualitatively correlated with both degree of epilepti<strong>for</strong>m activity as well as<br />
with impairments in <strong>the</strong> Morris water maze. These results indicate that VGSC alterations may<br />
underlie aberr<strong>an</strong>t brain activity, which is a general feature of AD mouse models that may<br />
contribute to cognitive impairments. Moreover, restoring neuronal activity by both regulating<br />
APP processing <strong>an</strong>d normalizing VGSCs may provide <strong>the</strong>rapeutic benefit <strong>for</strong> AD-related<br />
memory impairments.<br />
Disclosures: J. Chin, Wyeth Research, A. Employment (full or part-time); H. Ling, Wyeth<br />
Research, A. Employment (full or part-time); S.C. Leiser, Wyeth Research, A. Employment<br />
(full or part-time); J. Brown, None; R. Nagy, Wyeth Research, A. Employment (full or parttime);<br />
N. Breysse, Wyeth Research, A. Employment (full or part-time); D. Yu, Wyeth Research,<br />
A. Employment (full or part-time); M. P<strong>an</strong>galos, Wyeth Research, A. Employment (full or parttime);<br />
A. R<strong>an</strong>dall, Wyeth Research, A. Employment (full or part-time); A. Wood, Wyeth
Research, A. Employment (full or part-time); M.R. Bowlby, Wyeth Research, A. Employment<br />
(full or part-time); P. Reinhart, Wyeth Research, A. Employment (full or part-time).<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.14/I9<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: UTE project FIMA<br />
Sp<strong>an</strong>ish Ministry of Health (CIBERNED)<br />
Title: Chronic mild stress accelerates <strong>the</strong> onset <strong>an</strong> severety of Alzheimer´s disease symptoms<br />
Authors: M. CUADRADO-TEJEDOR, A. GARCIA-OSTA, A. RICOBARAZA, J. DEL RIO,<br />
D. FRECHILLA, *L. A. PEREZ-MEDIAVILLA;<br />
CIMA. Univ. of Navarra, Pamplona, Spain<br />
Abstract: Alzheimer disease (AD) is a neurodegenerative disorder characterized by a<br />
progressive loss of memory <strong>an</strong>d cognitive function. The pathological hallmarks of this disorder<br />
are deposits of amyloid-Β peptide (AΒ) <strong>an</strong>d phospho-tau (ptau). Although mutations in different<br />
genes, like hum<strong>an</strong> amyloid precursor protein (APP) or presenilin, are <strong>the</strong> responsible of some<br />
inheritable <strong>for</strong>ms of AD, <strong>the</strong> etiology of <strong>the</strong> more common cases (sporadic <strong>for</strong>ms), remains<br />
unknown. Chronic stress is one of <strong>the</strong> different environmental factors that have been suggested<br />
to influence pathogenesis of AD as it is linked with memory <strong>an</strong>d with o<strong>the</strong>r brain alterations.<br />
However, little is known about <strong>the</strong> relationship between chronic stress <strong>an</strong>d <strong>the</strong> onset of AD. In<br />
this study, we used Tg2576-mice or non-tr<strong>an</strong>sgenic mice to <strong>an</strong>alyze <strong>the</strong> effects of a chronic mild<br />
stress (CMS) on <strong>the</strong> onset <strong>an</strong>d severity of cognitive dysfunction <strong>an</strong>d on <strong>the</strong> pathological markers<br />
of AD. CMS procedure included a variety of unpredictable repeated mild stressors applied <strong>for</strong> 6<br />
weeks in a r<strong>an</strong>dom order. Learning per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d AD pathological markers were evaluated in<br />
<strong>the</strong> CMS-mice compared to non-stressed relative controls. To evaluate pathological AD markers<br />
we <strong>an</strong>alyzed <strong>the</strong> phosphorylation of tau <strong>an</strong>d <strong>the</strong> APP processing in <strong>the</strong> mice hippocampi. We<br />
found that CMS accelerates <strong>the</strong> onset <strong>an</strong>d severity of <strong>the</strong> cognitive dysfunctions in Tg2576 mice<br />
in <strong>the</strong> Morris water maze test. This behavior was accomp<strong>an</strong>ied with <strong>an</strong> increase in AΒ 42 <strong>an</strong>d<br />
AΒ 40 levels, carboxyl-terminal fragments (C99) of (APP-CTFs), <strong>an</strong>d extracellular amyloid<br />
deposits in <strong>the</strong> hippocampus of <strong>the</strong> CMS-Tg2576-mice. In addition, <strong>the</strong>se <strong>an</strong>imals also showed<br />
higher levels of p-tau compare to non-stressed mice. CMS-non-tr<strong>an</strong>sgenic mice showed a poor
learning per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> Morris water maze compare to <strong>the</strong>ir relative controls. We found <strong>an</strong><br />
increase in <strong>the</strong> levels of <strong>the</strong> murine carboxyl- terminal fragments (C83 <strong>an</strong>d C99) of APP <strong>an</strong>d ptau.<br />
These findings suggest that a mild chronic stress could be considered a risk factor <strong>for</strong> AD<br />
<strong>an</strong>d that it could play a role in <strong>the</strong> development <strong>an</strong>d progression of this disorder.<br />
Disclosures: M. Cuadrado-Tejedor, None; A. Garcia-Osta, None; A. Ricobaraza, None; J.<br />
Del Rio, None; D. Frechilla, None; L.A. Perez-Mediavilla, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.15/I10<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH Gr<strong>an</strong>t 2U54NS039407-09<br />
Title: Loss of dopaminergic neurons in <strong>the</strong> ventral tegmental area of aged dtg APP/PS1 mice<br />
Authors: *A. C. DREW 1 , J. O'NEIL 2 , D.-L. LEI 1 , P. R. MOUTON 3 , K. F. MANAYE 1 ;<br />
1 Physiol. <strong>an</strong>d Biophysics, Howard Univ. Col. of Med., Washington, DC; 2 Otolaryngology, Johns<br />
Hopkins Univ., Baltimore, MD; 3 Stereology Resource Ctr., Chester, MD<br />
Abstract: The characteristic neuropathology in Alzheimer’s disease (AD) includes β-amyloid<br />
plaques in cortical tissue <strong>an</strong>d neuron loss in <strong>the</strong> noradrenergic locus coeruleus (LC),<br />
dopaminergic ventral tegmental area (VTA), <strong>an</strong>d <strong>the</strong> pyramidal <strong>an</strong>d gr<strong>an</strong>ule layers of <strong>the</strong><br />
hippocampus. Our group <strong>an</strong>d o<strong>the</strong>rs have assessed <strong>the</strong> AD-type ch<strong>an</strong>ges in <strong>the</strong> dtg<br />
APPswe/PS1∆E9 (PrP promotor) line in <strong>the</strong> C57Bl/6 background. These dtg APP/PS1 mice<br />
accumulate amyloid plaques <strong>an</strong>d undergo neuron loss in LC <strong>an</strong>d CA regions of <strong>the</strong> hippocampus<br />
as in AD; however, neuropathology in <strong>the</strong> VTA that project <strong>from</strong> <strong>the</strong> ventral midbrain to <strong>the</strong><br />
neocortex has not been fully established. To address this question we <strong>an</strong>alyzed male dtg<br />
APP/PS1 mice in two different age groups, adult to middle-age (7-13 months) <strong>an</strong>d late middleage<br />
to old (18-24 mos), along with age-<strong>an</strong>d gender-matched wild-type, non-tg littermate controls.<br />
Mice were perfused with aldehydes <strong>an</strong>d brains removed, cryoprotected in sucrose, <strong>an</strong>d frozen<br />
sectioned at 40 µm. Sections were sampled <strong>an</strong>d immunostained with tyrosine hydroxylase (TH)<br />
to identify dopaminergic neurons in <strong>the</strong> VTA, <strong>an</strong>d total number <strong>an</strong>d me<strong>an</strong> cell size of THpositive<br />
neurons in VTA qu<strong>an</strong>tified using <strong>the</strong> optical fractionator <strong>an</strong>d nucleator techniques.<br />
There is a signific<strong>an</strong>t loss of TH-positive neurons in <strong>the</strong> older group of dtg APP/PS1 mice<br />
compared to age-matched controls [non-dtg [n=10] me<strong>an</strong> 6202 cells (SEM 406) vs. dtg [n=8]
me<strong>an</strong> 4845 (SEM 348). Apparent differences observed in <strong>the</strong> younger group that did not reach<br />
statistical signific<strong>an</strong>ce (p < 0.10), while no differences in cell sizes were observed <strong>for</strong> ei<strong>the</strong>r age<br />
group. Thus, both patients with AD <strong>an</strong>d older dtgAPP/PS1 mice undergo signific<strong>an</strong>t loss of<br />
dopaminergic neurons in VTA, a finding that helps to explain <strong>the</strong> clinical progression of AD, <strong>an</strong>d<br />
supports <strong>the</strong> use of dtg APP/PS1 mice <strong>for</strong> <strong>the</strong> development of novel <strong>the</strong>rapeutic strategies to slow<br />
or reverse <strong>the</strong> progression of this disease.<br />
Disclosures: A.C. Drew, None; J. O'neil, None; D. Lei, None; P.R. Mouton, None; K.F.<br />
M<strong>an</strong>aye, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.16/I11<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Wyeth<br />
Title: Aβ dimers precede memory deficits in PSAPP mice<br />
Authors: *E. NEEDLE 1 , R. G. W. STAAL 1 , N. BREYSSE 1 , R. CROZIER 1 , R. NAGY 1 , A.<br />
SUNG 1 , M. MONAGHAN 1 , R. MARTONE 1 , D. RIDDELL 1 , J. CHIN 1 , N. BOGDANOVIC 1 , D.<br />
M. WALSH 2 , M. N. PANGALOS 1 , P. H. REINHART 1 , J. S. JACOBSEN 1 , W. D. HIRST 1 ;<br />
1 2<br />
Neurosci., Wyeth Discovery Res., Princeton, NJ; Conway Inst. of Biomolecular <strong>an</strong>d Biomed.<br />
Res., Univ. Col. Dublin, Dublin, Irel<strong>an</strong>d<br />
Abstract: Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is<br />
characterized by elevated levels of <strong>the</strong> amyloid β-protein (Aβ). A number of studies have<br />
described <strong>the</strong> biochemical characterization of Aβ extracted <strong>from</strong> AD brain, demonstrating <strong>the</strong><br />
presence of monomer <strong>an</strong>d sodium dodecylsulfate (SDS)-stable dimers in both <strong>the</strong> water-soluble<br />
<strong>an</strong>d <strong>for</strong>mic acid-soluble fractions of brain. Moreover, recent studies have demonstrated that Aβ<br />
dimers isolated <strong>from</strong> AD brains inhibit long-term potentiation (LTP), reduce dendritic spine<br />
density in vitro <strong>an</strong>d upon direct injection into <strong>the</strong> brain of normal rats disrupt memory (Sh<strong>an</strong>kar<br />
et al., 2008). In <strong>the</strong> Tg2576 mouse line, that overexpress APP, endogenous soluble oligomeric<br />
(putative 12-mers, termed Aβ*56) species, but not dimers, have been isolated <strong>an</strong>d characterized<br />
(Lesne et al., 2006). In <strong>the</strong> current study we searched <strong>for</strong> Aβ oligomers in brains of PSAPP<br />
tr<strong>an</strong>sgenic mice. To this end whole cerebra were homogenized in Tris-buffered saline (TBS),<br />
centrifuged at high speed <strong>an</strong>d <strong>the</strong> supernat<strong>an</strong>t <strong>an</strong>alyzed using a sensitive
immunoprecipitation/Western blotting protocol (Sh<strong>an</strong>kar et al., 2008). Such <strong>an</strong>alysis of TBS<br />
extracts revealed abund<strong>an</strong>t Aβ monomers <strong>an</strong>d signific<strong>an</strong>t amounts of SDS-stable dimers,<br />
however, using <strong>the</strong> method described by Lesne et al., (2006) we were unable to detect SDSstable<br />
assemblies such as dimers, trimers, tetramers or Aβ*56. Dimers were evident as early as<br />
2.5 months, <strong>an</strong>d <strong>the</strong>ir appear<strong>an</strong>ce preceded impairments in spatial reference memory which only<br />
became apparent at ≥12 months. These data suggest that dimers, similar to those seen in AD<br />
patients, first appear at <strong>an</strong> early age in PSAPP mice but need to reach a critical threshold be<strong>for</strong>e<br />
memory deficits are observed.<br />
Disclosures: E. Needle, Wyeth, A. Employment (full or part-time); R.G.W. Staal, Wyeth, A.<br />
Employment (full or part-time); N. Breysse, Wyeth, A. Employment (full or part-time); R.<br />
Crozier, Wyeth, A. Employment (full or part-time); R. Nagy, Wyeth, A. Employment (full or<br />
part-time); A. Sung, Wyeth, A. Employment (full or part-time); M. Monagh<strong>an</strong>, Wyeth, A.<br />
Employment (full or part-time); R. Martone, Wyeth, A. Employment (full or part-time); D.<br />
Riddell, Wyeth, A. Employment (full or part-time); J. Chin, Wyeth, A. Employment (full or<br />
part-time); N. Bogd<strong>an</strong>ovic, Wyeth, A. Employment (full or part-time); D.M. Walsh, UCD, A.<br />
Employment (full or part-time); M.N. P<strong>an</strong>galos, Wyeth, A. Employment (full or part-time);<br />
P.H. Reinhart, Wyeth, A. Employment (full or part-time); J.S. Jacobsen, Wyeth, A.<br />
Employment (full or part-time); W.D. Hirst, Wyeth, A. Employment (full or part-time).<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.17/I12<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Evaluation of cerebral inflammation <strong>an</strong>d gliosis in Tg2576 mouse model of Alzheimer’s<br />
disease<br />
Authors: *L. TAHTIVAARA, J. PUOLIVÄLI, A. NURMI, R. PUSSINEN, T.-K. STENIUS,<br />
T. HEIKKINEN, A. POLUS, N. VARTIAINEN, J. YRJANHEIKKI;<br />
Cerebricon Ltd, Kuopio, Finl<strong>an</strong>d<br />
Abstract: Alzheimer’s disease (AD) is <strong>the</strong> most common cause of dementia in <strong>the</strong> elderly. One<br />
of <strong>the</strong> major hallmarks of <strong>the</strong> disease is <strong>the</strong> increased accumulation of amyloid beta (AB) in <strong>the</strong><br />
brain, but also brain inflammation <strong>an</strong>d gliosis have been suggested to play signific<strong>an</strong>t role in AD<br />
pathology. In this study we characterized inflammatory markers <strong>an</strong>d AB pathology over time in<br />
Tg2576 mouse model of AD. This widely used model carries a mutated <strong>for</strong>m of <strong>the</strong> hum<strong>an</strong>
amyloid precursor protein <strong>an</strong>d exhibits increased AB levels <strong>an</strong>d plaque deposition in <strong>the</strong> brain.<br />
At 5-6 <strong>an</strong>d 12-13 months of age we determined brain levels of soluble <strong>an</strong>d insoluble AB 1-42<br />
<strong>an</strong>d 1-40 <strong>an</strong>d AB plaque load in female Tg2576 mice. In order to study cerebral inflammation<br />
<strong>an</strong>d gliosis, expression of IL-6, COX-2, PGE-2, <strong>an</strong>d astrogliosis (GFAP) as well as<br />
microglia/monocyte/macrophage (CD68) accumulation were evaluated with ELISA <strong>an</strong>d<br />
immunohistochemistry, respectively.<br />
Results: At 5-6 months of age, Tg2576 mice showed highly increased levels of soluble AB 1-42<br />
<strong>an</strong>d 1-40 both in brain <strong>an</strong>d CSF, but as expected no AB plaques or insoluble AB. Increased astro-<br />
<strong>an</strong>d microgliosis was not present <strong>an</strong>d <strong>the</strong> expression of IL-6 <strong>an</strong>d PGE2 was not found to be<br />
signific<strong>an</strong>tly different between tg <strong>an</strong>d wt mice. At 12-13 months of age, <strong>the</strong> levels of soluble AB<br />
were slightly but signific<strong>an</strong>tly increased in tg mice when compared to wt mice, whereas <strong>the</strong><br />
levels of insoluble AB <strong>an</strong>d plaque load was robustly increased in cortex <strong>an</strong>d hippocampus of tg<br />
mice when compared to age matched wt mice. At 12-13 months of age GFAP <strong>an</strong>d COX-2<br />
immunoreactivity was found to be signific<strong>an</strong>tly higher both in cortex <strong>an</strong>d hippocampus in tg<br />
mice when compared to wt mice. However, CD68 immunoreactivity in tg mice was not found to<br />
be different <strong>from</strong> wt mice in cortex or hippocampus. Toge<strong>the</strong>r, <strong>the</strong>se data indicate that<br />
inflammation <strong>an</strong>d gliosis are not induced at <strong>the</strong> early age of Tg2576 mice when soluble amyloid<br />
beta is increased, but become a more pronounced factor at a later age when amyloid plaques<br />
appear.<br />
Disclosures: L. Tahtivaara, None; J. Puoliväli, None; A. Nurmi, None; R. Pussinen,<br />
None; T. Stenius, None; T. Heikkinen, None; A. Polus, None; N. Vartiainen, None; J.<br />
Yrj<strong>an</strong>heikki, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.18/I13<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: FAPESP<br />
CAPES<br />
Title: Effect of induced post-ischemic hyper<strong>the</strong>rmia in rats: Behavioral <strong>an</strong>d morphological study
Authors: *L. F. YAMASHITA, A. A. BORGES, C. GRASSL, C. GRASSL, R. C.<br />
SINIGAGLIA-COIMBRA, C. G. COIMBRA;<br />
Med., Federal Univ. of São Paulo, São Paulo, Brazil<br />
Abstract: Purpose: In a previous study, Sinigaglia-Coimbra et al (2002) reported <strong>the</strong> presence<br />
of senile plaques, neurofibrillary t<strong>an</strong>gles, activation of <strong>the</strong> complement system <strong>an</strong>d persistent<br />
gliosis in <strong>the</strong> brain of rats subjected to post-ischemic hyper<strong>the</strong>rmia induced by heating lamps at 2<br />
months of survival. The aim of this study was to evaluate both histopathological <strong>an</strong>d behavioral<br />
effects of post-ischemic hyper<strong>the</strong>rmia induced by heating lamps or heated air at six months of<br />
survival.<br />
Methods: Wistar H<strong>an</strong>over male rats (300-350g) were submitted to global brain ischemia<br />
(bilateral carotid occlusion combined with systemic hypotension of 50mmHg) <strong>for</strong> 10 minutes<br />
<strong>an</strong>d <strong>the</strong>n allocated into three groups: A) post-ischemic hyper<strong>the</strong>rmia, achieved with heating<br />
lamps focused directly on <strong>the</strong> <strong>an</strong>imal´s cage; B) post-ischemic hyper<strong>the</strong>rmia, achieved with <strong>the</strong><br />
circulation of pre-warmed air in a insulated <strong>an</strong>imal´s cage; <strong>an</strong>d C) post-ischemic normo<strong>the</strong>rmia,<br />
as a control group. Hyper<strong>the</strong>rmia (39 to 40° C) was induced in two consecutive periods (<strong>from</strong> 4h<br />
to 9-h recirculation <strong>an</strong>d <strong>from</strong> 21-h to 32-h recirculation). In order to evaluate spatial reference<br />
memory, <strong>the</strong> Morris water maze test was done along three different periods of survival: 15 days,<br />
2 months <strong>an</strong>d 6 months. After 6 months of survival, <strong>the</strong> <strong>an</strong>imal´s brain were perfusion-fixed.<br />
Subsequent immunohistochemical staining to neurodegenerative markers, such as βA-protein,<br />
neurofibrillary t<strong>an</strong>gles <strong>an</strong>d inflammatory cells. Hippocampal atrophy estimation was done using<br />
<strong>the</strong> Cavalieri´s principle.<br />
Results: Statistical <strong>an</strong>alysis showed that <strong>an</strong>imals submitted to two methods of hyper<strong>the</strong>rmia<br />
presented impaired cognitive per<strong>for</strong>m<strong>an</strong>ce (p
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: K01RR020360<br />
Title: Absence of genotypic effects on functional recovery or neuropathology within hum<strong>an</strong><br />
APOE-targeted-replacement mice<br />
Authors: *S. B. SHELTON 1 , S. A. SCOTT 2 , K. A. CRUTCHER 1 ;<br />
1 Univ. Cincinnati, Cincinnati, OH; 2 Ohio Univ., A<strong>the</strong>ns, OH<br />
Abstract: The APOE-ε4 allele is strongly associated with <strong>the</strong> risk of Alzheimer's disease <strong>an</strong>d<br />
worsened outcome after traumatic brain injury (TBI). Proteolytic fragments of apoE have been<br />
suggested to play a role in neuronal degeneration <strong>an</strong>d have been observed in Alzheimer's disease<br />
brain. To investigate <strong>the</strong> role of apoE, we studied hum<strong>an</strong> APOE-targeted-replacement mice<br />
expressing each of <strong>the</strong> hum<strong>an</strong> APOE alleles (ε2, ε3, ε4). We evaluated <strong>the</strong> APOE genotype<br />
response to TBI (controlled cortical impact). No genotypic differences in functional recovery,<br />
lesion cavity volume, GFAP immunoreactivity, neuronal loss, or brain apoE levels were found.<br />
Fur<strong>the</strong>rmore, no apoE fragments were observed. We evaluated conditions of proteolysis of<br />
hum<strong>an</strong> apoE in mouse brain in vitro. Incubation of APOE-targeted-replacement mouse brain<br />
tissue resulted in proteolysis of all 3 iso<strong>for</strong>ms. Of a p<strong>an</strong>el of 12 different protease inhibitors,<br />
proteolysis was inhibited only in <strong>the</strong> presence of <strong>the</strong> aspartic protease inhibitor pepstatin A. No<br />
genotypic differences in <strong>the</strong> rate of proteolysis were observed ei<strong>the</strong>r in <strong>the</strong> presence or absence<br />
of pepstatin A. As in hum<strong>an</strong> brain tissue, <strong>the</strong> results are consistent with a role <strong>for</strong> ca<strong>the</strong>psin D or<br />
similar protease in <strong>the</strong> degradation of apoE. The lack of genotypic effects may be <strong>the</strong> result of<br />
differences in protein interactions, expression, <strong>an</strong>d/or regulation of apoE in <strong>the</strong> mouse vs. <strong>the</strong><br />
hum<strong>an</strong> brain. Whatever <strong>the</strong> reason, <strong>the</strong> targeted-replacement mouse model <strong>for</strong> APOE genotype,<br />
does not provide clear parallels with hum<strong>an</strong> neuropathology but may provide a me<strong>an</strong>s to study<br />
<strong>the</strong> proteolytic fate of this protein.<br />
Disclosures: S.B. Shelton, None; S.A. Scott, None; K.A. Crutcher, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.20/I15<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Shapiro Foundation
AG10688<br />
Title: Neuronal colocalization of sex hormone receptors, beta amyloid, <strong>an</strong>d tau epitopes in <strong>the</strong><br />
<strong>for</strong>ebrain of 3xTgAD mice<br />
Authors: *C. R. OVERK, S. E. PEREZ, E. J. MUFSON;<br />
Rush Univ. Med. Ctr., Chicago, IL<br />
Abstract: Epidemiological studies have shown that decreased estrogen is a risk factor <strong>for</strong><br />
Alzheimer’s disease (AD). Tr<strong>an</strong>sgenic mouse models of amyloidosis, as well as <strong>the</strong> 3xTgAD<br />
mouse model of AD, which over expresses <strong>the</strong> FAD genes APPSWE, PS1M146V, <strong>an</strong>d TauP301L, have<br />
shown that decreased estrogen increases intra- <strong>an</strong>d extra-neuronal beta amyloid in <strong>the</strong> brain. The<br />
3xTgAD mice display beta amyloid <strong>an</strong>d tau pathology primarily in <strong>the</strong> hippocampus <strong>an</strong>d<br />
amygdala, regions which also display estrogen <strong>an</strong>d <strong>an</strong>drogen receptors (AR). However, whe<strong>the</strong>r<br />
estrogen receptors (ER) <strong>an</strong>d/or AR are expressed in beta amyloid - or abnormal tau-positive<br />
<strong>for</strong>ebrain neurons in 3xTgAD is unknown. To better underst<strong>an</strong>d <strong>the</strong> role of sex hormones in <strong>the</strong><br />
onset of AD pathology, <strong>for</strong>ebrain tissue was single or dual immunostained <strong>for</strong> ER alpha, ER<br />
beta, AR, amyloid precursor protein (APP)/ beta amyloid (6E10), <strong>an</strong>d <strong>the</strong> tau pathological<br />
staging markers (R1, Alz50, <strong>an</strong>d AT8) <strong>from</strong> 3, 9, <strong>an</strong>d 18 months old male <strong>an</strong>d female 3xTgAD<br />
<strong>an</strong>d non-tr<strong>an</strong>sgenic (ntg) mice. ER alpha-<strong>an</strong>d AR-, but not ER beta-positive nuclei were found in<br />
<strong>the</strong> CA1 field of <strong>the</strong> hippocampus across all ages <strong>an</strong>d genotypes examined. These three receptors<br />
were expressed throughout various amygdala subregions except in <strong>the</strong> basolateral amygdala, <strong>the</strong><br />
main nucleus affected by APP/ beta amyloid <strong>an</strong>d tau pathology in 3xTgAD mice. Although ER<br />
alpha <strong>an</strong>d ER beta distribution patterns were similar across gender, AR staining in males was<br />
more robust th<strong>an</strong> in females in both hippocampus <strong>an</strong>d amygdala. In <strong>the</strong> CA1 hippocampal region<br />
AR <strong>an</strong>d 6E10 colocalized in numerous CA1 pyramidal cells across <strong>the</strong> ages examined, while ER<br />
alpha only colocalized with a few 6E10-positive neurons in this hippocampal subfield. In<br />
addition, a few CA1/CA2 pyramidal neurons were dual immunopositive <strong>for</strong> ER alpha <strong>an</strong>d <strong>the</strong><br />
p<strong>an</strong> tau marker R1. However, AR failed to colocalize with <strong>the</strong> con<strong>for</strong>mational tau marker Alz50<br />
in <strong>the</strong> CA1 hippocampal field, although both coexisted in this region. Alternatively, ER alpha,<br />
ER beta, <strong>an</strong>d AR were present in <strong>the</strong> amygdala, but only AR <strong>an</strong>d 6E10 colocalized in a few cells<br />
in <strong>the</strong> central <strong>an</strong>d basomedial amygdala regions. Intriguingly, in <strong>the</strong> 9 <strong>an</strong>d 18 month old <strong>an</strong>imals<br />
ER beta was observed in <strong>the</strong> plaques. Moreover, tissue heated <strong>for</strong> <strong>an</strong>tigen retrieval displayed<br />
improved 6E10-immunoreactivity across all regions compared to untreated free-floating staining<br />
sections. These findings suggest AR <strong>an</strong>d ERs play a role in <strong>the</strong> modulation of beta amyloid <strong>an</strong>d<br />
tau pathology within <strong>the</strong> amygdala <strong>an</strong>d hippocampus possibly via a paracrine <strong>an</strong>d/or autocrine<br />
action in 3xTgAD mice.<br />
Disclosures: C.R. Overk, None; S.E. Perez, None; E.J. Mufson, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.21/I16<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NIH AG P10485<br />
NF/SG Veter<strong>an</strong>s Health System<br />
Title: Hippocampus-specific overexpression of inhibitor 1 of protein phosphatase 2A via adenoassociated<br />
virus serotype-5 reduces alternation behavior in wild-type <strong>an</strong>d APPSWE/PS1δE9<br />
tr<strong>an</strong>sgenic mice<br />
Authors: *M. A. KING 1 , M. L. MUSTROPH 2 , B. C. FITZGERALD 2 , A. C. HIRKO 2 ;<br />
2 Pharmacol. & Therapeut., 1 Univ. Florida, Gainesville, FL<br />
Abstract: Age-related inhibition of protein phosphatase 2A (PP2A) may play a key role in <strong>the</strong><br />
development of tauopathy in Alzheimer’s disease (AD). PP2A is a primary dephosphorylator of<br />
tau <strong>an</strong>d also regulates m<strong>an</strong>y tau kinases. It was hypo<strong>the</strong>sized that overexpression of I1PP2A<br />
(inhibitor 1 of protein phosphatase 2A), <strong>an</strong> endogenous inhibitor of PP2A, would introduce tau<br />
pathology to <strong>the</strong> Β-amyloid pathology in tr<strong>an</strong>sgenic APP mice, <strong>an</strong>d that this would accelerate <strong>the</strong><br />
cognitive decline observed in this AD model. Using a recombin<strong>an</strong>t adeno-associated virus<br />
serotype 5 (rAAV-5) vector, we tr<strong>an</strong>sduced I1PP2A or GFP (as a control) via bilateral<br />
hippocampal injections in 3-4 month old double-tr<strong>an</strong>sgenic APPSWE/PS1δE9 (APP) <strong>an</strong>d wildtype<br />
(WT) mice (Treatment Groups: APP+I1PP2A, APP+GFP, WT+I1PP2A, & WT+GFP).<br />
Animals were tested in a Y-maze <strong>for</strong> memory-related alternation behavior one <strong>an</strong>d two months<br />
after injections. One month following injections, 3 <strong>an</strong>imals <strong>from</strong> each group were examined<br />
histologically <strong>for</strong> amyloid (via 6E10 <strong>an</strong>tibody), hyperphoshorylated tau/paired helical filaments<br />
(AT8 <strong>an</strong>tibody), <strong>an</strong>d <strong>for</strong> thioflavin S labeling. Spont<strong>an</strong>eous alternation was signific<strong>an</strong>tly reduced<br />
one month after gene delivery in WT+I1PP2A, APP+I1PP2A, <strong>an</strong>d also APP+GFP. At two<br />
months, spont<strong>an</strong>eous alternation by <strong>the</strong> APP+GFP mice recovered to similar levels as <strong>the</strong><br />
WT+GFP, while both groups receiving I1PP2A continued to show deficits, with APP+I1PP2A<br />
showing <strong>the</strong> least amount of spont<strong>an</strong>eous alternation. After one month, no differences were<br />
detected among <strong>the</strong> groups <strong>for</strong> 6E10 staining. There was <strong>an</strong> apparent increase in AT8<br />
immunoreactivity in <strong>the</strong> APP+I1PP2A <strong>an</strong>d WT+I1PP2A groups. APP+GFP <strong>an</strong>d APP+I1PP2A<br />
mice showed only limited thioflavin S labeling at this age <strong>an</strong>d expression duration, but some<br />
intracellular thioflavin S staining was detected in <strong>the</strong> APP+I1PP2A group. The behavioral<br />
impairment indicates that hippocampus-specific inhibition of neuronal PP2A activity via I1PP2A<br />
upregulation accelerates or complements neuropathology associated with Β-amyloid<br />
overexpression. Active PP2A suppression may be a key mech<strong>an</strong>ism involved in neurofibrillary<br />
<strong>an</strong>d intracellular tr<strong>an</strong>sport pathology in AD, <strong>an</strong>d <strong>an</strong> import<strong>an</strong>t <strong>the</strong>rapeutic target.
Disclosures: M.A. King, None; M.L. Mustroph, None; B.C. Fitzgerald, None; A.C. Hirko,<br />
None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.22/I17<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: Anonymous Foundation<br />
Title: Mouse models <strong>for</strong> <strong>the</strong> study of Alzheimer’s disease<br />
Authors: *D. P. LIU, S. ROCKWOOD, C. M. LUTZ, M. SASNER;<br />
Genet. Resource Sci., Jackson Lab., Bar Harbor, ME<br />
Abstract: Mouse models are tools critical to <strong>the</strong> study of <strong>the</strong> basic pathogenesis of Alzheimer’s<br />
disease (AD) <strong>an</strong>d c<strong>an</strong> be used to develop new <strong>the</strong>rapies. The Alzheimer’s Disease Mouse Model<br />
Repository (ADMMR) was established within <strong>the</strong> mouse repository at The Jackson Laboratory<br />
to be a central resource <strong>for</strong> archiving <strong>an</strong>d distributing genetically engineered AD models at a<br />
high health status, to monitor genetic <strong>an</strong>d phenotypic stability, <strong>an</strong>d to provide in<strong>for</strong>mation<br />
supporting <strong>the</strong> selection <strong>an</strong>d use of mouse models in AD research. The ADMMR continues to<br />
seek new models, <strong>an</strong>d now distributes more th<strong>an</strong> 80 strains carrying mutations in 12 different<br />
genes directly relev<strong>an</strong>t to AD, including (Abca1), apolipoprotein E (Apoe), amyloid beta<br />
precursor protein (App), beta-site APP cleaving enzymes (Bace1 <strong>an</strong>d Bace2), a regulatory<br />
subunit of cyclin-dependent kinase 5 (Cdk5r1), microtubule-associated protein tau (Mapt), <strong>an</strong>d<br />
presenilins (Psen1 <strong>an</strong>d Psen2). Disease models include <strong>the</strong> 3XTg-FAD; APPswe/PS1dE9;<br />
5XFAD; R1.40; J20; BRI-Abeta42; <strong>an</strong>d APPSwDutIowa strains. Some of <strong>the</strong> more widely used<br />
disease models are now available as aged mice. In most cases studied to date, mice carrying<br />
combinations of mut<strong>an</strong>t alleles display a more severe disease phenotype; <strong>the</strong> repository maintains<br />
some of <strong>the</strong>se complex mut<strong>an</strong>ts (e.g. <strong>the</strong> APPSwDI/NOS2 line), <strong>an</strong>d facilitates <strong>the</strong> creation of<br />
new combinations of alleles. O<strong>the</strong>r strains carry alleles of AD modifier genes including clusterin<br />
(Clu), insulin degrading enzyme (IDE), membr<strong>an</strong>e metallo- endopeptidase (MME or neprilysin),<br />
plasminogen activator urokinase (Plau), <strong>an</strong>d sortilin-related receptor (Sorl1). The repository<br />
distributes tr<strong>an</strong>sgenic lines expressing mut<strong>an</strong>t <strong>for</strong>ms of hum<strong>an</strong> APP or CDK5R1 in a tet-inducible<br />
m<strong>an</strong>ner, as well as strains expressing <strong>the</strong> tetracycline regulated tr<strong>an</strong>sactivator (tTA) protein in<br />
various cell types. A variety of cre-expressing lines enable gene deletion in various neuronal cell<br />
types. Strains expressing reporter genes (including GFP, YFP, CFP <strong>an</strong>d lacZ) c<strong>an</strong> be useful tools
to assay neuronal viability <strong>an</strong>d disease progression. Submission of a strain to <strong>the</strong> repository<br />
fulfills requirements <strong>for</strong> sharing of mice in accord<strong>an</strong>ce with NIH’s policy <strong>for</strong> sharing of research<br />
reagents; strains c<strong>an</strong> be submitted using <strong>an</strong> online web <strong>for</strong>m. If necessary, donating investigators<br />
c<strong>an</strong> place licensing restrictions on <strong>the</strong> distribution <strong>an</strong>d use of <strong>the</strong>ir models. For more in<strong>for</strong>mation<br />
about <strong>the</strong> ADMMR including a list of strains, phenotype descriptions, allele <strong>an</strong>d genotyping<br />
in<strong>for</strong>mation <strong>an</strong>d associated references, see<br />
www.jax.org/jaxmice/research/neurobiology/alzheimers.<br />
Disclosures: D.P. Liu, None; S. Rockwood, None; C.M. Lutz, None; M. Sasner, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.23/I18<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Markers of early cerebrovascular inflammation in Alzheimer’s disease mouse models<br />
Authors: D. YU 1 , E. WANG 1 , *R. L. MARTONE 2 , P. REINHART 1 , S. CHO 1 , J. CHIN 1 ;<br />
1 Wyeth Res., Princeton, NJ; 2 Wyeth-Ayerst Res., Princeton, NJ<br />
Abstract: Alzheimer’s disease (AD) is characterized by senile plaques composed of amyloid<br />
beta (Aβ) peptides derived <strong>from</strong> <strong>the</strong> amyloid precursor protein (APP). Plaques induce a chronic<br />
inflammatory response associated with activated microglia <strong>an</strong>d reactive astrocytes, which could<br />
exacerbate neurodegeneration through <strong>the</strong> release of inflammatory cytokines, reactive oxygen<br />
species, <strong>an</strong>d o<strong>the</strong>r factors. Although much evidence suggests that inflammation contributes to<br />
neurodegeneration in later stages of AD, inflammation may also play a role in early disease<br />
pathogenesis. Progress in underst<strong>an</strong>ding <strong>the</strong> role of inflammation in AD has been hampered by<br />
<strong>the</strong> relative lack of robust markers of inflammation in AD mouse models. Increases in<br />
inflammatory cytokines in <strong>the</strong> brain have been documented in several lines of tr<strong>an</strong>sgenic mice,<br />
generally in aged, plaque-bearing mice. We thus sought to identify markers of early stages of<br />
inflammation that might also provide efficacy measures <strong>for</strong> <strong>the</strong>rapeutic strategies with <strong>an</strong>tiinflammatory<br />
effects. We report that expression of <strong>the</strong> endo<strong>the</strong>lial cell-specific <strong>an</strong>tigen MECA-<br />
32 is a marker of early stages of cerebrovascular inflammation in APP/PSEN1 (PSAPP)<br />
tr<strong>an</strong>sgenic mice.<br />
MECA-32 is expressed in both <strong>the</strong> central <strong>an</strong>d peripheral vasculature throughout development,<br />
but expression in <strong>the</strong> cerebral vasculature is down-regulated around E18 upon establishment of<br />
<strong>the</strong> blood-brain barrier (BBB). However, CNS inflammation triggers re-expression of MECA-32
in <strong>the</strong> compromised cerebral vasculature. Notably, increased MECA-32 immunostaining has<br />
been reported in AD brains (Ann NY Acad Sci 903:335-44). We found that PSAPP mice exhibit<br />
MECA-32 staining in <strong>the</strong> cerebral vasculature to a much greater extent th<strong>an</strong> nontr<strong>an</strong>sgenic mice,<br />
suggesting that <strong>the</strong> vasculature is compromised in PSAPP mice. The increased MECA-32<br />
staining was observed in young mice prior to plaque deposition as well as in aged mice,<br />
indicating that soluble Aβ c<strong>an</strong> trigger early cerebrovascular inflammation. In both nontr<strong>an</strong>sgenic<br />
<strong>an</strong>d PSAPP mice, MECA-32 staining was evident in <strong>the</strong> choroid plexus, as expected <strong>for</strong><br />
circumventricular org<strong>an</strong>s residing outside of <strong>the</strong> BBB. These results suggest that MECA-32<br />
expression may provide a measure of cerebrovascular inflammation in PSAPP mice.<br />
Disclosures: D. Yu, Wyeth Research, A. Employment (full or part-time); E. W<strong>an</strong>g, Wyeth<br />
Research, A. Employment (full or part-time); R.L. Martone, Wyeth Research, A. Employment<br />
(full or part-time); P. Reinhart, Wyeth Research, A. Employment (full or part-time); S. Cho,<br />
Wyeth Research, A. Employment (full or part-time); J. Chin, Wyeth Research, A. Employment<br />
(full or part-time).<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.24/I19<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: CSF Aβ40 <strong>an</strong>d Aβ42 levels <strong>an</strong>d cognitive dysfunction in senior beagles<br />
Authors: *C. DE RIVERA 1,2 , J. A. ARAUJO 1,2 , N. W. MILGRAM 1,2 ;<br />
1 Pharmacol, Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada; 2 C<strong>an</strong>Cog Technologies Inc., Toronto, ON,<br />
C<strong>an</strong>ada<br />
Abstract: Dogs model hum<strong>an</strong> cognitive decline <strong>an</strong>d pathological aging. With adv<strong>an</strong>ced age,<br />
dogs show deficits in memory, inhibitory control <strong>an</strong>d attention. Among aged <strong>an</strong>imals, individual<br />
differences in cognitive per<strong>for</strong>m<strong>an</strong>ce model those seen in aged hum<strong>an</strong>s <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e provide<br />
c<strong>an</strong>ine models of mild cognitive impairment <strong>an</strong>d dementia. Aged dogs also develop brain<br />
atrophy, ventricular enlargement, <strong>an</strong>d accumulate hum<strong>an</strong> type β-amyloid (Aβ) in plaques with<br />
development occurring earliest in <strong>the</strong> prefrontal cortex. Like hum<strong>an</strong>s, <strong>the</strong> predomin<strong>an</strong>t species of<br />
Aβ in <strong>the</strong> dog brain is <strong>the</strong> longer, more toxic fragment Aβ1-42, ra<strong>the</strong>r th<strong>an</strong> Aβ1-40, <strong>an</strong>d <strong>the</strong><br />
structure of both are identical to those in hum<strong>an</strong>s. The present study fur<strong>the</strong>r extends <strong>the</strong> c<strong>an</strong>ine<br />
model by: 1) assessing cerebrospinal fluid (CSF) Aβ levels in groups of dogs varying in age; <strong>an</strong>d<br />
2) correlating serum <strong>an</strong>d CSF Aβ levels to per<strong>for</strong>m<strong>an</strong>ce on tests of cognitive function. Lumbar
CSF samples were collected <strong>from</strong> over 50 Beagle dogs <strong>from</strong> three age groups: young (2-4 years);<br />
old (6-8 years); <strong>an</strong>d senior (≥ 10 years). Aβ40 <strong>an</strong>d Aβ42 levels were measured using ELISA.<br />
Levels of Aβ40 did not differ signific<strong>an</strong>tly between <strong>the</strong> three age groups. By contrast, Aβ42<br />
levels showed <strong>an</strong> inverted U-shaped relationship with age, with <strong>the</strong> senior <strong>an</strong>imals showing<br />
signific<strong>an</strong>tly decreased levels when compared to <strong>the</strong> old <strong>an</strong>imals (p < 0.05). We also measured<br />
plasma Aβ40; which increased progressively with age. These findings indicate a complex<br />
relationship between age <strong>an</strong>d soluble Aβ, as detected in CSF. We suggest that <strong>the</strong> lower levels of<br />
Aβ42 seen in senior dogs correlates with cognitive impairment seen in adv<strong>an</strong>ced age <strong>an</strong>d that <strong>the</strong><br />
decreased levels reflect greater cortical aggregation of Aβ42 in senior dogs th<strong>an</strong> in old or<br />
younger dogs. These findings are consistent with data <strong>from</strong> hum<strong>an</strong> subjects indicating lower<br />
Aβ42 levels in AD patients th<strong>an</strong> in aged matched controls. The current findings provide ex<strong>citing</strong><br />
new evidence of commonalties between hum<strong>an</strong> <strong>an</strong>d c<strong>an</strong>ine cognitive aging.<br />
Disclosures: C. de Rivera, C<strong>an</strong>Cog Technologies Inc., A. Employment (full or part-time); J.A.<br />
Araujo, C<strong>an</strong>Cog Technologies Inc., A. Employment (full or part-time); C<strong>an</strong>Cog Technologies<br />
Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); N.W.<br />
Milgram, C<strong>an</strong>Cog Technologies Inc., A. Employment (full or part-time); C<strong>an</strong>Cog Technologies<br />
Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.25/I20<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Ventricular infusion of Aβ25-35 causes deficits in spatial memory in Long-Ev<strong>an</strong>s rats<br />
Authors: *M. R. BLANKENSHIP, J. G. MCGARVEY, J. R. DEVRIES, M. S. PETERSON,<br />
A. L. COCHRAN, W. R. HAMLET;<br />
Western Illinois Univ., Macomb, IL<br />
Abstract: Amyloid beta (Aβ) peptide accumulation is thought to be a primary pathological<br />
characteristic associated with <strong>the</strong> memory impairments seen in Alzheimer’s disease (AD: Klein<br />
et al., 2007). Aβ’s role in memory disruption is not well understood, however <strong>the</strong>ories of Aβ<br />
action have shifted emphasis <strong>from</strong> toxicity <strong>for</strong> fibrillar <strong>for</strong>ms found in plaques to soluble,<br />
prefibrillar <strong>for</strong>ms. In vitro work with a wide r<strong>an</strong>ge of prefibrillar Aβ species has shown profound<br />
effects on synaptic spine morphology, accelerated synaptic degeneration, increased production of<br />
reactive oxygen species <strong>an</strong>d cell loss. Confirmation of Aβ’s disruptive effects on behavior <strong>an</strong>d
cognition has come <strong>from</strong> tr<strong>an</strong>sgenic Aβ overproducing mice (i.e. Tg2576). While <strong>the</strong>se models<br />
clearly reiterate <strong>the</strong> role of Aβ in <strong>the</strong> neuropathology of AD, models that allow more control of<br />
Aβ administration are also desirable. To this end, several groups have reported disruption of<br />
hippocampally-dependent memory following acute Aβ infusion. The hypo<strong>the</strong>sis that Aβ25-35<br />
infusion would result in long-term memory impairment was tested with injections into <strong>the</strong><br />
ventricles, followed by a 30-day recovery period. Twenty-eight male long Ev<strong>an</strong>s rats were<br />
administered bilateral infusions of 2µl (15nM Aβ25-35, incubated at 37°C <strong>for</strong> 72 hours) or<br />
sterilized distilled water (control). Following a 30-day recovery period, subjects were evaluated<br />
in a 12-arm radial maze using a win/shift task. A delay of 0, 2, or 4 hours was introduced<br />
between <strong>the</strong> win trial <strong>an</strong>d <strong>the</strong> shift trial. Increasing <strong>the</strong> delay interval has been shown to increase<br />
<strong>the</strong> hippocampal dependency of <strong>the</strong> radial maze task (Packard & White, 1991). The results found<br />
no difference between <strong>the</strong> Aβ <strong>an</strong>d control groups with a delay of 0 hours, however <strong>the</strong> Aβ group<br />
showed a signific<strong>an</strong>tly higher number of proactive errors when a delay of 2 or 4 hours was used.<br />
Given <strong>the</strong> long clear<strong>an</strong>ce time <strong>for</strong> <strong>the</strong> Aβ, it is likely that <strong>the</strong> observed results are based on<br />
neuronal degeneration. The data will be interpreted in comparison to current work in our lab <strong>an</strong>d<br />
o<strong>the</strong>rs involving acute Aβ treatment in o<strong>the</strong>rwise normal <strong>an</strong>imals.<br />
Disclosures: M.R. Bl<strong>an</strong>kenship, None; J.G. McGarvey, None; J.R. Devries, None; M.S.<br />
Peterson, None; A.L. Cochr<strong>an</strong>, None; W.R. Hamlet, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.26/I21<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: CADRD<br />
Title: Impaired glucose toler<strong>an</strong>ce precedes neuro<strong>an</strong>atomical identification of Aβ <strong>an</strong>d<br />
hyperphosphorylated tau accumulation in <strong>the</strong> 3xTg model of Alzheimer’s disease<br />
Authors: L. N. MACKLIN 1,2 , Y. CAI 1,2 , D. J. KANAK 1,2 , J. EBERSOLE 2 , A. E. CORBIN 2 , A.<br />
WOMACK 1 , D. WENDT 1 , S. ODDO 4 , F. M. LAFERLA 5 , X.-X. YAN 3,2 , G. J. BREWER 2,6,7 , R.<br />
STRUBLE 2,7 , G. M. ROSE 2,3 , A. D. STRADER 1,2 , *P. R. PATRYLO 1,2,3 ;<br />
1 Physiol., 2 Ctr. <strong>for</strong> Integrated Res. in Cognitivie <strong>an</strong>d Neural Sci., 3 Anat., SIUC, Carbondale, IL;<br />
4 Physiol., UTHSC, S<strong>an</strong> Antonio, TX; 5 Neurobio. <strong>an</strong>d Behavior, Univ. of Cali<strong>for</strong>nia Irvine,<br />
Irvine, CA; 6 Med. Microbiology, Immunol. Cell Biol., 7 Neurol. <strong>an</strong>d Ctr. <strong>for</strong> Alzheimer's Dis.,<br />
SIU, Springfield, IL
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by<br />
<strong>the</strong> deposition of Aβ intr<strong>an</strong>euronally as well as in plaques <strong>an</strong>d neurofibrillary t<strong>an</strong>gles in <strong>the</strong> brain.<br />
It has been suggested that diabetes is a risk factor <strong>for</strong> AD. To test this idea, we examined <strong>for</strong><br />
alterations in glucose toler<strong>an</strong>ce <strong>an</strong>d insulin secretion in <strong>the</strong> 3xTg mouse model of AD. We<br />
hypo<strong>the</strong>sized that glucose dysregulation precedes <strong>the</strong> appear<strong>an</strong>ce of brain pathology. To<br />
accomplish this, male 3xTg mice <strong>an</strong>d controls were examined at two ages: 2-3 or 8-10 months<br />
old. These age groups were chosen because 2-3 month old 3xTg mice are considered normal, <strong>an</strong>d<br />
show no plaques or t<strong>an</strong>gles, while abnormal Aβ <strong>an</strong>d tau deposition are present in <strong>the</strong> 8-10 month<br />
group. Basal plasma glucose <strong>an</strong>d insulin levels, as well as <strong>the</strong> response to glucose or insulin<br />
challenge, were measured. Baseline glucose levels in <strong>the</strong> 3xTg mice were elevated compared to<br />
<strong>the</strong> wild type control group at 2-3 months of age (WT 76 mg/dL; 3xTg 82 mg/dL). In <strong>the</strong> older<br />
mice, baseline glucose levels had proportionately increased in both groups (WT 98 mg/dL; 3xTg<br />
118 mg/dL). Young 3xTg mice also showed alterations in glucose toler<strong>an</strong>ce when compared to<br />
<strong>the</strong> control group (area under curve: AUC: WT 12085 ± 1400; 3xTg 20575 ± 1585). This<br />
measure also increased with age in both groups (AUC: WT 16964 ± 1844, 3xTg 24496 ± 2870).<br />
Baseline plasma insulin was lower in <strong>the</strong> 3xTg mice compared to <strong>the</strong> controls at both ages (2-3<br />
months: WT: 0.33 ± 0.14 ng/mL, 3xTg 0.17 ± 0.19; 8-10 months WT 0.95 ± 0 .14, 3xTg 0.39 ± 0<br />
.15). Fur<strong>the</strong>r, insulin secretion following a glucose bolus was lower <strong>for</strong> <strong>the</strong> 3xTg mice (2-3<br />
months: WT 0.64 ± 0.21 ng/mL, 3xTg 0.31 ± 0.28; 8-10 months: WT 2.03 ± 0.21, 3xTg 0.46 ±<br />
0.22). Immunohistochemistry verified that <strong>the</strong>re were no AD-like lesions in <strong>the</strong> 2-3 month old<br />
mice, but that Aβ <strong>an</strong>d hyper-phosphorylated tau immunoreactivities were present in cortical <strong>an</strong>d<br />
hippocampal pyramidal neurons in <strong>the</strong> 8-10 month group. Future experiments will entail<br />
examining whe<strong>the</strong>r insulin sensitivity differs between groups with age <strong>an</strong>d assessing whe<strong>the</strong>r<br />
metabolic m<strong>an</strong>ipulation c<strong>an</strong> affect pathogenesis in <strong>the</strong> 3xTg model. In conclusion, <strong>the</strong>se data<br />
indicate that alterations in glucose <strong>an</strong>d insulin levels <strong>an</strong>d regulation occur in <strong>the</strong> periphery prior<br />
to <strong>the</strong> appear<strong>an</strong>ce of abnormal Aβ <strong>an</strong>d tau deposition in 3xTg mice <strong>an</strong>d suggest that p<strong>an</strong>creatic βcell<br />
<strong>an</strong>d potentially skeletal muscle function is altered in this model of AD.<br />
Disclosures: L.N. Macklin, None; Y. Cai, None; D.J. K<strong>an</strong>ak, None; J. Ebersole, None; A.E.<br />
Corbin, None; A. Womack, None; D. Wendt, None; S. Oddo, None; F.M. LaFerla, None; X.<br />
Y<strong>an</strong>, None; G.J. Brewer, None; R. Struble, None; G.M. Rose, None; A.D. Strader,<br />
None; P.R. Patrylo, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.27/I22<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models
Support: NIH Gr<strong>an</strong>t RO1 AG20159<br />
Title: Microglia mediate <strong>the</strong> clear<strong>an</strong>ce of soluble Aß through both receptor-mediated<br />
phagocytosis <strong>an</strong>d fluid-phase pinocytosis<br />
Authors: *H. FU 1 , J. L. FROST 1 , S. HONG 1 , C. SHEN 2 , T. N. MAYADAS 3 , C. A. LEMERE 1 ;<br />
1 Dept. of Neurol., Ctr. For Neurologic Diseases, Brigham & Women's Hosp., Harvard Med.<br />
Sch., Boston, MA; 2 Harvard Univ., Cambridge, MA; 3 Dept. of Pathology, Brigham <strong>an</strong>d<br />
Women's Hospital, Harvard Med. Sch., Boston, MA<br />
Abstract: Amyloid-beta (Aß) peptides are <strong>the</strong> major constituents of <strong>the</strong> senile plaque, which is<br />
one of <strong>the</strong> pathological hallmarks of Alzheimer's disease (AD). Both fibrillar <strong>for</strong>m (fAß) <strong>an</strong>d<br />
soluble <strong>for</strong>m of Abeta (sAß) are considered to play import<strong>an</strong>t roles in <strong>the</strong> pathogenesis of AD.<br />
Microglia, <strong>the</strong> principle immune effector <strong>an</strong>d phagocytic cells in <strong>the</strong> CNS, are found to be<br />
associated with senile plaques in AD patients <strong>an</strong>d to phagocytose both fAß <strong>an</strong>d sAß. The<br />
clear<strong>an</strong>ce of fAß by microglia has been widely reported to be through receptor-mediated<br />
phagocytosis, however, <strong>the</strong> mech<strong>an</strong>isms underlying microglia-mediated clear<strong>an</strong>ce of sAß are<br />
unclear. In this study, we found that both primary microglia <strong>an</strong>d immortal murine microglial cell<br />
lines were able to take up sAß42 in a time-dependent m<strong>an</strong>ner using flow cytometry <strong>an</strong>d confocal<br />
laser sc<strong>an</strong>ning microscope. The distribution of sAß42 taken up by microglia colocalized with <strong>the</strong><br />
immunostaining of <strong>the</strong> late endosomal marker LAMP-1. Uptake of sAß42 by primary microglia<br />
derived <strong>from</strong> mouse pups genetically deficient <strong>for</strong> complement component C3 (C3) or its<br />
receptor, CR3 (Mac-1), was signific<strong>an</strong>tly inhibited when compared with that of primary<br />
microglia derived <strong>from</strong> wild type C57BL/6 mouse pups. However, knockdown of C3 or Mac-1<br />
in BV-2 or N9 microglial cells by tr<strong>an</strong>sient tr<strong>an</strong>sfection of C3 or Mac-1 siRNA did not<br />
signific<strong>an</strong>tly inhibit sAß42 uptake when compared to cells tr<strong>an</strong>sfected with control siRNA.<br />
Fur<strong>the</strong>rmore, it was found that deficiency or knockdown of C3 or Mac-1 signific<strong>an</strong>tly enh<strong>an</strong>ced<br />
<strong>the</strong> ability of <strong>the</strong> scavenger receptor inhibitor (Fucoid<strong>an</strong>) to inhibit <strong>the</strong> uptake of sAß42 by<br />
primary microglia <strong>an</strong>d N9 microglial cells. In addition, sAß42 uptake was dependent on both<br />
actin <strong>an</strong>d tubulin dynamics, <strong>an</strong>d membr<strong>an</strong>e cholesterol in primary microglia <strong>an</strong>d N9 microglial<br />
cells; whereas it was independent of receptor-mediated phagocytosis <strong>an</strong>d membr<strong>an</strong>e cholesterol<br />
in BV-2 microglial cells. Taken toge<strong>the</strong>r, <strong>the</strong>se results demonstrate that both receptor-mediated<br />
phagocytosis <strong>an</strong>d fluid-phase pinocytosis are involved in <strong>the</strong> clear<strong>an</strong>ce of sAß induced by<br />
microglia, which may be beneficial <strong>for</strong> underst<strong>an</strong>ding how sAß is removed <strong>from</strong> <strong>the</strong> AD brain by<br />
microglia <strong>an</strong>d <strong>for</strong> developing new <strong>the</strong>rapies aimed at removing sAß.<br />
Disclosures: H. Fu, None; J.L. Frost, None; S. Hong, None; C. Shen, None; T.N. Mayadas,<br />
None; C.A. Lemere, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.28/I23<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Support: NS036647<br />
Title: The role of BDNF in <strong>the</strong> triple tr<strong>an</strong>sgenic Alzheimer's disease mouse model<br />
Authors: *J. ZHANG 1,2 , M. W. VONDRAN 1,2 , L. L. LERCHER 1 , F. M. LAFERLA 3 , C. F.<br />
DREYFUS 1 ;<br />
1 Neurosci & Cell Bio, 2 Grad. Sch. of Biomed. Sci., UMDNJ, Piscataway, NJ; 3 Dept. of<br />
Neurobio. <strong>an</strong>d Behavior, Univ. of Cali<strong>for</strong>nia, Irvine, Irvine, CA<br />
Abstract: Alzheimer’s disease (AD) is associated with loss of basal <strong>for</strong>ebrain (BF) cholinergic<br />
neurons (Wilcock et al 1982) as well as <strong>the</strong>ir myelin ensheathment (Bartzokis 2004). The<br />
identification of molecules that impact <strong>the</strong>se processes is critical. We hypo<strong>the</strong>size that one such<br />
molecule is brain derived neurotrophic factor (BDNF). Previous studies in culture found that<br />
BDNF through <strong>the</strong> trkB receptor increases DNA syn<strong>the</strong>sis <strong>an</strong>d differentiation of oligodendrocyte<br />
(OLG) lineage cells in <strong>the</strong> BF (Du et al 2006). Mice with reduced levels of BDNF exhibit<br />
decreases in OLG progenitors <strong>an</strong>d myelin traits (Wood et al 2005). Moreover, BDNF increases<br />
<strong>the</strong> survival <strong>an</strong>d function of cultured BF cholinergic neurons (Friedm<strong>an</strong> et al 1993). The current<br />
work investigated <strong>the</strong> effects of BDNF in a triple tr<strong>an</strong>sgenic AD mouse model (3xTg-AD, Oddo<br />
et al 2003). We found that <strong>the</strong>se mice, at 1 year of age, exhibit a reduction in BDNF in <strong>the</strong> BF by<br />
Western blot <strong>an</strong>alysis. Immunohistochemical <strong>an</strong>alysis indicates that numbers of choline<br />
acetyltr<strong>an</strong>sferase+ neurons in <strong>the</strong> BF are decreased. In addition, <strong>the</strong> 3xTg-AD mice exhibit<br />
decreases in NG2+ OLG progenitor cell numbers <strong>an</strong>d protein as well as decreases in numbers of<br />
CC1+ mature OLGs <strong>an</strong>d myelin basic protein (MBP). These data suggest that <strong>the</strong>re is a<br />
correlative loss of BDNF, cholinergic neurons <strong>an</strong>d OLGs. To evaluate <strong>the</strong> potential <strong>the</strong>rapeutic<br />
effects of BDNF, exogenous BDNF (0.5ug/ul) or vehicle, artificial cerebrospinal fluid (aCSF)<br />
was stereotaxically injected into <strong>the</strong> lateral ventricle. 3xTgAD mice injected with BDNF exhibit<br />
increased BDNF protein levels <strong>an</strong>d MBP levels in <strong>the</strong> BF after a week compared to 3xTgAD<br />
mice injected with aCSF. In contrast, NG2 protein levels were similar in 3xTgAD mice injected<br />
with BDNF or aCSF. These findings suggest that exogenous BDNF may impact mature OLGs<br />
but not progenitors in AD mice. Future studies will evaluate whe<strong>the</strong>r exogenous BDNF may<br />
affect cholinergic neurons.<br />
Disclosures: J. Zh<strong>an</strong>g, None; M.W. VonDr<strong>an</strong>, None; L.L. Lercher, None; F.M. LaFerla,<br />
None; C.F. Dreyfus, None.<br />
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.29/I24<br />
Topic: C.02.l. Neuropharmacology <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: Croati<strong>an</strong> Ministry of Science, Education <strong>an</strong>d Sports, 098-0982522-2457<br />
Title: Dopamine beta-hydroxylase (DBH) activity <strong>an</strong>d -1021C/T polymorphism of DBH gene in<br />
Alzheimer’s disease<br />
Authors: M. MUSTAPIC 1 , P. PRESECKI 2 , N. MIMICA 3 , N. PIVAC 1 , V. FOLNEGOVIC -<br />
SMALC 3 , *M. DIKSIC 4 , D. MÜCK-SELER 1 ;<br />
1 Div. of Mol. Med., Ruđer Boskovic Inst., Zagreb, Croatia; 2 Dept. of Psychiatry, Gen. Hosp.<br />
Pula, Pula, Croatia; 3 Dept. of Psychiatry, Psychiatric Hosp. Vrapce, Zagreb, Croatia; 4 Dept<br />
Neurol & Neurosurg, McGill Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Background: Alzheimer’s disease (AD) is complex <strong>an</strong>d polygenic disorder.<br />
Polymorphisms within <strong>the</strong> dopamine beta-hydroxylase (DBH) gene could be related to etiology<br />
of Alzheimer’s disease (AD), given <strong>the</strong> well-documented ch<strong>an</strong>ges in <strong>the</strong> catecholamine-mediated<br />
neurotr<strong>an</strong>smission that occurs in this disorder. The aim of <strong>the</strong> present study was to investigate<br />
DBH -1021C/T gene polymorphism <strong>an</strong>d plasma DBH activity between patients with AD <strong>an</strong>d<br />
healthy controls.<br />
Methods: Plasma DBH activity <strong>an</strong>d DBH -1021C/T polymorphism were determined in 155<br />
patients (me<strong>an</strong> ± SD age 66.3 ± 11.2 years; MMSE = 13.1 ± 8.1) with AD (NINCDS-ADRDA<br />
<strong>an</strong>d DSM-IV-TR criteria) <strong>an</strong>d 188 healthy controls (66.3 ± 11.2 years). The patients were<br />
subdivided into two subgroups according to <strong>the</strong> presence or absence of psychotic features <strong>an</strong>d<br />
according to <strong>the</strong> early (F00.0) or late (F00.1) onset AD. Plasma DBH activity was determined by<br />
a photometric method <strong>an</strong>d DBH genotype by st<strong>an</strong>dard RFLP technique.<br />
Results: Among AD patients 62%, 31% <strong>an</strong>d 6.5% were carrying CC, CT <strong>an</strong>d TT genotype, while<br />
61.5%, 33,5% <strong>an</strong>d 5.3% of healthy controls were carrying CC, CT <strong>an</strong>d TT genotype,<br />
respectively. DBH genotype (Chi-square=0.38; df=2; p=0.825) <strong>an</strong>d allele (Chi-square=0.038;<br />
df=1; p=0.90) frequencies were similarly distributed between healthy controls <strong>an</strong>d patients with<br />
AD, between patients with or without psychotic features (Chi-square=1.90; df=2; p=0.386) <strong>an</strong>d<br />
between patients with early- <strong>an</strong>d late-onset AD (Chi-square=3.07; df=2; p=0.215). A<br />
signific<strong>an</strong>tly (p
Poster<br />
528. Alzheimer's Disease: Experimental Models <strong>an</strong>d Disease Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 528.30/I25<br />
Topic: C.02.c. Abeta toxicity<br />
Support: NIH-AG028072<br />
NIH-P30 AG013280<br />
Title: Mitochondrially targeted catalase increases <strong>the</strong> mRNA expression of longevity genes <strong>an</strong>d<br />
extend <strong>the</strong> lifesp<strong>an</strong>: Evidence <strong>from</strong> a global gene expression <strong>an</strong>alysis<br />
Authors: *P. H. REDDY 1 , P. MAO 1 , M. MANCZAK 1 , P. RABINOVITCH 3 , R. SEARLES 2 , A.<br />
P. REDDY 1 ;<br />
1 Primate Ctr., Oregon Hlth. Sci. Univ., Beaverton, OR; 2 Gene Microarray Shared Resource,<br />
Oregon Hlth. Sci. Univ., Portl<strong>an</strong>d, OR; 3 Univ. of Washington, Seattle, WA<br />
Abstract: BACKGROUND<br />
Increasing evidence suggests that mitochondrially targeted <strong>an</strong>tioxid<strong>an</strong>ts reduce oxidative<br />
damage, boost mitochondrial function, <strong>an</strong>d protect neurons <strong>from</strong> various toxic insults. Recently,<br />
over-expressing mitochondrial targeted catalase (MCAT) were found to exhibit reduced reactive<br />
oxygen species <strong>an</strong>d <strong>an</strong> extended lifesp<strong>an</strong> (Schriner et al., 2005). The purpose of our study was to<br />
better underst<strong>an</strong>d cellular ch<strong>an</strong>ges that extend <strong>the</strong> lifesp<strong>an</strong> of mice <strong>an</strong>d delay disease progression<br />
in amyloid precursor protein (APP) tr<strong>an</strong>sgenic mice.<br />
METHODS<br />
To determine whe<strong>the</strong>r MCAT reduces reactive oxygen species, mitochondrial toxicity, <strong>an</strong>d<br />
amyloid beta toxicity in AD, we crossed MCAT mice with <strong>the</strong> APP mice. Total RNA was<br />
isolated <strong>from</strong> cerebral cortex <strong>an</strong>d skeletal muscle tissues of 2-, 12-, 24-, <strong>an</strong>d 34-month-old<br />
MCAT mice, non-tr<strong>an</strong>sgenic wild-type (WT) mice (control), double tr<strong>an</strong>sgenic mice<br />
(MCATxAPP) <strong>an</strong>d APP mice (19 months). mRNA expressions were measured in <strong>the</strong> tissues<br />
using <strong>an</strong> Illumina mouse array (V6-chip) that contains 46,000 mouse tr<strong>an</strong>scripts. Gene<br />
expression data were <strong>an</strong>alyzed pair-wise using GenSifter software <strong>an</strong>d were verified using qRT-<br />
PCR, in situ hybridization, <strong>an</strong>d immunohistochemistry techniques.<br />
RESULTS<br />
Our pair-wise gene expression <strong>an</strong>alysis of 2-, 12-, 24-, <strong>an</strong>d 34-month-old MCAT mice <strong>an</strong>d agematched<br />
WT mice revealed that genes related to axon cargo tr<strong>an</strong>sport, vesicle mediated<br />
tr<strong>an</strong>sport, dopamine receptor signaling, mitochondrial fission, <strong>an</strong>d <strong>the</strong> APP catabolic pathway
were down-regulated. In contrast, genes related to <strong>the</strong> mRNA metabolic process,<br />
neuroprotection/neuronal remodeling, <strong>the</strong> uniquitin-dependent protein catabolic process, <strong>an</strong>d<br />
longevity were up-regulated in <strong>the</strong> MCAT mice relative to age-matched WT mice. Similar gene<br />
expression patterns were found in <strong>the</strong> skeletal muscle tissues. Interestingly, in double tr<strong>an</strong>sgenic<br />
mice (MCATxAPP), up-regulation was found in neuroprotection/neuronal remodeling, uniquitindependent<br />
protein catabolic process, <strong>an</strong>d longevity genes relative to <strong>the</strong> APP mice, suggesting<br />
that MCAT may reduce mut<strong>an</strong>t APP <strong>an</strong>d amyloid beta toxicity. Fur<strong>the</strong>r, our HPLC <strong>an</strong>alysis of<br />
oxidative DNA damage (8OHdG) in <strong>the</strong> cerebral cortex tissues <strong>from</strong> <strong>the</strong> 12-month-old MCAT<br />
mice, APP mice, MCATxAPP mice, <strong>an</strong>d age-matched WT mice revealed decreased levels of<br />
8OHdG in only <strong>the</strong> MCAT <strong>an</strong>d MCATxAPP mice, fur<strong>the</strong>r supporting our gene expression<br />
findings.<br />
CONCLUSION<br />
Our global gene expression <strong>an</strong>alysis of MCAT, APP, MCATxAPP, <strong>an</strong>d age-matched WT mice<br />
suggests that MCAT effectively protects neurons <strong>from</strong> toxicities related to aging <strong>an</strong>d amyloid<br />
beta.<br />
Disclosures: P.H. Reddy, None; P. Mao, None; M. M<strong>an</strong>czak, None; P. Rabinovitch,<br />
None; R. Searles, None; A.P. Reddy, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.1/I26<br />
Topic: C.02.o. Therapies<br />
Support: NIA R01 AG022462<br />
Title: Nicotine treatment of mild cognitive impairment (MCI): Efects of APOE4 genotype<br />
Authors: *H. L. WILKINS 1 , E. LEVIN 2 , E. CODERRE 1 , H. WHITE 2 , D. HOWARD 1 , P.<br />
AISEN 3 , K. KELLAR 3 , K. WESNES 4 , P. NEWHOUSE 1 ;<br />
1 Univ. Vermont, Burlington, VT; 2 Duke Univ., Durham, NC; 3 Georgetown Univ., Washington,<br />
DC; 4 Cognitive Drug Res. Ltd., Goring-on-Thames, United Kingdom<br />
Abstract: A strong genetic risk factor <strong>for</strong> late onset Alzheimer's disease (AD) <strong>an</strong>d mild<br />
cognitive impairment (MCI) is <strong>the</strong> presence of <strong>the</strong> E4 allele of <strong>the</strong> apolipoprotein E (APOE4)<br />
gene which encodes a protein involved in cholesterol metabolism <strong>an</strong>d contributes to disease<br />
pathogenesis by modulating <strong>the</strong> metabolism of Aβ peptide. APOE4 status has been variably
elated to treatment effects in AD <strong>an</strong>d MCI <strong>an</strong>d has been reported to interact with nicotinic<br />
receptor activation. Mild Cognitive Impairment (MCI) may be <strong>an</strong> appropriate target <strong>for</strong> nicotinic<br />
treatment <strong>an</strong>d thus we explored <strong>the</strong> effect of APOE genotype on <strong>the</strong> response to tr<strong>an</strong>sdermal<br />
treatment with nicotine in MCI patients. 74 non-smoking subjects <strong>from</strong> 3 sites who met criteria<br />
<strong>for</strong> amnestic MCI were r<strong>an</strong>domized to receive ei<strong>the</strong>r double-blind tr<strong>an</strong>sdermal nicotine (NIC) at<br />
15 mg/day or placebo patch <strong>for</strong> <strong>the</strong> first 6 months of <strong>the</strong> study <strong>an</strong>d open label nicotine patch <strong>for</strong><br />
<strong>the</strong> second 6 months of <strong>the</strong> study. Subjects were cognitively assessed at baseline, 91, <strong>an</strong>d 182<br />
days utilizing <strong>the</strong> Cognitive Drug Research (CDR) battery, Conners CPT task <strong>an</strong>d o<strong>the</strong>rs. 32 of<br />
72 subjects had at least one E4 allele. APOE4 genotype effects were seen on a CDR battery<br />
items including <strong>the</strong> power of attention composite measure (NIC x APOE, p=.048) with E4/E4<br />
double allele subjects showing a highly signific<strong>an</strong>t (p=.02) NIC treatment benefit compared to<br />
single allele E4/E3 or E3/E3 subjects. For <strong>the</strong> Digit Vigil<strong>an</strong>ce task, <strong>the</strong>re was a similar pattern<br />
with E4/E4 subjects showing a signific<strong>an</strong>t positive effect of nicotine compared to o<strong>the</strong>r<br />
genotypes on speed (p=.01) <strong>an</strong>d false alarms (p=.03). Signific<strong>an</strong>t nicotine treatment (p
Title: Nicotine treatment of mild cognitive impairment (MCI): A six-month multi-center pilot<br />
study<br />
Authors: *P. A. NEWHOUSE 1 , E. LEVIN 2 , H. WHITE 2 , E. CODERRE 1 , H. WILKINS 1 , K.<br />
KELLAR 3 , P. AISEN 3 , B. K. SAXBY 4 , K. WESNES 4 ;<br />
1 Clin. Neurosci Res. Unit, Univ. Vermont Col. Med., Burlington, VT; 2 Duke Univ., Durham,<br />
NC; 3 Georgetown Univ., Washington, DC; 4 Cognitive Drug Res. Ltd, Goring-on-Thames,<br />
United Kingdom<br />
Abstract: Interest in nicotinic treatment of Alzheimer’s disease (AD) developed following<br />
recognition of <strong>the</strong> loss of nicotinic receptors <strong>an</strong>d that short-term administration of nicotine<br />
improved cognitive per<strong>for</strong>m<strong>an</strong>ce in AD. Mild Cognitive Impairment (MCI) may also be <strong>an</strong><br />
appropriate target <strong>for</strong> nicotinic treatment as patients will have greater numbers of nicotinic<br />
receptors th<strong>an</strong> AD patients. We hypo<strong>the</strong>sized that nicotine treatment would improve cognitive<br />
per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d clinical ratings, <strong>an</strong>d that treatment would be safe <strong>an</strong>d well tolerated. 74 nonsmoking<br />
subjects <strong>from</strong> 3 sites who met criteria <strong>for</strong> amnestic MCI were r<strong>an</strong>domized to receive<br />
ei<strong>the</strong>r double-blind tr<strong>an</strong>sdermal nicotine (15 mg/day) or placebo patch <strong>for</strong> <strong>the</strong> first 6 months of<br />
<strong>the</strong> study <strong>an</strong>d open label nicotine patch <strong>for</strong> <strong>the</strong> second 6 months of <strong>the</strong> study. Subjects were<br />
cognitively assessed at baseline, 91, <strong>an</strong>d 182 days utilizing <strong>the</strong> Cognitive Drug Research battery,<br />
Conners continuous per<strong>for</strong>m<strong>an</strong>ce task (CPT), non-computer verbal <strong>an</strong>d visual tasks, <strong>an</strong>d clinical<br />
global impression of ch<strong>an</strong>ge (CGIC). For cognitive per<strong>for</strong>m<strong>an</strong>ce, <strong>an</strong>alysis showed that nicotine<br />
treatment produced signific<strong>an</strong>t (p
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.3/I28<br />
Topic: C.02.o. Therapies<br />
Support: ADRC gr<strong>an</strong>t P50AG025711<br />
Funds <strong>from</strong> <strong>the</strong> Byrd Institute<br />
Title: The <strong>the</strong>rapeutic effects of caffeine <strong>an</strong>d coffee in Alzheimer’s disease<br />
Authors: *C. CAO 1 , X. LIN 1 , L. WANG 2 , M. MAMCARZ 2 , M. RUNFELDT 1 , G. W.<br />
ARENDASH 2 ;<br />
1 Mol. Pharmacol. <strong>an</strong>d Physiol., 2 Biol., Univ. of South Florida, Tampa, FL<br />
Abstract: The premise that caffeine/coffee may provide a safe, effective, readily available, <strong>an</strong>d<br />
inexpensive <strong>the</strong>rapeutic against Alzheimer’s Disease (AD) is underscored by a growing body of<br />
epidemiologic literature showing protective effects against memory impairment <strong>an</strong>d AD. For<br />
example, mid-life coffee consumption has been linked to a 65% decreased risk of AD. Our own<br />
work in AD tr<strong>an</strong>sgenic mice has shown that caffeine both protects against <strong>an</strong>d reverses cognitive<br />
impairment by directly affecting Aβ pathogenesis. To determine <strong>the</strong> extent to which noncaffeinergic<br />
components of coffee may be involved in <strong>the</strong> cognitive benefits of coffee, we have<br />
conducted a long-term study involving three months daily treatment by gavage with regular<br />
coffee or decaffeinated coffee to APP/PS1 (Tg) mice. Both coffee <strong>an</strong>d decaffeinated coffee were<br />
prepared in <strong>the</strong> same way <strong>an</strong>d <strong>the</strong> caffeine concentration of each was measured be<strong>for</strong>e<br />
administrated. Behavior testing near <strong>the</strong> end of <strong>the</strong> treatment period revealed that coffee treated<br />
Tg mice had signific<strong>an</strong>tly improved working memory compared to control Tg mice. By contrast,<br />
<strong>the</strong> decaffeinated coffee treatment group of Tg mice exhibited no such beneficial effects. Plasma<br />
cytokine <strong>an</strong>alysis none<strong>the</strong>less indicated that both caffeine <strong>an</strong>d non-caffeinergic compounds in<br />
coffee c<strong>an</strong> provide beneficial effects <strong>an</strong>d act synergistically. We also <strong>an</strong>alyzed 208 hum<strong>an</strong><br />
plasma samples <strong>for</strong> caffeine levels <strong>from</strong> two clinical sites of <strong>the</strong> Florida Alzheimer’s Disease<br />
Research Center (119 <strong>from</strong> Miami <strong>an</strong>d 89 <strong>from</strong> Tampa), divided into AD, MCI <strong>an</strong>d normal aged<br />
individuals. Our result show that spont<strong>an</strong>eous caffeine levels in normal aged subject are<br />
signific<strong>an</strong>tly higher th<strong>an</strong> AD subjects at <strong>the</strong> Tampa site, but no signific<strong>an</strong>t difference compared<br />
with MCI, while caffeine levels in normal is higher th<strong>an</strong> MCI <strong>an</strong>d AD in subjects at <strong>the</strong> Miami<br />
site. Thus, our results <strong>from</strong> both <strong>an</strong>imal studies <strong>an</strong>d hum<strong>an</strong> habitual caffeine intake show a<br />
consistent association of caffeine with normal or maintained cognitive function. Additionally, in
oth AD Tg mice <strong>an</strong>d young adult/aged hum<strong>an</strong>s, we find that blood Aβ levels are modified <strong>for</strong><br />
hours following a single administration of caffeine or coffee. This is consistent with our previous<br />
data showing that caffeine c<strong>an</strong> quickly <strong>an</strong>d directly inhibit Aβ production. Our studies<br />
collectively suggest that mainten<strong>an</strong>ce of habitual coffee intake (around 5 cups per day)<br />
throughout adult hood is very import<strong>an</strong>t <strong>for</strong> prevention of AD development.<br />
Disclosures: C. Cao, None; X. Lin, None; L. W<strong>an</strong>g, None; M. Mamcarz, None; M. Runfeldt,<br />
None; G.W. Arendash, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.5/I29<br />
Topic: C.02.o. Therapies<br />
Title: Treatment of Alzheimer's model mice with AMPK-activating drug, met<strong>for</strong>min<br />
Authors: *K. A. DITACCHIO 1 , R. R. NOFSINGER 2 , G. DZIEWCZAPOLSKI 1 , S. F.<br />
HEINEMANN 1 ;<br />
1 Mol. Neurobio. Laboratory-Heinem<strong>an</strong>n, The Salk Insitute, La Jolla, CA; 2 Gene Expression<br />
Laboratory-Ev<strong>an</strong>s, The Salk Inst., La Jolla, CA<br />
Abstract: Epidemiological studies in hum<strong>an</strong> patients suggest that exercise may delay <strong>the</strong><br />
progression of Alzheimer’s disease (AD), while studies in <strong>an</strong>imals additionally support caloric<br />
restriction as protective. Both of <strong>the</strong>se interventions create a shortage of cellular energy whereby<br />
available ATP is converted to AMP <strong>an</strong>d <strong>the</strong> AMP-activated protein kinase (AMPK) is activated.<br />
While <strong>the</strong> role of AMPK activation has been well established under <strong>the</strong>se conditions, its function<br />
in <strong>the</strong> progression of AD has been neglected. To address this, we have used <strong>the</strong> AMPKactivating<br />
drug, met<strong>for</strong>min, in <strong>an</strong> attempt to pharmacologically mimic conditions that are thought<br />
to be protective in AD. Mice were treated <strong>for</strong> six months with met<strong>for</strong>min in <strong>the</strong>ir drinking water<br />
at 2mg/ml resulting in consumption of ~300mg/kg/day. Here we show surprising alteration to <strong>the</strong><br />
progression of Alzheimer’s-like phenotypes in APP tr<strong>an</strong>sgenic mice following this treatment<br />
with potentially import<strong>an</strong>t consequences <strong>for</strong> hum<strong>an</strong> patients.<br />
Disclosures: K.A. DiTacchio, None; R.R. Nofsinger, None; G. Dziewczapolski, None; S.F.<br />
Heinem<strong>an</strong>n, None.
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.6/I30<br />
Topic: C.02.o. Therapies<br />
Support: MIUR<br />
Title: The richness of <strong>the</strong> environment bidirectionally affects <strong>the</strong> progression of <strong>the</strong> disease in a<br />
mouse model of Alzheimer’s disease, <strong>the</strong> AD11 mice<br />
Authors: *A. POLI 1 , C. BRASCHI 2 , N. BERARDI 3,2 , S. CAPSONI 4 , A. CATTANEO 1 , L.<br />
MAFFEI 1,3 ;<br />
1 Lab. of Neurobiology, Scuola Normale Superiore, Pisa, Italy; 2 Dept. Psychology, Florence<br />
Univ., Florence, Italy; 3 Inst. of Neurosci. CNR, Pisa, Italy; 4 EBRI, Rome, Italy<br />
Abstract: Studies in <strong>an</strong>imal models of neurological diseases, including Alzheimer’s disease<br />
(AD), have recently suggested that EE could be a potential non invasive <strong>the</strong>rapeutic strategy. In<br />
particular, we have demonstrated that EE starting at two months of age, that is be<strong>for</strong>e <strong>the</strong> onset<br />
of cognitive deficits, completely prevents <strong>the</strong> onset of visual <strong>an</strong>d spatial memory deficits in<br />
AD11 mice, a comprehensive mouse model <strong>for</strong> AD, reducing Abeta burden <strong>an</strong>d rescuing <strong>the</strong><br />
cholinergic deficit. AD11 mice, which express <strong>an</strong>ti nerve growth factor (NGF) <strong>an</strong>tibodies,<br />
develop <strong>an</strong> age dependent neurodegeneration which encompasses all hallmarks of hum<strong>an</strong> AD.<br />
Exposing AD11 mice to EE after 7 months of age, that is after <strong>the</strong> onset of behavioural deficits<br />
<strong>an</strong>d <strong>the</strong> appear<strong>an</strong>ce of AD neuropathological hallmarks rescues visual memory deficits <strong>an</strong>d<br />
reduces <strong>the</strong> cholinergic deficits <strong>an</strong>d Abeta accumulation.<br />
If <strong>the</strong> richness of <strong>the</strong> environment has <strong>an</strong> impact on <strong>the</strong> disease progression, a faster cognitive<br />
decline might be found in <strong>an</strong>imal models of AD kept under “impoverished” conditions.<br />
To assess <strong>the</strong> effects of impoverishment we have exposed AD11 mice to isolated housing <strong>from</strong> 2<br />
to 7 months of age (I-AD11); <strong>an</strong>imals have been housed individually in cages with opaque walls<br />
<strong>an</strong>d with a roof filter to prevent exch<strong>an</strong>ge of odours. At <strong>the</strong> end of <strong>the</strong> isolation period, we have<br />
assessed per<strong>for</strong>m<strong>an</strong>ce in a spatial memory test (Morris water maze); Abeta burden was assessed<br />
immunohistochemically. As controls, 7 months old AD11 mice kept in st<strong>an</strong>dard cages were used.<br />
We found that I-AD11 mice per<strong>for</strong>med signific<strong>an</strong>tly worse th<strong>an</strong> control AD11 mice in <strong>the</strong> Morris<br />
water maze <strong>an</strong>d <strong>the</strong>y showed a signific<strong>an</strong>tly higher presence of Abeta clusters.<br />
These results have <strong>an</strong> immediate implication <strong>for</strong> hum<strong>an</strong> AD, since <strong>the</strong>y suggest that <strong>the</strong> level of<br />
environmental stimulation affects bidirectionally <strong>the</strong> progression of <strong>the</strong> disease.<br />
Disclosures: A. Poli, None; C. Braschi, None; N. Berardi, None; S. Capsoni, None; A.<br />
Catt<strong>an</strong>eo, None; L. Maffei, None.
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.7/I31<br />
Topic: C.02.o. Therapies<br />
Support: Alzheimer <strong>Society</strong> UK research gr<strong>an</strong>t<br />
Title: Novel GLP-1 <strong>an</strong>alogues improve memory <strong>for</strong>mation - A link between diabetes <strong>an</strong>d<br />
Alzheimer’s disease<br />
Authors: *S. F. MCGOVERN, P. MCCLEAN, V. A. GAULT, C. HOLSCHER;<br />
Sch. of Biomed. Sci., Ulster Univ., Coleraine, United Kingdom<br />
Abstract: Type 2 diabetes mellitus (T2DM) has been identified as a risk factor <strong>for</strong> developing<br />
Alzheimer’s disease (AD). Disturb<strong>an</strong>ces in insulin signaling is a common impairment shared by<br />
both AD <strong>an</strong>d T2DM, <strong>an</strong>d insulin receptors have been found to be desensitised in <strong>the</strong> brains of<br />
AD patients, which has been linked to <strong>the</strong> development of impairments in cognitive<br />
per<strong>for</strong>m<strong>an</strong>ce. In this study we test what effects of novel glucose-like peptide-1 (GLP-1)<br />
<strong>an</strong>alogues (that are protease resist<strong>an</strong>t) have on memory <strong>for</strong>mation. These <strong>an</strong>alogues have been<br />
developed as a treatment <strong>for</strong> T2DM <strong>an</strong>d have been shown to cross <strong>the</strong> blood brain barrier <strong>an</strong>d<br />
increase synaptic plasticity in <strong>the</strong> brain (see related posters at this meeting). The GLP-1 <strong>an</strong>alogue<br />
(Val8) GLP-1 has been tested in <strong>an</strong> open field test <strong>an</strong>d in memory tasks. Male C57/Bl6 mice had<br />
been injected ip. with (Val8)GLP-1 (25nmol/kg) or saline 30 min be<strong>for</strong>e training. In <strong>the</strong> open<br />
field test, <strong>the</strong>re was no difference between groups in exploration or <strong>an</strong>xiety parameters, but<br />
(Val8) GLP-1 showed increased speed (p
<strong>the</strong> novel GLP-1 <strong>an</strong>alogue Liraglutide. These results show that <strong>the</strong> GLP-1 <strong>an</strong>alogue (Val8) GLP-<br />
1 has supportive effects on memory <strong>for</strong>mation. Taken toge<strong>the</strong>r with <strong>the</strong> results that (Val8) GLP-1<br />
crosses <strong>the</strong> blood-brain barrier <strong>an</strong>d improves synaptic plasticity in a mouse model of Alzheimer’s<br />
disease, such protease resist<strong>an</strong>t GLP-1 <strong>an</strong>alogues show promise as a treatment <strong>for</strong><br />
neurodegenerative diseases such as AD.<br />
Disclosures: S.F. McGovern, None; P. McCle<strong>an</strong>, Alzheimer <strong>Society</strong> UK gr<strong>an</strong>t, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received); V.A. Gault, None; C. Holscher, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.8/I32<br />
Topic: C.02.o. Therapies<br />
Support: Alzheimer <strong>Society</strong> UK research gr<strong>an</strong>t<br />
Title: Novel GLP-1 <strong>an</strong>alogues cross <strong>the</strong> blood brain barrier: A link between diabetes <strong>an</strong>d<br />
Alzheimer’s disease<br />
Authors: *P. MCCLEAN, K. FUNG, R. MCCURTIN, V. A. GAULT, C. HOLSCHER;<br />
Ulster Univ., Coleraine, United Kingdom<br />
Abstract: Type 2 diabetes has been identified as a risk factor <strong>for</strong> developing Alzheimer’s<br />
disease. Disturb<strong>an</strong>ces in insulin signalling is a common impairment shared by both Alzheimer’s<br />
<strong>an</strong>d diabetes. The desensitisation of insulin receptors may play a role in <strong>the</strong> development of<br />
neurodegenerative disease, by disrupting insulin’s neuroprotective actions in brain. The incretin<br />
signalling pathway runs in parallel with <strong>the</strong> insulin signalling pathway, offering <strong>an</strong> alternative<br />
route to improve insulin sensitivity. Incretin receptors (GLP-1 <strong>an</strong>d GIP) are expressed in <strong>the</strong><br />
brain. Recently, <strong>the</strong> incretin glucagon-like peptide 1 (GLP-1) has been shown to have<br />
neuroprotective properties. Accordingly we tested whe<strong>the</strong>r novel GLP-1 <strong>an</strong>alogues that are<br />
resist<strong>an</strong>t to protease degradation <strong>an</strong>d are developed as a treatment <strong>for</strong> type 2 diabetes c<strong>an</strong> cross<br />
<strong>the</strong> blood brain barrier (BBB) <strong>an</strong>d <strong>the</strong>reby may have effects on neuronal communication <strong>an</strong>d<br />
synaptic plasticity (LTP) in <strong>the</strong> brain (see related poster on this by C. Hölscher et. al.). We tested<br />
two novel protease -resist<strong>an</strong>t GLP-1 <strong>an</strong>alogues, Liraglutide <strong>an</strong>d Val(8)GLP-1. In order to<br />
determine <strong>the</strong> peptides ability to cross <strong>the</strong> BBB, male mice were injected intraperitoneally (ip.)<br />
with a 25nmol/kg or 250nmol/kg dose of GLP-1, Val(8)GLP-1, or Liraglutide. Brains were
excised 30 minutes <strong>an</strong>d 3 hours after peptide administration <strong>an</strong>d <strong>an</strong>alysed using protein<br />
separation <strong>an</strong>d purification steps <strong>an</strong>d a RIA <strong>an</strong>alysis kit. Data were <strong>an</strong>alysed using 1-way<br />
ANOVAs or t-tests.<br />
Both Val(8)GLP-1 <strong>an</strong>d Liraglutide crossed <strong>the</strong> BBB. Injection of 25nmol/kg or 250 nmol/kg of<br />
ei<strong>the</strong>r peptide resulted in a signific<strong>an</strong>t increase in <strong>the</strong> brain 30 min or 3 h post-injection.<br />
Liraglutide 25nmol/kg at 30 min: p
<strong>an</strong>alogues were tested on 2 mouse models of diabetes: (i) obese diabetic (ob/ob) mice in which<br />
leptin syn<strong>the</strong>sis is impaired causing hyperphagia <strong>an</strong>d development of mild diabetes <strong>an</strong>d<br />
hyperinsulinaemia; <strong>an</strong>d (ii) db/db mice with leptin receptor deficiency resulting in reduced<br />
insulin levels <strong>an</strong>d severe diabetes. Animals were administered once-daily subcut<strong>an</strong>eous<br />
injections of exendin-4 or Liraglutide (each at 25 nmol/kg bw) or saline (0.9%(w/v) NaCl) over<br />
6-8 weeks. Animals were administered a single dose of BrdU (50 mg/kg bw) prior to tissue<br />
collection (24 hr). Cells positive <strong>for</strong> BrdU were counted throughout <strong>the</strong> DG in <strong>the</strong> gr<strong>an</strong>ule cell<br />
layer, <strong>an</strong>d differences between treatment groups <strong>an</strong>alysed using one-way ANOVA <strong>an</strong>d post-hoc<br />
tests. The number of BrdU positive cells in control ob/ob (40% increase, p
Authors: *C. HOLSCHER 1 , V. A. GAULT 1 , P. HARRIOTT 2 , S. GENGLER 1 ;<br />
1 2<br />
Sch. Biomed Sci., Univ. Ulster, Coleraine, United Kingdom; Sch. of Biol. <strong>an</strong>d Biochem,<br />
Queen's Univ., Belfast, United Kingdom<br />
Abstract: Type 2 diabetes is a known risk factor <strong>for</strong> Alzheimer’s disease (AD), with<br />
impairments in insulin signalling contributing to neurodegeneration. To treat diabetes, proteaseresist<strong>an</strong>t<br />
<strong>an</strong>alogues of Glucagon-like peptide-1 (GLP-1) have been developed. Import<strong>an</strong>tly, GLP-<br />
1 also plays a role in neuronal activity <strong>an</strong>d brain functions <strong>an</strong>d has been shown to have<br />
neuroprotective properties. We <strong>the</strong>re<strong>for</strong>e tested whe<strong>the</strong>r novel GLP-1 <strong>an</strong>alogues have effects on<br />
neuronal communication <strong>an</strong>d synaptic plasticity (LTP) in <strong>the</strong> brain, indicating that <strong>the</strong>y also<br />
could have positive effects on neurodegenerative diseases. GLP-1 <strong>an</strong>alogues with alterations at<br />
amino acid position 7, 8, or 9 had been developed. Additionally, glycation at position 7, or<br />
addition of fatty acids to <strong>the</strong> GLP-1 molecule to enh<strong>an</strong>ce <strong>the</strong> half life of <strong>the</strong> peptide have been<br />
engineered as novel <strong>an</strong>alogues. One of <strong>the</strong>se peptides is Liraglutide, which is about to be<br />
released onto <strong>the</strong> market as a treatment. Rats were injected with GLP-1 <strong>an</strong>alogues at a<br />
concentration of 15 nmol in 5µl i.c.v. Rats were <strong>an</strong>aes<strong>the</strong>tised with ureth<strong>an</strong>e <strong>an</strong>d electrodes<br />
lowered into <strong>the</strong> hippocampus area CA1 to record field excitatory field potentials (fEPSPs)<br />
elicited by high-frequency stimulation of <strong>the</strong> Schaffer collaterals. The agonists Liraglutide,<br />
asp(7)GLP-1, Val(8)GLP-1, N-glyc-GLP-1, <strong>an</strong>d Pro(9)GLP-1, <strong>an</strong>d <strong>the</strong> <strong>an</strong>tagonist exendin-4(9-<br />
36) were tested. The novel GLP-1 <strong>an</strong>alogue Liraglutide enh<strong>an</strong>ced LTP, a two-way ANOVA<br />
showed a difference between <strong>the</strong> drug group <strong>an</strong>d saline control (p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.11/I35<br />
Topic: C.02.o. Therapies<br />
Support: Alzheimer Research Trust UK project gr<strong>an</strong>t<br />
Title: Impairment of synaptic plasticity <strong>an</strong>d learning in GLP-1 receptor Knockout mice<br />
Authors: *E. FAIVRE 1 , T. ABBAS 2 , D. J. DRUCKER 3 , C. HOLSCHER 2 ;<br />
1 Ulster Univ., Portstewart, United Kingdom; 2 Ulster Univ., Coleraine, United Kingdom; 3 Dept.<br />
of Medicine, B<strong>an</strong>ting <strong>an</strong>d Best Diabetes Ctr., Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Type 2 diabetes mellitus has been identified as a risk factor <strong>for</strong> Alzheimer’s disease<br />
(AD). A desensitization of insulin receptors has been found in <strong>the</strong> brain of AD. Insulin acts as<br />
growth factor <strong>an</strong>d supports neuronal repair, dendritic sprouting <strong>an</strong>d differentiation. Glucagonlike<br />
peptide-1 (GLP1), <strong>an</strong> incretin hormone, normalizes insulin signaling by facilitating insulin<br />
release <strong>an</strong>d re-sensitising insulin receptors. Import<strong>an</strong>tly, GLP-1 acts as a growth factor <strong>an</strong>d<br />
possesses neuroprotective effects. We previously showed that stimulation of neuronal GLP1<br />
receptors plays <strong>an</strong> import<strong>an</strong>t role in regulating neuronal tr<strong>an</strong>smission <strong>an</strong>d synaptic plasticity, <strong>an</strong>d<br />
demonstrate that GLP1 <strong>an</strong>alogues protect synapses <strong>from</strong> <strong>the</strong> detrimental effects of beta-amyloid<br />
(see posters at this meeting). The aim of this study was to elucidate <strong>the</strong> specific role of GLP1<br />
receptors in synaptic plasticity <strong>an</strong>d cognitive processes in <strong>the</strong> GLP1 receptor Knockout (GLP-1R<br />
KO) mouse model. Male GLP-1R KO mice <strong>an</strong>d age-matched control Wild-type (WT) <strong>an</strong>imals<br />
with <strong>the</strong> same C57/Bl6 genetic background were used to assess exploratory behavior <strong>an</strong>d<br />
learning <strong>an</strong>d memory in Open-field, object recognition (ORT) <strong>an</strong>d location (OLT) task as well as<br />
spatial Morris water maze (MWM) task. A small increase in speed of movement in GLP-1R KO<br />
mice (p
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.12/I36<br />
Topic: C.02.o. Therapies<br />
Support: NIH NRSA<br />
AHAF # A20009063<br />
Title: CNS PPARγ mediates rosiglitazone reversal of cognitive deficits in <strong>the</strong> Alzheimer’s<br />
disease mouse model, Tg2576<br />
Authors: *J. RODRIGUEZ-RIVERA 1 , K. T. DINELEY 2 , L. DENNER 3 ;<br />
1 2 3<br />
Ctr. <strong>for</strong> Addiction Res., Univ. Texas Med. Br., Galveston, TX; Neurol., Univ. of Texas Med.<br />
Br., Galveston, TX<br />
Abstract: Clinical studies have provided evidence to suggest that treatment with rosiglitazone<br />
(RTZ), a peroxiosome proliferator-activated receptor gamma (PPARγ) agonist, may improve<br />
cognition in Alzheimer's disease (AD) patients. The mech<strong>an</strong>isms by which RTZ achieves <strong>the</strong>se<br />
effects are unknown. Our recent studies in <strong>the</strong> AD mouse model (Tg2576) have shown that RTZ<br />
reverses <strong>the</strong> associative learning deficit exhibited by <strong>the</strong>se <strong>an</strong>imals at 9 months of age, when<br />
Tg2576 first exhibit gluco-regulatory abnormalities. Intracerebroventricular (i.c.v.) injections<br />
with a selective irreversible <strong>an</strong>tagonist of PPAR γ (GW9662) blocked <strong>the</strong> cognitive-enh<strong>an</strong>cing<br />
effects of RTZ in Tg2576 mice, providing evidence that central PPAR γ contributes to cognition<br />
in Tg2576. As such, cognitive rescue by PPAR γ agonism <strong>an</strong>d its reversal with PPAR γ<br />
<strong>an</strong>tagonism provide a special opportunity to identify <strong>the</strong> connectivity between <strong>the</strong> biochemical<br />
<strong>an</strong>d behavioral events by distinguishing those associated with cognitive per<strong>for</strong>m<strong>an</strong>ce <strong>from</strong> those<br />
that are causally related. Thus, while m<strong>an</strong>y ch<strong>an</strong>ges have been described to occur after cognitive<br />
impairment, it has been extremely challenging, if not impossible, to determine which ch<strong>an</strong>ges are<br />
really import<strong>an</strong>t, but only correlative. In our studies, only a subset of molecular <strong>an</strong>d<br />
electrophysiological ch<strong>an</strong>ges affected by subsequent cognitive rescue will be c<strong>an</strong>didates <strong>for</strong><br />
playing a causal role. We c<strong>an</strong> fur<strong>the</strong>r focus this subset because only some will be subsequently<br />
affected by inhibition with PPAR γ <strong>an</strong>tagonism. Our approach is to take <strong>the</strong> entire set of<br />
biochemical <strong>an</strong>d electrophysiological mech<strong>an</strong>isms implicated in cognitive decline, focusing only<br />
on those affected by RTZ rescue, <strong>an</strong>d <strong>the</strong>n fur<strong>the</strong>r focusing to those affected by GW <strong>an</strong>tagonism.<br />
In order to underst<strong>an</strong>d how PPAR γ agonism reverses cognitive deficits in Tg2576, we have<br />
initiated studies in which we measure functional readouts of CNS PPAR γ activity including
DNA binding, recruitment of heterodimeric binding partners <strong>an</strong>d target gene expression in RTZtreated<br />
Tg2576 mice.<br />
Disclosures: J. Rodriguez-Rivera, None; K.T. Dineley, None; L. Denner, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.13/I37<br />
Topic: C.02.o. Therapies<br />
Title: Anti-oxidative stress effects of apomorphine mediated via activation of glutathione<br />
peroxidase<br />
Authors: *Y. OHYAGI, L. MA, N. SAKAE, K. MOTOMURA, E. HIMENO, N. SOEJIMA, J.-<br />
I. KIRA;<br />
Dept Neurol, Kyushu Univ., Fukuoka 812-8582, Jap<strong>an</strong><br />
Abstract: Oxidative stress contributes various neurodegenerative diseases such as Alzheimer’s<br />
disease (AD) <strong>an</strong>d Parkinson’s disease (PD). Apomorphine hydrochloride (APO) is a dopamine<br />
agonist being used <strong>for</strong> PD patients has a protective effect against oxidative stress; however, <strong>the</strong><br />
mech<strong>an</strong>ism behind this action is unclear. We have found remarkable efficacy <strong>for</strong> <strong>an</strong> AD mouse<br />
model, 3XTg (Himeno et al., in this meeting). In cultured neuroblastoma SH-SY5Y cells, APO<br />
treatment protected cells <strong>from</strong> oxidative stress induced by hydrogen peroxide (H2O2), but did not<br />
protect against toxicity of staurosporine, etoposide, thapsigargin <strong>an</strong>d MG132. We next<br />
investigated <strong>the</strong> actions of APO on intracellular redox systems such as <strong>the</strong> glutathione cycle <strong>an</strong>d<br />
catalase. Cell viability was decreased concomit<strong>an</strong>t with upregulation of p53 <strong>an</strong>d p21 protein<br />
expression when treated with H2O2. We <strong>the</strong>n treated cells with specific inhibitors <strong>for</strong> glutathione<br />
peroxidase (GPx), mercaptosuccinic acid (MCS); <strong>for</strong> glutathione reductase (GR), 1,3-bis (2chlorethyl)-1-nitrosourea<br />
(BCNU); <strong>an</strong>d catalase, 3-amino-1,2,4-triazole (ATZ). Treatments with<br />
MCS or BCNU, but not ATZ, signific<strong>an</strong>tly counteracted <strong>the</strong> protective effects of APO. However,<br />
<strong>the</strong> attenuation of APO effects by MCS <strong>an</strong>d BCNU was incomplete, indicating that <strong>the</strong> protective<br />
effects of APO may be mediated via multiple mech<strong>an</strong>isms. After treatment cells with H2O2,<br />
GPx1 protein <strong>an</strong>d activity were promptly decreased <strong>an</strong>d <strong>the</strong>n gradually recovered. Suppression of<br />
GPx1 protein expression <strong>an</strong>d activity was counteracted by APO treatment, suggesting <strong>the</strong><br />
specific upregulation of GPx1 by APO treatment. APO treatment activated GPx activity<br />
signific<strong>an</strong>tly, but did not increase GPx1 protein expression. While, GR <strong>an</strong>d catalase activity was<br />
not elevated by APO treatment. Treatment with a dopamine D4 <strong>an</strong>tagonist U-101958 did not
inhibit <strong>the</strong> protective action of APO. Thus, APO enh<strong>an</strong>ces intracellular <strong>an</strong>ti-oxid<strong>an</strong>t system by<br />
stimulating GPx activity through a dopamine D4 receptor-independent pathway. Anti-oxidative<br />
stress mech<strong>an</strong>ism of APO is a unique one <strong>for</strong> protecting neurons, <strong>an</strong>d APO may be a c<strong>an</strong>didate<br />
drug <strong>for</strong> various neurodegenerative diseases including AD.<br />
Disclosures: Y. Ohyagi, None; L. Ma, None; N. Sakae, None; K. Motomura, None; E.<br />
Himeno, None; N. Soejima, None; J. Kira, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.14/I38<br />
Topic: C.02.o. Therapies<br />
Support: Fumi Yamamura Memorial Foundation <strong>for</strong> Female Natural Scientists<br />
Title: CNS delivery of peripherally administered colivelin peptide: A time-domain optical<br />
imaging study<br />
Authors: *M. YAMADA, S. AISO, T. CHIBA;<br />
KEIO Univ. Schl Med., Tokyo, Jap<strong>an</strong><br />
Abstract: Peripherally administered colivelin (CLN) appears to be delivered to <strong>the</strong> central<br />
nervous system (CNS) because intraperitoneal (i.p.) injection of colivelin peptide suppresses<br />
Alzheimer's disease (AD)-related memory impairment. Direct evidence of CLN delivery,<br />
however, had not been provided. In this study, we show that i.p. injection of CLN (7 nmol)<br />
signific<strong>an</strong>tly suppressed spatial working memory impairment in Y-maze (YM) caused by 3quinuclidinyl<br />
benzilate. The memory-improving effect of s.c. injection of CLN at <strong>the</strong> same dose<br />
was equal to those of i.p. treatment. To examine <strong>the</strong> pharmacokinetics of CLN, we labeled CLN<br />
peptide with a near-infrared fluorescence dye, Alexa Fluor 680 (CLN-A680). We injected 7<br />
nmol of CLN-A680 via i.p. or s.c. <strong>an</strong>d observed <strong>the</strong> brain regions with <strong>an</strong> in vivo optical imager,<br />
Optix MX2. Optical imaging of CLN-A680 revealed subst<strong>an</strong>tial fluorescence intensity in CLN-<br />
A680-injected brain regions of both i.p.- <strong>an</strong>d s.c.-injected mice at 30 min after <strong>the</strong> injection.<br />
Thus, peripherally administered CLN was delivered to <strong>the</strong> CNS <strong>an</strong>d elicited a pharmacological<br />
effect. Time-domain optical imaging constitutes a novel method <strong>for</strong> pharmacokinetics.<br />
Disclosures: M. Yamada, None; S. Aiso, None; T. Chiba, None.
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.15/J1<br />
Topic: C.02.o. Therapies<br />
Support: Washington State funds<br />
Pacific Northwest Biotechnology funds<br />
Title: Development of orally active Angiotensin IV <strong>an</strong>alogs as <strong>an</strong>ti-dementia drugs<br />
Authors: J. W. HARDING, J. W. WRIGHT, C. C. BENOIST, *P. C. MEIGHAN, B. J.<br />
YAMAMOTO, G. A. WAYMAN, M. D. VARNUM;<br />
Washington State Univ., Pullm<strong>an</strong>, WA<br />
Abstract: Angiotensin IV (AngIV; VYIHPF) <strong>an</strong>alogs have long been recognized to possess procognitive,<br />
<strong>an</strong>ti-dementia properties. Studies with multiple dementia models, which employed<br />
Nle1-AngIV, a high affinity AT4 receptor agonist, demonstrated that its acute application could<br />
reverse spatial learning deficits that resulted <strong>from</strong>: 1) treatment with scopolamine; 2) kainic acid<br />
injections into <strong>the</strong> hippocampus; 3) per<strong>for</strong><strong>an</strong>t path cuts or, 4) ischemia resulting <strong>from</strong> a tr<strong>an</strong>sient<br />
four-vessel occlusion. Despite <strong>the</strong> promising behavioral impact of AT4 receptor peptide agonists<br />
in various dementia models, two major impediments precluded <strong>the</strong>ir development into clinically<br />
relev<strong>an</strong>t drugs; namely, <strong>the</strong>ir lack of metabolic stability <strong>an</strong>d <strong>the</strong>ir inability to penetrate <strong>the</strong> bloodbrain<br />
barrier (BBB). In <strong>the</strong> last two years we have engaged in a major syn<strong>the</strong>tic ef<strong>for</strong>t to produce<br />
agonists that are both stable <strong>an</strong>d permeable enough to enable <strong>an</strong> effective concentration of drug<br />
to reach <strong>the</strong> brain following oral administration. The initial data presented here describe our<br />
rational approach to <strong>the</strong> design of such drugs based on a Nle1-AngIV template. These syn<strong>the</strong>tic<br />
activities focused on reducing size, increasing hydrophobicity, <strong>an</strong>d limiting <strong>the</strong> hydrogenbonding<br />
potential of putative drugs while maintaining pro-cognitive activity. This ef<strong>for</strong>t has<br />
resulted in <strong>the</strong> generation of a library of hydrophobic, metabolically stable <strong>an</strong>alogs with<br />
impressive cognitive enh<strong>an</strong>cing activity, one of which, Dihexa, will be highlighted in this<br />
presentation. Orally applied Dihexa was found to reverse spatial learning deficits produced ei<strong>the</strong>r<br />
acutely with scopolamine application or as a result of long-term accumulation of biochemical<br />
<strong>an</strong>d structural damage evident in aged rats. Mech<strong>an</strong>istic studies determined that <strong>the</strong> Dihexa was<br />
both a potent enh<strong>an</strong>cer of LTP in <strong>the</strong> hippocampus as well a spectacular inducer of dendrite<br />
arborization, dendritic spine growth, <strong>an</strong>d spine head enlargement in cultured hippocampal<br />
neurons. These data are consistent with a mode of action <strong>for</strong> Dihexa that involves <strong>an</strong> exp<strong>an</strong>sion
of connectivity among neurons resulting in improved synaptic communication. Fur<strong>the</strong>rmore,<br />
<strong>the</strong>se studies suggest that Dihexa <strong>an</strong>d related compounds may have general <strong>the</strong>rapeutic utility in<br />
neurodegenerative disease characterized by neuronal death <strong>an</strong>d/or a loss of neuronal<br />
connectivity.<br />
Disclosures: J.W. Harding, Pacific Northwest Biotechnology, LLC, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); J.W. Wright, Pacific Northwest<br />
Biotechnology, LLC, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); C.C. Benoist, None; P.C. Meigh<strong>an</strong>, Pacific Northwest Biotechnology, LLC, C. O<strong>the</strong>r<br />
Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); Pacific<br />
Northwest Biotechnology, LLC, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); B.J. Yamamoto, Pacific Northwest Biotechnology, LLC, E. Ownership<br />
Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); G.A. Waym<strong>an</strong>, None; M.D.<br />
Varnum, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.16/J2<br />
Topic: C.02.o. Therapies<br />
Support: NIH Gr<strong>an</strong>t R01 AG031311<br />
NIH Gr<strong>an</strong>t R01 NS056051<br />
Title: Medicinal chemistry refinement of a proof-of-concept lead compound into a CNS clinical<br />
c<strong>an</strong>didate that targets <strong>the</strong> protein kinase p38α MAPK<br />
Authors: S. M. ROY 1 , L. K. CHICO 1,3 , A. S. BORDERS 1 , *L. J. VAN ELDIK 2 , D. M.<br />
WATTERSON 1 ;<br />
1 Feinberg Sch. of Med., 2 Northwestern Univ., Chicago, IL; 3 InnovatePharma, Chicago, IL<br />
Abstract: Protein kinases represent a newer drug target class that has seen growing success,<br />
especially in oncology indications after <strong>the</strong> Gleevec precedent. Targeting kinases <strong>for</strong> CNS<br />
disorders has received less attention, in part due to <strong>the</strong> combination of challenges associated with<br />
targeting protein kinases <strong>an</strong>d <strong>the</strong> unique challenges associated with CNS drug discovery. In this<br />
regard, p38 MAPKs are attractive because <strong>the</strong>y are established <strong>the</strong>rapeutic targets <strong>for</strong> peripheral<br />
tissue disorders, <strong>an</strong>d are key signal tr<strong>an</strong>sduction regulators of cellular stress or injury responses.
Data <strong>from</strong> cell culture studies, post-mortem pathology, <strong>an</strong>d <strong>an</strong>imal physiology investigations<br />
raise <strong>the</strong> hypo<strong>the</strong>sis that p38α MAPK may be a viable CNS discovery target through its<br />
involvement in injury-induced neuronal dysfunction <strong>an</strong>d excessive, injurious proinflammatory<br />
cytokine production by glia. For example, p38α MAPK regulates <strong>the</strong> increased, stressor-induced<br />
cytokine production by microglia via its key role in <strong>the</strong> intracellular pathways that converge on<br />
control of cytokine protein biosyn<strong>the</strong>sis. However, a major roadblock to progress in testing if<br />
p38α MAPK is a druggable CNS target, <strong>an</strong>d generating viable starting points <strong>for</strong> drug<br />
development campaigns, has been <strong>the</strong> lack of bioavailable, CNS-penetr<strong>an</strong>t, efficacious p38<br />
MAPK inhibitors. We recently reported a proof-of-concept study (J Neuroinfl 4:21 (2007)) in<br />
which a novel, orally bioavailable, CNS-penetr<strong>an</strong>t, selective small molecule p38α MAPK<br />
inhibitor was shown to be efficacious in <strong>an</strong> <strong>an</strong>imal model of hum<strong>an</strong> Aβ-induced injury <strong>an</strong>d<br />
behavioral deficits. Oral administration of <strong>the</strong> inhibitor attenuated hippocampal proinflammatory<br />
cytokine overproduction <strong>an</strong>d improved synaptic dysfunction <strong>an</strong>d behavioral endpoints. These<br />
data provided <strong>an</strong> initial in vivo causative linkage between brain p38 MAPK activation, increased<br />
proinflammatory cytokines, <strong>an</strong>d synaptic dysfunction. A recursive medicinal chemistry<br />
refinement campaign is underway to develop a clinical c<strong>an</strong>didate based on this efficacious lead<br />
compound. The focus is on retention of selective target activity <strong>an</strong>d efficacy while improving<br />
ADMET properties <strong>an</strong>d <strong>the</strong>rapeutic index, or lack of observable adverse effects at high doses<br />
compared to lower efficacious doses. Poor <strong>the</strong>rapeutic index has been a problem in development<br />
of p38-targeted drugs <strong>for</strong> peripheral tissue disorders, with adverse pharmacology being<br />
correlated with <strong>the</strong> specifics of <strong>the</strong> small molecule inhibitor. Potential project deliverables<br />
include a possible new class of disease-modifying <strong>the</strong>rapies <strong>for</strong> CNS disorders as well as insight<br />
into desired vs undesired pharmacology of small molecule p38α MAPK inhibitor drugs.<br />
Disclosures: S.M. Roy, None; L.K. Chico, None; A.S. Borders, None; L.J. V<strong>an</strong> Eldik,<br />
None; D.M. Watterson, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.17/J3<br />
Topic: C.02.o. Therapies<br />
Support: NIH Gr<strong>an</strong>t U01 AG028561<br />
NIH Gr<strong>an</strong>t R01 AG031311
Title: Addressing <strong>the</strong> challenge of small molecule CNS drug discovery: Database mining <strong>an</strong>d<br />
case studies reveal key multi-property considerations <strong>for</strong> success<br />
Authors: L. K. CHICO 1,2 , W. HU 3 , H. A. BEHANNA 1 , S. M. ROY 1 , L. J. VAN ELDIK 1 , *D.<br />
WATTERSON 1 ;<br />
1 Feinberg Sch. of Med., Northwestern Univ., Chicago, IL; 2 InnovatePharma, Chicago, IL;<br />
3 Gu<strong>an</strong>gzhou Inst. of Biomedicine <strong>an</strong>d Hlth., Gu<strong>an</strong>gzhou, China<br />
Abstract: Successful drug delivery across <strong>the</strong> blood-brain barrier (BBB) represents a major<br />
hurdle in <strong>the</strong> development of CNS <strong>the</strong>rapeutics. The magnitude of <strong>the</strong> problem is exemplified by<br />
estimates that 98% of small molecule drugs fail to cross <strong>the</strong> BBB. Cytochrome P450 (CYP)mediated<br />
metabolism fur<strong>the</strong>r limits brain uptake <strong>an</strong>d drug efficacy by reducing drug<br />
bioavailability, as well as potentially compromising safety. The underlying influence of small<br />
molecule properties on <strong>the</strong>se biological phenomena represents critical features affecting both<br />
pharmacokinetics <strong>an</strong>d pharmacodynamics across discovery plat<strong>for</strong>ms. The overall hypo<strong>the</strong>sis<br />
addressed by this research is that <strong>an</strong>alyses of small molecule properties c<strong>an</strong> generate project<br />
m<strong>an</strong>agement tools in CNS-focused drug discovery campaigns. First, we created <strong>an</strong>d <strong>an</strong>alyzed<br />
molecular properties trends in a new small molecule CYP2D6 substrate database. Metabolism by<br />
CYP2D6 is disproportionately high <strong>for</strong> CNS drugs compared to o<strong>the</strong>r marketed drugs, making it<br />
<strong>an</strong> import<strong>an</strong>t consideration in CNS drug discovery. Second, <strong>the</strong> database construction <strong>an</strong>d<br />
in<strong>for</strong>matics studies were complemented by experimental investigations of <strong>the</strong> molecular<br />
properties <strong>an</strong>d molecular basis <strong>for</strong> differential CYP2D6 substrate status between two structurally<br />
related CNS <strong>the</strong>rapeutics. Third, we assembled a robustly parsed small molecule database of<br />
CNS-penetr<strong>an</strong>t compounds <strong>an</strong>d <strong>an</strong>alyzed molecular properties trends within this new database.<br />
The combination of trends <strong>from</strong> <strong>the</strong> unique databases <strong>an</strong>d experimental results <strong>from</strong> discrete case<br />
studies provides insight into prior successes <strong>an</strong>d failures as well as a foundation <strong>for</strong> <strong>the</strong><br />
development of <strong>for</strong>ecasting tools <strong>for</strong> use in future CNS drug discovery campaigns.<br />
Disclosures: L.K. Chico, None; W. Hu, None; H.A. Beh<strong>an</strong>na, None; S.M. Roy, None; L.J.<br />
V<strong>an</strong> Eldik, None; D. Watterson, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.18/J4<br />
Topic: C.02.o. Therapies<br />
Support: NIH Gr<strong>an</strong>t UO1 AG028561
NIH Gr<strong>an</strong>t R01 NS056051<br />
Title: An update on <strong>the</strong> Minozac family of small molecule compounds: A potential CNS diseasemodifying<br />
<strong>the</strong>rapeutic plat<strong>for</strong>m that selectively attenuates injury-induced glia proinflammatory<br />
cytokine up-regulation with improvement in neurologic outcomes<br />
Authors: *B. DESAI 1 , S. M. ROY 1 , L. K. CHICO 1,2 , W. HU 3 , H. BEHANNA 1 , L. J. VAN<br />
ELDIK 1 , D. M. WATTERSON 1 ;<br />
1 Feinberg Sch. of Med., Northwestern Univ., Chicago, IL; 2 InnovatePharma, Chicago, IL;<br />
3 Gu<strong>an</strong>gzhou Inst. of Biomedicine <strong>an</strong>d Hlth., Gu<strong>an</strong>gzhou, China<br />
Abstract: Overproduction of proinflammatory cytokines <strong>from</strong> activated glia in <strong>the</strong> CNS has<br />
been implicated as <strong>an</strong> import<strong>an</strong>t contributor to pathophysiology progression in neurodegenerative<br />
diseases, based on <strong>the</strong> ability of high levels of cytokines to induce synaptic dysfunction <strong>an</strong>d<br />
neuronal damage. This raises <strong>the</strong> possibility that restoring homeostasis by attenuating excessive<br />
proinflammatory cytokine production back towards basal, without p<strong>an</strong>-suppression of glia<br />
responses, might be one approach to development of disease-modifying <strong>the</strong>rapies <strong>for</strong> CNS<br />
disorders. Towards this goal, we established <strong>an</strong>d validated several years ago a de novo, novel<br />
compound discovery engine <strong>for</strong> CNS drug discovery. For <strong>the</strong> Minozac family of compounds, <strong>the</strong><br />
project started with <strong>an</strong> inactive pyridazine molecular fragment that was subjected to focused<br />
chemical diversifications. The project was driven by a series of decision gates derived <strong>from</strong> a<br />
knowledgebase of molecular features that correlate with a higher probability of a small molecule<br />
to be orally bioavailable, brain-penetr<strong>an</strong>t, <strong>an</strong>d lacking in fr<strong>an</strong>k adverse pharmacology <strong>an</strong>d<br />
toxicology. The diversification chemistry relied heavily on facile, high yield syn<strong>the</strong>tic reactions<br />
<strong>an</strong>d a common precursor in which restricted chemical space could be sampled. The approach<br />
yielded a focused set of compounds with high potential <strong>for</strong> favorable ADMET, <strong>an</strong>d rapid<br />
movement directly to in vivo efficacy screens in <strong>an</strong>imal models. The Minozac family campaign<br />
yielded <strong>an</strong> efficacious lead compound (J Neurosci 26:662 (2006)), which was subjected to<br />
medicinal chemistry refinement to improve molecular properties <strong>an</strong>d ADMET, yielding<br />
Minozac, a water-soluble, novel drug c<strong>an</strong>didate (BMCL17:414 (2007)). The Minozac family of<br />
compounds are efficacious in <strong>an</strong>imal models of diverse CNS disorders that have in common<br />
proinflammatory cytokine overproduction as a component of disease progression. These include<br />
models of Alzheimer’s disease, traumatic brain injury, seizures, <strong>an</strong>d <strong>the</strong> EAE model <strong>for</strong> multiple<br />
sclerosis. A syn<strong>the</strong>tic chemistry production scheme <strong>for</strong> large-scale production under FDA GMP<br />
production constraints was developed, allowing rapid production of clinical material at <strong>the</strong> multikg<br />
level. Continuing medicinal chemistry refinement has focused on generation of several<br />
closely related Minozacs that vary in pharmacokinetic properties with retention of CNS<br />
pharmacodynamics (efficacy). This is in <strong>an</strong>ticipation of <strong>the</strong> need to pursue different disease<br />
indications <strong>an</strong>d administration paradigms. We report a summary of progress <strong>an</strong>d <strong>the</strong> first report<br />
of a Minozac with extended bioavailability properties with retention of in vivo efficacy.<br />
Disclosures: B. Desai, None; S.M. Roy, None; L.K. Chico, None; W. Hu, None; H. Beh<strong>an</strong>na,<br />
None; L.J. V<strong>an</strong> Eldik, None; D.M. Watterson, None.
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.19/J5<br />
Topic: C.02.o. Therapies<br />
Title: Apomorphine treatment <strong>for</strong> Alzheimer’s disease: A novel approach<br />
Authors: E. HIMENO, Y. OHYAGI, N. SAKAE, K. MOTOMURA, L. MA, N. SOEJIMA,<br />
*J.-I. KIRA;<br />
Dept of Neurology, Kyushu Univ., Fukuoka City, Jap<strong>an</strong><br />
Abstract: It is well known that amyloid β pratein (Aβ) is neurotoxic <strong>an</strong>d deeply involved in <strong>the</strong><br />
pathogenesis of Alzheimer’s disease (AD). Various pathogeneses of intracellular Aβ such as<br />
mitochondrial damage, proteasomal damage, synaptic damage <strong>an</strong>d promotion of p53 cascade<br />
have been suggested. We found a c<strong>an</strong>didate drug, apomorphine (APO), to promote degradation<br />
of intracellular Aβ. Also, we disclosed a unique <strong>an</strong>ti-oxidative mech<strong>an</strong>ism of APO to protect<br />
neurons (Ohyagi et al., in this meeting). To investigate efficacy of APO <strong>for</strong> AD, we treated <strong>an</strong><br />
AD mouse model, 3XTg (APP-KM670/671NL, Tau-P301L, PS1-M146V), with APO <strong>an</strong>d<br />
checked cognitive function <strong>an</strong>d AD-related pathologies. Since memory impairment <strong>an</strong>d<br />
intr<strong>an</strong>euronal Aβ42 accumulation are observed at 6-month-old 3XTg, we injected APO at 0, 5 or<br />
10 mg/kg to 6-month-old heterozygote (h-3XTg) <strong>an</strong>d homozygote (H-3XTg) subcut<strong>an</strong>eously<br />
once a week <strong>for</strong> 1 month (total 4 injections until 7-month-old), <strong>an</strong>d evaluated memory function<br />
using Morris water maze <strong>an</strong>alysis (MWM) be<strong>for</strong>e/after treatment <strong>an</strong>d AD pathologies by<br />
immunocytochemistry <strong>an</strong>d western blotting of Aβ <strong>an</strong>d phosphorylated tau protein (p-tau). In both<br />
h-3XTg (n=4) <strong>an</strong>d H-3XTg (n=8), APO treatment signific<strong>an</strong>tly improved learning function<br />
(latencies to reach <strong>the</strong> goal) <strong>an</strong>d memory function (exploring patterns 24 or 48 hours after<br />
learning <strong>the</strong> goal location) on MWM. After evaluation by MWM, those mice brains were<br />
collected <strong>an</strong>d used <strong>for</strong> pathological <strong>an</strong>alysis. Both immunocytochemistry <strong>an</strong>d western blotting<br />
demonstrated decrease in Aβ42 <strong>an</strong>d p-tau protein levels signific<strong>an</strong>tly. All <strong>the</strong>se results indicated<br />
efficacy of APO to attenuate or even recover <strong>the</strong> AD progression. Interestingly, <strong>the</strong> effect of<br />
APO treatment in 5 mg/kg APO-injected group was more signific<strong>an</strong>t th<strong>an</strong> that in 10 mg/kg APOinjected<br />
groups. Moreover, intr<strong>an</strong>euronal Aβ-clear<strong>an</strong>ce effects of APO was not so clearly<br />
correlated with cognitive improvement, suggesting that APO effects on cognitive improvement<br />
may not be only due to intr<strong>an</strong>euronal Aβ clear<strong>an</strong>ce. Since APO is currently injected <strong>for</strong> patients<br />
with Parkinson’s disease at off period <strong>an</strong>d safety is approved, APO would become a powerful<br />
c<strong>an</strong>didate drug <strong>for</strong> AD in <strong>the</strong> near future.<br />
Disclosures: E. Himeno, None; Y. Ohyagi, None; N. Sakae, None; K. Motomura, None; L.<br />
Ma, None; N. Soejima, None; J. Kira, None.
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.20/J6<br />
Topic: C.02.o. Therapies<br />
Support: JSPS-20599017<br />
Fumi Yamamura Memorial Foundation <strong>for</strong> Female Natural Scientists (2008-<strong>2009</strong>)<br />
Title: [Gly(14)]-Hum<strong>an</strong>in suppresses memory impairment in ovariectomized mice by<br />
upregulating <strong>the</strong> local estrogen biosyn<strong>the</strong>sis<br />
Authors: *T. CHIBA 1 , M. YAMADA 2 , S. AISO 2 ;<br />
1 2<br />
Anat., Keio Univ, Schl Med., Tokyo, Jap<strong>an</strong>; Dept. of Anat., KEIO University, Sch. of Med.,<br />
Tokyo, Jap<strong>an</strong><br />
Abstract: Estrogen insufficiency after menopause has been implicated in <strong>the</strong> increased risk of<br />
Alzheimer's disease (AD) in females. Consistently, ovariectomy (OVX) has been reported not<br />
only to impair cognitive function in both rodents <strong>an</strong>d primates but also to accelerate AD-like<br />
pathology in murine AD models. Hum<strong>an</strong>in (HN), a short peptide identified <strong>from</strong> <strong>an</strong> occipital<br />
lobe of <strong>an</strong> AD patient, suppresses memory impairment related to AD. In this study, we examined<br />
<strong>the</strong> effect of <strong>an</strong> HN derivative [Gly(14)]-HN (HNG) on memory impairment induced by OVX.<br />
HNG ameliorated memory impairment induced by OVX without increasing serum estrogen<br />
levels. Multi-spot ELISA assay revealed that phospho- (p-) MEK, p-ERK <strong>an</strong>d p-STAT3 were<br />
upregulated in <strong>the</strong> hippocampal lysates of OVX mice with HNG treatment. Real-time q-PCR<br />
<strong>an</strong>alyses fur<strong>the</strong>r revealed that HNG upregulated genes related to <strong>the</strong> local estrogen biosyn<strong>the</strong>sis<br />
such as aromatase (CYP19a1) <strong>an</strong>d CYP17a1 in hippocampal neurons presumably through p-<br />
STAT3-mediated tr<strong>an</strong>scriptional activation. This provides a novel insight into <strong>the</strong>rapy <strong>for</strong><br />
postmenopausal cognitive decline <strong>an</strong>d AD.<br />
Disclosures: T. Chiba, None; M. Yamada, None; S. Aiso, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.21/J7<br />
Topic: C.02.o. Therapies<br />
Support: NIH R01ES014826<br />
COBRE 5P20RR017699<br />
Title: Leptin signaling in <strong>the</strong> cholesterol-fed rabbit, a model system of Alzheimer’s disease<br />
Authors: *G. A. MARWARHA, J. SCHOMMER, O. GHRIBI;<br />
Pharmacology, Physiol. & Therapeut., Univ. of North Dakota, Gr<strong>an</strong>d Forks, ND<br />
Abstract: Alzheimer’s disease (AD) is characterized by <strong>the</strong> deposition of extracellular beta<br />
amyloid (Aß) plaques <strong>an</strong>d intracellular tau protein neurofibrillary t<strong>an</strong>gles. Elevated cholesterol<br />
levels increase Aβ in cellular <strong>an</strong>d <strong>an</strong>imal models of AD, <strong>an</strong>d drugs that reduce cholesterol levels<br />
lower Aβ in <strong>the</strong>se models. 27-hydroxycholesterol (27-OHC), <strong>an</strong> oxysterol metabolite of<br />
cholesterol, is also able to produce AD hallmarks in org<strong>an</strong>otypic hippocampal slices <strong>an</strong>d cultured<br />
differentiated SH-SY5Y cells. Leptin is a 16 KDa adipocytokine necessary <strong>for</strong> cell survival in<br />
<strong>the</strong> hippocampus <strong>an</strong>d is involved in learning <strong>an</strong>d spatial memory. Leptin has also been shown to<br />
decrease Tau hyperphosphorylation <strong>an</strong>d to correct <strong>the</strong> dysregulation in glucose metabolism, <strong>an</strong><br />
event that is involved in <strong>the</strong> pathophysiology of AD. However, <strong>the</strong> effects of Leptin on Aβ<br />
generation <strong>an</strong>d Tau hyperphsophorylation induced by high cholesterol are yet to be determined.<br />
The aim of our study was to elucidate <strong>the</strong> role that Leptin signaling plays in<br />
hypercholesterolemia induced AD-like pathology. In this study we determined <strong>the</strong> effect of<br />
hypercholesterolemia <strong>an</strong>d 27-OHC on Leptin expression <strong>an</strong>d <strong>the</strong> extent to which Leptin<br />
treatment affects cholesterol - induced AD-like pathology. Fur<strong>the</strong>rmore, we determined <strong>the</strong><br />
molecular pathway involved in Leptin regulatory effects on Aβ accumulation, Tau<br />
phosphorylation <strong>an</strong>d cell death<br />
Our results show that hypercholesterolemia <strong>an</strong>d treatment with 27-OHC reduce <strong>the</strong> expression<br />
levels of Leptin. Leptin treatment of org<strong>an</strong>otypic slices decreases <strong>the</strong> 27-OHC - induced increase<br />
in Aβ, p-Tau <strong>an</strong>d pro-apoptotic protein levels. Our results indicate that decreased Leptin levels<br />
<strong>an</strong>d expression may underlie <strong>the</strong> mech<strong>an</strong>ism by which cholesterol <strong>an</strong>d its metabolite 27-OHC<br />
induce AD-like pathology.<br />
Disclosures: G.A. Marwarha, None; J. Schommer, None; O. Ghribi, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.22/J8<br />
Topic: C.02.o. Therapies<br />
Title: The identification of CX1837 a potent positive AMPA receptor modulator with potential<br />
use <strong>for</strong> treatment of neurological diseases<br />
Authors: L. J. STREET, *S. ZHONG, R. MUELLER, S. RACHWAL, S. JOHNSON, P.<br />
HAROLDSEN, T. HERBST, J. ULAS, L. YONG, A. HAMPSON, M. VARNEY;<br />
Cortex Pharmaceuticals Inc, Irvine, CA<br />
Abstract: The AMPA receptor is a tetrameric tr<strong>an</strong>smembr<strong>an</strong>e protein structure consisting of four<br />
closely related subunits, termed GluR 1-4, each of which exists in two different alternately<br />
spliced <strong>for</strong>ms, termed flip or flop. All AMPA receptor subunits have a common allosteric<br />
modulatory binding site, which has been well characterized <strong>an</strong>d is often called <strong>the</strong> ‘cyclothiazide<br />
site’. Ampakine ® compounds are positive allosteric modulators of <strong>the</strong> AMPA-type glutamate<br />
receptor <strong>an</strong>d enh<strong>an</strong>ce synaptic activity mediated by <strong>the</strong> AMPA-type glutamate receptor, which is<br />
<strong>the</strong> most prevalent excitatory neurotr<strong>an</strong>smitter receptor in <strong>the</strong> central nervous system. This leads<br />
to enh<strong>an</strong>ced communication between neurons, enh<strong>an</strong>ced synaptic plasticity underlying learning<br />
<strong>an</strong>d memory, <strong>an</strong>d also leads to <strong>the</strong> use-dependent upregulation of neurotrophic factors, such as<br />
brain-derived neurotrophic factor (BDNF). Normal function of <strong>the</strong> brain is dependent on<br />
sufficient <strong>an</strong>d coordinated synaptic activity in <strong>the</strong> vast, interconnected neuronal networks that<br />
principally consist of excitatory <strong>an</strong>d inhibitory neurons. Imbal<strong>an</strong>ces in synaptic activity between<br />
or within key brain regions often produce <strong>the</strong> symptoms defining neurologic <strong>an</strong>d psychiatric<br />
diseases. This c<strong>an</strong> result <strong>from</strong> synapse or neuron loss due to neurodegenerative disease, or <strong>from</strong><br />
synaptic defects in a particular brain region, leading to <strong>an</strong> imbal<strong>an</strong>ce. For example, cognitive<br />
deficits <strong>an</strong>d severe memory impairments in Alzheimer’s disease are due primarily to a loss of<br />
synapses <strong>an</strong>d neurons in hippocampus, entorhinal cortex <strong>an</strong>d temporal cortex, key brain regions<br />
<strong>for</strong> intellectual function.<br />
The objectives of <strong>the</strong> Cortex Ampakine program are to identify potent orally bioavailable<br />
compounds with good activity in behavioral cognition models <strong>an</strong>d suitable saftey margin <strong>for</strong><br />
hum<strong>an</strong> POC studies. The lead compound <strong>from</strong> <strong>the</strong> program, CX1837, meets <strong>the</strong> criteria we set<br />
<strong>an</strong>d has good in vitro <strong>an</strong>d in vivo activity in hippocampal electrophysiology <strong>an</strong>d excellent oral<br />
bioavailability in rat <strong>an</strong>d monkey. The effect of CX1837 was examined on hippocampal LTP<br />
measured in <strong>an</strong> <strong>an</strong>es<strong>the</strong>tized rat in vivo. CX1837 at 1.0 mg/kg i.p. signific<strong>an</strong>tly facilitated <strong>the</strong><br />
amplitude of evoked potentials after tet<strong>an</strong>ic stimulation. In behavioral cognition models CX1837<br />
has activity down to 0.01 mg/kg i.p. Based on this data CX1837 has been targeted <strong>for</strong> <strong>the</strong><br />
treatment of hum<strong>an</strong> neurological diseases. The identification <strong>an</strong>d full behavioral profile of <strong>the</strong><br />
AMPAKINE CX1837 will be presented.
Disclosures: L.J. Street, None; S. Zhong , None; R. Mueller, None; S. Rachwal, None; S.<br />
Johnson, None; P. Haroldsen, None; T. Herbst, None; J. Ulas, None; L. Yong, None; A.<br />
Hampson, None; M. Varney, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.23/J9<br />
Topic: C.02.o. Therapies<br />
Support: AG02219 to GMP<br />
Title: Deep brain stimulation as a potential <strong>the</strong>rapeutic strategy to enh<strong>an</strong>ce cognitive function in<br />
a mouse model of Alzheimer's disease<br />
Authors: *I. ARRIETA-CRUZ 1 , C. PAVLIDES 2 , G. M. PASINETTI 1 ;<br />
1 Psychiatry, Mount Sinai Sch. Med., New York, NY; 2 The Rockefeller Univ., New York, NY<br />
Abstract: There is evidence suggesting that deep brain stimulation (DBS) promotes certain<br />
cognitive processes raising <strong>the</strong> interest in developing DBS as a me<strong>an</strong>s of mitigating cognitive<br />
impairments in neurodegenerative disorders. DBS is known to influence motor <strong>an</strong>d limbic<br />
circuits, <strong>the</strong>re<strong>for</strong>e, it may be possible to apply electrical stimulation to modulate memory<br />
function through <strong>the</strong> activation of excitatory <strong>an</strong>d inhibitory circuits. This will also allow us to<br />
gain a better underst<strong>an</strong>ding of <strong>the</strong> neural substrates of memory <strong>an</strong>d its possible <strong>the</strong>rapeutic role in<br />
neurodegenerative disease with cognitive impairment such as Alzheimer’s disease (AD).<br />
Considerable evidence indicates that electrical stimulation of <strong>the</strong> <strong>an</strong>terior nucleus of <strong>the</strong> thalamus<br />
(ANT) in rats activates <strong>the</strong> ANT-cingulate cortex-hippocampus brain circuit involved in<br />
processing of memory <strong>for</strong> contextual in<strong>for</strong>mation. Using <strong>an</strong> in vivo study, we will investigate <strong>the</strong><br />
<strong>the</strong>rapeutic impact of ANT electrical stimulation in re-establishing cognitive function in a<br />
tr<strong>an</strong>sgenic AD mouse model. Five weeks-old TgCRND8 mice will be bilaterally impl<strong>an</strong>ted with<br />
acute or chronic stimulating <strong>an</strong>d recording electrodes in <strong>the</strong> ANT or medial per<strong>for</strong><strong>an</strong>t path (mPP)<br />
<strong>for</strong> stimulating <strong>an</strong>d <strong>an</strong>terior cingulate cortex (Cg1) or dentate gyrus (DG) <strong>for</strong> recording of field<br />
potentials. The ANT <strong>an</strong>d mPP will receive high frequency stimulation (HFS) to induce shortterm<br />
<strong>an</strong>d long-term potentiation measured by ch<strong>an</strong>ges in <strong>the</strong> amplitude of <strong>the</strong> population spike<br />
<strong>an</strong>d fEPSP slope. Immediately after finishing field recordings <strong>the</strong> <strong>an</strong>imals will be sacrificed to<br />
collect brain tissue <strong>for</strong> neuropathological <strong>an</strong>alysis. Comparisons will be made between<br />
stimulated hemisphere by HFS <strong>an</strong>d non-stimulated hemisphere (low frequency control). To test<br />
whe<strong>the</strong>r inhibitory pathways are predomin<strong>an</strong>tly activated in <strong>the</strong> thalamo-cortico-hippocampal
circuit by HFS, we will use paired-pulse stimulation in <strong>the</strong> ANT or mPP area. Based on <strong>the</strong><br />
in<strong>for</strong>mation ga<strong>the</strong>red, we will test <strong>the</strong> potential efficacy of DBS to attenuate cognitive<br />
deterioration in TgCRND8 through long-term electrical stimulation of ANT. TgCRND8 mice<br />
will be tested behaviorally to determine spatial learning <strong>an</strong>d memory, freezing behavior <strong>an</strong>d<br />
locomotor activity. Data generated <strong>from</strong> our ongoing study is expected to provide support <strong>for</strong> <strong>the</strong><br />
development of DBS as a novel <strong>the</strong>rapeutic strategy in <strong>the</strong> treatment of AD.<br />
Disclosures: I. Arrieta-Cruz, None; C. Pavlides, None; G.M. Pasinetti, None.<br />
Poster<br />
529. Treatments: Alzheimer's <strong>an</strong>d Neurodegeneration<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 529.24/J10<br />
Topic: C.02.n. Animal <strong>an</strong>d experimental models<br />
Title: Evaluation of <strong>the</strong> effect of molsidomine on nitregic system in <strong>an</strong> experimental model of<br />
cognitive impairment<br />
Authors: *M. A. HERNANDEZ 1,2 , J. B. PINEDA 2 , L. DEL VALLE-MONDRAGÓN 3 , M.<br />
ALCARAZ-ZUBELDIA 2 , C. RÍOS 2 , F. PÉREZ-SEVERIANO 2 ;<br />
1 Mexico City, Mexico; 2 Neurochemistry, Natl. Inst. of Neurol. <strong>an</strong>d Neurosurg. “Dr. M<strong>an</strong>uel<br />
Velasco Suárez”., Mexico City, Mexico; 3 Pharmacol., Natl. Institue of Cardiol., Mexico City,<br />
Mexico<br />
Abstract: The relationship between nitric oxide (NO) <strong>an</strong>d cholinergic system in brain has been<br />
evidenced by using inhibitors of <strong>the</strong> nitric oxide synthase (NOS) that blocked cognition, while<br />
NO donors c<strong>an</strong> facilitate it. Never<strong>the</strong>less, <strong>the</strong> participation of NO in <strong>the</strong> recovery of cholinergic<br />
deficit due to <strong>the</strong> administration of a selective cholinergic immunotoxin, 192 IgG saporin (SAP)<br />
has not been studied. The aim in this work was to evaluate <strong>the</strong> modulation of <strong>the</strong> nitrergic system<br />
after <strong>the</strong> damage induced by SAP <strong>an</strong>d to measure <strong>the</strong> response to <strong>the</strong> administration of a NO<br />
donor, molsidomine (MOL). We used adult male Wistar rats allocated into ei<strong>the</strong>r one of 4<br />
groups: 1) vehicle PBS, 0.1M pH 7.4, 2) intraseptal administration of SAP (0.22 µg), 3) MOL ip<br />
4 mg/kg, 4) SAP+MOL. Striatum, prefrontal cortex <strong>an</strong>d hippocampus were dissected out at<br />
different times after treatment <strong>an</strong>d qu<strong>an</strong>tification of nitrites, NOS activity <strong>an</strong>d expression were<br />
per<strong>for</strong>med. Our results show that SAP induces a reduction on <strong>the</strong> constitutive NOS activity in<br />
prefrontal cortex <strong>an</strong>d striatum (54%, 64% respectively compared with control p
signific<strong>an</strong>tly. <strong>When</strong> <strong>the</strong> nitrites levels were <strong>an</strong>alyzed, ch<strong>an</strong>ges were region-specific. We conclude<br />
that administration of <strong>the</strong> NO donor promotes <strong>the</strong> recovery of cNOS activity in <strong>the</strong> model of<br />
cholinergic denervation associated to 192 IgG SAP. Fur<strong>the</strong>r cognitive studies are being carried<br />
out in order to demonstrate <strong>the</strong> cholinergic recovery by MOL.<br />
Disclosures: M.A. Hern<strong>an</strong>dez, None; J.B. Pineda, None; L. del Valle-Mondragón, None; M.<br />
Alcaraz-Zubeldia, None; C. Ríos, None; F. Pérez-Severi<strong>an</strong>o, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.1/J11<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Title: Pathological modulation of stretch reflex gain in Parkinson’s disease<br />
Authors: V. STANISLAUS 1,2 , *M. KNIKOU 3,1,4 , J. A. BURNE 2 ;<br />
1 Sensory Motor Per<strong>for</strong>m<strong>an</strong>ce Program, Rehabil. Inst. of Chicago, Chicago, IL; 2 Fac. of<br />
Medicine, Discipline of Biomed. Sci., The Univ. of Sydney, Sydney, Australia; 3 Hlth. Sci.<br />
Doctoral Prgm, CUNY, Staten Isl<strong>an</strong>d, NY; 4 Physical Med. <strong>an</strong>d Rehabil., Northwestern Univ.,<br />
Chicago, IL<br />
Abstract: Objectives: The objective of this study was to investigate stretch reflex excitability<br />
during static <strong>an</strong>d dynamic motor tasks in Parkinson’s disease (PD) patients.<br />
Methods: The experimental protocol was approved by <strong>the</strong> Sydney University ethics committee<br />
<strong>an</strong>d a written consent <strong>for</strong>m was acquired <strong>from</strong> each particip<strong>an</strong>t be<strong>for</strong>e testing. Twenty healthy<br />
subjects <strong>an</strong>d 15 PD patients participated. The first metacarpal joint of <strong>the</strong> index finger was<br />
perturbed at low amplitude sinusoidally at 5 Hz using a computer controlled servo motor. In<br />
addition, subjects varied <strong>the</strong>ir background contraction continuously between rest <strong>an</strong>d 50% of<br />
maximum by tracing a tri<strong>an</strong>gular wave<strong>for</strong>m on <strong>the</strong> computer screen with a signal that represented<br />
<strong>the</strong> low-pass filtered rectified EMG of <strong>the</strong> first dorsal interosseous (FDI) muscle. Reflex EMG<br />
data were also collected <strong>from</strong> <strong>the</strong> FDI in trials where <strong>the</strong> contraction was maintained at const<strong>an</strong>t<br />
levels (0, 10, 20, 30, 40, <strong>an</strong>d 50 %) of isometric contraction. Cross correlation <strong>an</strong>alysis was<br />
per<strong>for</strong>med between perturbation <strong>an</strong>d EMG so to compare <strong>the</strong> stretch reflex during static <strong>an</strong>d<br />
dynamic motor tasks.<br />
Results: In <strong>the</strong> control group, <strong>the</strong> stretch reflex gain was signific<strong>an</strong>tly increased during <strong>the</strong><br />
dynamic task compared to <strong>the</strong> static. In contrast, no ch<strong>an</strong>ge in <strong>the</strong> stretch reflex gain was<br />
established between <strong>the</strong> static <strong>an</strong>d dynamic tasks in PD patients, <strong>an</strong>d it remained low throughout
<strong>the</strong> dynamic task when compared to <strong>the</strong> control group.<br />
Conclusions: Reflex gain (input/output relationship) remained low <strong>an</strong>d un-modulated during <strong>the</strong><br />
dynamic task in PD patients, suggesting that poor movement per<strong>for</strong>m<strong>an</strong>ce may be related to<br />
inadequate recruitment/de-recruitment of motoneurons as required in <strong>the</strong> dynamic task. This<br />
mech<strong>an</strong>ism may be involved in <strong>the</strong> complex phenomenon of bradykinesia in <strong>the</strong>se patients but<br />
more research is needed.<br />
Disclosures: V. St<strong>an</strong>islaus, None; M. Knikou , None; J.A. Burne, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.2/J12<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: NIH Gr<strong>an</strong>t R01-NS-52318<br />
NIH Gr<strong>an</strong>t R01-NS-28127<br />
NIH Gr<strong>an</strong>t R01-NS-40902<br />
NIH Gr<strong>an</strong>t R01-NS-58487<br />
Title: Hypoactivity in all basal g<strong>an</strong>glia nuclei in early stage, drug naïve Parkinson’s disease<br />
depends on <strong>the</strong> presence of visual feedback<br />
Authors: *J. PRODOEHL 1 , M. B. SPRAKER 2 , D. M. CORCOS 1 , C. L. COMELLA 3 , D. E.<br />
VAILLANCOURT 1 ;<br />
1 Dept. of Kinesiology <strong>an</strong>d Nutr., Univ. of Illinois at Chicago, Chicago, IL; 2 Dept Bioengeering,<br />
Univ. Illinois Chicago, Chicago, IL; 3 Rush Univ. Med. Ctr., Chicago, IL<br />
Abstract: Over <strong>the</strong> past decade, <strong>the</strong> study of <strong>the</strong> visual control of movement <strong>an</strong>d <strong>for</strong>ce<br />
generation has shown signific<strong>an</strong>t involvement of <strong>the</strong> visual cortex, parietal lobes, cerebellum,<br />
<strong>an</strong>d also <strong>the</strong> basal g<strong>an</strong>glia (BG). Two lines of evidence suggest that <strong>the</strong> nuclei of <strong>the</strong> BG are<br />
involved in <strong>the</strong> visual control of movement <strong>an</strong>d <strong>for</strong>ce generation. First, BG activation, as<br />
measured by <strong>the</strong> blood oxygenation level dependent (BOLD) signal, has been demonstrated in a<br />
variety of movement tasks that require visual processing. Second, patients with Parkinson’s<br />
disease (PD) have consistently shown impaired movement per<strong>for</strong>m<strong>an</strong>ce relative to controls in <strong>the</strong>
absence of vision. The present study was designed to compare <strong>the</strong> BOLD response in a precision<br />
grip <strong>for</strong>ce task with <strong>an</strong>d without vision in patients with early stage PD <strong>an</strong>d in healthy controls.<br />
We hypo<strong>the</strong>sized that <strong>the</strong>re would be <strong>an</strong> increase in <strong>the</strong> BOLD signal in <strong>the</strong> BG when healthy<br />
individuals used vision compared to <strong>the</strong> no vision condition <strong>an</strong>d that this increase in <strong>the</strong> vision<br />
condition would be less in patients with PD. The BOLD signal in <strong>the</strong> BG was compared between<br />
15 early-stage PD patients (tested prior to beginning <strong>an</strong>y <strong>an</strong>tiparkinsoni<strong>an</strong> medication) <strong>an</strong>d 15<br />
healthy controls during a precision grip <strong>for</strong>ce task using fMRI at 3T. Subjects produced ten, 2s<br />
<strong>for</strong>ce pulses to a target at 15% of <strong>the</strong>ir maximum voluntary contraction under two conditions:<br />
<strong>for</strong>ce with vision <strong>an</strong>d <strong>for</strong>ce without vision. Regions of interest <strong>an</strong>alysis included <strong>the</strong> caudate,<br />
putamen, internal <strong>an</strong>d external segments of <strong>the</strong> globus pallidus, <strong>an</strong>d <strong>the</strong> subthalamic nucleus.<br />
There was a signific<strong>an</strong>t group by vision interaction <strong>for</strong> BOLD activation in each BG nucleus.<br />
Healthy individuals <strong>an</strong>d patients with PD had a similar BOLD response in <strong>the</strong> no vision<br />
condition. Healthy individuals showed <strong>an</strong> increased BOLD response in <strong>the</strong> presence of vision<br />
when compared to <strong>the</strong> no vision condition. In contrast, patients with PD showed no ch<strong>an</strong>ge in <strong>the</strong><br />
BOLD response in <strong>the</strong> presence of vision. These data suggest that even very early in <strong>the</strong> disease,<br />
patients with PD are unable to fully utilize basal g<strong>an</strong>glia circuitry to take adv<strong>an</strong>tage of <strong>the</strong><br />
presence of visual in<strong>for</strong>mation during <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of a motor task.<br />
Disclosures: J. Prodoehl, None; M.B. Spraker, None; D.M. Corcos, None; C.L. Comella,<br />
None; D.E. Vaill<strong>an</strong>court, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.3/J13<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Title: D<strong>an</strong>ce video game training <strong>an</strong>d falling risks in Parkinson’s disease<br />
Authors: *J. A. NOAH 1 , S. BRONNER 2 ;<br />
1 ADAM Ctr., 2 Adam center, Long Isl<strong>an</strong>d Univ., Brooklyn, NY<br />
Abstract: Falling is a common occurrence <strong>an</strong>d <strong>the</strong> leading cause of accidental death in people<br />
over <strong>the</strong> age of 65 years. The cost of treatment <strong>for</strong> injuries sustained in falls exceeded $20 billion<br />
in 2000. Individuals with Parkinson’s disease (PD) are nine times more likely to fall th<strong>an</strong> healthy<br />
adults of <strong>the</strong> same age due to a number of factors including: loss of bal<strong>an</strong>ce, decrease in strength<br />
<strong>an</strong>d endur<strong>an</strong>ce, delayed reaction time, deficits in vision, impaired attention, <strong>an</strong>d lack of<br />
confidence. Studies on treadmill training, d<strong>an</strong>ce, <strong>an</strong>d traditional <strong>the</strong>rapy have shown some
positive results including increases in bal<strong>an</strong>ce <strong>an</strong>d self confidence. An issue is that <strong>the</strong>se<br />
interventions tend to target only one or two modifiable risk factors <strong>an</strong>d often have not indicated<br />
long-term benefits to subjects. A multimodal intervention that is effective in addressing multiple<br />
impairments simult<strong>an</strong>eously would be beneficial to <strong>the</strong> patient <strong>an</strong>d cost effective. We piloted a<br />
novel training program using <strong>an</strong> off-<strong>the</strong> shelf d<strong>an</strong>ce video game, D<strong>an</strong>ce D<strong>an</strong>ce Revolution<br />
(DDR). The purpose of this study was to measure <strong>the</strong> efficacy of DDR training in reducing<br />
falling risks in PD. We trained two 78-year old subjects <strong>for</strong> 24 sessions: one PD subject (Hoehn<br />
<strong>an</strong>d Yahr level 1) <strong>an</strong>d one healthy age matched control. Pre <strong>an</strong>d post training, subjects were<br />
tested on a number of outcome measures <strong>for</strong> ch<strong>an</strong>ges in bal<strong>an</strong>ce, functional per<strong>for</strong>m<strong>an</strong>ce,<br />
reaction time, <strong>an</strong>d health <strong>an</strong>d well-being. Both subjects improved in all behavioral tests,<br />
including a dual task reaction time step test. Additionally, subjects had 100% compli<strong>an</strong>ce <strong>an</strong>d<br />
indicated enjoyment of <strong>the</strong> training. The high level of compli<strong>an</strong>ce <strong>an</strong>d improvement may be<br />
related to <strong>the</strong> real time feedback of temporal <strong>an</strong>d spatial accuracy of foot placement <strong>an</strong>d<br />
knowledge of results about overall accuracy with game scores. Fur<strong>the</strong>rmore, behavioral<br />
improvements may be related to <strong>the</strong> external rhythmic cuing provided through <strong>the</strong> virtual<br />
environment of DDR. External cuing strategies may encourage neuroplasticity via cerebellarthalamo-cortical<br />
circuits <strong>an</strong>d thus bypass <strong>the</strong> defective basal g<strong>an</strong>glia. This research suggests that<br />
<strong>the</strong> complex interactive environment created by video games stimulates multiple senses <strong>an</strong>d<br />
trains particip<strong>an</strong>ts to coordinate effective sensory-motor responses that are relev<strong>an</strong>t to <strong>the</strong> real<br />
world. Future research will conduct a r<strong>an</strong>domized clinical trial with pre, post, <strong>an</strong>d retention tests<br />
that include brain imagery.<br />
Disclosures: J.A. Noah, None; S. Bronner, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.4/J14<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: NIH NCRR RR024992<br />
Americ<strong>an</strong> Parkinson Disease Association Adv<strong>an</strong>ced Research Center <strong>for</strong> Parkinson<br />
Disease at Washington University<br />
Greater St. Louis Chapter of <strong>the</strong> Americ<strong>an</strong> Parkinson Disease Association
Title: PIB binding in Parkinson disease dementia: Relationship with clinical <strong>an</strong>d<br />
neuropsychological features<br />
Authors: *E. R. FOSTER 1 , M. C. CAMPBELL 1 , M. A. BURACK 2 , A. GOULDING 1 , H.<br />
FLORES 1 , J. HARTLEIN 1 , T. HERSHEY 1 , J. S. PERLMUTTER 1 ;<br />
1 Washington Univ. Sch. Med., St Louis, MO; 2 Univ. of Rochester Med. Ctr., Rochester, NY<br />
Abstract: Dementia is common in Parkinson disease (PD) <strong>an</strong>d likely is caused by comorbid<br />
Alzheimer disease (AD; amyloid plaques, neurofibrillary t<strong>an</strong>gles) or dementia with Lewy bodies<br />
(DLB; diffuse cortical alpha-synuclein aggregates). Currently, <strong>the</strong> only reliable method <strong>for</strong><br />
distinguishing AD <strong>from</strong> DLB in PD patients is post-mortem <strong>an</strong>alysis. Pittsburgh Compound B<br />
(PIB) PET imaging allows us to measure amyloid burden in vivo as <strong>an</strong> indicator of AD pathology<br />
<strong>an</strong>d may enable better characterization of <strong>the</strong> neuropathologic subtypes of dementia in PD. The<br />
purpose of this pilot study was to investigate PIB binding <strong>an</strong>d its association with clinical <strong>an</strong>d<br />
neuropsychological features in PD patients with <strong>an</strong>d without cognitive impairment.<br />
Particip<strong>an</strong>ts (N=47) underwent PIB PET imaging <strong>an</strong>d in-depth clinical examinations. We used<br />
<strong>the</strong> Clinical Dementia Rating scale (CDR) to determine global cognitive status <strong>an</strong>d<br />
neuropsychological tests to assess memory, attention, executive <strong>an</strong>d visuospatial function. The<br />
specific binding potential of PIB <strong>for</strong> predefined regions (occipital, prefrontal, temporal,<br />
precuneus, gyrus rectus, caudate) <strong>an</strong>d <strong>the</strong> average cortical binding potential (>0.2 is considered<br />
elevated, PIB+) were calculated <strong>for</strong> each particip<strong>an</strong>t.<br />
The sample consisted of 9 healthy controls (HC), 8 PD with no cognitive impairment (PD-noCI),<br />
9 PD with mild cognitive impairment (PD-MCI), <strong>an</strong>d 21 PD with dementia (PDD) patients.<br />
Groups were equivalent in age (M=72, SD=7 years; p=0.61). Of <strong>the</strong> PDD patients, 6 had onset of<br />
cognitive symptoms within 1 year of motor symptoms <strong>an</strong>d met criteria <strong>for</strong> DLB. There were no<br />
signific<strong>an</strong>t differences in PIB binding across groups (p>0.13). One HC, 1 PD-noCI, 1 PD-MCI<br />
<strong>an</strong>d 5 PDD (2 DLB) particip<strong>an</strong>ts were classified as PIB+. Within PDD, <strong>the</strong> PIB+ particip<strong>an</strong>ts had<br />
signific<strong>an</strong>tly worse MMSE scores (p=0.02) <strong>an</strong>d tended to have worse CDR scores <strong>an</strong>d more<br />
reported cognitive fluctuations (p
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.5/J15<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: NIH Gr<strong>an</strong>t UL1RR024982<br />
Title: Identification of biomarkers <strong>for</strong> Parkinson’s disease in peripheral blood<br />
Authors: *C. M. TAYLOR 1 , M. A. MARVIN 1 , K. W. SEAMANS 1 , P. E.<br />
O'SUILLEABHAIN 1 , R. B. DEWEY 1 , S. CHITNIS 1 , M. S. GOLDBERG 1,2 ;<br />
1 Neurol., 2 Psychiatry, Univ. of Texas Southwestern Med. Ctr., Dallas, TX<br />
Abstract: Parkinson’s disease (PD) is a progressive neurodegenerative disorder that presents<br />
with features of muscle rigidity, tremor <strong>an</strong>d bradykinesia. The clinical symptoms are primarily<br />
caused by degeneration of dopaminergic neurons in <strong>the</strong> subst<strong>an</strong>tia nigra pars compacta. While<br />
oral L-DOPA c<strong>an</strong> alleviate some of <strong>the</strong> clinical symptoms, <strong>the</strong>re are currently no treatments that<br />
are proven to slow down <strong>the</strong> progressive nigral cell loss in PD. Although <strong>the</strong> cause of PD is<br />
uncertain, numerous studies point to mitochondrial dysfunction <strong>an</strong>d oxidative damage both in <strong>the</strong><br />
subst<strong>an</strong>tia nigra <strong>an</strong>d in peripheral blood cells. Because <strong>the</strong>re are currently no diagnostic tests <strong>for</strong><br />
PD <strong>an</strong>d no ways to objectively measure disease progression that would be practical <strong>for</strong> clinical<br />
trials to test neuroprotective compounds, we propose do identify surrogate markers of PD in<br />
peripheral blood cells. Because mitochondrial respiration defects have been found in peripheral<br />
blood platelets <strong>an</strong>d leukocytes of PD patients, we hypo<strong>the</strong>size that markers of <strong>the</strong> disease c<strong>an</strong> be<br />
identified in peripheral blood platelets by <strong>an</strong>alyzing mitochondrial proteins using twodimensional<br />
gel electrophoresis <strong>an</strong>d western <strong>an</strong>alysis. We fur<strong>the</strong>r hypo<strong>the</strong>size that <strong>the</strong><br />
mitochondrial respiration defects are caused by oxidative damage to mitochondrial proteins that<br />
c<strong>an</strong> be resolved by two-dimensional gel electrophoresis <strong>an</strong>d detected by western blotting with<br />
oxidative damage specific markers or known mitochondrial complex I <strong>an</strong>tibodies. Here we<br />
present data on our <strong>an</strong>alysis of mitochondria isolated <strong>from</strong> peripheral blood platelets of PD<br />
patients <strong>an</strong>d controls.<br />
Disclosures: C.M. Taylor, None; M.A. Marvin, None; K.W. Seam<strong>an</strong>s, None; P.E.<br />
O'Suilleabhain, None; R.B. Dewey, None; S. Chitnis, None; M.S. Goldberg, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.6/J16<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Title: Visual vertical <strong>an</strong>d olfactory perception in Parkinson’s disease<br />
Authors: S. DOCHERTY 1 , *J. BAGUST 2 , A. KHATTAB 3 , K. AMAR 4 ;<br />
2 Preclinical Sci., 1 Anglo-Europe<strong>an</strong> Col. Chiropractic, Bournemouth, United Kingdom; 3 Sch. of<br />
Hlth. <strong>an</strong>d Social Care, Bournemouth Univ., Bournemouth, United Kingdom; 4 Bournemouth <strong>an</strong>d<br />
Christchurch NHS Fndn. Trust, Bournemouth, United Kingdom<br />
Abstract: Parkinson’s Disease (PD) is characterised by a mixture of motor <strong>an</strong>d non-motor<br />
features, where olfactory dysfunction is reported to be present in <strong>the</strong> majority of PD patients.<br />
Impairment of <strong>the</strong> sense of smell is thought to develop early in <strong>the</strong> course of <strong>the</strong> disease during<br />
<strong>the</strong> pre-motor phase 1 . It would be useful to establish if <strong>the</strong> impairment of olfactory perception<br />
results <strong>from</strong> a generalised deterioration of perceptual skills, or is specific to <strong>the</strong> sense of smell.<br />
As a result, this study compared <strong>the</strong> perception of visual vertical, as measured by <strong>the</strong> Computer<br />
Road <strong>an</strong>d Frame (CRAF) test, <strong>an</strong>d sense of smell between PD patients <strong>an</strong>d <strong>an</strong> age <strong>an</strong>d gender<br />
matched non-PD control group.<br />
Forty seven patients were recruited <strong>from</strong> a PD clinic at Christchurch Hospital (UK) <strong>an</strong>d were<br />
compared to 33 age <strong>an</strong>d gender matched non PD subjects. Sense of smell was tested using <strong>the</strong><br />
12-item University of Pennsylv<strong>an</strong>ia Smell Identification Test. The number of smells correctly<br />
identified was used to compare <strong>the</strong> olfactory acuity of <strong>the</strong> control <strong>an</strong>d PD subjects.<br />
Perception of visual vertical was measured using <strong>the</strong> CRAF test. A diagonal white line inside a<br />
white frame displayed on a black background was viewed through video glasses. Subjects rotated<br />
<strong>the</strong> rod using switches until it appeared to be vertical, when <strong>the</strong> computer recorded <strong>the</strong> error of<br />
<strong>the</strong> position of <strong>the</strong> line relative to vertical. Each test consisted of 4 trials with <strong>the</strong> frame square<br />
(0°), rotated clockwise (+18°) <strong>an</strong>d rotated <strong>an</strong>ticlockwise (-18°), in a r<strong>an</strong>dom order. The me<strong>an</strong><br />
value <strong>for</strong> <strong>the</strong> absolute (unsigned) errors <strong>for</strong> <strong>the</strong> two frame-tilted conditions was compared<br />
between <strong>the</strong> two subject groups.<br />
The me<strong>an</strong> (± SD) correct smell test score in <strong>the</strong> PD patients was 5.94 ± 3.47, compared to 9.21 ±<br />
2.09 in <strong>the</strong> control group, confirming <strong>the</strong> presence of <strong>an</strong> olfactory deficit in <strong>the</strong> PD patients P<<br />
.001, t-test). The absolute errors in <strong>the</strong> CRAF test were medi<strong>an</strong>: 2.63° (0.5°-18.5°), in PD<br />
patients <strong>an</strong>d medi<strong>an</strong>: 2.19° (0.5°-18.50°) in <strong>the</strong> control subjects. These were not signific<strong>an</strong>tly<br />
different (P = 0.597, M<strong>an</strong>n Whitney U test).<br />
These results confirm that <strong>the</strong> PD patients in this study exhibited <strong>an</strong> olfactory deficit. However,<br />
<strong>the</strong> CRAF results showed that <strong>the</strong> PD patients’ ability to estimate visual vertical was not<br />
signific<strong>an</strong>tly different <strong>from</strong> <strong>an</strong> age <strong>an</strong>d gender matched control group. This suggests that <strong>the</strong> loss<br />
of olfactory ability in <strong>the</strong>se patients is not part of a generalised disturb<strong>an</strong>ce of perceptual ability.<br />
1. Hawkes CH <strong>an</strong>d Deeb J (2006) Predicting Parkinson’s disease: worthwhile but are we <strong>the</strong>re<br />
yet? Practical Neurology 6: 272-277.
Disclosures: S. Docherty, None; J. Bagust , None; A. Khattab, None; K. Amar, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.7/J17<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institute of Health Research MOP-64423<br />
Title: Deep brain stimulation of <strong>the</strong> subthalamic nucleus improves motor functions at <strong>the</strong><br />
expense of response inhibition. A PET study<br />
Authors: *B. BALLANGER 1 , T. VAN EIMEREN 2 , E. MORO 3 , A. M. LOZANO 4,5 , C.<br />
HAMANI 4,5 , P. BOULINGUEZ 1 , G. PELLECCHIA 2 , S. HOULE 2 , Y. POON 3 , A. E. LANG 3,5 ,<br />
A. P. STRAFELLA 3,5,2 ;<br />
1 Ctr. De Neurosciences Cognitives UMR CNRS 5229 ISC, Bron Cedex, Fr<strong>an</strong>ce; 2 PET Imaging<br />
Centre,, Ctr. <strong>for</strong> Addiction <strong>an</strong>d Mental Hlth. (CAMH), Toronto, ON, C<strong>an</strong>ada; 3 Movement<br />
Disorders Ctr., 4 Dept. of Neurosurg., Toronto Western Hosp., Toronto, ON, C<strong>an</strong>ada; 5 Div. of<br />
Brain, Imaging <strong>an</strong>d Behaviour - Systems Neuroscience, BIB-SN, Toronto Western Res. Inst.,<br />
Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Objective: In Parkinson’s disease (PD) patients, deep brain stimulation (DBS) of <strong>the</strong><br />
subthalamic nucleus (STN) may contribute to certain impulsive behaviour during high-conflict<br />
decisions. A neurocomputational model of <strong>the</strong> basal g<strong>an</strong>glia has recently proposed that this<br />
behavioural aspect may be related to <strong>the</strong> role played by <strong>the</strong> STN in relaying a “hold your horses”<br />
signal intended to allow more time to settle on <strong>the</strong> best option. The aim of <strong>the</strong> present study was<br />
twofold: (i) to extend <strong>the</strong>se observations by providing evidence that <strong>the</strong> STN may influence <strong>an</strong>d<br />
prevent <strong>the</strong> execution of <strong>an</strong>y response even during low-conflict decisions, <strong>an</strong>d (ii) to identify <strong>the</strong><br />
neural correlates of this effect.<br />
Method: We measured regional cerebral blood flow (rCBF) during a Go/NoGo <strong>an</strong>d a control<br />
(Go) task to study <strong>the</strong> motor improvement <strong>an</strong>d response-inhibition deficits associated with STN-<br />
DBS in 7 patients with PD.<br />
Results: While improving UPDRS motor ratings <strong>an</strong>d inducing a global decrease in reaction time<br />
during task per<strong>for</strong>m<strong>an</strong>ce, STN-DBS impaired response-inhibition as revealed by <strong>an</strong> increase in<br />
commission errors in NoGo trials. These behavioral effects were accomp<strong>an</strong>ied by ch<strong>an</strong>ges in<br />
synaptic activity consisting in a reduced activation in <strong>the</strong> cortical network responsible <strong>for</strong><br />
reactive <strong>an</strong>d proactive response inhibition (e.g., precuneus, posterior <strong>an</strong>d <strong>an</strong>terior cingulate
cortex, pre-supplementary motor area).<br />
Interpretation: The present results suggest that, while improving motor functions in PD patients,<br />
modulation of STN hyperactivity with DBS may tend at <strong>the</strong> same time to favour <strong>the</strong> appear<strong>an</strong>ce<br />
of impulsive behaviour by acting on <strong>the</strong> gating mech<strong>an</strong>ism involved in response initiation..<br />
Based on <strong>the</strong>se observations, we propose that impairment of <strong>the</strong> response inhibition network<br />
plays <strong>an</strong> import<strong>an</strong>t role in both akinesia (which may be viewed as a global difficulty release<br />
proactive inhibition OFF stimulation, i.e. to “release <strong>the</strong> horses”) <strong>an</strong>d impulsivity (which may<br />
conversely be considered as a global difficulty to lock movement triggering processes ON<br />
stimulation, i.e., to “hold <strong>the</strong> horses”). In o<strong>the</strong>r words, akinesia <strong>an</strong>d impulsivity could represent<br />
opposite sides of <strong>the</strong> same coin. We believe that this new <strong>the</strong>oretical approach may provide new<br />
insights <strong>an</strong>d better underst<strong>an</strong>ding of movement initiation disorders <strong>an</strong>d <strong>the</strong> motor <strong>an</strong>d behavioral<br />
effects of STN-DBS.<br />
Disclosures: B. Ball<strong>an</strong>ger, None; T. v<strong>an</strong> Eimeren, None; E. Moro, None; A.M. Loz<strong>an</strong>o,<br />
None; C. Ham<strong>an</strong>i, None; P. Boulinguez, None; G. Pellecchia, None; S. Houle, None; Y. Poon,<br />
None; A.E. L<strong>an</strong>g, None; A.P. Strafella, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.8/J18<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Krembil Neuroscience Fund<br />
Title: Early signs of nigrostriatal dysfunction in <strong>an</strong> <strong>an</strong>imal model of Parkinson’s diseasesynucleinopathy<br />
following AAV1/2 viral vector expression of hum<strong>an</strong> A53T alpha-synuclein<br />
Authors: J. B. KOPRICH, T. H. JOHNSTON, *G. REYES, X. S. SUN, J. M. BROTCHIE;<br />
Toronto Western Res. Inst., Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Parkinson’s disease (PD) is a synucleinopathy characterized by a progressive loss of<br />
dopaminergic function <strong>an</strong>d <strong>the</strong> presence of alpha-synuclein aggregates in <strong>the</strong> nigrostriatal<br />
pathway. To model this aspect of <strong>the</strong> disease we overexpressed mutated alpha-synuclein in <strong>the</strong><br />
SN using viral vectors. AAV1/2 A53T hum<strong>an</strong> alpha-synuclein or GFP (5.1 x 10exp12 gp/ml<br />
under control of <strong>the</strong> CMV/CBA promoter <strong>for</strong> each) were injected unilaterally into <strong>the</strong> SN of rats.<br />
Three weeks following injection, >90% of remaining tyrosine hydroxylase (TH) positive neurons<br />
of <strong>the</strong> entire SN were immunoreactive with hum<strong>an</strong> alpha-synuclein or GFP. Expression of TH in
<strong>the</strong> SN of AAV-synuclein injected rats was reduced by >40%, in <strong>the</strong> absence of overt cell loss,<br />
compared to <strong>the</strong> non-injected side. Notably, TH expression, without cell loss, was also reduced<br />
by >30% in AAV-GFP injected rats, indicating that GFP at this titer is potentially toxic. At <strong>the</strong><br />
level of <strong>the</strong> striatum, widespread immunoreactivity of GFP was evident throughout its entire<br />
extent <strong>an</strong>d to a lesser degree, alpha-synuclein immunoreactivity, likely representing a tr<strong>an</strong>sport<br />
deficit. Fur<strong>the</strong>rmore, AAV-synuclein injected <strong>an</strong>imals alone showed dystrophic <strong>an</strong>d bulging<br />
neurites in effected axons. Levels of TH in <strong>the</strong> striatum were reduced by 30% <strong>an</strong>d 10% in AAVsynuclein<br />
<strong>an</strong>d -GFP injected rats respectively. In conclusion, overexpression of hum<strong>an</strong> A53T<br />
alpha-synuclein using AAV1/2 produces widespread levels of protein expression in <strong>the</strong> SN <strong>an</strong>d<br />
striatum that c<strong>an</strong> be seen within 3 weeks of treatment <strong>an</strong>d is associated with a reduction in<br />
nigrostriatal TH expression <strong>an</strong>d <strong>the</strong> generation of neurite pathology.<br />
Disclosures: J.B. Koprich, None; T.H. Johnston, None; G. Reyes, None; X.S. Sun,<br />
None; J.M. Brotchie, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.9/J19<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: Krembil Foundation<br />
Cure Parkinson's Trust<br />
De<strong>an</strong>'s New staff gr<strong>an</strong>t, University of Toronto<br />
Safra Foundation<br />
Title: Serotonergic 5HT2A receptor levels are increased in <strong>the</strong> temporal lobe of Parkinson’s<br />
disease patients with visual hallucinations<br />
Authors: P. HUOT 1 , T. H. JOHNSTON 1 , L.-N. HAZRATI 2 , J. M. BROTCHIE 1 , *S. H. FOX 1 ;<br />
1 2<br />
Toronto Western Res. Inst., Toronto, ON, C<strong>an</strong>ada; Ctr. <strong>for</strong> research in Neurodegenerative Dis.,<br />
Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Background<br />
Visual hallucinations (VH) c<strong>an</strong> affect up to 50% of patients with Parkinson’s disease (PD). The
aetiology of VH in PD is unknown .However, <strong>the</strong> inferior temporal cortex is involved in<br />
complex visual processing <strong>an</strong>d shares intimate connections with <strong>the</strong> basal g<strong>an</strong>glia. The inferior<br />
temporal cortex may participate in <strong>the</strong> genesis of VH in PD, as it is targeted by <strong>the</strong> disease<br />
process.<br />
5HT2A receptors are linked to psychotic symptoms in several o<strong>the</strong>r disorders <strong>an</strong>d <strong>the</strong> efficacy of<br />
<strong>the</strong> atypical <strong>an</strong>tipsychotic drugs in reducing hallucinations is believed to be mediated via <strong>the</strong>ir<br />
affinity <strong>for</strong> <strong>the</strong> 5HT2A receptors. We hypo<strong>the</strong>size that 5HT2A receptors in <strong>the</strong> inferior temporal<br />
cortex are involved in <strong>the</strong> neural mech<strong>an</strong>isms underlying VH in PD.<br />
Methods<br />
The distribution <strong>an</strong>d density of 5HT2A receptors were determined by autoradiographic binding.<br />
Brain sections (12 µM thick) <strong>from</strong> six PD subjects with VH, six PD subjects without VH, <strong>an</strong>d<br />
five normal age-matched controls were <strong>an</strong>alyzed. For each brain, <strong>the</strong> frontal, motor, <strong>an</strong>d inferior<br />
temporal cortices, striatum, pallidum, <strong>an</strong>d subst<strong>an</strong>tia nigra were studied. [ 3 H]ket<strong>an</strong>serin, a 5HT2A<br />
receptor <strong>an</strong>tagonist, was used as <strong>the</strong> radiolig<strong>an</strong>d. Prazosin <strong>an</strong>d tetrabenazine were added to <strong>the</strong><br />
incubation solution, to prevent <strong>the</strong> binding of [ 3 H]ket<strong>an</strong>serin to <strong>the</strong> alpha-1 adrenoreceptors <strong>an</strong>d<br />
<strong>the</strong> vesicular monoamine tr<strong>an</strong>sporter. Non-specific binding was determined in <strong>the</strong> presence of<br />
spiperone. Images were obtained by apposing sections to [ 3 H]sensitive films with [ 3 H]microscale<br />
st<strong>an</strong>dards. Me<strong>an</strong> optical densities of regions of interest were <strong>an</strong>alyzed <strong>an</strong>d converted to fmol of<br />
bound radiolig<strong>an</strong>d per mg of tissue protein.<br />
Results<br />
Specific 5HT2A receptor binding was increased by 46%, in <strong>the</strong> inferior temporal cortex of PD<br />
patients with VH (54.29 ± 5.21 fmol/mg), when compared to PD patients without VH (37.28 ±<br />
4.31 fmol/mg). (P < 0.05 one way ANOVA test, followed by Tukey’s multiple comparison test).<br />
In contrast, specific 5HT2A receptor binding in <strong>the</strong> inferior temporal cortex of control brains<br />
(42.71 ± 5.37 fmol/mg) was not signific<strong>an</strong>tly different to that found in <strong>the</strong> brains of PD subjects<br />
without VH.<br />
Conclusions<br />
Enh<strong>an</strong>ced 5HT2A receptor-mediated tr<strong>an</strong>smission in <strong>the</strong> temporal lobe may underlie VH in PD.<br />
Selectively targeting of 5HT2A receptors could potentially alleviate VH in PD.<br />
Disclosures: P. Huot, None; T.H. Johnston, None; L. Hazrati, None; J.M. Brotchie,<br />
None; S.H. Fox, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.10/J20<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies
Support: These studies were supported by Sepracor Inc.<br />
Title: Anti-parkinsoni<strong>an</strong> actions of <strong>the</strong> mixed monoamine re-uptake inhibitors SEP-228791 <strong>an</strong>d<br />
SEP-226330 in <strong>the</strong> MPTP-lesioned macaque model of Parkinson’s disease<br />
Authors: T. H. JOHNSTON 1 , S. H. FOX 1,2 , J. MA 3 , L. SHAO 3 , U. CAMPBELL 3 , *J. M.<br />
BROTCHIE 1 ;<br />
1 Atuka Ltd, Toronto, ON, C<strong>an</strong>ada; 2 Movement Disorders Clin., Toronto Western Hosp.,<br />
Toronto, ON, C<strong>an</strong>ada; 3 Sepracor Inc., Marlborough, MA<br />
Abstract: Objective: To assess <strong>the</strong> ability of <strong>the</strong> mixed monoamine re-uptake inhibitors SEP-<br />
228791 <strong>an</strong>d SEP-226330 to alleviate parkinsoni<strong>an</strong> symptoms as mono<strong>the</strong>rapy <strong>an</strong>d to modify <strong>the</strong><br />
actions of L-DOPA in <strong>the</strong> MPTP-lesioned macaque model of Parkinson’s disease (PD).<br />
Background: Long-term L-DOPA <strong>the</strong>rapy in PD patients results in motor complications<br />
including dyskinesia <strong>an</strong>d shortening of duration of <strong>an</strong>ti-parkinsoni<strong>an</strong> action, ‘wearing-off’.<br />
Dopamine re-uptake inhibitors have potential to ameliorate such complications ei<strong>the</strong>r as<br />
mono<strong>the</strong>rapy or in combination with L-DOPA. SEP-228791 <strong>an</strong>d SEP-226330 are novel<br />
monoamine re-uptake inhibitors with differing affinities <strong>for</strong> <strong>the</strong> monamine tr<strong>an</strong>sporters, DAT,<br />
NET <strong>an</strong>d SERT.<br />
Methods: Six MPTP-lesioned cynomolgus monkeys (Macaca fascicularis) were treated with L-<br />
DOPA (25 mg/kg p.o. bid) <strong>for</strong> at least 3 months until stable dyskinesia <strong>an</strong>d wearing-off were<br />
induced. SEP-228791 or SEP-226330 were <strong>the</strong>n administered acutely alone (1, 3 or 10 mg/kg,<br />
p.o.) or in combination with low (LDl) <strong>an</strong>d high (LDh)-doses of L-DOPA (s.c.). For comparison,<br />
<strong>the</strong> actions of acute oral L-DOPA (15, 25 <strong>an</strong>d 35 mg/kg, as Madopar) <strong>an</strong>d ropinirole (0.3, 1<br />
<strong>an</strong>d 3 mg/kg, as Requip) as mono<strong>the</strong>rapy were also assessed. Quality <strong>an</strong>d duration of ‘on-time’<br />
were assessed <strong>for</strong> up to 8h via post hoc <strong>an</strong>alysis of parkinsoni<strong>an</strong> disability <strong>an</strong>d dyskinesia by a<br />
rater blinded to treatment.<br />
Results: SEP-228791 mono<strong>the</strong>rapy (3 mg/kg) provided <strong>an</strong>ti-parkinsoni<strong>an</strong> benefit of similar<br />
duration to that seen with high dose L-DOPA alone (258 cf. 295 min respectively). The quality<br />
of this benefit was much greater th<strong>an</strong> with L-DOPA alone, with less th<strong>an</strong> 3% of total on-time<br />
associated with dyskinesia. In contrast, 71% of L-DOPA (35 mg/kg) <strong>an</strong>d 37% of ropinirole (3<br />
mg/kg) on-time was associated with dyskinesia. SEP-228791 did not signific<strong>an</strong>tly alter <strong>the</strong> <strong>an</strong>tiparkinsoni<strong>an</strong><br />
actions of L-DOPA.<br />
SEP-226330 (1, 3 or 10 mg/kg, p.o.) showed no signific<strong>an</strong>t effect as mono<strong>the</strong>rapy. In contrast,<br />
SEP-226330 (10 mg/kg) enh<strong>an</strong>ced <strong>the</strong> effects of L-DOPA, when combined with LDl, increasing<br />
on time by 85%. In <strong>the</strong>se <strong>an</strong>imals, on-time was not associated with a increase in disabling<br />
dyskinesia <strong>an</strong>d <strong>the</strong> amount of on-time <strong>for</strong> which dyskinesia was absent was signific<strong>an</strong>tly<br />
increased by 200%.<br />
Conclusions: SEP-228791 <strong>an</strong>d SEP-226330 have potential as <strong>the</strong>rapeutics in Parkinson’s<br />
disease. SEP-228971 may be of use as mono<strong>the</strong>rapy while SEP-226330 offers <strong>an</strong> opportunity to<br />
increase <strong>the</strong> quality <strong>an</strong>d duration of <strong>an</strong>ti-parkinsoni<strong>an</strong> action of L-DOPA. The <strong>the</strong>rapeutic<br />
benefits of mixed monoamine re-uptake inhibitors may depend on <strong>the</strong>ir relative actions at <strong>the</strong><br />
different monoamine tr<strong>an</strong>sporters.<br />
Disclosures: T.H. Johnston, None; S.H. Fox, None; J. Ma, JM is <strong>an</strong> employee of Sepracor<br />
Inc., A. Employment (full or part-time); L. Shao, LS is <strong>an</strong> employee of Sepracor Inc., A.
Employment (full or part-time); U. Campbell, UC is <strong>an</strong> employee of Sepracor Inc., A.<br />
Employment (full or part-time); J.M. Brotchie, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.11/J21<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Title: Recognition of emotional prosody is disrupted after subthalamic nucleus deep brain<br />
stimulation in Parkinson’s disease<br />
Authors: *J. PERON 1 , D. GRANDJEAN 5 , F. LE JEUNE 6 , P. SAULEAU 2 , C. HAEGELEN 3 ,<br />
D. DRAPIER 7 , T. ROUAUD 4 , S. DRAPIER 4 , M. VERIN 4 ;<br />
2 Physiol., 3 Neurosurg., 4 Neurol., 1 Univ. Hosp. Pontchaillou, Rennes, Fr<strong>an</strong>ce; 5 Swiss Ctr. <strong>for</strong><br />
Affective Sci., Geneva, Switzerl<strong>an</strong>d; 6 Nuclear Med., CAC Eugene Marquis, Rennes, Fr<strong>an</strong>ce;<br />
7 Psychiatry, CH Guillaume Regnier, Rennes, Fr<strong>an</strong>ce<br />
Abstract: Background: The recognition of facial emotions is impaired following subthalamic<br />
nucleus (STN) deep brain stimulation (DBS) in Parkinson’s disease (PD). These ch<strong>an</strong>ges have<br />
been related to a disturb<strong>an</strong>ce of <strong>the</strong> STN’s limbic territory, thought to be involved in emotional<br />
processing. This was confirmed by a recent PET study where <strong>the</strong>se emotional modifications were<br />
correlated with ch<strong>an</strong>ges in glucose metabolism in <strong>the</strong> limbic system. Never<strong>the</strong>less, <strong>the</strong> question<br />
as to whe<strong>the</strong>r <strong>the</strong>se emotional ch<strong>an</strong>ges induced by STN DBS in PD are modality specific has yet<br />
to be <strong>an</strong>swered. The objective of this study was to examine <strong>the</strong> effects of STN DBS in PD on <strong>the</strong><br />
recognition of emotional prosody.<br />
Methodology/Principal findings: An original emotional prosody paradigm was administered to<br />
twenty-one post-operative PD patients, twenty-one pre-operative PD patients <strong>an</strong>d twenty-one<br />
matched controls. An <strong>an</strong>alysis of <strong>the</strong>ir continuous judgments revealed that <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong><br />
post-operative group compared with that of <strong>the</strong> o<strong>the</strong>r two groups was characterized by a<br />
systematic bias to attribute more emotions (i. e., fear <strong>an</strong>d sadness) to neutral utter<strong>an</strong>ces <strong>an</strong>d to<br />
perceive negative emotions more strongly.<br />
Conclusion/Signific<strong>an</strong>ce: These results suggest that <strong>the</strong> impaired recognition of emotions is not<br />
specific to <strong>the</strong> visual modality but is also present when emotions are expressed through <strong>the</strong><br />
hum<strong>an</strong> voice, demonstrating <strong>for</strong> <strong>the</strong> first time <strong>the</strong> involvement of <strong>the</strong> STN in <strong>the</strong> recognition of<br />
emotional prosody.
Disclosures: J. Peron, None; D. Gr<strong>an</strong>dje<strong>an</strong>, None; F. Le Jeune, None; P. Sauleau, None; C.<br />
Haegelen, None; D. Drapier, None; T. Rouaud, None; S. Drapier, None; M. Verin, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.12/J22<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: Renschler Foundation<br />
Lathum Foundation<br />
Title: Increased chemokine CXCL12 <strong>an</strong>d its receptor CXCR4 expressions in <strong>the</strong> nigro-striatal<br />
system of Parkinson's disease<br />
Authors: *M. SHIMOJI 1 , F. PAGAN 2 , E. B. HEALTON 2 , I. MOCCHETTI 1 ;<br />
1 2<br />
Neurosci, Georgetown Univ. Med.Ctr., Washington D.C., DC; Neurol., Georgetown Univ.<br />
Hosp., Washington D.C., DC<br />
Abstract: Except <strong>for</strong> a h<strong>an</strong>dful of inherited cases related to known gene defects, Parkinson’s<br />
disease (PD) is a sporadic neurodegenerative disease of unknown etiology. There is increasing<br />
evidence that inflammation <strong>an</strong>d proliferation of microglia may contribute to <strong>the</strong> neuronal damage<br />
seen in <strong>the</strong> nigro-striatal dopaminergic system of PD patients. Microglia events that participate in<br />
neuronal injury include <strong>the</strong> release of pro-inflammatory <strong>an</strong>d neurotoxic factors. Characterizing<br />
<strong>the</strong>se factors may help prevent <strong>the</strong> exacerbation of PD symptoms or remediate <strong>the</strong> disease<br />
progression. In rodents, <strong>the</strong> nigro-striatal system exhibits high expression of <strong>the</strong> chemokine<br />
receptor CXCR4. Its natural lig<strong>an</strong>d CXCL12 c<strong>an</strong> promote neuronal apoptosis. There<strong>for</strong>e, <strong>the</strong><br />
present study investigated <strong>the</strong> expression of CXCR4 <strong>an</strong>d CXCL12 in post-mortem brains of PD<br />
<strong>an</strong>d control (non-PD) individuals <strong>an</strong>d in <strong>an</strong> <strong>an</strong>imal model of PD. In <strong>the</strong> hum<strong>an</strong> subst<strong>an</strong>tia nigra<br />
(SN), CXCR4 immunoreactivity was high in dopaminergic neurons. Interestingly, <strong>the</strong> SN of PD<br />
subjects exhibited higher expression of CXCR4 expression <strong>an</strong>d CXCL12 th<strong>an</strong> control subjects<br />
despite <strong>the</strong> loss of dopamine (DA) neurons. This effect was accomp<strong>an</strong>ied by <strong>an</strong> increase in<br />
activated microglia. However, results <strong>from</strong> post-mortem brains may not provide indication as to<br />
whe<strong>the</strong>r CXCL12/CXCR4 c<strong>an</strong> cause <strong>the</strong> degeneration of DA neurons. To examine <strong>the</strong> role of<br />
<strong>the</strong>se chemokines, we determined <strong>the</strong> levels of CXCL12 <strong>an</strong>d CXCR4 in <strong>the</strong> SN of MPTP-treated<br />
mice. MPTP produced a time-dependent up-regulation of CXCR4 that preceded <strong>the</strong> loss of DA
neurons. These results suggest that CXCL12/CXCR4 may participate in <strong>the</strong> etiology of PD <strong>an</strong>d<br />
indicate a new possible target molecule <strong>for</strong> PD.<br />
Disclosures: M. Shimoji, None; F. Pag<strong>an</strong>, None; E.B. Healton, None; I. Mocchetti, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.13/J23<br />
Topic: C.03.a. Genetic determin<strong>an</strong>ts<br />
Support: Charitable Hertie Foundation to L.F.B.<br />
Fortuene Gr<strong>an</strong>t to R.K. (F.1313061)<br />
Federal Ministry <strong>for</strong> Education <strong>an</strong>d Research (BMBF, NGFNplus, 01GS08134)<br />
Title: Identification <strong>an</strong>d functional characterization of a novel mutation in <strong>the</strong> mortalin/GRP75<br />
gene in Germ<strong>an</strong> Parkinson disease patients<br />
Authors: L. F. BURBULLA 1,2 , C. SCHELLING 1 , B. MAURER 1 , P. BAUER 3 , L. SCHÖLS 1 ,<br />
O. RIESS 3 , *R. KRUGER 4,1 ;<br />
1<br />
Ctr. of Neurol. <strong>an</strong>d Hertie Inst. <strong>for</strong> Clin. Brain Research, Univ. of Tübingen, Tübingen,<br />
Germ<strong>an</strong>y; 2 Grad. Sch. of Cell. & Mol. Neuroscience, Univ. of Tübingen, Tübingen, Germ<strong>an</strong>y;<br />
3 4<br />
Dept. of Med. Genetics, Univ. of Tübingen, Tübingen, Germ<strong>an</strong>y; Univ. of Tubingen,<br />
Tubingen, Germ<strong>an</strong>y<br />
Abstract: Using <strong>an</strong> unbiased proteomic approach, we identified mortalin/GRP75 as a<br />
mitochondrial DJ-1-interacting protein that is responsible <strong>for</strong> cellular stress response.<br />
Mortalin/GRP75 has been linked to Parkinson’s disease (PD) pathogenesis due to signific<strong>an</strong>tly<br />
reduced levels of mortalin/GRP75 in affected brain regions of PD patients indicating a potential<br />
role in neurodegeneration. Moreover loss of DJ-1 function leads to reduced mitochondrial<br />
membr<strong>an</strong>e potential, suggesting a potential relev<strong>an</strong>ce of its interaction with <strong>the</strong><br />
intramitochondrial stress response protein mortalin/GRP75 <strong>for</strong> mitochondrial homeostasis.<br />
To define <strong>the</strong> relev<strong>an</strong>ce of mortalin/GRP75 in DJ-1-mediated mitochondrial protection we<br />
per<strong>for</strong>med a detailed mutation <strong>an</strong>alysis in a large sample of 286 Germ<strong>an</strong> sporadic <strong>an</strong>d familial<br />
PD patients. Using DHPLC <strong>an</strong>alyses <strong>for</strong> high throughput mutation screening <strong>an</strong>d subsequent<br />
direct sequencing, we defined sequence variations that were compared to a group of 290 healthy
controls. Identified sequence variations that lead to amino acid ch<strong>an</strong>ges of <strong>the</strong> protein were<br />
subsequently <strong>an</strong>alyzed concerning differential interaction, mitochondrial morphology <strong>an</strong>d<br />
mitochondrial function, reactive oxygen species production <strong>an</strong>d cell death in different cellular<br />
models.<br />
In total we identified 8 sequence variations, including 4 silent base substitutions in <strong>the</strong> coding<br />
region <strong>an</strong>d 4 intronic polymorphisms. One mutation in exon 12 of <strong>the</strong> mortalin/GRP75 gene<br />
leads to <strong>an</strong> amino acid exch<strong>an</strong>ge in a highly conserved region of <strong>the</strong> peptide sequence. This<br />
vari<strong>an</strong>t was not present in more th<strong>an</strong> 500 control chromosomes <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e may contribute to<br />
PD. Using immunoprecipitation we found no evidence <strong>for</strong> differential interaction of <strong>the</strong> mut<strong>an</strong>t<br />
protein with DJ-1. However first functional studies argue in favour of subcellular mislocalization<br />
caused by mut<strong>an</strong>t mortalin/GRP75 in stably tr<strong>an</strong>sfected HEK293 cells. Fur<strong>the</strong>rmore, cells<br />
overexpressing mut<strong>an</strong>t mortalin/GRP75 displayed a higher susceptibility towards proteolytic<br />
stress.<br />
Mutations in <strong>the</strong> mortalin/GRP75 gene in PD are a rare event. We identified <strong>for</strong> <strong>the</strong> first time a<br />
mutation in <strong>the</strong> hum<strong>an</strong> mortalin/GRP75 gene that links this DJ-1-interacting mitochondrial<br />
protein with PD. Future studies focus on functional effects of <strong>the</strong> identified vari<strong>an</strong>t in neurons to<br />
determine <strong>the</strong> relev<strong>an</strong>ce of mortalin/GRP75 in neurodegeneration. Our functional <strong>an</strong>alysis will<br />
be relev<strong>an</strong>t <strong>for</strong> <strong>the</strong> underst<strong>an</strong>ding of this novel mutation <strong>an</strong>d its effects in relation to<br />
neurodegeneration.<br />
Disclosures: L.F. Burbulla, None; C. Schelling, None; B. Maurer, None; P. Bauer, None; L.<br />
Schöls, None; O. Riess, None; R. Kruger, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.14/J24<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Title: Effect of subthalamic deep brain stimulation on emotional cognition in patients with<br />
Parkinson's disease<br />
Authors: *F. YOKOCHI 1 , M. TANIGUCHI 2 , N. YOSHIMURA 3 , M. KAWAMURA 4 ;<br />
2 Neurosurg., 1 Tokyo Metro Neurolog Hosp, Tokyo 183-0042, Jap<strong>an</strong>; 3 Neurol., Tokyo Ebara<br />
Hosp., Tokyo, Jap<strong>an</strong>; 4 Neurol., Showa Univ. Sch. of Med., Tokyo, Jap<strong>an</strong><br />
Abstract: Background: Subthalamic nucleus deep brain stimulation (STN-DBS) is a valuable<br />
treatment to improve cardinal parkinsoni<strong>an</strong> motor symptoms <strong>an</strong>d adverse symptoms caused by
long-term levodopa <strong>the</strong>rapy. It has been reported that <strong>the</strong> adverse effects of STN-DBS on<br />
cognitive function evaluated by psychological tests are not severe in most patients with<br />
Parkinson's disease. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, divorce <strong>an</strong>d suicidal thoughts are not rare in parkinsoni<strong>an</strong><br />
patients treated by STN-DBS. The relationship between patients <strong>an</strong>d <strong>the</strong>ir relatives or <strong>the</strong> mood<br />
of patients may ch<strong>an</strong>ge following chronic STN-DBS. The emotional cognition of parkinsoni<strong>an</strong><br />
patients treated with STN-DBS was examined by assessing <strong>the</strong>ir emotional ch<strong>an</strong>ges.<br />
Subjects <strong>an</strong>d methods: Twenty patients without STN-DBS as <strong>the</strong> control group <strong>an</strong>d twenty<br />
patients with STN-DBS as <strong>the</strong> STN-DBS group were examined. The STN-DBS group was<br />
examined both under conditions of on-stimulation (on-DBS) <strong>an</strong>d off-stimulation (off-DBS) <strong>from</strong><br />
six months to two years after starting DBS treatment. All <strong>the</strong> patients were examined using<br />
psychological <strong>an</strong>d emotional cognition tests, <strong>an</strong>d UPDRS. (1) Mood <strong>an</strong>d executive cognitive<br />
function were examined using a psychological test battery as follows: SDS, MMSE, AVLT,<br />
WCST, Stroop test, <strong>an</strong>d RCPM. (2) Regarding emotional cognition, six basic emotions<br />
(happiness, sadness, fear, surprise, disgust, <strong>an</strong>d <strong>an</strong>ger) were examined by presenting pictures of<br />
faces, videos of faces, <strong>an</strong>d vocalized short me<strong>an</strong>ingful <strong>an</strong>d me<strong>an</strong>ingless sentences by tape<br />
recorder. Patients were instructed to select one emotion that matched <strong>the</strong> presented item. The<br />
order of examination under <strong>the</strong> condition of on-DBS or off-DBS was determined r<strong>an</strong>dom in each<br />
patient. The examination during <strong>the</strong> condition of off-DBS was per<strong>for</strong>med three hours after<br />
turning off DBS.<br />
Results: Recognition of expression of fear <strong>an</strong>d disgust were reduced in both groups. Percentages<br />
of correct <strong>an</strong>swers <strong>for</strong> <strong>the</strong> items on fear were <strong>the</strong> lowest in both groups <strong>an</strong>d among four tests of<br />
emotional cognition, <strong>an</strong>d were particularly low <strong>for</strong> pictures of faces in <strong>the</strong> patients with on-DBS.<br />
Recognition of fear using pictures was reduced in 60% of <strong>the</strong> condition with on-DBS compared<br />
with off-DBS. Percentages of correct <strong>an</strong>swers of fear did not correlate with o<strong>the</strong>r psychological<br />
results.<br />
Conclusion: A selective reduction of recognition of emotion was observed in patients with<br />
Parkinson’s disease treated with STN-DBS. It is speculated that STN-DBS affects limbic<br />
function.<br />
Disclosures: F. Yokochi, None; M. T<strong>an</strong>iguchi, None; N. Yoshimura, None; M. Kawamura,<br />
None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.15/J25<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies
Title: The effect of bilateral subthalamic deep brain stimulation on visuo-motor sequence<br />
learning <strong>an</strong>d decision making in Parkinson’s disease<br />
Authors: *Y. SHIMO 1 , S. NATORI 1 , G. OYAMA 1 , M. NAKAJIMA 2 , N. HATTORI 1 ;<br />
1 Neurol., Juntendo Univ., Bunkyo-ku/Tokyo, Jap<strong>an</strong>; 2 Neurosurg., Juntendo Univ., Tokyo, Jap<strong>an</strong><br />
Abstract: Background: Parkinson’s disease (PD) patients show a variety of non-motor<br />
symptoms, including mood, cognitive <strong>an</strong>d impulse control disorders. Deep brain stimulation<br />
(DBS) of <strong>the</strong> bilateral subthalamic nucleus (STN) has been widely accepted <strong>for</strong> <strong>the</strong> treatment of<br />
Parkinson’s disease with motor fluctuation, however, it is still in debate <strong>for</strong> <strong>the</strong> efficacy <strong>an</strong>d/or<br />
side-effects to non-motor symptoms. Several studies have been done on <strong>the</strong> effect of STN-DBS<br />
<strong>for</strong> cognitive function, however, <strong>the</strong>re are not m<strong>an</strong>y studies about <strong>the</strong> effect <strong>for</strong> visuo-motor skill<br />
learning <strong>an</strong>d decision making. In previous studies, it has shown that <strong>the</strong> frontal cortex (dorsolateral<br />
prefrontal cortex: DLPFC, pre supplementary motor area: pre-SMA, supplementary motor<br />
area: SMA) <strong>an</strong>d <strong>an</strong>terior part of <strong>the</strong> caudate play import<strong>an</strong>t role in early stage of this visuo-motor<br />
skill learning, <strong>an</strong>d orbito-frontal cortex may play <strong>an</strong> import<strong>an</strong>t role in decision making. In this<br />
report, we assessed <strong>the</strong> effect of <strong>the</strong> STN-DBS on visuo-motor skill learning <strong>an</strong>d decision<br />
making.<br />
Method:<br />
We applied a sequential bottom press task (BPT) (Hikosaka et al 2002) <strong>an</strong>d Iowa gambling task<br />
(IGT) to 15 PD patients be<strong>for</strong>e <strong>an</strong>d 3-4 weeks after bilateral STN-DBS operation. In BPT,<br />
patients per<strong>for</strong>med <strong>the</strong> same sequence <strong>for</strong> three days. We <strong>an</strong>alyzed <strong>the</strong> number of error trials <strong>an</strong>d<br />
<strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce time. We also apply IGT to <strong>the</strong> same patients at <strong>the</strong> same time. The result of<br />
IGT was indicated by <strong>the</strong> number of choosing good deck - choosing bad deck. All patients<br />
showed marked improvement in UPDRS especially in off time period. All tests were done at <strong>the</strong><br />
best on medication be<strong>for</strong>e <strong>the</strong> operation, <strong>an</strong>d STN stimulation ON with optimized medication<br />
after <strong>the</strong> operation.<br />
Results: The score of UPDRS PART 3, which evaluate motor functions of parkinsonism, showed<br />
no difference between be<strong>for</strong>e <strong>an</strong>d after <strong>the</strong> operation during per<strong>for</strong>ming tasks. In BPT, we found<br />
that STN-DBS increased <strong>the</strong> number of error trials (p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.16/J26<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: NIH/NIEHS ESO15777<br />
NIH Gr<strong>an</strong>t P30NS 055077<br />
Title: RNF11 gene expression levels are elevated in Parkinson's disease<br />
Authors: *N. V. DALAL 1 , A. L. ORR 1 , E. L. PRANSKI 1 , D. S. COOPER 1 , H. D. REES 1 , M.<br />
GEARING 1,2 , Y. M. GOZAL 1 , J. J. FRITZ 1 , J. J. LAH 1 , A. I. LEVEY 1 , R. S. BETARBET 1 ;<br />
1 2<br />
Dept of Neurology, Ctr. <strong>for</strong> Neurodegenerative Dis., Dept. of Pathology & Lab. Med.,<br />
EMORY UNIVERSITY, Atl<strong>an</strong>ta, GA<br />
Abstract: Parkinson’s disease (PD) is a slow progressing, neurodegenerative disorder<br />
characterized by selective loss of dopaminergic neurons in <strong>the</strong> subst<strong>an</strong>tia nigra <strong>an</strong>d <strong>the</strong> <strong>for</strong>mation<br />
of cytoplasmic proteinaceous inclusions called Lewy bodies. The underlying cause <strong>for</strong> majority<br />
of PD cases is not known, although both environmental <strong>an</strong>d genetic factors appear to play crucial<br />
roles. The PARK-10 locus on chromosome 1p32 has been linked to <strong>an</strong> increased susceptibility to<br />
PD. We previously reported that <strong>the</strong> PARK-10 locus c<strong>an</strong>didate Ring Finger Protein 11 (RNF11),<br />
<strong>an</strong> E3 ubiquitin ligase, is expressed by vulnerable neurons <strong>an</strong>d localizes to Lewy bodies in<br />
Parkinson’s disease patients. In <strong>the</strong> current study, we fur<strong>the</strong>r explore <strong>the</strong> role of RNF11 in PD<br />
pathology by comparative <strong>an</strong>alysis of RNF11 gene expression levels in postmortem brain tissue<br />
<strong>from</strong> PD <strong>an</strong>d PD with DLB (dementia with Lewy body) cases using TaqM<strong>an</strong> based qu<strong>an</strong>titative<br />
real-time PCR (qrtPCR) by <strong>the</strong> delta-delta CT method. To determine <strong>the</strong> specificity of PDassociated<br />
ch<strong>an</strong>ges in RNF11 expression levels, we included age-matched non-disease controls<br />
as well as cases of o<strong>the</strong>r neurodegenerative diseases such as DLB <strong>an</strong>d Alzheimer’s disease (AD).<br />
Our preliminary <strong>an</strong>alysis included <strong>an</strong> evaluation of 32 c<strong>an</strong>didate “housekeeping” genes <strong>an</strong>d<br />
GUSB was selected as <strong>the</strong> most stable control gene <strong>for</strong> post-mortem brain tissue. The expression<br />
level of RNF11 was qu<strong>an</strong>tified relative to <strong>the</strong> expression of GUSB gene. Analysis of RNF11<br />
mRNA levels revealed a 7-10 fold increase in PD <strong>an</strong>d PD/DLB cases as compared to agematched<br />
AD <strong>an</strong>d controls cases respectively. This specific increase in RNF11 mRNA level in PD<br />
brain tissue suggests a direct link between RNF11 <strong>an</strong>d PD pathogenesis. Fur<strong>the</strong>r investigations<br />
will be necessary to underst<strong>an</strong>d <strong>the</strong> function of RNF11 in Parkinson’s disease associated<br />
neurodegeneration.<br />
Disclosures: N.V. Dalal, None; A.L. Orr, None; E.L. Pr<strong>an</strong>ski, None; D.S. Cooper,<br />
None; H.D. Rees, None; M. Gearing, None; Y.M. Gozal, None; J.J. Fritz, None; J.J. Lah,<br />
None; A.I. Levey, None; R.S. Betarbet, None.<br />
Poster
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.17/J27<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Krembil Foundation<br />
Cure Parkinson's Trust<br />
De<strong>an</strong>'s New staff gr<strong>an</strong>t, University of Toronto<br />
Safra Foundation<br />
Title: A novel mixed monoamine uptake inhibitor, UWA-0121, increases quality <strong>an</strong>d duration of<br />
L-dopa action in <strong>the</strong> MPTP-lesioned primate model of Parkinson’s disease<br />
Authors: *P. HUOT 1 , T. H. JOHNSTON 1 , M. REYES 1 , K. D. LEWIS 2 , S. H. FOX 1 , M. J.<br />
PIGGOTT 2 , J. M. BROTCHIE 1 ;<br />
1 Toronto Western Hosp., Toronto, ON, C<strong>an</strong>ada; 2 Univ. of Western Australia, Perth, Australia<br />
Abstract: Background<br />
L-dopa remains <strong>the</strong> mainstay of treatment <strong>for</strong> <strong>the</strong> motor symptoms of Parkinson’s disease (PD).<br />
However, with increasing duration of <strong>the</strong>rapy, L-dopa use is associated with development of<br />
motor complications, including dyskinesia <strong>an</strong>d wearing-off. Racemic UWA-0101 ((RS)-2-(1,3benzodioxol-5-yl)-1-cyclopropyl-N-methyleth<strong>an</strong>aminium<br />
chloride), a mixed 5HT <strong>an</strong>d dopamine<br />
tr<strong>an</strong>sport inhibitor, increases <strong>the</strong> quality of on-time provided by L-dopa in parkinsoni<strong>an</strong> primates.<br />
In <strong>the</strong> present study, we syn<strong>the</strong>sised <strong>the</strong> R <strong>an</strong>d S en<strong>an</strong>tiomers of UWA-0101, UWA-0121 <strong>an</strong>d<br />
UWA-0122, respectively, <strong>an</strong>d determined <strong>the</strong>ir binding affinities at a r<strong>an</strong>ge of 5HT receptor <strong>an</strong>d<br />
monoamine tr<strong>an</strong>sporter sites. In addition, <strong>the</strong> effects of UWA-0121 on <strong>an</strong>ti-parkinsoni<strong>an</strong> actions<br />
of L-dopa were assessed in <strong>the</strong> MPTP-treated common marmoset.<br />
Methods<br />
The affinity of UWA-0121/2 <strong>for</strong> serotonergic 5HT2A receptors, dopamine (DAT), serotonin<br />
(SERT), <strong>an</strong>d norepinephrine (NET) tr<strong>an</strong>sporters, was assessed using homogenate binding.<br />
[ 3 H]Ket<strong>an</strong>serin (5HT2A), [ 3 H]GBR12935 (DAT), [ 3 H]citalopram (SERT), [ 3 H]nisoxetine (NET)<br />
were <strong>the</strong> radiolig<strong>an</strong>ds.<br />
Six female common marmosets were rendered parkinsoni<strong>an</strong> by injection of MPTP (2.0 mg/kg<br />
s.c. daily, <strong>for</strong> five consecutive days). Motor complications, including dyskinesia <strong>an</strong>d wearing-off,<br />
were induced by twice daily doses of L-dopa/benserazide (15/3.75 mg/kg p.o.) <strong>for</strong> at least one<br />
month. In single acute challenges, L-dopa with benserazide (15/3.75 mg/kg s.c.) was <strong>the</strong>n coadministered<br />
with vehicle or UWA-0121 (1, 3, or 10 mg/kg s.c.). Behaviour, including<br />
parkinsoni<strong>an</strong> disability, duration of on-time, <strong>an</strong>d L-dopa-induced dyskinesias, was assessed over
a 6 h period by a neurologist blinded to treatment.<br />
Results<br />
UWA-0121 displayed high affinity <strong>for</strong> DAT (Ki = 198 nM) <strong>an</strong>d moderate affinity <strong>for</strong> SERT (Ki<br />
= 4.02 µM). UWA121 showed only low affinity <strong>for</strong> <strong>the</strong> 5HT2A receptor (Ki > 90 µM) <strong>an</strong>d o<strong>the</strong>r<br />
sites.<br />
L-dopa in combination with UWA-0121 (10 mg/kg) increased total duration of on-time by 30%<br />
compared to that seen following L-dopa/vehicle (268 vs. 191 min, p < 0.05). UWA-0121 also<br />
increased <strong>the</strong> duration of on-time without dyskinesias by over 300% (81 vs. 25 min, p < 0.05).<br />
UWA-0121 did not increase <strong>the</strong> duration of on-time with non-disabling dyskinesias. At no time<br />
did combination with UWA-0121 increase <strong>the</strong> duration or severity of L-dopa induced dyskinesia.<br />
ConclusionsUWA-0121 may have <strong>the</strong>rapeutic potential in PD by offering <strong>an</strong> opportunity to<br />
increase <strong>the</strong> duration <strong>an</strong>d quality of <strong>an</strong>ti-parkinsoni<strong>an</strong> action of L-dopa. The benefits of agents<br />
such as UWA-0121 may depend on <strong>the</strong>ir relative actions at <strong>the</strong> different monoamine tr<strong>an</strong>sporter<br />
sites.<br />
Disclosures: P. Huot, None; T.H. Johnston, None; M. Reyes, None; K.D. Lewis, None; S.H.<br />
Fox, None; M.J. Piggott, None; J.M. Brotchie, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.18/J28<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Michael J. Fox Foundation<br />
Title: PYM50028, <strong>an</strong> orally active neurotrophic factor inducer, restores <strong>an</strong>d protects nigrostriatal<br />
dopaminergic function following MPTP-administration in a mouse model of Parkinson’s disease<br />
Authors: *T. H. JOHNSTON 1 , J. B. KOPRICH 1 , M. G. REYES 1 , X. S. SUN 1 , T. M.<br />
GIFFORD 1 , J. M. BROTCHIE 1 , P. A. HOWSON 2 ;<br />
1 Atuka Ltd., Toronto, ON, C<strong>an</strong>ada; 2 Phytopharm plc, Godm<strong>an</strong>chester, United Kingdom<br />
Abstract: Objective: This study assessed <strong>the</strong> ability of PYM50028 to reverse dopaminergic loss<br />
after MPTP administration in mice.<br />
Background: PYM50028, administered orally, protects against loss of dopaminergic phenotype<br />
in MPTP-mice, a model of Parkinson’s disease (PD; Vis<strong>an</strong>ji et al. 2008). However, <strong>the</strong> effect of
PYM50028 when given after development of MPTP-induced damage is unknown.<br />
Methods: 10 wk old mice received MPTP (25 mg/kg, i.p.) <strong>for</strong> 5 days (D1-D5). Daily<br />
PYM50028 treatment (10 mg/kg/day, p.o.) was started during (D2), or after MPTP treatment<br />
(D12), <strong>an</strong>d continued until D72. Three extra groups were used; unlesioned mice receiving ei<strong>the</strong>r<br />
vehicle or PYM50028 (D2-72; control mice) or MPTP-lesioned mice receiving vehicle (D2-72).<br />
On D72, 2 h after PYM50028, plasma <strong>an</strong>d brain levels of drug were measured, <strong>an</strong>imals killed<br />
<strong>an</strong>d striatal dopamine tr<strong>an</strong>sporter (DAT) <strong>an</strong>d dopamine (DA) levels qu<strong>an</strong>tified. Stereological<br />
estimates of total nigral (NeuN +ve ) <strong>an</strong>d nigral dopaminergic neuron numbers (TH +ve ) were made.<br />
To define kinetics of DA marker loss using this model, <strong>the</strong> above parameters were assessed in<br />
mice treated with MPTP (D1-D5) <strong>an</strong>d killed on D6, D12 or D24.<br />
Results: Loss of dopaminergic phenotype was apparent <strong>for</strong> striatal DAT <strong>from</strong> D6, <strong>for</strong> DA <strong>from</strong><br />
D12 <strong>an</strong>d TH +ve cells <strong>from</strong> D24 onwards. At no time did this MPTP regimen lead to a signific<strong>an</strong>t<br />
loss of total nigral neuron number (NeuN +ve ). By D72, MPTP decreased DAT, DA <strong>an</strong>d TH +ve<br />
cell counts compared to control mice (veh-veh cf. MPTP-veh; DAT: 436 cf. 158 nCi/g***, DA:<br />
337 cf. 118 pmol/mg***, TH: 2438 cf. 1525 cells*). In MPTP-lesioned mice receiving<br />
PYM50028 (D12 start) <strong>the</strong>re was no difference in TH +ve cells compared to control mice (veh-veh<br />
cf. MPTP-PYM50028); TH: 2438 cf. 1892 cells). Fur<strong>the</strong>rmore, PYM50028 increased DAT <strong>an</strong>d<br />
DA (MPTP-veh cf. MPTP-PYM50028; DAT: 158 cf. 280 nCi/g**, DA: 118 cf. 208 pmol/mg*).<br />
Similar results were obtained in <strong>the</strong> PYM50028 (D2) group. In unlesioned mice PYM50028 did<br />
not alter DAT, DA or TH +ve cell count. Plasma <strong>an</strong>d brain levels of PYM50028 were 413 ng/ml<br />
<strong>an</strong>d 3734 ng/g (D2 <strong>an</strong>imals) <strong>an</strong>d 439 ng/ml <strong>an</strong>d 3894 ng/g (D12 <strong>an</strong>imals) respectively.<br />
Conclusions: The MPTP regimen used causes loss of dopaminergic function but does not kill<br />
nigral neurons. It thus models a population of neurons previously described in hum<strong>an</strong> PD by<br />
Hirsch et al. (1988) as a tr<strong>an</strong>sitional stage prior to death <strong>an</strong>d thus represent <strong>an</strong> attractive target <strong>for</strong><br />
restorative <strong>the</strong>rapies. PYM50028 restores dopaminergic function when given after a deficit has<br />
been established supporting <strong>the</strong> continued development of PYM50028 as a disease-modifying<br />
treatment <strong>for</strong> PD.<br />
References:<br />
Vis<strong>an</strong>ji et al. (2008) FASEB J. 22(7): 2488-97<br />
Hirsch et al. (1988) Nature 28; 334: 345-8<br />
Disclosures: T.H. Johnston, None; J.B. Koprich, None; M.G. Reyes, None; X.S. Sun,<br />
None; T.M. Gif<strong>for</strong>d, None; J.M. Brotchie, None; P.A. Howson, PAH is <strong>an</strong> employee of<br />
Phytopharm plc., A. Employment (full or part-time).<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 530.19/J29<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Krembil Neuroscience Fund<br />
Cure Parkinson's Trust<br />
Title: Anti-dyskinetic effects of <strong>the</strong> selective metabotropic glutamate receptor 5 (mGlu5)<br />
<strong>an</strong>tagonist MTEP in <strong>the</strong> MPTP-lesioned macaque model of Parkinson’s disease<br />
Authors: T. H. JOHNSTON 1,2 , S. H. FOX 3 , M. J. MCILDOWIE 4 , *M. J. PIGGOTT 5 , J. M.<br />
BROTCHIE 1,2 ;<br />
1 Toronto Western Res. Inst., Toronto, ON, C<strong>an</strong>ada; 2 Atuka Ltd., Toronto, ON, C<strong>an</strong>ada;<br />
3 Movement Disorders Clin., Toronto Western Hosp., Toronto, ON, C<strong>an</strong>ada; 4 The Univ. of<br />
Western Australia, Perth, Australia; 5 The Univ. of Western Australia, Perth, WA, Australia<br />
Abstract: Objective: To assess <strong>the</strong> pharmacokinetic profile <strong>an</strong>d ability to reduce L-DOPAinduced<br />
dyskinesia (LID) <strong>an</strong>d to alleviate parkinsoni<strong>an</strong> symptoms, of <strong>the</strong> orally-active, selective<br />
mGlu5 <strong>an</strong>tagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP), in <strong>the</strong> MPTP-lesioned<br />
macaque model of Parkinson’s disease (PD).<br />
Background: Whilst being <strong>the</strong> subject of on-going clinical development in PD patients (e.g.<br />
AFQ056, ADX48621), no data have been published to demonstrate <strong>an</strong> ability of selective mGlu5<br />
<strong>an</strong>tagonism to reduce LID in primates. Here, we assess <strong>the</strong> pharmacokinetic profile <strong>an</strong>d<br />
corresponding effects of MTEP on LID in <strong>the</strong> MPTP-lesioned macaque.<br />
Methods: MTEP hydrochloride was syn<strong>the</strong>sized using a novel methodology as described in<br />
McIldowie et al. (in press). Six parkinsoni<strong>an</strong> cynomolgus monkeys (Macaca fascicularis) were<br />
treated with L-DOPA (25 mg/kg p.o. bid) <strong>for</strong> at least 3 months until <strong>the</strong> development of stable<br />
dyskinesia, typified by mixed chorea <strong>an</strong>d dystonia,. MTEP (4.5 - 36 mg/kg, p.o.), or vehicle, was<br />
administered ei<strong>the</strong>r alone or in combination with L-DOPA (Madopar, 33 ± 1 mg/kg, p.o.).<br />
Total motor activity, which includes both normal <strong>an</strong>d dyskinetic movements, was assessed by<br />
infra-red activity monitoring throughout <strong>the</strong> whole 6h observation period. Anti-parkinsoni<strong>an</strong><br />
action <strong>an</strong>d dyskinesia was assessed by post hoc <strong>an</strong>alysis of recorded behaviour by a movement<br />
disorder neurologist rater (SHF) blinded to treatment. Plasma drug levels were assessed by LC-<br />
MS.<br />
Results: MTEP had no <strong>an</strong>ti-parkinsoni<strong>an</strong> action as mono<strong>the</strong>rapy. However, following<br />
administration of L-DOPA in combination with MTEP (36 mg/kg), total motor activity was<br />
signific<strong>an</strong>tly reduced (by 39% at peak) compared to that following L-DOPA alone. This effect<br />
reflected a 90% reduction in LID (LID score; L-DOPA / vehicle cf. L-DOPA / MTEP, 10 cf. 1,<br />
medi<strong>an</strong> values). MTEP (36 mg/kg) did not reduce peak <strong>an</strong>ti-parkinsoni<strong>an</strong> benefit af<strong>for</strong>ded by L-<br />
DOPA (parkinsoni<strong>an</strong> disability; L-DOPA / vehicle cf. L-DOPA / MTEP, 0 cf. 0). The effects of<br />
MTEP in reducing LID represented a reduction in severity, <strong>from</strong> disabling to non-disabling, or<br />
<strong>an</strong> absence of dyskinesia. MTEP reduced both chorei<strong>for</strong>m <strong>an</strong>d dystonic dyskinesia.<br />
Conclusions: Selective inhibitors of mGlu5 have potential to ameliorate dyskinesia without<br />
compromising, or enh<strong>an</strong>cing, <strong>the</strong> <strong>an</strong>ti-parkinsoni<strong>an</strong> actions of L-DOPA.
Reference: McIldowie MJ et al. (<strong>2009</strong>). Physical <strong>an</strong>d crystallographic characterisation of <strong>the</strong><br />
mGlu5 <strong>an</strong>tagonist, MTEP, <strong>an</strong>d its monohydrochloride. J Pharmaceutical Sci. (in press).<br />
Disclosures: T.H. Johnston, None; S.H. Fox, None; M.J. McIldowie, None; M.J. Piggott,<br />
None; J.M. Brotchie, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.20/J30<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Adolor Corp.<br />
Title: The selective mu-opioid receptor <strong>an</strong>tagonist, ADL5510, reduces L-DOPA induced<br />
dyskinesia, without affecting <strong>an</strong>ti-parkinsoni<strong>an</strong> action, in <strong>the</strong> MPTP macaque model of<br />
Parkinson’s disease<br />
Authors: *J. B. KOPRICH 1 , S. H. FOX 2 , T. H. JOHNSTON 1 , A. J. GOODMAN 3 , B. LE<br />
BOURDONNEC 3 , R. E. DOLLE 3 , R. N. DEHAVEN 3 , D. L. DEHAVEN-HUDKINS 3 , P.<br />
LITTLE 3 , J. M. BROTCHIE 1 ;<br />
1 2<br />
Atuka Ltd, Toronto, ON, C<strong>an</strong>ada; Movement Disorders Clin., Toronto Western Hosp, Toronto,<br />
ON, C<strong>an</strong>ada; 3 Adolor Corp., Exton, PA<br />
Abstract: L-DOPA <strong>the</strong>rapy remains <strong>the</strong> gold st<strong>an</strong>dard <strong>for</strong> alleviating <strong>the</strong> motor symptoms of<br />
Parkinson’s disease (PD). However, over time, disabling dyskinesias develop <strong>for</strong> which no<br />
completely effective treatment is available. We examined <strong>the</strong> potential <strong>for</strong> a selective mu-opioid<br />
receptor (MOR) <strong>an</strong>tagonist (ADL5510) to reduce L-DOPA induced dyskinesia (LID) in <strong>the</strong><br />
MPTP macaque model of PD. Cynomolgus monkeys (Macaca fascicularis), rendered<br />
parkinsoni<strong>an</strong> by repeated administration of MPTP <strong>an</strong>d with established LID, received acute<br />
challenges with L-DOPA (s.c.) in combination with ei<strong>the</strong>r vehicle, ADL5510 (0.1, 1, 3 <strong>an</strong>d 10<br />
mg/kg, p.o.) or with <strong>the</strong> non-subtype selective opioid receptor <strong>an</strong>tagonist naltrexone (1, 3 <strong>an</strong>d 10<br />
mg/kg, s.c.). Immediately following treatments, behavior was monitored <strong>for</strong> six hours;<br />
parameters assessed were total activity, parkinsonism <strong>an</strong>d dyskinesia, rated in a blinded m<strong>an</strong>ner<br />
by a movement disorder neurologist. ADL5510 (1, 3 <strong>an</strong>d 10 mg/kg) reduced activity without<br />
interfering with <strong>the</strong> <strong>an</strong>ti-parkinsoni<strong>an</strong> actions of L-DOPA. ADL5510 reduced LID whe<strong>the</strong>r it<br />
was choreic or dystonic in nature. The <strong>an</strong>ti-dyskinetic effect showed a U-shaped dose-response<br />
relationship, being inactive at 0.1 mg/kg, highly efficacious at 1 <strong>an</strong>d 3 mg/kg (72% <strong>an</strong>d 40%
eductions, respectively, in LID to levels no longer signific<strong>an</strong>tly different <strong>from</strong> vehicle controls),<br />
but less effective at 10 mg/kg. Moreover, because ADL5510 was able to reduce dyskinesia<br />
without compromising <strong>an</strong>ti-parkinsoni<strong>an</strong> benefit, <strong>the</strong>re was <strong>an</strong> improvement in <strong>the</strong> quality of ontime<br />
produced by L-DOPA. Thus, “good quality” on-time, i.e. on-time without disabling<br />
dyskinesia, was increased by 30% <strong>an</strong>d 26%, <strong>for</strong> L-DOPA in combination with 1 mg/kg <strong>an</strong>d 3<br />
mg/kg respectively, compared to L-DOPA alone. Naltrexone had no signific<strong>an</strong>t effect on LID or<br />
on <strong>the</strong> <strong>an</strong>ti-parkinsoni<strong>an</strong> actions of L-DOPA. Selective MOR <strong>an</strong>tagonists thus have signific<strong>an</strong>t<br />
potential to <strong>for</strong>m <strong>the</strong> basis of a novel treatment <strong>for</strong> Parkinson’s disease as <strong>an</strong> adjunct to dopamine<br />
replacement <strong>the</strong>rapy.<br />
Disclosures: J.B. Koprich, None; S.H. Fox, None; T.H. Johnston, None; A.J. Goodm<strong>an</strong>,<br />
Adolor Corp, A. Employment (full or part-time); B. Le Bourdonnec, Adolor Corp, A.<br />
Employment (full or part-time); R.E. Dolle, Adolor Corp, A. Employment (full or part-time);<br />
R.N. DeHaven, Adolor Corp, A. Employment (full or part-time); D.L. DeHaven-Hudkins,<br />
Adolor Corp, A. Employment (full or part-time); P. Little, Adolor Corp, A. Employment (full or<br />
part-time); J.M. Brotchie, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.21/J31<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: NIEHS Gr<strong>an</strong>t ES015777<br />
NIH Gr<strong>an</strong>t P30 NS 055077<br />
Title: Comparative <strong>an</strong>alysis of α-synuclein <strong>an</strong>d RNF11 associated neuropathology in<br />
Parkinson’s disease<br />
Authors: *R. BETARBET 1 , D. N. KARMALI 1 , D. S. COOPER 1 , M. GEARING 1,2 , J. J. LAH 1 ,<br />
A. I. LEVEY 1 , A. L. ORR 1 ;<br />
1 2<br />
Dept of Neurology, Ctr. <strong>for</strong> Neurodegenerative Dis., Dept. of Pathology <strong>an</strong>d Lab. Med., Emory<br />
Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Parkinson’s disease (PD) is a slowly progressive, multisystem neurodegenerative<br />
disorder characterized by both <strong>the</strong> selective loss of dopaminergic neurons in <strong>the</strong> subst<strong>an</strong>tia nigra<br />
<strong>an</strong>d <strong>the</strong> more widespread appear<strong>an</strong>ce of proteinaceous inclusions containing α-synuclein. Braak
et al. (Cell Tissue Res, 318, 121, 2004) have suggested that <strong>the</strong>se synuclein-positive neurites <strong>an</strong>d<br />
inclusions first appear in <strong>the</strong> medulla be<strong>for</strong>e adv<strong>an</strong>cing to <strong>the</strong> pons, midbrain <strong>an</strong>d cerebral cortex.<br />
We have previously reported that RNF11, <strong>an</strong> E3 ubiquitin ligase, localizes to Lewy bodies in PD<br />
brains. To develop a comprehensive underst<strong>an</strong>ding of RNF11-associated neuropathology in PD<br />
patients we per<strong>for</strong>med a comparative <strong>an</strong>alysis of <strong>the</strong> distribution of α-synuclein <strong>an</strong>d RNF11<br />
pathology in brain regions associated with <strong>the</strong> progression of PD. Age-matched control, PD, <strong>an</strong>d<br />
PD/AD (PD with Alzheimer's Disease pathology) brain tissues were obtained <strong>from</strong> Emory’s<br />
brain b<strong>an</strong>k. Adjacent 50-µm thick free-floating sections through <strong>the</strong> brain stem (BSt) regions,<br />
subst<strong>an</strong>tia nigra (SN) <strong>an</strong>d cingulate cortex (Ctx) were processed <strong>for</strong> α-synuclein or RNF11<br />
immunohistochemistry. α-synuclein pathology was revealed using a p<strong>an</strong>-α-synuclein <strong>an</strong>tibody<br />
<strong>an</strong>d <strong>an</strong> <strong>an</strong>tibody specific to oxidized <strong>for</strong>ms of α-synuclein. RNF11 was immunolabeled using <strong>an</strong><br />
<strong>an</strong>tibody against <strong>the</strong> full-length protein. Immunolabeled sections were scored semiqu<strong>an</strong>titatively<br />
<strong>from</strong> 0 to 5, with 5 representing <strong>the</strong> most severe pathology. No α-synuclein- or RNF11-positive<br />
inclusions were detected in control cases used in this study. Extensive (scores=4-5) α-synucleinpositive<br />
Lewy bodies <strong>an</strong>d Lewy neurites were detected in BSt followed by moderate (scores=3-<br />
4) pathology in SN <strong>an</strong>d mild (scores=1-2) pathology in Ctx, in most (7 of 11) but not all PD <strong>an</strong>d<br />
PD/AD cases. Three cases had more extensive α-synuclein pathology in <strong>the</strong> SN <strong>an</strong>d Ctx th<strong>an</strong> in<br />
BSt; one case had mild (1) pathology in all <strong>the</strong> regions examined. The distribution of RNF11<br />
pathology paralleled that of α-synuclein pathology in all 11 cases. However, <strong>the</strong> degree of<br />
RNF11 pathology was less in 5 cases, greater in two cases, <strong>an</strong>d similar in four cases to αsynuclein<br />
pathology. Our data indicate that RNF11-positive neuropathology follows a pattern of<br />
regional distribution similar to PD-related α-synuclein pathology, streng<strong>the</strong>ning <strong>the</strong> association<br />
of RNF11 with PD.<br />
Disclosures: R. Betarbet, None; D.N. Karmali, None; D.S. Cooper, None; M. Gearing,<br />
None; J.J. Lah, None; A.I. Levey, None; A.L. Orr, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.22/J32<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: NIH/NIEHS ES015777<br />
Title: Genetic m<strong>an</strong>ipulations of RNF11 <strong>an</strong>d α-synuclein aggregation
Authors: *E. L. PRANSKI, N. V. DALAL, J. J. LAH, A. I. LEVEY, R. S. BETARBET;<br />
Dept. of Neurology, Ctr. <strong>for</strong> Neurodegenerative Dis., Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is<br />
characterized pathologically by selective loss of dopaminergic neurons in <strong>the</strong> subst<strong>an</strong>tia nigra<br />
<strong>an</strong>d <strong>the</strong> presence of Lewy bodies, protein accumulations containing alpha-synuclein, in <strong>the</strong><br />
cytoplasm of surviving neurons. We have previously demonstrated that RNF11, a c<strong>an</strong>didate gene<br />
at <strong>the</strong> PARK 10 locus, is localized to cortical <strong>an</strong>d nigral Lewy bodies in PD brain tissue. RNF11<br />
has E3 ubiquitin ligase activity that is dependent on <strong>the</strong> RING domain <strong>an</strong>d is believed to have a<br />
role in protein degradation. An impaired protein degradation system, leading to protein<br />
aggregation, has been implicated in PD pathogenesis. In this study, we investigated <strong>the</strong><br />
colocalization of RNF11 <strong>an</strong>d alpha-synuclein in normal conditions <strong>an</strong>d following genetic<br />
m<strong>an</strong>ipulations in a cell culture model. SN4741 cells are mouse midbrain dopaminergic<br />
progenitor cells, <strong>an</strong>d were tr<strong>an</strong>siently tr<strong>an</strong>sfected to <strong>for</strong>m four groups: wild type alpha-synuclein<br />
with wild-type RNF11, alpha-synuclein with <strong>the</strong> A53T mutation with wild-type RNF11, wildtype<br />
alpha-synuclein with <strong>the</strong> RING mut<strong>an</strong>t of RNF11, <strong>an</strong>d alpha-synuclein with <strong>the</strong> A53T<br />
mutation with <strong>the</strong> RING mut<strong>an</strong>t of RNF11. Mutations within <strong>the</strong> RING domain of RNF11 tends<br />
to <strong>for</strong>m aggresomes, while alpha-synuclein with <strong>the</strong> A53T mutation is known to aggregate more<br />
rapidly th<strong>an</strong> wild-type protein. RNF11 <strong>an</strong>d alpha-synuclein colocalization was per<strong>for</strong>med using<br />
confocal images of cells immunofluorescently labeled <strong>for</strong> <strong>the</strong> two proteins. Aggregated alphasynuclein,<br />
both wild-type <strong>an</strong>d A53T mut<strong>an</strong>t, colocalized with wild-type RNF11 as well as with<br />
aggregated, mut<strong>an</strong>t RNF11. Colocalization between A53T alpha-synuclein <strong>an</strong>d RING mut<strong>an</strong>t<br />
RNF11 was more extensive suggesting a possible role of RNF11 in alpha-synuclein aggregation.<br />
Investigating <strong>the</strong> role of RNF11 in alpha-synuclein aggregation <strong>an</strong>d toxicity will be critical in<br />
underst<strong>an</strong>ding its function in PD pathogenesis.<br />
Disclosures: E.L. Pr<strong>an</strong>ski, None; N.V. Dalal, None; J.J. Lah, None; A.I. Levey, None; R.S.<br />
Betarbet, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.23/J33<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: Magistro APTA Foundation Gr<strong>an</strong>t
Title: Ch<strong>an</strong>ges in corticomotor excitability in response to intense exercise are associated with<br />
improved motor per<strong>for</strong>m<strong>an</strong>ce in individuals with Parkinson’s disease<br />
Authors: *Y.-C. CHUNG 1 , B. G. FARLEY 2 , A. D. WU 3 , G. M. PETZINGER 4 , J. YIP 1 , B. E.<br />
FISHER 1 ;<br />
1 Univ. Sou<strong>the</strong>rn Cali<strong>for</strong>nia, Div. Biokinesiol & Physical Therapy, Los Angeles, CA; 2 Dept. of<br />
Physiol., Univ. of Arizona, Tucson, AZ; 3 Dept. of Neurol., Univ. of Cali<strong>for</strong>nia Los Angeles, Los<br />
Angeles, CA; 4 Dept. of Neurol., USC, Los Angeles, CA<br />
Abstract: Background <strong>an</strong>d purpose<br />
Parkinson's disease (PD) is a progressive disease with debilitating motor features including<br />
tremor, generalized slowness, stiffness, bal<strong>an</strong>ce <strong>an</strong>d gait dysfunction. In addition to movement<br />
abnormalities, PD has been shown to alter brain excitability, as measured by tr<strong>an</strong>scr<strong>an</strong>ial<br />
magnetic stimulation. People with PD demonstrate larger resting motor evoked potential (MEP)<br />
amplitude <strong>an</strong>d shorter cortical silent period (CSP) duration when compared to age-matched<br />
controls, suggesting corticomotor (CM) hyper-excitability in PD. We previously demonstrated<br />
that CM hyperexcitability was signific<strong>an</strong>tly correlated with greater disease severity, as measured<br />
by <strong>the</strong> Unified Parkinson’s Disease Rating Scale (UPDRS). Additionally, we demonstrated<br />
normalization of CM excitability in individuals with PD undergoing intense exercise. The<br />
purpose of our ongoing study is to determine whe<strong>the</strong>r ch<strong>an</strong>ges in CM excitability in response to<br />
intense exercise are associated with improved UPDRS scores in individuals with PD.<br />
Methods<br />
To date, twelve individuals within 3 years of PD diagnosis, Hoehn <strong>an</strong>d Yahr stages 1-2, have<br />
been recruited <strong>an</strong>d assigned to ei<strong>the</strong>r <strong>an</strong> exercise or non-exercise group. The exercise group<br />
received 18 1-hour sessions of high intensity locomotor training. Outcome variables measured<br />
pre <strong>an</strong>d post exercise included <strong>the</strong> lateralized Unified Parkinson’s Disease Rating Scales<br />
(UPDRS) <strong>for</strong> <strong>the</strong> more affected side, which consisted of side-specific UPDRS motor items, <strong>an</strong>d<br />
corticomotor excitability (resting MEP) of <strong>the</strong> first dorsal interosseous <strong>an</strong>d tibialis <strong>an</strong>terior<br />
muscles on <strong>the</strong> side of <strong>the</strong> body most affected by PD.<br />
Results <strong>an</strong>d discussion<br />
After 6-weeks of high intensity locomotor exercise, 58% of particip<strong>an</strong>ts demonstrated<br />
improvement in <strong>the</strong> lateralized UPDRS <strong>for</strong> <strong>the</strong> more affected side. Of those with improved<br />
lateralized UPDRS score, 71% demonstrated decreased resting MEP amplitude in both <strong>the</strong> first<br />
dorsal interosseous <strong>an</strong>d tibialis <strong>an</strong>terior.<br />
Conclusion<br />
This finding suggests that improved CM excitability in response to intensive exercise may be<br />
associated with decreased disease severity in individuals with PD. Fur<strong>the</strong>rmore, normalization of<br />
CM excitability may be <strong>an</strong> import<strong>an</strong>t mech<strong>an</strong>ism underlying <strong>the</strong> beneficial effects of exercise in<br />
individuals with PD.<br />
Disclosures: Y. Chung, None; B.G. Farley, None; A.D. Wu, None; G.M. Petzinger, None; J.<br />
Yip, None; B.E. Fisher, None.<br />
Poster
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.24/J34<br />
Topic: C.03.k. Dopamine <strong>an</strong>d non-dopamine pathways<br />
Support: CIBERNED<br />
Title: Reciprocal relationship between <strong>the</strong> striatum bilaterally <strong>an</strong>d compensation of<br />
dopaminergic depletion in early Parkinson’s disease. An 18F-FDOPA PET study<br />
Authors: C. JURI 1 , J. ARBIZU 4 , J. BLESA 1 , M. RODRÍGUEZ-OROZ 1 , J. GURIDI 1 , E.<br />
IGLESIAS 1 , E. PRIETO 4 , J. MARTI 4 , M. COLLANTES 2 , J. RICHTER 4 , I. PEÑUELAS 2 , *J. A.<br />
OBESO 3 ;<br />
1 Neurosciences, 2 MicroPET Unit, 3 CIMA, Pamplona, Spain; 4 Nuclear Med., Clinica<br />
Universitaria. Univ. of Navarra, Pamplona, Spain<br />
Abstract: Parkinson’s disease (PD) is characterized by progressive dopaminergic depletion.<br />
Although numerous dopaminergic compensatory mech<strong>an</strong>isms have been proposed, <strong>the</strong> role in<br />
hum<strong>an</strong>s of contralateral striatal dopaminergic activity in <strong>the</strong> onset <strong>an</strong>d progression of PD remains<br />
elusive. This study assesses <strong>the</strong> bilateral interdependence of striatal innervation in early PD.<br />
Fifteen clinically asymmetric (i.e. unilateral) PD patients were compared with 15 patients with<br />
bilateral motor features paired by age <strong>an</strong>d years of evolution. A group of normal subjects was<br />
used as control. 6-[18F] fluoro-Ldopa (FDOPA) positron emission tomography (PET) studies<br />
were conducted <strong>an</strong>d whole-brain parametric images of specific FDOPA uptake were created with<br />
<strong>an</strong> occipital reference input function to generate <strong>the</strong> const<strong>an</strong>t of decarboxilation (Ki). ROI<br />
<strong>an</strong>alysis was conducted using a fixed object map template after stereotaxic tr<strong>an</strong>s<strong>for</strong>mation of<br />
images into st<strong>an</strong>dard space <strong>an</strong>d voxel based statistical <strong>an</strong>alysis was done by SPM2. For <strong>an</strong>alysis,<br />
<strong>the</strong> images were realigned to compare primarily affected (PA) <strong>an</strong>d secondarily affected (SA)<br />
striatum according to <strong>the</strong> onset of motor symptoms <strong>an</strong>d <strong>an</strong> asymmetry index (AI) was calculated<br />
to evaluate <strong>the</strong> relation between striata . Additionally, to evaluate <strong>the</strong> role of asymmetry in a<br />
progressive MPTP model a surgical lesion was done in <strong>the</strong> nigrostriatal pathway in one monkey,<br />
generating a 30% of asymmetrical dopaminergic depletion measured by PET. Subsequently,<br />
systemic MPTP (0.25 mg/kg each 2 weeks) was progressively administered. Motor test <strong>an</strong>d<br />
video qu<strong>an</strong>tification of movement were done subsequently <strong>an</strong>d FDOPA <strong>an</strong>d 11C-DTBZ PET<br />
were acquired during progression.<br />
The me<strong>an</strong> Ki (*1000) was reduced in PD patients compared to normal controls in both groups in<br />
both sides. Notably, <strong>the</strong>re was no statistically signific<strong>an</strong>t difference in <strong>the</strong> Ki between <strong>the</strong> SA <strong>an</strong>d<br />
<strong>the</strong> PA affected side in both groups. It was evident that all patients with AI over 35% <strong>an</strong>d Ki<br />
higher th<strong>an</strong> 4 in <strong>the</strong> SA putamen were in <strong>the</strong> Unilateral group, while those subjects with AI in <strong>the</strong><br />
lower r<strong>an</strong>ge (AI
progressive dopaminergic depletion preserving <strong>the</strong> striatal asymmetry without revealing motor<br />
abnormalities until <strong>an</strong> adv<strong>an</strong>ced degree of dopaminergic depletion.<br />
Our results suggest that <strong>the</strong> onset of motor symptoms related to dopaminergic depletion is related<br />
to dopaminergic status of contralateral striatum in hum<strong>an</strong>s <strong>an</strong>d non hum<strong>an</strong> primates, providing <strong>an</strong><br />
“in vivo” demonstration of interdependence of both sides in <strong>the</strong> compensation of early PD in<br />
hum<strong>an</strong>s.<br />
Disclosures: C. Juri, None; J. Arbizu, None; J. Blesa, None; M. Rodríguez-Oroz, None; J.<br />
Guridi, None; E. Iglesias, None; E. Prieto, None; J. Marti, None; M. Coll<strong>an</strong>tes, None; J.<br />
Richter, None; I. Peñuelas, None; J.A. Obeso, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.25/J35<br />
Topic: C.03.g. Clinical <strong>the</strong>rapies<br />
Support: Funded by Health Science Faculty <strong>from</strong> <strong>the</strong> University of Ottawa<br />
Title: Modulation of cortico-motor excitability <strong>an</strong>d rehabilitation effect on bal<strong>an</strong>ce after 6 weeks<br />
of virtual reality training with <strong>the</strong> nintendo WII <strong>an</strong>d WII Fit on patients with Parkinson’s disease<br />
Authors: J.-F. ESCULIER 1 , P. BÉRIAULT 1 , J. VAUDRIN 1 , K. GAGNON 1 , *L. E.<br />
TREMBLAY 3,2 ;<br />
2 1 3<br />
Sch. of Rehabil. Sci., Univ. of Ottawa, Ottawa, ON, C<strong>an</strong>ada; Hum<strong>an</strong> Sci., Univ. Du Québec À<br />
Chicoutimi, Chicoutimi, QC, C<strong>an</strong>ada<br />
Abstract: Parkinson’s disease (PD) is known to affect <strong>the</strong> ability to generate movement due to a<br />
decreased activity of <strong>the</strong> basal g<strong>an</strong>glia. The objectives of this study are to examine <strong>the</strong> effects of<br />
a 6 weeks training program using virtual reality <strong>the</strong>rapy with <strong>the</strong> Nintendo WII console <strong>an</strong>d <strong>the</strong><br />
WII fit bal<strong>an</strong>ce board on modulation of corticospinal excitability during observation of action<br />
(OBS), motor imagery (MI) <strong>an</strong>d imitation of action, <strong>an</strong>d to determine if this program c<strong>an</strong><br />
improve <strong>the</strong> bal<strong>an</strong>ce of PD subjects. 20 subjects (11 medicated PD (61.9 ± 11 yrs, me<strong>an</strong> UPDRS<br />
motor score of 2,2) <strong>an</strong>d 9 healthy (HS) (63.5 ± 12 yrs) subjects participated in <strong>the</strong> study.<br />
Corticospinal excitability (CE) was measured using evoked potentials (MEPs) by EMG on motor<br />
points of Quadriceps femoris (QF), Tibialis <strong>an</strong>terior (TA) <strong>an</strong>d Soleus (SOL) muscles <strong>an</strong>d<br />
expressed in % of control values induced by Tr<strong>an</strong>scr<strong>an</strong>ial Magnetic Stimulation (TMS) with<br />
Magstim 200 using a double-c<strong>an</strong>e-coil. Be<strong>for</strong>e <strong>an</strong>d after <strong>the</strong> training program using <strong>the</strong>Wii
console, CE was measured into 5 conditions (1-rest, 2-OBS of static position, 3-OBS of dynamic<br />
action, 4-MI, 5-IMIT ) using a task shown by <strong>the</strong> Wii Fit game. Bal<strong>an</strong>ce <strong>an</strong>d general abilities<br />
were measured with common rehabilitation tests: Activities Specific Bal<strong>an</strong>ce Confidence Scale<br />
(ABC), Community Bal<strong>an</strong>ce <strong>an</strong>d Mobility Scale (CBM), velocity <strong>an</strong>d displacement of center of<br />
gravity using a <strong>for</strong>ce plat<strong>for</strong>m device, Time Up <strong>an</strong>d Go (TUG), Sit to St<strong>an</strong>d Test (STST), Tinetti<br />
Per<strong>for</strong>m<strong>an</strong>ce-Oriented Mobility Assessment (POMA) <strong>an</strong>d 10 meters walk test. PD subjects<br />
presented a signific<strong>an</strong>t deficit of QF in OBS when compared to HS be<strong>for</strong>e treatment (120% ± 18<br />
vs. 154% ± 12), but was improved by <strong>the</strong> training program (148% ± 12 vs. 118% ± 10). They<br />
presented a similar deficit of 45% <strong>for</strong> <strong>the</strong> SOL during MI compared to HS, which was also<br />
improved by <strong>the</strong> Wii training (162% ± 14 vs. 195% ± 80). For <strong>the</strong> IMIT condition, PD presented<br />
a signific<strong>an</strong>t deficit on QF <strong>an</strong>d SOL compared to HS, respectively 396% ± 20 vs. 737% ± 25 <strong>an</strong>d<br />
285% ± 10 vs. 248% ± 9. Training improved TA muscle only on PD (205% ± 15 vs 270% ± 12).<br />
Finally, training effect was shown <strong>for</strong> all three muscles. Results were improved after <strong>the</strong> 6 weeks<br />
program respectively <strong>for</strong> PD <strong>an</strong>d HS on <strong>the</strong> CBM (21% vs 7%), STST (45% vs 22%) <strong>an</strong>d TUG<br />
(23% vs 16%). As revealed by TMS results, <strong>the</strong>re is a good potential to use <strong>the</strong> Nintendo WII<br />
<strong>an</strong>d WII Fit <strong>for</strong> <strong>the</strong> rehabilitation of people affected by PD. This virtual reality equipment c<strong>an</strong><br />
help to rehabilitate mirror neurons system (OBS) <strong>an</strong>d to improve <strong>the</strong> internal model of action<br />
(MI) <strong>an</strong>d <strong>the</strong> cortico-motor recruitment capacity (IMIT). These results demonstrate that using <strong>the</strong><br />
Nintendo Wii <strong>an</strong>d Wii Fit is <strong>an</strong> effective rehabilitative treatment <strong>for</strong> PD patients <strong>an</strong>d aging<br />
subjects.<br />
Disclosures: J. Esculier, None; P. Bériault, None; J. Vaudrin, None; K. Gagnon, None; L.E.<br />
Tremblay, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.26/J36<br />
Topic: C.03.b. Hum<strong>an</strong> studies<br />
Support: Department of Veter<strong>an</strong>s Affairs<br />
Northwest Network Mental Illness Research, Education, <strong>an</strong>d Clinical Center<br />
Title: Tyrosine hydroxylase <strong>an</strong>d dopamine-beta-hydroxylase mRNA expression in <strong>the</strong> LC<br />
noradrenergic neurons in Parkinson's disease
Authors: *P. SZOT 1,2 , S. S. WHITE 1 , A. FRANKLIN 2 , M. A. RASKIND 2,1 ;<br />
1 2<br />
Dept Psychiat & Behav Sci., Univ. Washington, Seattle, WA; Mirecc, VA Puget Sound Hlth.<br />
Care Syst., Seattle, WA<br />
Abstract: Severe degeneration <strong>an</strong>d loss of midbrain subst<strong>an</strong>tia nigra dopaminergic neurons <strong>an</strong>d<br />
<strong>the</strong> result<strong>an</strong>t brain dopaminergic deficiency has been defined as <strong>the</strong> major pathophysiology of<br />
Parkinson’s disease (PD) <strong>an</strong>d cause <strong>the</strong> hallmark motor dysfunction, <strong>the</strong> major symptom.<br />
However, less well recognized <strong>an</strong>d understood is <strong>the</strong> equally extensive degeneration <strong>an</strong>d loss of<br />
<strong>the</strong> pontine locus coeruleus (LC) neurons in PD. The consequence of this neuronal loss in PD is<br />
unknown. However, <strong>the</strong> loss of noradrenergic neurons in PD may precede <strong>the</strong> loss of<br />
dopaminergic neurons. Recently, my laboratory showed that <strong>the</strong> loss of noradrenergic neurons in<br />
<strong>the</strong> LC of Alzheimer’s disease (AD), <strong>an</strong>o<strong>the</strong>r neurodegenerative disorder, results in<br />
compensatory ch<strong>an</strong>ges in <strong>the</strong> surviving neurons. In AD, <strong>the</strong>re is a signific<strong>an</strong>t loss of<br />
noradrenergic neurons in <strong>the</strong> LC, but <strong>the</strong> surviving neurons demonstrate a signific<strong>an</strong>t increase in<br />
mRNA <strong>for</strong> tyrosine hydroxylase (TH), <strong>the</strong> rate limiting enzyme in <strong>the</strong> syn<strong>the</strong>sis of NE. The goal<br />
of this study is to determine if similar compensatory ch<strong>an</strong>ges are observed in <strong>the</strong> LC of PD<br />
subjects. LC control (n=8) <strong>an</strong>d PD (n=6) tissue was obtained <strong>from</strong> University of Washington<br />
Alzheimer’s disease Research Center, University of S<strong>an</strong> Diego, Emory <strong>an</strong>d Brain <strong>an</strong>d Tissue<br />
B<strong>an</strong>k <strong>for</strong> Developmental Disorders. TH <strong>an</strong>d dopamine β-hydroxylase (DBH) mRNA was<br />
measured at two different levels of <strong>the</strong> LC in control <strong>an</strong>d PD subjects using in situ hybridization.<br />
The number of neurons positively labeled <strong>an</strong>d <strong>the</strong> amount per neurons was determined <strong>for</strong> both<br />
in situ probes in each subject. Both TH <strong>an</strong>d DBH expression indicate <strong>an</strong> approximate 90%<br />
neuronal loss at <strong>the</strong> mid-LC region <strong>an</strong>d <strong>an</strong> approximate 70% neuronal loss at <strong>the</strong> caudal portion<br />
of <strong>the</strong> LC. The loss of noradrenergic neurons is greater in PD subjects th<strong>an</strong> what was observed in<br />
AD subjects. The surviving noradrenergic neurons in <strong>the</strong> LC of PD subjects also do not<br />
demonstrate compensatory expression of TH mRNA, but it does result in <strong>an</strong> increase in DBH<br />
mRNA. The consequence of <strong>an</strong> increased DBH mRNA upon <strong>the</strong> syn<strong>the</strong>sis of NE is unclear since<br />
DBH is not <strong>the</strong> rate-limiting enzyme. What <strong>the</strong>se results do indicate is that <strong>the</strong> loss of<br />
noradrenergic neurons in PD subjects results in a different response of <strong>the</strong> surviving neurons th<strong>an</strong><br />
what is observed in AD.<br />
Disclosures: P. Szot , None; S.S. White, None; A. Fr<strong>an</strong>klin, None; M.A. Raskind, None.<br />
Poster<br />
530. Parkinson's Disease: Hum<strong>an</strong> Related<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 530.27/J37<br />
Topic: C.03.b. Hum<strong>an</strong> studies
Support: NIH Gr<strong>an</strong>t 5T32GM007507<br />
NIH T32 AG000213<br />
Title: The neurophysiological effects of medication <strong>an</strong>d ef<strong>for</strong>t during bim<strong>an</strong>ual symmetrical<br />
index finger tapping in Parkinson’s disease<br />
Authors: *Y. GULLER 1,2 , D. G. MCLAREN 5,1,3 , S. T. WITT 6,2 , J. C. LAZARUS 4 , M. E.<br />
MEYERAND 2 ;<br />
1 Neurosci. Training Program, 2 Dept. of Med. Physics, 3 Dept. of Med., 4 Dept. of Kinesiology,<br />
Univ. of Wisconsin, Madison, WI; 5 Geriatric Res. Educ. <strong>an</strong>d Clin. Ctr., William S. Middleton<br />
Mem. Veter<strong>an</strong>s Hosp., Madison, WI; 6 Olin Neuropsychiatry Res. Ctr., Hart<strong>for</strong>d, CT<br />
Abstract: Introduction<br />
Parkinson’s disease (PD) is a progressive neurodegenerative disorder primarily characterized by<br />
motor impairments such as bradykinesia <strong>an</strong>d resting tremor. Difficulties in pl<strong>an</strong>ning <strong>an</strong>d<br />
initiating movements are some of <strong>the</strong> more disabling symptoms of PD. However, if patients<br />
attempt to put ef<strong>for</strong>t into specific components of movement, <strong>the</strong>y are able to increase <strong>the</strong><br />
amplitude <strong>an</strong>d coordination of <strong>the</strong> movement. This suggests that PD motor deficits may be more<br />
related to ef<strong>for</strong>t th<strong>an</strong> motor ability.<br />
We hypo<strong>the</strong>sized that <strong>the</strong> amount of ef<strong>for</strong>t PD patients exerted during bim<strong>an</strong>ual symmetrical<br />
finger tapping would affect <strong>the</strong> blood oxygen level dependent (BOLD) response in areas<br />
responsible <strong>for</strong> pl<strong>an</strong>ning <strong>an</strong>d initiating movement. Secondly, we investigated <strong>the</strong> effect of<br />
dopamine (DA) treatment on <strong>the</strong> task-related BOLD responses.<br />
Methods<br />
Functional magnetic reson<strong>an</strong>ce imaging sc<strong>an</strong>s (1.5 Tesla, Signa LX; GE Medical Systems) were<br />
acquired in 8 right-h<strong>an</strong>ded, left-side affected PD patients (6 female) while per<strong>for</strong>ming a bim<strong>an</strong>ual<br />
symmetrical index finger tapping task with <strong>an</strong>d without ef<strong>for</strong>t. Each task was per<strong>for</strong>med in<br />
separate runs consisting of eleven 20 second blocks alternating between tapping <strong>an</strong>d rest<br />
conditions. Patients were sc<strong>an</strong>ned in two sessions on different days: one while on medication <strong>an</strong>d<br />
one after <strong>an</strong> 8-12 hour washout period.<br />
Results<br />
<strong>When</strong> patients were on medication, ef<strong>for</strong>tful finger tapping evoked a larger response th<strong>an</strong> nonef<strong>for</strong>tful<br />
finger tapping in <strong>the</strong> left frontal inferior operculum, left supramarginal gyrus, tri<strong>an</strong>gular<br />
part of <strong>the</strong> inferior frontal gyrus, <strong>an</strong>d right middle frontal area. Signific<strong>an</strong>t differences were not<br />
observed between ef<strong>for</strong>tful <strong>an</strong>d non-ef<strong>for</strong>tful tasks after <strong>the</strong> washout period. Compared to <strong>the</strong><br />
washout period, medication caused greater BOLD activity during ef<strong>for</strong>tful finger tapping in right<br />
primary motor cortex, right supramarginal gyrus, <strong>an</strong>d right frontal middle area. All statistics were<br />
per<strong>for</strong>med at P
in pl<strong>an</strong>ning <strong>an</strong>d initiating movement <strong>an</strong>d potentially explains <strong>the</strong> decreases in patients’ difficulty<br />
in initiating bim<strong>an</strong>ual movement when on medication.<br />
Disclosures: Y. Guller, None; D.G. McLaren, None; S.T. Witt, None; J.C. Lazarus,<br />
None; M.E. Meyer<strong>an</strong>d, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.1/J38<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Michael J. Fox Foundation<br />
NINDS<br />
NIA<br />
New York Stem Cell Foundation<br />
K<strong>an</strong>ae Foundation <strong>for</strong> <strong>the</strong> Promotion of Medical Science<br />
Research Foundation ITSUU Laboratory<br />
Title: Atg7-deficient dopaminergic neurons display tau pathology <strong>an</strong>d neurodegeneration<br />
Authors: *K. INOUE 1 , J. RISPOLI 1 , A. ABELIOVICH 2 ;<br />
1 Pathology & Cell Biol., Col. of Physici<strong>an</strong>s & Surgeons, Columbia Univ., New York, NY;<br />
2 Departments of Pathology <strong>an</strong>d Neurology, Taub Institute, Columbia Univ., New York, NY<br />
Abstract: Abstract<br />
Macroautophagy (herein autophagy) is <strong>an</strong> evolutionarily conserved mech<strong>an</strong>ism <strong>for</strong> bulk<br />
intracellular degradation of proteins <strong>an</strong>d org<strong>an</strong>elles. Pathological studies have implicated<br />
defective autophagy in neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s<br />
disease, <strong>an</strong>d tauopathies, but <strong>the</strong> causal relationship remains to be established. Here we generate<br />
mice deficient in <strong>an</strong> essential autophagy component, Atg7, within mature CNS neuron<br />
populations that are associated with <strong>the</strong>se hum<strong>an</strong> neurodegenerative syndromes, including<br />
midbrain dopamine <strong>an</strong>d hippocampal pyramidal neurons. In <strong>the</strong>se cell populations, autophagy
deficiency leads specifically to tauopathy in <strong>the</strong> absence of synucleinopathy or o<strong>the</strong>r<br />
degenerative findings. Activated GSK3β, a downstream effector of <strong>the</strong> PI3 kinase signaling<br />
pathway <strong>an</strong>d a major tau kinase, co-localizes with phospho-tau inclusions in Atg7-deficient<br />
neurons. Fur<strong>the</strong>rmore, genetic induction of <strong>the</strong> PI3 kinase signaling cascade suppresses<br />
neurodegeneration in <strong>the</strong> context of Atg7 deficiency. These data implicate autophagy deficiency<br />
in tauopathies, <strong>an</strong>d suggest PI3K pathway activation as a <strong>the</strong>rapeutic strategy.<br />
Disclosures: K. Inoue, None; J. Rispoli, None; A. Abeliovich, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.2/K1<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Sp<strong>an</strong>ish Ministerio de S<strong>an</strong>idad y Consumo, # PI07-1073,<br />
<strong>an</strong>d by PNSD<br />
Title: Genetic inactivation of dopamine D1, not D2 receptors inhibits L-DOPA-induced<br />
dyskinesia <strong>an</strong>d chromatin modification<br />
Authors: *R. MORATALLA, S. DARMOPIL, I. RUIZ DE DIEGO, S. ARES, N. GRANADO,<br />
T. LIU;<br />
Cajal Institute, CSIC, Madrid, Spain<br />
Abstract: Pharmacological studies have implicated dopamine D1-like receptors in <strong>the</strong><br />
development of L-DOPA-induced dyskinesias <strong>an</strong>d associated molecular ch<strong>an</strong>ges in<br />
hemiparkinsoni<strong>an</strong> mice. However, pharmacological agents <strong>for</strong> D1 or D2 receptors also recognize<br />
o<strong>the</strong>r receptor family members. Genetic inactivation of ei<strong>the</strong>r <strong>the</strong> dopamine D1 or D2 receptor<br />
was used to define <strong>the</strong> involvement of <strong>the</strong>se receptor subtypes. Dyskinesias were assessed during<br />
a three-week period after daily treatment with 25mg/kg L-DOPA. Ch<strong>an</strong>ges in expression of<br />
signaling molecules <strong>an</strong>d o<strong>the</strong>r proteins in <strong>the</strong> lesioned striatum were examined<br />
immunohistochemically, 2 h after <strong>the</strong> last L-DOPA injection. Chronic L-DOPA treatment<br />
gradually induced rotational behavior <strong>an</strong>d dyskinesia in wild type hemiparkinsoni<strong>an</strong> mice.<br />
Dyskinetic symptoms were associated with increased FosB <strong>an</strong>d dynorphin expression,<br />
phosphorylation of ERK <strong>an</strong>d phosphoacetylation of histone 3 (H3) in <strong>the</strong> lesioned striatum.<br />
These molecular ch<strong>an</strong>ges were restricted to striatal areas with complete dopaminergic
denervation <strong>an</strong>d occurred only in dynorphin-containing neurons of <strong>the</strong> direct pathway. D1<br />
receptor inactivation completely abolished L-DOPA-induced dyskinesias <strong>an</strong>d <strong>the</strong> associated<br />
molecular ch<strong>an</strong>ges. Inactivation of <strong>the</strong> D2 receptor had no signific<strong>an</strong>t effect on <strong>the</strong> behavioral or<br />
molecular response to chronic L-DOPA. Our results demonstrate that <strong>the</strong> dopamine D1 receptor<br />
is critical <strong>for</strong> <strong>the</strong> development of L-DOPA-induced dyskinesias in mice <strong>an</strong>d in <strong>the</strong> underlying<br />
molecular ch<strong>an</strong>ges in <strong>the</strong> denervated striatum, <strong>an</strong>d that <strong>the</strong> D2 receptor has little or no<br />
involvement. In addition, we demonstrate that H3 phosphoacetylation, a read out <strong>for</strong> chromatin<br />
modification, is blocked by D1 receptor inactivation, suggesting that inhibitors of H3 acetylation<br />
<strong>an</strong>d/or phosphorylation may be useful in preventing or reversing dyskinesia <strong>an</strong>d <strong>the</strong> chromatin<br />
modification involved. Funded by <strong>the</strong> Sp<strong>an</strong>ish Ministerio de S<strong>an</strong>idad y Consumo, # PI07-1073,<br />
<strong>an</strong>d by PNSD<br />
Disclosures: R. Moratalla, None; S. Darmopil, None; I. Ruiz de Diego, None; S. Ares,<br />
None; N. Gr<strong>an</strong>ado, None; T. Liu, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.3/K2<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIA PO1 AG023630<br />
Title: Repeated lipopolysaccaride exposure during adulthood exacerbates <strong>the</strong> accelerated decline<br />
in motor function <strong>an</strong>d nigrostriatal dopamine system in GDNF heterozygous mice<br />
Authors: *H. A. BOGER, C. D. UMPHLET, L. D. MIDDAUGH, A.-C. GRANHOLM;<br />
Neurosciences, Med. Univ. of South Carolina, Charleston, SC<br />
Abstract: Inflammation has been implicated in <strong>the</strong> pathology of several neurodegenerative<br />
diseases, including Parkinson’s disease (PD). Inflammation is thought to contribute to <strong>the</strong> supply<br />
of neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF), by<br />
increasing its expression/excretion <strong>from</strong> activated microglia/astrocytes. Studies using <strong>the</strong><br />
endotoxin lipopolysaccharide (LPS), a potent inflammogen, show that systemic insults c<strong>an</strong><br />
trigger prolonged microglial activation <strong>an</strong>d pro-inflammatory cytokine production leading to<br />
delayed <strong>an</strong>d progressive death of subst<strong>an</strong>tia nigra (SN) dopamine (DA) neurons mimicking<br />
idiopathic PD. Previous studies <strong>from</strong> our laboratory demonstrate that mice with a partial loss of<br />
GDNF (GDNF +/- ) have increased neuroinflammation <strong>an</strong>d phosho-p38 MAPK as early as 3
months of age, with no differences in <strong>the</strong> nigrostriatal DA system. There<strong>for</strong>e, <strong>the</strong> focus of <strong>the</strong><br />
current study was to determine if <strong>the</strong> heightened state of inflammation present in GDNF +/- mice<br />
exacerbates <strong>the</strong> DAergic toxicity associated with repeated inflammatory-induced administration<br />
of LPS. To test this hypo<strong>the</strong>sis, we administered LPS (1mg/kg, i.p.) once a month <strong>for</strong> 3 months<br />
starting at 7 months of age to male wildtype (WT) <strong>an</strong>d GDNF +/- mice. One month following <strong>the</strong><br />
last administration, LPS-treated GDNF +/- mice demonstrated a signific<strong>an</strong>t reduction in locomotor<br />
activity compared to LPS-treated WT mice <strong>an</strong>d saline-treated GDNF +/- mice. Fur<strong>the</strong>rmore, LPStreated<br />
GDNF +/- mice displayed <strong>the</strong> greatest reduction of SN tyrosine hydroxylase-positive<br />
neurons compared to <strong>the</strong> o<strong>the</strong>r treatment groups. Follow-up <strong>an</strong>alysis is underway to determine<br />
<strong>the</strong> inflammatory response to LPS in GDNF +/- mice <strong>an</strong>d <strong>the</strong> relationship between <strong>the</strong> heightened<br />
inflammation <strong>an</strong>d DAergic damage observed in <strong>the</strong>se mice. The data presented thus far<br />
demonstrate a potential role of inflammation in <strong>the</strong> exacerbated DAergic damage observed as a<br />
result of a partial loss of GDNF. This work is supported by NIA PO1 AG023630.<br />
Disclosures: H.A. Boger, None; C.D. Umphlet, None; L.D. Middaugh, None; A. Gr<strong>an</strong>holm,<br />
None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.4/K3<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Swedish Research Council Gr<strong>an</strong>t 20715, 13482 <strong>an</strong>d 14862<br />
Parkinson Foundation in Sweden<br />
Hjärnfonden<br />
The Wenner-Gren Foundations<br />
USAMRAA/DOD Gr<strong>an</strong>ts<br />
Title: L-DOPA activates ERK signaling <strong>an</strong>d phosphorylates histone H3 in <strong>the</strong> striatonigral<br />
medium spiny neurons of hemiparkinsoni<strong>an</strong> mice<br />
Authors: *E. SANTINI 1 , C. ALCACER 2,4,5 , S. CACCIATORE 1 , M. HEIMAN 6 , D.<br />
HERVÉ 2,4,5 , P. GREENGARD 6 , J.-A. GIRAULT 2,4,5 , E. VALJENT 1,3,4,5 , G. FISONE 1 ;
1 Karolinska Inst., Stockholm, Sweden; 2 UMR-S839, 3 Umr-s839, INSERM, Paris, Fr<strong>an</strong>ce; 4 Univ.<br />
Pierre et Marie Curie, Paris, Fr<strong>an</strong>ce; 5 Inst. du Fer à Moulin, Paris, Fr<strong>an</strong>ce; 6 The Rockefeller<br />
Univ., New York, NY<br />
Abstract: Administration of <strong>the</strong> <strong>an</strong>tiparkinsoni<strong>an</strong> drug, L-DOPA, produces <strong>an</strong> abnormal<br />
activation of <strong>the</strong> extracellular signal-regulated kinase (ERK) tr<strong>an</strong>sduction cascade, in <strong>the</strong><br />
dopamine-depleted striatum. Here, we have examined <strong>the</strong> effect produced by L-DOPA on ERK<br />
signaling at <strong>the</strong> level of <strong>the</strong> two major populations of striatal neurons, <strong>the</strong> mediums spiny<br />
neurons (MSNs) of <strong>the</strong> striatopallidal <strong>an</strong>d striatonigral pathway. To this purpose, we have<br />
employed tr<strong>an</strong>sgenic mice expressing EGFP under <strong>the</strong> control of <strong>the</strong> promoter <strong>for</strong> <strong>the</strong> dopamine<br />
D2 receptor (D2R) (D2dr-EGFP mice) or <strong>the</strong> dopamine D1 receptor (Drd1a-EGFP mice), which<br />
are expressed in striatopallidal <strong>an</strong>d striatonigral MSNs, respectively. We show that, in 6-OHDAlesioned<br />
mice, L-DOPA increases <strong>the</strong> state of phosphorylation of ERK, <strong>an</strong>d of its nuclear target,<br />
mitogen- <strong>an</strong>d stress-activated kinase 1 (MSK1), selectively in striatonigral MSNs. An identical<br />
distribution is observed at <strong>the</strong> level of <strong>the</strong> phosphorylation of <strong>the</strong> acetylated <strong>for</strong>m of histone H3,<br />
which is a major substrate <strong>for</strong> MSK1. The effect of L-DOPA is prevented by blockade of<br />
dopamine D1 receptors (D1Rs), which are specifically expressed in striatonigral MSNs. The<br />
same pattern of activation of ERK signaling is maintained even after repeated administration of<br />
L-DOPA, which results in <strong>the</strong> development of abnormal involuntary movements, or dyskinesia.<br />
Overall, <strong>the</strong>se results indicate that, in a mouse model of Parkinson’s disease, acute or repeated<br />
administration of L-DOPA activates ERK specifically in striatonigral MSNs <strong>an</strong>d that this effect<br />
is accomp<strong>an</strong>ied by phosphorylation of MSK1 <strong>an</strong>d histone H3 in <strong>the</strong> same neuronal cell type. This<br />
abnormal regulation may result in nucleosomal responses linked to ch<strong>an</strong>ges in gene expression<br />
<strong>an</strong>d ultimately to <strong>the</strong> development of L-DOPA-induced dyskinesia.<br />
Disclosures: E. S<strong>an</strong>tini, None; C. Alcacer, None; S. Cacciatore, None; M. Heim<strong>an</strong>, None; D.<br />
Hervé, None; P. Greengard, None; J. Girault, None; E. Valjent, None; G. Fisone, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.5/K4<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: P50 NS38367<br />
PO1 ES016732
Title: Dietary selenium deficiency leads to nigrostriatal dysfunction in DJ-1 knockout mice<br />
Authors: *F. MORTAZAVI 1 , S. M. FLEMING 1 , F. NIK-AHD 1 , C. K. MULLIGAN 1 , P. C.<br />
SIOSHANSI 1 , M.-F. CHESSELET 1,2 ;<br />
1 Dept Neurol, 2 Neurobio, UCLA, Los Angeles, CA<br />
Abstract: The etiology of Parkinson’s disease (PD) remains uncertain, however, a large amount<br />
of evidence implicates a role <strong>for</strong> mitochondrial dysfunction <strong>an</strong>d oxidative stress in contributing<br />
to <strong>the</strong> selective vulnerability of dopaminergic neurons. A loss-of-function mutation in <strong>the</strong> DJ-1<br />
gene has been linked to autosomal recessive early-onset parkinsonism. Surprisingly, mice with<br />
similar loss-of-function mutations in DJ-1 do not show age-dependent loss of nigral<br />
dopaminergic neurons, but have increased dopamine tr<strong>an</strong>sporter (DAT) expression in <strong>the</strong><br />
striatum. DJ-1 is a putative <strong>an</strong>ti-apoptotic protein <strong>an</strong>d has <strong>an</strong>ti-oxid<strong>an</strong>t properties. Selenium is <strong>an</strong><br />
essential constituent of a number of enzymes, some of which have potent <strong>an</strong>tioxid<strong>an</strong>t functions.<br />
Selenium deficiency has been shown to increase dopamine turnover <strong>an</strong>d upregulate tyrosine<br />
hydroxylase (TH) <strong>an</strong>d c<strong>an</strong> lead to <strong>an</strong> increase in oxidative stress. The goal of <strong>the</strong> present study<br />
was to determine whe<strong>the</strong>r dietary selenium deficiency combined with <strong>the</strong> loss of function of DJ-<br />
1 has deleterious effects on <strong>the</strong> nigrostrial pathway. DJ-1 KO <strong>an</strong>d wildtype (WT) littermates<br />
were fed selenium deficient or control diet <strong>from</strong> 2 months to 14 months of age. Mice were<br />
weighed <strong>an</strong>d tested <strong>for</strong> spont<strong>an</strong>eous motor activity in <strong>the</strong> cylinder at 14 months, followed by<br />
intracardiac perfusion <strong>for</strong> immunohistochemical <strong>an</strong>alysis. Selenium deficiency resulted in<br />
reduced body weights in DJ-1 KO mice but not WT mice. Both DJ-1 KO <strong>an</strong>d WT mice exposed<br />
to selenium-deficient diet showed reduced <strong>for</strong>elimb <strong>an</strong>d hindlimb stepping, <strong>an</strong>d rearing in <strong>the</strong><br />
cylinder, however <strong>the</strong> effects were more pronounced in <strong>the</strong> DJ-1 KO mice. TH<br />
immunofluorescence in <strong>the</strong> striatum was increased in DJ-1 KO mice compared to WT<br />
littermates; this effect was abolished in DJ-1 KO mice exposed to <strong>the</strong> selenium deficient diet.<br />
Unbiased stereological <strong>an</strong>alysis of TH+ neurons in <strong>the</strong> subst<strong>an</strong>tia nigra compacta is currently<br />
underway. Taken toge<strong>the</strong>r, <strong>the</strong> results suggest that nutritional factors, especially <strong>the</strong> level of<br />
<strong>an</strong>tioxid<strong>an</strong>t in <strong>the</strong> diet, modulate <strong>the</strong> effects of DJ-1 deficiency on <strong>the</strong> nigrostriatal dopaminergic<br />
neurons.<br />
Disclosures: F. Mortazavi, None; S.M. Fleming, None; F. Nik-Ahd, None; C.K. Mullig<strong>an</strong>,<br />
None; P.C. Siosh<strong>an</strong>si, None; M. Chesselet, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.6/K5
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Title: Targeted increase of dopamine levels in <strong>the</strong> subst<strong>an</strong>tia nigra of A53T α-synuclein<br />
tr<strong>an</strong>sgenic mice<br />
Authors: *E. TSIKA 1,2 , J. R. MAZZULLI 3 , J. H. WOLFE 1,4,5 , R. SANDALTZOPOULOS 2 , H.<br />
ISCHIROPOULOS 1,5 ;<br />
1 Childrens Hosp. Philadephia, Philadelphia, PA; 2 Mol. Biol. <strong>an</strong>d Genet., Democritus Univ. of<br />
Thrace, Alex<strong>an</strong>droupolis, Greece; 3 Neurology, Massachusetts Gen. Hosp., Harvard Med. School,<br />
MassGeneral Inst. <strong>for</strong> Neurodegenerative Dis. (MIND), Charlestown, MA; 4 Dept. of<br />
Pathobiology, Sch. of Vet. Med., Philadelphia, PA; 5 Pediatrics <strong>an</strong>d Pharmacol., Sch. of<br />
Medicine, Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Accumulation of α-synuclein (α-syn) positive inclusions <strong>an</strong>d <strong>the</strong> preferential loss of<br />
<strong>the</strong> dopaminergic neurons of <strong>the</strong> subst<strong>an</strong>tia nigra are features of Parkinson’s disease (PD). Even<br />
though α-syn aggregation is considered a key event in <strong>the</strong> development of <strong>the</strong> disease, <strong>the</strong><br />
molecular processes that affect it are not thoroughly defined. Studies in cell free <strong>an</strong>d cellular<br />
model systems have described that dopamine <strong>an</strong>d its metabolic products c<strong>an</strong> modulate <strong>the</strong><br />
polymerization process of α-syn. In particular, oxidized catechols appear to arrest <strong>the</strong> fibrillation<br />
process of α-syn, while stabilizing triton-soluble higher molecular weight species of α-syn. The<br />
purpose of this study is to describe this interaction in vivo. To this end we have used tr<strong>an</strong>sgenic<br />
mice that overexpress <strong>the</strong> disease associated mutated <strong>for</strong>m of α-syn, A53T, under <strong>the</strong> mouse<br />
prion promoter; a broadly used <strong>an</strong>d extensively characterized model of α-synucleinopathies. In<br />
order to study <strong>the</strong> interaction of <strong>the</strong> protein with dopamine we have utilized a lentiviral vector to<br />
deliver <strong>the</strong> coding sequence of tyrosine hydroxylase (TH) into <strong>the</strong> dopaminergic neurons of <strong>the</strong><br />
subst<strong>an</strong>tia nigra. Specifically, tr<strong>an</strong>sduction with a mut<strong>an</strong>t <strong>for</strong>m of TH, where Arg37-Arg38 is<br />
replaced by Glu37-Glu38 (TH(RREE)) that lacks dopamine feedback inhibition, led to increased<br />
concentration of dopamine in <strong>the</strong> striatum of <strong>the</strong>se mice. Even though <strong>the</strong> behavioral assessment<br />
<strong>an</strong>d <strong>the</strong> age of phenotype onset are not ch<strong>an</strong>ged due to <strong>the</strong> increased levels of dopamine, <strong>the</strong><br />
biochemical <strong>an</strong>alysis has verified accumulation of distinct triton soluble higher molecular weight<br />
α-syn species in <strong>the</strong> nigra of <strong>the</strong>se mice along with <strong>the</strong> presence of insoluble α-syn.<br />
Immunohistochemical <strong>an</strong>alysis <strong>for</strong> selective cell death, presence of α-syn positive inclusions, <strong>an</strong>d<br />
o<strong>the</strong>r pathological ch<strong>an</strong>ges were also per<strong>for</strong>med. The results of this study indicate that targeted<br />
increase of dopamine in <strong>the</strong> nigral neurons may profoundly alter α-syn aggregation.<br />
Disclosures: E. Tsika, None; J.R. Mazzulli, None; J.H. Wolfe, None; R. S<strong>an</strong>daltzopoulos,<br />
None; H. Ischiropoulos, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.7/K6<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: JCCM Gr<strong>an</strong>t PI-2006/15<br />
JCCM Gr<strong>an</strong>t PCC08-0064<br />
Title: α-synucleinopathy in <strong>the</strong> olfactory system in Parkinson’s tr<strong>an</strong>sgenic mouse model<br />
(B6C3H-Tg SNCA83Vle/J)<br />
Authors: *I. UBEDA-BANON 1 , D. SAIZ-SANCHEZ 1 , C. DE LA ROSA-PRIETO 1 , L.<br />
ARGANDOÑA-PALACIOS 2 , S. ARGANDONA-PALACIOS 2 , A. MARTINEZ-MARCOS 1 ;<br />
1 2<br />
Univ. Castilla-La M<strong>an</strong>cha, Albacete, Spain; Complejo Hospitalario de Albacete, Servicio de<br />
Neurologia, Albacete, Spain<br />
Abstract: Idiopathic Parkinson Disease is characterized by a preclinical/premotor phase that<br />
includes symptoms such as hyposmia. This dysfunction has a neuropathological correlate.<br />
Pathological accumulations of α-synuclein (α-synucleinopathy) c<strong>an</strong> be observed in <strong>the</strong> hum<strong>an</strong><br />
olfactory bulb <strong>an</strong>d <strong>an</strong>terior olfactory nucleus in Braak’s stage 1; whereas it does not appear in <strong>the</strong><br />
subst<strong>an</strong>tia nigra up to Braak’s stage 3. This work tries to investigate <strong>the</strong> distribution of αsynucleinopathy<br />
in <strong>the</strong> olfactory system of a Parkinson’s tr<strong>an</strong>sgenic mouse model (B6C3H-Tg<br />
SNCA83Vle/J, The Jackson Laboratory). The olfactory bulb, <strong>an</strong>terior olfactory nucleus,<br />
olfactory tubercle <strong>an</strong>d piri<strong>for</strong>m, amygdaloid <strong>an</strong>d entorhinal cortices have been <strong>an</strong>alyzed at 2, 4, 6,<br />
8 <strong>an</strong>d 10 months of age in homozygote <strong>an</strong>imals <strong>an</strong>d compared with <strong>the</strong> corresponding controls.<br />
Experimental procedures were carried out according to <strong>the</strong> guidelines of <strong>the</strong> Europe<strong>an</strong><br />
Community on Welfare of Research Animals (Directive 86/69/EEC) <strong>an</strong>d <strong>the</strong> Ethical Committee<br />
of Animal Research of <strong>the</strong> University of Castilla-La M<strong>an</strong>cha (gr<strong>an</strong>t PI-2006/15). Sections were<br />
incubated with primary <strong>an</strong>tibodies <strong>for</strong> α-synuclein (1:50, mouse monoclonal <strong>an</strong>tibody,<br />
Novocastra) combined with tyrosine hydroxylase (1:1000, rabbit polyclonal <strong>an</strong>tibody, Millipore),<br />
somatostatin (1:1000, goat policlonal <strong>an</strong>tibody, S<strong>an</strong>ta Cruz), calretinin (1:1000, rabbit policlonal<br />
<strong>an</strong>tibody, Sw<strong>an</strong>t) or subst<strong>an</strong>ce-P (1:1000, rabbit policlonal <strong>an</strong>tibody, Chemicon); <strong>an</strong>d<br />
subsequently incubated with biotinylated (1:200, Vector) or fluorescent (Alexas 488 or 568,<br />
Molecular Probes) secondary <strong>an</strong>tibodies. The preparations have been <strong>an</strong>alyzed under optical <strong>an</strong>d<br />
confocal microscopy. α-synucleinopathy progressively affects olfactory structures including <strong>the</strong><br />
olfactory bulb <strong>an</strong>d olfactory cortices. Double labeling studies reveal some of <strong>the</strong> cell types<br />
affected by neurodegeneration such as mitral cells. These data are somewhat comparable to <strong>the</strong><br />
spatial <strong>an</strong>d temporal distribution of α-synucleinopathy in <strong>the</strong> hum<strong>an</strong> brain during Parkinson<br />
Disease. Authors th<strong>an</strong>k Dr. Calvo <strong>for</strong> helping in genotyping <strong>an</strong>imals. Supported by <strong>the</strong><br />
Autonomous Government of Castilla-La M<strong>an</strong>cha-FEDER (Gr<strong>an</strong>ts PI-2006/15 <strong>an</strong>d PCC08-0064).<br />
Disclosures: I. Ubeda-B<strong>an</strong>on, None; D. Saiz-S<strong>an</strong>chez, None; C. de la Rosa-Prieto, None; L.<br />
Arg<strong>an</strong>doña-Palacios, None; S. Arg<strong>an</strong>dona-Palacios, None; A. Martinez-Marcos, None.
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.8/K7<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIH Gr<strong>an</strong>t 1R21NSD58375-01A2<br />
Title: Nigrostriatal dopamine neurotr<strong>an</strong>smission: Effects of aging <strong>an</strong>d <strong>the</strong> Nurr1-null<br />
heterozygous genotype<br />
Authors: *J. B. EELLS, A. BELL;<br />
Mississippi St Univ., Mississippi State, MS<br />
Abstract: Parkinson’s disease is a progressive neurodegenerative disorder that results primarily<br />
<strong>from</strong> loss of nigrostriatal dopamine neurons <strong>an</strong>d <strong>the</strong> subsequent reduction in striatal dopamine. In<br />
<strong>the</strong>se nigrostriatal dopamine neurons, <strong>the</strong> tr<strong>an</strong>scription factor Nurr1 is essential <strong>for</strong> dopamine<br />
neuron differentiation <strong>an</strong>d is import<strong>an</strong>t in maintaining dopamine syn<strong>the</strong>sis <strong>an</strong>d neurotr<strong>an</strong>smission<br />
in <strong>the</strong> adult. Previous data has indicated that reduced expression of Nurr1, as shown by <strong>the</strong><br />
Nurr1-null heterozygous mice, impacts dopamine neuron function. Specifically, <strong>the</strong> Nurr1-null<br />
genotype reduces <strong>the</strong> survival of nigrostriatal dopamine neurons after toxic<strong>an</strong>t treatment <strong>an</strong>d with<br />
aging, decreases dopamine syn<strong>the</strong>sis <strong>an</strong>d reduces extracellular DOPAC. Additional studies have<br />
found that Nurr1 levels in dopamine neurons are reduced with aging <strong>an</strong>d that Nurr1-null<br />
heterozygous mice have reduced tissue levels of dopamine after approximately 15 months, a<br />
parameter that is not altered in young adults. In order to fur<strong>the</strong>r investigate <strong>the</strong> effects of aging<br />
<strong>an</strong>d <strong>the</strong> Nurr1-null heterozygous genotype, microdialysis in <strong>the</strong> striatum was used to measure<br />
basal <strong>an</strong>d potassium (100 mM) stimulated dopamine overflow. Fur<strong>the</strong>rmore, <strong>the</strong> function of <strong>the</strong><br />
dopamine D2 autoreceptor regulation of potassium stimulated dopamine release was assessed.<br />
Microdialysis c<strong>an</strong>nula were impl<strong>an</strong>ted into <strong>the</strong> striatum. After 48 h of recovery, a microdialysis<br />
probe was inserted <strong>an</strong>d fractions were collected every 20 minutes. Artificial cerebral spinal fluid<br />
(CSF) was infused <strong>for</strong> 140 min to achieve stable basal levels of dopamine <strong>an</strong>d metabolites. A<br />
depolarizing concentration of potassium (100 mM) in CSF was infused <strong>for</strong> 60 min. After<br />
returning to CSF <strong>for</strong> recovery <strong>an</strong>d to return to basal levels, 200 microM of <strong>the</strong> dopamine D2<br />
receptor agonist quinpirole was infused 20 min prior to <strong>an</strong>d concurrent with <strong>an</strong>o<strong>the</strong>r 60 min of<br />
100 mM potassium infusion. Dopamine <strong>an</strong>d <strong>the</strong> metabolites DOPAC <strong>an</strong>d HVA in microdialysis<br />
fractions were measured using HPLC with electrochemical detection. Preliminary results show<br />
that D2 receptor activation reduces potassium stimulated dopamine overflow. Additionally,<br />
comparison of old (14-16 months of age) <strong>an</strong>d young (3-4 months) mice suggest that young mice
have greater autoreceptor inhibition of dopamine release th<strong>an</strong> old mice. This suggests that a<br />
potential compensatory mech<strong>an</strong>ism that occurs with aging to maintain dopamine levels is a<br />
reduction in autoreceptor inhibition of stimulated dopamine release. Fur<strong>the</strong>r studies are<br />
underway to determine <strong>the</strong> role of <strong>the</strong> Nurr1-null heterozygous genotype on dopamine<br />
neurotr<strong>an</strong>smission <strong>an</strong>d aging.<br />
Disclosures: J.B. Eells, None; A. Bell, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.9/K8<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIH Gr<strong>an</strong>t P50NS38367<br />
Americ<strong>an</strong> Parkinson Disease Association<br />
Title: Alterations in heart rate variability in tr<strong>an</strong>sgenic mice overexpressing hum<strong>an</strong> wildtype<br />
alpha synuclein<br />
Authors: *S. M. FLEMING 1 , J. G. HOLDEN 3 , P. C. SIOSHANSI 1 , M. C. JORDAN 2 , E.<br />
MASLIAH 4 , K. P. ROOS 2 ;<br />
1 2 3<br />
Dept Neurol., Physiol., UCLA, Los Angeles, CA; Psychology, Univ. of Cincinnati, Cincinnati,<br />
OH; 4 Pathology, UCSD, S<strong>an</strong> Diego, CA<br />
Abstract: Autonomic dysfunction is a common non-motor symptom associated with<br />
synucleinopathies such as Parkinson’s disease (PD). Heart rate variability, <strong>an</strong> index of autonomic<br />
function, is reduced in patents with PD <strong>an</strong>d may actually occur early in <strong>the</strong> development of <strong>the</strong><br />
disease. Tr<strong>an</strong>sgenic mice that overexpress alpha synuclein under <strong>the</strong> Thy1 promoter (Thy1-aSyn)<br />
have high levels of alpha synuclein expression throughout <strong>the</strong> brain including <strong>the</strong> cortex,<br />
thalamus, subst<strong>an</strong>tia nigra, <strong>an</strong>d brainstem (Rockenstein et al., 2002), <strong>an</strong>d <strong>the</strong>y exhibit proteinase<br />
K-resist<strong>an</strong>t alpha synuclein aggregates. Behaviorally <strong>the</strong>y display early <strong>an</strong>d progressive<br />
<strong>an</strong>omalies in sensorimotor function (Fleming et al., 2004) <strong>an</strong>d impairments in olfaction <strong>an</strong>d<br />
gastrointestinal function (Fleming et al. 2008; W<strong>an</strong>g et al., 2008), both early non-motor<br />
symptoms associated with PD. In this study we assessed heart rate variability in Thy1-aSyn <strong>an</strong>d<br />
wildtype mice. Male Thy1-aSyn (n= 4) <strong>an</strong>d wildtype (n= 3) mice at 3-4 months of age were<br />
impl<strong>an</strong>ted with arterial radio telemetry tr<strong>an</strong>sducers. Following recovery, blood pressure
ecordings were taken <strong>for</strong> 20 seconds every 30 minutes over eight days. In addition, continuous<br />
recordings were taken <strong>for</strong> 60 minutes in <strong>the</strong> homecage, following <strong>an</strong> injection of saline, <strong>an</strong>d after<br />
administration of <strong>the</strong> muscarinic <strong>an</strong>tagonist atropine (1mg/kg, ip). Inter-beat-intervals (IBIs)<br />
were determined <strong>from</strong> pressure peak intervals in both experiments. Probability density functions<br />
of <strong>the</strong> 8-day experiment revealed altered variability in Thy1-aSyn mice compared to wildtype<br />
mice. A bootstrapping <strong>an</strong>alysis that compared <strong>the</strong> vari<strong>an</strong>ce between <strong>the</strong> two distributions<br />
indicated lower overall vari<strong>an</strong>ce in <strong>the</strong> Thy1-aSyn compared to wildtype mice (p
DFG Tr<strong>an</strong>sregio SFB TR3 A11 <strong>an</strong>d D12<br />
Title: Aging of Pink1-knock-out mice results in progressive mitochondrial dysfunction with<br />
parkinsoni<strong>an</strong> movement <strong>an</strong>d dopamine deficit, but no neurodegeneration<br />
Authors: S. GISPERT 1 , F. RICCIARDI 1 , A. KURZ 1 , M. AZIZOV 1 , H.-H. HOEPKEN 1 , D.<br />
BECKER 3 , W. VOOS 3 , K. LEUNER 4 , W. MUELLER 4 , A. KUDIN 5 , W. KUNZ 5 , A.<br />
ZIMMERMANN 6 , J. ROEPER 6 , D. WENZEL 7 , M. JENDRACH 1 , M. GARCIA-ARENCIBIA 8 ,<br />
J. FERNANDEZ-RUIZ 8 , L. HUBER 9 , H. ROHRER 9 , M. BARRERA 10 , A. REICHERT 10 , U.<br />
RUEB 11 , A. CHEN 12 , R. NUSSBAUM 12 , *G. W. AUBURGER 2 ;<br />
1 Goe<strong>the</strong> Univ. Med. School, Exptl. Neurol., Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y; 2 Goe<strong>the</strong> Univ. Med.<br />
School, Exptl. Neurol., Fr<strong>an</strong>kfurt Am Main, Germ<strong>an</strong>y; 3 Univ. Bonn, Inst. fuer Biochemie und<br />
Molekularbiologie, Bonn, Germ<strong>an</strong>y; 4 Dept. Pharmacology, Biocenter Niederursel, Goe<strong>the</strong> Univ.,<br />
Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y; 5 Dept. of Epileptology, Univ. Bonn Med. Ctr., Bonn, Germ<strong>an</strong>y;<br />
6 Inst. of Physiology, Goe<strong>the</strong> Univ. Med. Sch., Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y; 7 Max-Pl<strong>an</strong>ck-<br />
Institut für biophysikalische Chemie, Goettingen, Germ<strong>an</strong>y; 8 Dept. Biochem. <strong>an</strong>d Mol. Biol. <strong>an</strong>d<br />
Ctr. de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),<br />
Madrid, Spain; 9 Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Brain Res., Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y; 10 CEF<br />
Makromolekulare Komplexe, Mitochondriale Biologie, Goe<strong>the</strong> Univ. Med. Sch., Fr<strong>an</strong>kfurt am<br />
Main, Germ<strong>an</strong>y; 11 Dept. of Clin. Neuro<strong>an</strong>atomy, Goe<strong>the</strong> Univ., Fr<strong>an</strong>kfurt am Main, Germ<strong>an</strong>y;<br />
12 Natl. Hum<strong>an</strong> Genome Res. Institute, Natl. Inst. of Hlth., Be<strong>the</strong>sda, MD<br />
Abstract: Parkinson’s disease (PD) is <strong>the</strong> second most common adult-onset neurodegenerative<br />
movement disorder. Its etiology is largely unknown. Loss-of-function mutations of <strong>the</strong><br />
mitochondrial protein kinase PINK1 cause <strong>the</strong> autosomal recessive PARK6 vari<strong>an</strong>t of PD. To<br />
study <strong>the</strong> physiological function of PINK1 <strong>an</strong>d to model <strong>the</strong> disease, we generated a Pink1-<br />
Knock-Out mouse <strong>an</strong>d detected onset of Parkinsoni<strong>an</strong> deficits around 18 months of age. Similar<br />
to PD, abnormal dopamine levels in <strong>the</strong> nigrostriatal projection were found toge<strong>the</strong>r with reduced<br />
sponatneous movements <strong>an</strong>d a progressive body weight reduction. In contrast to PD but possibly<br />
compatible with <strong>the</strong> specific syndrome of PARK6, a decreased lifesp<strong>an</strong>, dysfunction of<br />
brainstem <strong>an</strong>d sympa<strong>the</strong>tic nerves, visible aggregates of α-synuclein within<br />
Lewy bodies or nigrostriatal neurodegeneration were undetectable in old Pink1-Knock-Out mice.<br />
At earlier ages, we showed a progressive reduction in mitochondrial preprotein import toge<strong>the</strong>r<br />
with defects of core mitochondrial functions like ATP-generation <strong>an</strong>d respiration in Pink1<br />
deficient mice. In spite of <strong>the</strong> strong PINK1 effect on Drosophila mel<strong>an</strong>ogaster mitochondrial<br />
dynamics, we demonstrate reduced fission <strong>an</strong>d increased aggregation of mitochondria in Pink1-<br />
Knock-Out neurons to occur only under stress, concomit<strong>an</strong>t with reduced expression of fission<br />
factor Mtp18.<br />
Disclosures: S. Gispert, None; F. Ricciardi, None; A. Kurz, None; M. Azizov, None; H.<br />
Hoepken, None; D. Becker, None; W. Voos, None; K. Leuner, None; W. Mueller, None; A.<br />
Kudin, None; W. Kunz, None; A. Zimmerm<strong>an</strong>n, None; J. Roeper, None; D. Wenzel,<br />
None; M. Jendrach, None; M. Garcia-Arencibia, None; J. Fern<strong>an</strong>dez-Ruiz, None; L. Huber,<br />
None; H. Rohrer, None; M. Barrera, None; A. Reichert, None; U. Rueb, None; A. Chen,<br />
None; R. Nussbaum, None; G.W. Auburger, None.
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.11/K10<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIH gr<strong>an</strong>t NS038065<br />
NIH gr<strong>an</strong>t NS0380377<br />
NIH gr<strong>an</strong>t NS055776<br />
Title: Endoplasmic reticulum stress <strong>an</strong>d microsomes aggregated α-synuclein are associated with<br />
<strong>the</strong> α-synucleinopathies in vivo<br />
Authors: *E. COLLA 1 , Y. LIU 2 , W. STIRLING 2 , P. H. JENSEN 3 , T. IWATSUBO 4 , M. K.<br />
LEE 2 ;<br />
1 Dept Pathol, Johns Hopkins Univ. SOM, Baltimore, MD; 2 Pathology, Johns Hopkins Sch. of<br />
Med., Baltimore, MD; 3 Inst. of Med. Biochem., Univ. of Aarhus, Aarhus, Denmark; 4 Dept. of<br />
Neuropathology <strong>an</strong>d Neurosci., Univ. of Tokyo, Tokyo, Jap<strong>an</strong><br />
Abstract: Accumulation of aggregated α-synuclein (αS) in Lewy bodies is a pathological<br />
hallmark of α-synucleinopathies. Because cellular accumulation of misfolded proteins c<strong>an</strong> cause<br />
endoplasmic reticulum (ER) stress <strong>an</strong>d unfolded protein response (UPR), we examined if ER<br />
stress was associated with α-synucleinopathy in A53T hum<strong>an</strong> αS tr<strong>an</strong>sgenic mice. We found that<br />
α-synucleinopathy in mice is associated with induction of ER chaperons (BIP/Grp78, Grp94,<br />
PDI). However, <strong>the</strong>re was lack of <strong>the</strong> expected coordinated increase in eIF2α phosphorylation<br />
<strong>an</strong>d subsequent CHOP activation, a downstream effector of <strong>the</strong> phospho-eIF2 dependent<br />
protection <strong>from</strong> ER stress toxicity. This abnormal UPR response in αS tr<strong>an</strong>genic mice was<br />
associated with increased levels of cleaved caspase 12, <strong>an</strong> ER stress-related caspase, <strong>an</strong>d cleaved<br />
caspase 9, a downstream target of cleaved caspase 12. These UPR signs coincide with disease<br />
<strong>an</strong>d were absent in not affected areas.<br />
Concomit<strong>an</strong>t with ER stress, <strong>the</strong>re was increased association of αS monomers but not βS, <strong>an</strong>d αS<br />
aggregates with <strong>the</strong> ER-enriched microsomes. While most of <strong>the</strong> αS monomer was present<br />
within <strong>the</strong> lumen of <strong>the</strong> microsomal vesicles, microsome associated αS aggregates appeared to be<br />
associated with <strong>the</strong> cytosolic side of <strong>the</strong> ER membr<strong>an</strong>e. Never<strong>the</strong>less ER-associated αS<br />
aggregates appeared to be biochemical distinguishable <strong>from</strong> αS aggregates localized in <strong>the</strong><br />
cytosol. In particular, <strong>the</strong> ER-associated aggregates were highly enriched in phospho-Ser129 but
poorly ubiquitinated when compared to αS cytosolic aggregates. Microsomal αS was found to<br />
bind ER luminal chaperones BIP/Grp78 <strong>an</strong>d Grp94, confirming that αS is indeed <strong>an</strong> ER resident<br />
protein. Signific<strong>an</strong>tly, hum<strong>an</strong> PD cases also show increased microsomal αS compared to controls<br />
cases.<br />
We hypo<strong>the</strong>size that in α-synucleinopathies, increased ER αS level <strong>an</strong>d <strong>the</strong> presence of<br />
aggregated αS in ER triggers chronic ER stress that contributes to neurodegeneration. Fur<strong>the</strong>r,<br />
presence of αS aggregates in ER raises <strong>the</strong> possibility that <strong>the</strong> αS aggregates are secreted <strong>from</strong><br />
affected neurons to negatively impact <strong>the</strong> biology of neighboring glia <strong>an</strong>d neurons.<br />
Disclosures: E. Colla, None; Y. Liu, None; W. Stirling, None; P.H. Jensen, None; T.<br />
Iwatsubo, None; M.K. Lee, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.12/K11<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIH Gr<strong>an</strong>t 5P50NS038375-090003<br />
The Michael J. Fox Foundation<br />
National Parkinson Foundation<br />
Title: The R1441C mutation in LRRK2 affects dopaminergic neurotr<strong>an</strong>smission in mice<br />
Authors: *Y. TONG 1 , A. PISANI 2 , G. MARTELLA 2 , M. KAROUANI 3 , H. YAMAGUCHI 1 ,<br />
E. N. POTHOS 3 , J. SHEN 1 ;<br />
1 Brigham & Women's Hosp Harvard Med. Sch., Boston, MA; 2 Univ. of Rome Tor Vergata <strong>an</strong>d<br />
Fondazione S<strong>an</strong>ta Lucia, Rome, Italy; 3 Tufts Univ., Boston, MA<br />
Abstract: Domin<strong>an</strong>tly inherited missense mutations in <strong>the</strong> leucine-rich repeat kinase 2<br />
(LRRK2) gene are a common genetic cause of Parkinson’s disease (PD). Of <strong>the</strong> five<br />
pathogenic mutations identified to date, two occur at <strong>the</strong> R1441 residue (R1441C, R1441G)<br />
within <strong>the</strong> GTPase domain, highlighting <strong>the</strong> import<strong>an</strong>ce of this arginine residue in <strong>the</strong><br />
pathogenesis. We <strong>the</strong>re<strong>for</strong>e generated a LRRK2 knock-in (KI) mouse, in which <strong>the</strong> R1441C<br />
mutation is expressed under <strong>the</strong> control of <strong>the</strong> endogenous regulatory elements.<br />
Homozygous LRRK2 R1441C KI mice are viable <strong>an</strong>d fertile <strong>an</strong>d appear grossly normal.
These mice show reduced response in locomotor activity to amphetamine stimulation, but<br />
<strong>the</strong>y do not develop dopaminergic neurodegeneration or exhibit alterations in steady-state<br />
levels of striatal dopamine <strong>an</strong>d its major metabolites by two years of age. The stimulated<br />
catecholamine release is signific<strong>an</strong>tly reduced in cultured chromaffin cells of LRRK2<br />
R1441C KI mice. The KI mice are also less sensitive in locomotor activity to <strong>the</strong> inhibition<br />
by quinpirole, a dopamine D2 receptor agonist, suggesting partial impairment of dopamine<br />
D2 receptor signaling. Fur<strong>the</strong>rmore, nigral neurons of LRRK2 R1441C KI mice show<br />
reduced responses in firing to <strong>the</strong> inhibitory effect of quinpirole, dopamine, as well as<br />
amphetamine. Taken toge<strong>the</strong>r, our data suggest that <strong>the</strong> R1441C mutation in LRRK2<br />
causes abnormal regulation of activity-dependent dopamine neurotr<strong>an</strong>smission, which may<br />
precede dopaminergic degeneration in patients.<br />
Disclosures: Y. Tong, None; A. Pis<strong>an</strong>i, None; G. Martella, None; M. Karou<strong>an</strong>i, None; H.<br />
Yamaguchi, None; E.N. Pothos, None; J. Shen, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.13/K12<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: H.C., Z01-AG000959-05<br />
Title: Generation <strong>an</strong>d characterization of Parkinson’s disease-related leucine-rich repeat kinase 2<br />
tr<strong>an</strong>sgenic mice<br />
Authors: *X. GU, C.-X. LONG, X. LIN, L. WANG, L. SUN, H. SHIM, C. XIE, L.<br />
PARISIADOU, H. CAI;<br />
Natl. Inst. of Hlth., Be<strong>the</strong>sda, MD<br />
Abstract: Parkinson’s disease (PD), <strong>the</strong> most common age-related movement disorder, is<br />
pathologically characterized by a relatively selective loss of midbrain dopaminergic neurons <strong>an</strong>d<br />
<strong>the</strong> presence of α-synuclein-positive protein aggregates within neurons. Although most cases of<br />
PD are sporadic, increasing number of genetic mutations has been linked to familial PD,<br />
including autosomal domin<strong>an</strong>t mutations in Leucine-rich Repeat Kinase 2 (LRRK2). The<br />
G2019S missense mutation in LRRK2 represents one of <strong>the</strong> most frequent mutations in both<br />
sporadic <strong>an</strong>d familial PD. To study <strong>the</strong> pathogenic mech<strong>an</strong>ism of G2019S LRRK2 in vivo, we<br />
generated <strong>an</strong>d characterized hum<strong>an</strong> LRRK2 wild-type <strong>an</strong>d G2019S inducible tr<strong>an</strong>sgenic mice
using <strong>the</strong> “tet-off” system. <strong>When</strong> G2019S LRRK2 was selectively expressed in <strong>the</strong> <strong>for</strong>ebrain<br />
regions, <strong>the</strong> spont<strong>an</strong>eous ambulatory activities of mut<strong>an</strong>t mice were signific<strong>an</strong>tly increased<br />
starting at 12 months of age. However, no obvious neurodegeneration was detected in <strong>the</strong> brain<br />
of G2019S tr<strong>an</strong>sgenic mice up to 24 months of age. Interestingly, LRRK2 <strong>an</strong>d ubiquitin-positive<br />
protein aggregates were frequently detected in <strong>the</strong> cytoplasm of neurons in G2019S mice at 6<br />
months of age, indicating a potential impairment of ubiquitin-proteasome system. We now<br />
extend <strong>the</strong> above studies to mice that selectively expressed G2019S LRRK2 in midbrain<br />
dopaminergic neurons. These in vivo findings <strong>from</strong> G2019S LRRK2 tr<strong>an</strong>sgenic mice may shed<br />
light on <strong>the</strong> pathogenic mech<strong>an</strong>ism of this ra<strong>the</strong>r common <strong>for</strong>m of PD.<br />
Disclosures: X. Gu, None; C. Long, None; X. Lin, None; L. W<strong>an</strong>g, None; L. Sun, None; H.<br />
Shim, None; C. xie, None; L. Parisiadou, None; H. cai, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.14/K13<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIA AG023630<br />
NIH Training Gr<strong>an</strong>t 1T32 DA022738<br />
DA017186<br />
AG13494<br />
NSF EEC-0310723<br />
USPHS NS39787<br />
Title: Twelve month old glial cell derived neurotrophic factor heterozygous mice show increased<br />
dopamine release <strong>an</strong>d dopamine uptake in <strong>the</strong> striatum<br />
Authors: *O. M. LITTRELL 1 , H. BOGER 2 , P. HUETTL 1 , F. POMERLEAU 1 , G.<br />
QUINTERO 1 , A.-C. GRANHOLM 2 , G. GERHARDT 1 ;<br />
1 2<br />
Dept Anat & Neurobiol, Univ. Kentucky, Lexington, KY; Med. Univ. of South Carolina,<br />
Charleston, SC
Abstract: Glial cell derived neurotrophic factor (GDNF) directs early developmental processes<br />
in <strong>the</strong> CNS <strong>an</strong>d o<strong>the</strong>r systems. Emerging evidence also support GDNF’s involvement in postnatal<br />
mainten<strong>an</strong>ce <strong>an</strong>d survival of various neurotr<strong>an</strong>smitter systems. Previous studies established<br />
that deletion of one GDNF allele (GDNF+/-) results in signific<strong>an</strong>t GDNF protein reductions (34-<br />
35%) in <strong>the</strong> mouse striatum when compared to age matched wild-type (WT) counterparts. At 12<br />
months of age, GDNF+/- mice show <strong>an</strong> accelerated age-related decline of tyrosine hydroxylase<br />
immunoreactivity in <strong>the</strong> subst<strong>an</strong>tia nigra as well as in locomotor activity. To fur<strong>the</strong>r investigate<br />
<strong>the</strong> effects of this partial GDNF depletion in <strong>the</strong> nigrostriatal dopamine (DA) pathway, we used<br />
carbon fiber microelectrodes coupled with chronoamperometry to characterize functional<br />
ch<strong>an</strong>ges in <strong>the</strong> striatum of 12 month old GDNF+/- mice. This technique provides high spatial<br />
(microns) <strong>an</strong>d temporal (seconds) resolution to evaluate subtle ch<strong>an</strong>ges in DA release <strong>an</strong>d uptake<br />
kinetics within discrete striatal regions. We measured evoked DA release <strong>an</strong>d uptake in 2<br />
recording tracts, (a) rostral <strong>an</strong>d (b) caudal striatum, with 5 recording sites each along <strong>the</strong> dorsalventral<br />
axis of <strong>the</strong> striatum. GDNF+/- mice showed a signific<strong>an</strong>t (p < 0.05) spatially discrete<br />
increase in potassium (120 mM, isotonic, pH = 7.2 - 7.4) evoked DA release in 3 of <strong>the</strong> 5<br />
recording depths in <strong>the</strong> caudal recording tract (me<strong>an</strong> ± SEM (uM)): Depth 1 WT: 3.14 ± 0.94;<br />
GDNF+/-: 9.77 ± 1.03. Depth 2 WT: 2.54 ± 1.03; GDNF+/-: 9.73 ± 3.57. Depth 3 WT: 0.94 ±<br />
0.51; GDNF+/-: 8.25 ± 2.58, n = 4 -7). We did not identify signific<strong>an</strong>t differences in <strong>the</strong> rostral<br />
tract. Local application of DA solution (100 uM, in 0.9% NaCl, pH = 7.2 - 7.4) was used<br />
similarly to investigate ch<strong>an</strong>ges in signal parameters associated with DA tr<strong>an</strong>sporter function.<br />
Uptake data was combined <strong>for</strong> all depths of each recording tract. In <strong>the</strong> rostral recording site<br />
increased clear<strong>an</strong>ce rate (Tc, <strong>the</strong> slope of <strong>the</strong> linear portion of <strong>the</strong> decay curve, nM/sec) <strong>an</strong>d<br />
shorter decay time (T80, time <strong>from</strong> maximum amplitude <strong>for</strong> signal to decay 80%, sec) in DA<br />
signals of equivalent amplitude suggest increased DA tr<strong>an</strong>sporter activity in GDNF+/- mice<br />
(me<strong>an</strong> ± SEM: Tc WT: 23 ± 3; GDNF+/-: 42 ± 7 T80 WT: 60.5 ± 7.3; GDNF+/-: 30.3 ± 4.9,<br />
WT: n = 31, GDNF+/-: n = 19, p < 0.01, both). A partial depletion of GDNF appears to alter<br />
nigrostriatal DA neurotr<strong>an</strong>smission in both <strong>the</strong> capacity to release DA <strong>an</strong>d signal duration. These<br />
paradoxical findings suggest complex compensatory mech<strong>an</strong>isms involved in this DA<br />
dysfunction mouse model. Ongoing studies aim to determine regional ch<strong>an</strong>ges in DA storage <strong>an</strong>d<br />
metabolism to explain <strong>the</strong> increase in release amplitude.<br />
Disclosures: O.M. Littrell, None; H. Boger, None; P. Huettl, None; F. Pomerleau, None; G.<br />
Quintero, None; A. Gr<strong>an</strong>holm, None; G. Gerhardt, None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.15/K14
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Parkinson <strong>Society</strong> C<strong>an</strong>ada<br />
NSERC<br />
Title: Hum<strong>an</strong> A53T α-synuclein tr<strong>an</strong>sgenes rescue affective phenotypes <strong>an</strong>d monoamine<br />
tr<strong>an</strong>smitter depletion in α-synuclein-deficient mice<br />
Authors: *H. T. MOUNT 1,2,3 , E. M. CUMYN 1,2 , J. YANG 1 , A. TANDON 1,2,3 ;<br />
1 CRND, 2 Inst. of Med. Sci., 3 Dept. Med. (Div. Neurol.), Univ. Toronto, Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Parkinsoni<strong>an</strong> patients often suffer <strong>from</strong> <strong>an</strong>xiety <strong>an</strong>d depression that may be related to<br />
altered monoamine tr<strong>an</strong>smission. Evidence <strong>from</strong> <strong>an</strong>imal models has suggested that mood c<strong>an</strong> be<br />
affected by tissue alpha-synuclein (SNCA) load. Previously, our group assessed affect <strong>an</strong>d<br />
monoamine content in mouse brains expressing hum<strong>an</strong> wild-type (WT) <strong>an</strong>d pathogenic A53T or<br />
A30P SNCA tr<strong>an</strong>sgenes (Tgs), but found severe impairments only in A30P-Tg mice. We have<br />
now re-examined effects of <strong>the</strong>se Tgs in alpha-synuclein-knockout (Snca -/- ) mice, to determine<br />
whe<strong>the</strong>r presence of <strong>the</strong> endogenous murine protein might confound behavioural phenotypes<br />
associated with hum<strong>an</strong> SNCA Tg expression. In <strong>the</strong> tail suspension test Snca -/- , WT-Tg*Snca -/-<br />
<strong>an</strong>d A30P-Tg*Snca -/- mice were immobile longer th<strong>an</strong> non-Tg wild type controls, indicating<br />
increased behavioural despair in <strong>the</strong>se groups. In contrast, <strong>the</strong> A53T-Tg*Snca -/- mice were<br />
indistinguishable <strong>from</strong> non-Tg wild type controls in this test. In <strong>the</strong> elevated zero maze, mice<br />
expressing WT, A53T <strong>an</strong>d A30P Tgs all spent more time in <strong>the</strong> open sectors of <strong>the</strong> plat<strong>for</strong>m th<strong>an</strong><br />
Snca -/- mice, indicating a strong reduction of <strong>an</strong>xiety by <strong>the</strong> tr<strong>an</strong>sgenes. To explore <strong>the</strong><br />
neurochemical basis of <strong>the</strong> behavioural ch<strong>an</strong>ges, regional monoamine tr<strong>an</strong>smitter levels were<br />
assayed by HPLC-ED. We found cortical, hippocampal <strong>an</strong>d striatal norepinephrine (NE) deficits<br />
in Snca -/- mice that were partially rescued by <strong>the</strong> Tgs. In cerebellum, expression of <strong>the</strong> A53T Tg<br />
completely rescued NE levels. It is possible that correction of cerebellar function contributes to<br />
<strong>the</strong> reduced immobility of this group in <strong>the</strong> tail suspension test. Serotonin (5-HT) levels were low<br />
in Snca -/- cortex, hippocampus, cerebellum <strong>an</strong>d striatum. All three Tgs rescued regional 5-HT<br />
deficits in <strong>the</strong> Snca -/- brain. However, in temporoparietal cortex, only expression of <strong>the</strong> A53T Tg<br />
restored 5-HT to control levels. This cortical restoration of 5-HT may also contribute to <strong>the</strong><br />
marked reduction in immobility of A53T-Tg*Snca -/- mice in <strong>the</strong> tail suspension test. In sum,<br />
mice deficient in murine alpha-synuclein display affective disturb<strong>an</strong>ces that appear to be<br />
associated with tissue NE <strong>an</strong>d 5-HT levels. The Ala53Thr substitution, while pathogenic in<br />
hum<strong>an</strong>s, is <strong>the</strong> normal amino acid residue <strong>for</strong> <strong>the</strong> murine protein. The results point to a critical<br />
role <strong>for</strong> 53Thr in <strong>the</strong> functional regulation of monoaminergic tr<strong>an</strong>smission in mice.<br />
Disclosures: H.T. Mount, None; E.M. Cumyn, None; J. Y<strong>an</strong>g, None; A. T<strong>an</strong>don, None.<br />
Poster<br />
531. Parkinson's Disease: Models III
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.16/K15<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Michael J. Fox Foundation <strong>for</strong> Parkinson’s Research<br />
NIH Gr<strong>an</strong>t K08 NS48858<br />
Emory Neuroscience NINDS Core Facilities (ENNCF) Gr<strong>an</strong>t (P30 NS055077)<br />
Title: Evaluation of gastrointestinal motility in tr<strong>an</strong>sgenic mice expressing hum<strong>an</strong> mut<strong>an</strong>t alpha-<br />
synuclein<br />
Authors: *A. NOORIAN 1 , G. M. TAYLOR 1 , S. SRINIVASAN 2 , J. G. GREENE 1 ;<br />
1 Neurol., 2 Medicine/ Gastroenterology, Emory Univ. Sch. Med., Atl<strong>an</strong>ta, GA<br />
Abstract: Gastrointestinal (GI) symptoms are some of <strong>the</strong> most common non-motor symptoms<br />
in Parkinson’s disease (PD). The exact pathologic mech<strong>an</strong>ism of <strong>the</strong>se symptoms is unknown,<br />
but abnormal function <strong>an</strong>d pathology in <strong>the</strong> enteric nervous system (ENS) <strong>an</strong>d dorsal motor<br />
nucleus of <strong>the</strong> vagus (DMV) are thought to play <strong>an</strong> import<strong>an</strong>t role. Mutations in alpha-synuclein<br />
(a-SYN) cause a rare familial <strong>for</strong>m of parkinsonism, <strong>an</strong>d even in sporadic PD, a-SYN is a<br />
primary structural component of Lewy bodies, a pathologic hallmark of <strong>the</strong> disorder. These<br />
aggregates have been described frequently in <strong>the</strong> ENS <strong>an</strong>d DMV of PD patients. It has been<br />
shown that tr<strong>an</strong>sgenic mice expressing hum<strong>an</strong> mut<strong>an</strong>t (A53T) a-SYN (TG a-SYN mice) develop<br />
age-related motor disturb<strong>an</strong>ces <strong>an</strong>d synuclein accumulation similar in some ways to PD. We<br />
have evaluated GI function in TG a-SYN mice <strong>from</strong> behavioral, pathological, <strong>an</strong>d<br />
electrophysiological st<strong>an</strong>dpoints compared to wild type (WT) mice in different age groups.<br />
Behavioral tests showed delayed gastric emptying <strong>an</strong>d increased colon tr<strong>an</strong>sit time in a-SYN<br />
tr<strong>an</strong>sgenic mice at 20 months of age. These differences were not seen in younger mice, although<br />
immunohistochemical staining demonstrated increased a-SYN reactivity in certain enteric neural<br />
processes in TG a-SYN mice as early as 3 months of age. Using additional behavioral <strong>an</strong>d<br />
physiological measures, no o<strong>the</strong>r differences were found in distal colon motility or in vitro<br />
response to electrical stimulation between <strong>the</strong> groups. Except <strong>for</strong> increased a-SYN, <strong>the</strong>re is no<br />
dramatic difference in ENS morphology or number of neuron subtypes between <strong>the</strong> groups.<br />
Fur<strong>the</strong>r studies are ongoing at older ages to delineate <strong>an</strong>y age-related effects of overexpression of<br />
mut<strong>an</strong>t a-SYN on ENS structure <strong>an</strong>d function.<br />
Disclosures: A. Noori<strong>an</strong>, None; G.M. Taylor, None; S. Srinivas<strong>an</strong>, None; J.G. Greene, None.<br />
Poster
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.17/K16<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: NIH Gr<strong>an</strong>t 5RO1AG028108<br />
Title: Behavior <strong>an</strong>d neurochemical <strong>an</strong>alyses in a53t alpha-synuclein tr<strong>an</strong>sgenic mouse<br />
Authors: *D. R. GRAHAM FINCH, A. SIDHU;<br />
Georgetown Univ., Washington, DC<br />
Abstract: Parkinson’s Disease (PD) is characterized by <strong>the</strong> <strong>for</strong>mation of Lewy bodies (LB) <strong>an</strong>d<br />
<strong>the</strong> accumulation of α-synuclein deposits concentrated in <strong>the</strong> neurons of <strong>the</strong> subst<strong>an</strong>tia nigra <strong>an</strong>d<br />
<strong>the</strong> brain stem nuclei. In fact, aggregated α-synuclein is believed to play a key role in <strong>the</strong> process<br />
of neuronal toxicity, degeneration as seen in PD as well as m<strong>an</strong>y o<strong>the</strong>r neurodegenerative<br />
diseases. As indicated by <strong>the</strong> disease-causing mutation (A53T), α-synuclein plays a key role in<br />
<strong>the</strong> genesis of synucleopathies. We have previously investigated locomotor activity <strong>an</strong>d behavior<br />
phenotype in 8 month old hA53T-tg mice. Our results suggest that <strong>the</strong>re are subtle ch<strong>an</strong>ges in<br />
behavior occurring between 8 month tg <strong>an</strong>imals in comparison to wild-type. Neurochemical<br />
<strong>an</strong>alysis show that 8 month old hA53T-tg mice have increased levels of tau <strong>an</strong>d associated<br />
kinases. In this study our aim was to investigate this link in <strong>the</strong> A53T α-synuclein tr<strong>an</strong>sgenic<br />
mice at 12 months of age, using behavioral tests, in addition to molecular <strong>an</strong>alyses by western<br />
blots. The data shows that tr<strong>an</strong>sgenic mice had markedly different behavioral phenotype th<strong>an</strong><br />
wild-type mice. Total locomotor activity was slightly higher in 12 month tr<strong>an</strong>sgenic mice in<br />
comparison to wild-type mice. Zone <strong>an</strong>alysis of locomotor activity in <strong>the</strong> open field shows<br />
differences between <strong>the</strong> tr<strong>an</strong>sgenic <strong>an</strong>d wild-type mice, where dist<strong>an</strong>ce traveled <strong>an</strong>d speed are<br />
slightly higher in tr<strong>an</strong>sgenic mice th<strong>an</strong> wild-type mice which may suggest hyper-activity.<br />
Western blot <strong>an</strong>alyses shows neurochemical ch<strong>an</strong>ges in <strong>the</strong> striata of hA53T-tg mice that result<br />
in increased levels of hyperphoshorylated tau <strong>an</strong>d its associated kinases in comparison to normal<br />
mice. These findings that <strong>the</strong> mice display different patterns of behavioral <strong>an</strong>d molecular ch<strong>an</strong>ges<br />
suggest that <strong>the</strong>y may provide relev<strong>an</strong>t model <strong>for</strong> studying mech<strong>an</strong>isms underlying PD.<br />
Disclosures: D.R. Graham Finch, None; A. Sidhu, None.<br />
Poster<br />
531. Parkinson's Disease: Models III
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.18/K17<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Anonymous Foundation<br />
Michael J Fox Foundation<br />
Title: Mouse resources <strong>for</strong> <strong>the</strong> study of Parkinson’s disease<br />
Authors: *M. SASNER, C. M. LUTZ, S. F. ROCKWOOD;<br />
Genet. Resource Sci., The Jackson Lab., Bar Harbor, ME<br />
Abstract: Mouse models are valuable tools to underst<strong>an</strong>d <strong>the</strong> basic pathogenesis of Parkinson’s<br />
disease (PD), to study <strong>the</strong> role of environmental factors in PD, <strong>an</strong>d to be used as disease models<br />
to identify <strong>the</strong>rapeutic targets <strong>an</strong>d develop new <strong>the</strong>rapies <strong>for</strong> PD. The Parkinson’s Disease Mouse<br />
Model Repository (PDMMR) was established within <strong>the</strong> mouse repository at The Jackson<br />
Laboratory to be a central resource <strong>for</strong> archiving <strong>an</strong>d distributing genetic models of PD. This<br />
includes: rederiving strains to a high health status; moving key alleles to st<strong>an</strong>dard genetic<br />
backgrounds; routine monitoring of tr<strong>an</strong>sgenic copy number <strong>an</strong>d phenotype; <strong>an</strong>d distribution of<br />
aged mice <strong>for</strong> certain strains. Currently <strong>the</strong> resource contains alpha-, beta- <strong>an</strong>d gamma-synuclein<br />
knockout lines, <strong>an</strong>d a collection of tr<strong>an</strong>sgenic lines expressing hum<strong>an</strong> alpha-synuclein with<br />
various mutations driven by different promoters. Three new synuclein lines express <strong>the</strong> A53T-,<br />
E46K-, or Syn119- truncation mutations in a cre-dependent m<strong>an</strong>ner, enabling regulated<br />
expression of hum<strong>an</strong> disease alleles. Some of <strong>the</strong>se synuclein mut<strong>an</strong>ts display movement<br />
disorders, alpha-synuclein inclusions <strong>an</strong>d neurodegeneration similar to that seen in PD patients.<br />
O<strong>the</strong>r PD models distributed include Park2 (parkin), Park7 (DJ-1), Htra2 (Park 13) <strong>an</strong>d Pitx3<br />
mut<strong>an</strong>ts. Also available are strains that are specifically sensitive or resist<strong>an</strong>t to toxins such as<br />
MPTP that c<strong>an</strong> be used to study mech<strong>an</strong>isms of neuronal death in PD. The repository also<br />
distributes various “research tool” strains, such as tr<strong>an</strong>sgenic lines that express cre <strong>an</strong>d <strong>the</strong><br />
tetracycline regulated tr<strong>an</strong>sactivator (tTA) protein to enable regulated gene expression, or that<br />
express reporter genes in a neuronal-specific m<strong>an</strong>ner. We are actively seeking both disease<br />
model <strong>an</strong>d research tool strains. Submission of a strain to <strong>the</strong> repository, which c<strong>an</strong> be initiated<br />
via a web <strong>for</strong>m, fulfills requirements <strong>for</strong> sharing of mice in accord<strong>an</strong>ce with NIH’s policy <strong>for</strong><br />
sharing of research reagents. If necessary, donating investigators c<strong>an</strong> place licensing restrictions<br />
on <strong>the</strong> distribution <strong>an</strong>d use of <strong>the</strong>ir models. The PDMMR will serve as a central resource <strong>for</strong><br />
in<strong>for</strong>mation on <strong>the</strong> selection <strong>an</strong>d use of PD mouse models. For more in<strong>for</strong>mation about <strong>the</strong><br />
PDMMR including a list of strains, phenotype descriptions, allele <strong>an</strong>d genotyping in<strong>for</strong>mation<br />
<strong>an</strong>d associated references, see<br />
www.jax.org/jaxmice/research/neurobiology/parkinsons<br />
Disclosures: M. Sasner, None; C.M. Lutz, None; S.F. Rockwood, None.
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.19/K18<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: Parkinson <strong>Society</strong> C<strong>an</strong>ada<br />
NSERC<br />
Title: Altered affect <strong>an</strong>d disrupted bioenergetic homeostasis in mice expressing hum<strong>an</strong> A30P αsynuclein<br />
Authors: *E. M. CUMYN 1,2 , J. YANG 1 , B. H. ROBINSON 4 , H. T. J. MOUNT 1,2,3 ;<br />
1 Ctr. <strong>for</strong> Res. in Neurodegenerative Dis., 2 Inst. of Med. Sci., 3 Dept. of Med. (Division of<br />
Neurology), Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada; 4 Hosp. <strong>for</strong> Sick Children, Toronto, ON,<br />
C<strong>an</strong>ada<br />
Abstract: Familial Parkinson’s disease (PD) is <strong>an</strong> alpha-synucleinopathy that c<strong>an</strong> be caused by<br />
A53T or A30P point mutations in <strong>the</strong> alpha-synuclein gene (SNCA), or by multiplication of <strong>the</strong><br />
gene locus. M<strong>an</strong>y PD patients suffer <strong>from</strong> depression <strong>an</strong>d <strong>an</strong>xiety. Evidence <strong>from</strong> experimental<br />
models suggests that this affective state may be related to SNCA load. We studied mice<br />
expressing hum<strong>an</strong> wild-type (WT) or mut<strong>an</strong>t (A53T, A30P) SNCA tr<strong>an</strong>sgenes (Tgs) to explore a<br />
possible relationship between pathogenic SNCA vari<strong>an</strong>t expression <strong>an</strong>d affective behaviours. To<br />
probe <strong>the</strong> cause of behavioural ch<strong>an</strong>ges, we assessed regional monoamine tr<strong>an</strong>smitter levels as<br />
well as bioenergetic homeostasis. A30P-Tg mice exhibited increased behavioural despair in <strong>the</strong><br />
tail suspension test <strong>an</strong>d decreased <strong>an</strong>xiety in <strong>the</strong> elevated zero maze. These mice also displayed<br />
increased serotonin (5-HT) turnover in cortex <strong>an</strong>d striatum, consistent with <strong>an</strong> altered affective<br />
state. Bioenergetic dysfunction has been implicated in PD. We measured high-energy phosphate<br />
donors in microwave-fixed brain tissue <strong>an</strong>d found reduced cortical ATP in A30P-Tg mice. ATP<br />
levels were preserved in WT-Tg <strong>an</strong>d A53T-Tg cortex, but elevated in WT-Tg hippocampus <strong>an</strong>d<br />
cerebellum. Creatine, a buffer of intracellular ATP stores, tracked closely with ATP content. To<br />
determine whe<strong>the</strong>r reduced ATP biosyn<strong>the</strong>sis was driving ch<strong>an</strong>ges in ATP level, regional<br />
complex I+III activities were assessed in flash frozen tissue. Consistent with ATP measurements,<br />
complex I+III activity was decreased in A30P-Tg cortex, but elevated in several WT-Tg brain<br />
areas. Activity of pyruvate dehydrogenase (PDH), <strong>an</strong>o<strong>the</strong>r ATP-generating enzyme, tracked less<br />
well with ATP levels. PDH activity was unaltered in A30P-Tg cortex, but decreased in A30P-Tg<br />
hippocampus <strong>an</strong>d cerebellum. The depletion of ATP in <strong>the</strong> A30P-Tg mice was associated with
altered 5-HT tr<strong>an</strong>smission <strong>an</strong>d increased behavioural despair. Conversely, increased ATP <strong>an</strong>d<br />
mitochondrial enzyme activity in WT-Tg mice were reflected in signific<strong>an</strong>tly improved<br />
per<strong>for</strong>m<strong>an</strong>ce of certain tasks, even relative to non-Tg littermates. Our results reveal linkages<br />
between expression of hum<strong>an</strong> A30P SNCA, bioenergetic impairment <strong>an</strong>d diminished affect that<br />
precedes fr<strong>an</strong>k evidence of synucleinopathy <strong>an</strong>d cell loss.<br />
Disclosures: E.M. Cumyn, None; J. Y<strong>an</strong>g, None; B.H. Robinson, None; H.T.J. Mount,<br />
None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.20/K19<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Title: In vivo modeling of LRRK2-dependent neuronal dysfunction in mice<br />
Authors: M. HERZIG, P. SCHMID, G. ROVELLI, K. KAUPMANN, C. BARSKE, M.<br />
MUELLER, B. KINZEL, H. VAN DER PUTTEN, *D. R. SHIMSHEK;<br />
Novartis Pharma AG, Basel, Switzerl<strong>an</strong>d<br />
Abstract: Mutations in <strong>the</strong> multidomain protein leucine-rich repeat kinase 2 (LRRK2) are <strong>the</strong><br />
major known genetic cause of Parkinson’s disease (PD). G2019S, <strong>the</strong> most frequent LRRK2<br />
mutation, leads to late-onset autosomal-domin<strong>an</strong>t PD in 4% of familial PD patients which is<br />
clinically indistinguishable <strong>from</strong> idiopathic PD but may m<strong>an</strong>ifest in pleomorphic<br />
neuropathology. In addition, LRRK2 G2019S is found in 1% of sporadic PD. This pathogenic<br />
mutation is located in <strong>the</strong> kinase domain <strong>an</strong>d is thought to increase LRRK2 kinase activity,<br />
suggesting a <strong>the</strong>rapeutic avenue <strong>for</strong> selective kinase inhibitors. To study <strong>the</strong> physiological<br />
function of wild-type LRRK2 <strong>an</strong>d <strong>the</strong> pathological role of G2019S-mutated LRRK2 in PD, we<br />
have generated several genetically engineered mouse lines. These mice ei<strong>the</strong>r (1) highly overexpress<br />
wild-type or G2019S-mutated hum<strong>an</strong> LRRK2 in neurons driven by murine Thy1<br />
regulatory sequences, (2) harbor <strong>the</strong> pathogenic G2019S mutation within <strong>the</strong> endogenous,<br />
murine LRRK2 locus (knock-in mice), or (3) do not express LRRK2 protein at all (knock-out<br />
mice). We are currently characterizing <strong>the</strong>se mice by behavioral, biochemical, <strong>an</strong>d<br />
immunohistochemical me<strong>an</strong>s. These mice will hopefully provide insights into <strong>the</strong> function of<br />
LRRK2 <strong>an</strong>d <strong>the</strong> pathomech<strong>an</strong>isms underlying LRRK2-associated PD.
Disclosures: M. Herzig, None; P. Schmid, None; G. Rovelli, None; K. Kaupm<strong>an</strong>n, None; C.<br />
Barske, None; M. Mueller, None; B. Kinzel, None; H. v<strong>an</strong> der Putten, None; D.R. Shimshek,<br />
None.<br />
Poster<br />
531. Parkinson's Disease: Models III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 531.21/K20<br />
Topic: C.03.d. Tr<strong>an</strong>sgenic <strong>an</strong>d related mouse models<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research<br />
Parkinson's <strong>Society</strong> C<strong>an</strong>ada<br />
<strong>the</strong> Parkinson's Disease Foundation (USA)<br />
Parkinson's Research Consortium<br />
Title: Analysis of conditional knock-out of calpain small subunit (capn4) in mouse brain<br />
Authors: *M. AMINI 1 , C.-L. MA 1 , J. VANDERLUIT 2 , J. SAVITT 3 , P. GREER 4 , R. SLACK 1 ,<br />
R. BERGERON 1 , D. PARK 1 ;<br />
1 Univ. of Ottawa, Ottawa, ON, C<strong>an</strong>ada; 2 Mem. Univ. of Newfoundl<strong>an</strong>d, St. John’s, NL, C<strong>an</strong>ada;<br />
3 Johns Hopkins Univ. Sch. of Med., Baltimore, MD; 4 Queen's Univ., Kingston, ON, C<strong>an</strong>ada<br />
Abstract: Calpains, calcium-dependent proteases, have been implicated in basic cellular<br />
processes <strong>an</strong>d neuropathological events. The ubiquitous calpains, µ <strong>an</strong>d m-calpain, are<br />
heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 <strong>an</strong>d capn2, respectively,<br />
<strong>an</strong>d a common 28-kDa regulatory subunit encoded by capn4. However, most evidence<br />
implicating calpains in physiological <strong>an</strong>d pathophysiological events are based on indirect<br />
findings or <strong>the</strong> use of calpain inhibitors which have insufficient specificity. Germline disruption<br />
of capn4, which eliminates both ubiquitous calpain activities, causes early embryonic lethality,<br />
hampering <strong>the</strong> use of those mouse models to explore <strong>the</strong> physiological <strong>an</strong>d pathophysiological<br />
functions of calpains in <strong>the</strong> CNS. Here we describe <strong>the</strong> use of a capn4-loxP/nestin-cre<br />
conditional mouse mut<strong>an</strong>t to explore calpain functions in a mouse model of Parkinson’s disease<br />
<strong>an</strong>d ischemic stroke. Using <strong>the</strong> Nestin promoter to drive Cre expression we have targeted capn4<br />
in early neuronal progenitors <strong>an</strong>d selectively eliminated ubiquitous calpain activities in <strong>the</strong> CNS.<br />
In <strong>the</strong> absence of calpain activity, mice were born at Mendeli<strong>an</strong> ratios <strong>an</strong>d survived to adulthood.
They did not show <strong>an</strong>y gross abnormality in morphology in <strong>the</strong> CNS ei<strong>the</strong>r at embryonic or adult<br />
periods. Interestingly, however, <strong>the</strong> mut<strong>an</strong>t mice exhibited signific<strong>an</strong>t decrease in Long term<br />
potentiation (LTP) <strong>for</strong>mation; <strong>an</strong>d alternation in expression of some related cytoskeletal<br />
components, glutamate receptors subunits <strong>an</strong>d <strong>the</strong>ir <strong>an</strong>choring proteins. Moreover, neurons<br />
obtained <strong>from</strong> <strong>the</strong>se mut<strong>an</strong>t mice were resist<strong>an</strong>t to <strong>the</strong> Parkinson’s disease related toxin MPP+,<br />
hypoxia, <strong>an</strong>d glutamate in vitro. We also targeted capn4 in tyrosine hydroxylase (TH) cells using<br />
TH promoter <strong>an</strong>d showed that dopaminergic neurons obtained <strong>from</strong> capn4-loxP/TH-cre<br />
conditional mouse mut<strong>an</strong>ts were resist<strong>an</strong>t to MPP+ as well. In summary, <strong>the</strong>se observations<br />
suggest that calpains play more prominent roles in CNS disease th<strong>an</strong> in CNS development.<br />
Moreover, this conditional genetic strategy will allow us to examine <strong>the</strong> precise functions of<br />
calpains in LTP generation <strong>an</strong>d neurodegeneration in adult in vivo settings in future experiments.<br />
Disclosures: M. Amini, None; C. Ma, None; J. V<strong>an</strong>derluit, None; J. Savitt, None; P. Greer,<br />
None; R. Slack, None; R. Bergeron, None; D. Park, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.1/K21<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: Americ<strong>an</strong> Parkinson's Disease Association<br />
NIH Udall Center <strong>for</strong> <strong>the</strong> Study of Parkinson's Disease (NS38367)<br />
NIH Center <strong>for</strong> Gene-Environment Studies of Parkinson's Disease (ES16732)<br />
Title: Over-expression of <strong>the</strong> vesicular monoamine tr<strong>an</strong>sporter as a protective strategy against<br />
alpha-synuclein over-expression in rat dopamine neurons in vitro <strong>an</strong>d in vivo<br />
Authors: B. KHODAYARI 1 , H. A. LAM 1 , F. RICHTER 2 , M. F. CHESSELET 2 , D. E.<br />
KRANTZ 1 , *W. M. WALWYN 3 , N. T. MAIDMENT 1 ;<br />
1 Dept. of Psychiatry <strong>an</strong>d Biobehavioral Sci., 2 Dept. of Neurol., UCLA, Los Angeles, CA;<br />
3 UCLA-NPI, Los Angeles, CA<br />
Abstract: The previously hypo<strong>the</strong>sized contribution of cytosolic dopamine (DA) to <strong>the</strong><br />
pathophysiology of Parkinson’s disease (PD) suggests a specific <strong>an</strong>d relatively straight<strong>for</strong>ward<br />
neuroprotective strategy. The build-up of cytosolic DA <strong>an</strong>d result<strong>an</strong>t generation of reactive
oxygen species in neurons is prevented under normal circumst<strong>an</strong>ces by packaging into synaptic<br />
vesicles, mediated by <strong>the</strong> vesicular monoamine tr<strong>an</strong>sporter (VMAT2). We find that lentiviral<br />
mediated expression of VMAT2-GFP protects nigral DA neurons <strong>from</strong> <strong>the</strong> toxic effects of<br />
lentiviral-mediated hum<strong>an</strong> A30P alpha-synuclein (aSyn) expression in rats. Unilateral injection<br />
of A30P aSyn lentivirus plus GFP virus into <strong>the</strong> subst<strong>an</strong>tia nigra (SN) induced progressive<br />
asymmetrical <strong>for</strong>epaw use over a 6-week period. Post-mortem <strong>an</strong>alysis revealed a reduction in<br />
TH-positive immunoreactive fiber staining in <strong>the</strong> striatum <strong>an</strong>d lower numbers of dopaminergic<br />
neurons in <strong>the</strong> SN on <strong>the</strong> injected side compared with <strong>an</strong>imals injected with GFP-expressing<br />
virus alone. Such effects were markedly reduced or absent in <strong>an</strong>imals injected with aSyn<br />
lentivirus plus VMAT2-GFP virus. Parallel experiments in primary cultures of DA neurons<br />
revealed <strong>an</strong> increase in <strong>the</strong> number of TH-positive cells surviving 1-week after exposure to<br />
VMAT2-GFP lentivirus compared with untreated cultures or cultures exposed to GFP virus.<br />
These data point to <strong>the</strong> potential of drugs targeting VMAT2 expression as a neuroprotective<br />
strategy in <strong>the</strong> treatment of PD.<br />
Disclosures: B. Khodayari, None; H.A. Lam, None; F. Richter, None; M.F. Chesselet,<br />
None; D.E. Kr<strong>an</strong>tz, None; W.M. Walwyn, None; N.T. Maidment, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.2/K22<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: NSERC - CRSNG<br />
C<strong>an</strong>ada Research Chairs- Molecular neuropharmacology<br />
Title: Partial dopamine depletion impairs <strong>the</strong> acquisition of skilled behaviour but not motor<br />
control<br />
Authors: L. CHAGNIEL, F. LABARRE, G. BUREAU, *M. CYR;<br />
U. Quebec a Trois-Rivieres, Trois-Rivieres, QC, C<strong>an</strong>ada<br />
Abstract: Recent findings suggest that <strong>the</strong> nigrostriatal dopamine (DA) system plays a role in<br />
motor control <strong>an</strong>d <strong>the</strong> acquisition of habits <strong>an</strong>d skills. We hypo<strong>the</strong>sized that <strong>the</strong> striatum-based<br />
function underlying <strong>the</strong> acquisition of skilled behaviours might be more vulnerable to DA<br />
depletion th<strong>an</strong> <strong>the</strong> motor control. To test this hypo<strong>the</strong>sis, we investigated whe<strong>the</strong>r motor skilled
learning is impaired in partially DA-depleted mice. Three protocols using <strong>the</strong> neurotoxin 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine<br />
(MPTP) were per<strong>for</strong>med to decrease DA syn<strong>the</strong>sis<br />
to various extents: {1} 4 injections (i.p.) of 9 mg/kg in one day, {2} 4 injections (i.p.) of 15<br />
mg/kg in one day or {3} 5 injections (i.p.) of 30 mg/kg in five days. Degrees of DA-depletion<br />
were estimated by <strong>the</strong> evaluation of tyrosine hydroxylase (TH) levels in <strong>the</strong> striatum, using<br />
western blot technique, <strong>an</strong>d by counting TH positive cells in <strong>the</strong> subst<strong>an</strong>tia nigra, using TH<br />
immunofluorescence. These protocols were associated with a loss of striatal TH levels of {1}<br />
30%, {2} 50% <strong>an</strong>d {3} 85%, respectively. All three protocols of DA depletion in mice were<br />
associated with relatively intact motor functions as revealed by <strong>the</strong> wire suspension <strong>an</strong>d <strong>the</strong> pole<br />
tests. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, however, impaired acquisition of skilled behaviour occurred in mice<br />
with 85% decreased in TH levels. Motor skill learning was evaluated by using <strong>the</strong> accelerating<br />
rotarod test, which accelerates <strong>from</strong> 4 to 40 rpm in 300 seconds, <strong>for</strong> 10 trials on days 1, 2, 3 <strong>an</strong>d<br />
8. Mice <strong>from</strong> protocol {3} exhibited signific<strong>an</strong>tly lower scores within <strong>the</strong> first trials of <strong>the</strong> first<br />
training day as well as a much slower progression in <strong>the</strong> following days in comparison to vehicle<br />
treated mice. The two o<strong>the</strong>rs groups of DA-depleted mice showed normal motor skill learning.<br />
Based on <strong>the</strong>se results, we concluded that <strong>the</strong> striatum-based function underlying <strong>the</strong> acquisition<br />
of skilled behaviours may be more vulnerable to DA depletion th<strong>an</strong> <strong>the</strong> motor control. These<br />
experiments suggest that a decrease in dopaminergic function might underlie <strong>the</strong> reduced ability<br />
to <strong>for</strong>m new motor memories with training in normal aging.<br />
Disclosures: L. Chagniel, None; F. Labarre, None; G. Bureau, None; M. Cyr, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.3/K23<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: Health-2007-22918 - Replaces<br />
Title: Distinct levels of dopaminergic denervation differentially alter striatal synaptic plasticity<br />
<strong>an</strong>d NMDA receptor subunit composition: Implications <strong>for</strong> Parkinson’s disease <strong>the</strong>rapy<br />
Authors: B. PICCONI 1 , V. BAGETTA 1 , V. PAILLÉ 1,2 , V. GHIGLIERI 1 , C. SGOBIO 1 , I.<br />
BARONE 1 , M. DI FILIPPO 3,1 , M. VISCOMI 1 , F. GARDONI 4 , G. BERNARDI 1 , P.<br />
GREENGARD 5 , M. DI LUCA 4 , *P. CALABRESI 3,1 ;<br />
1 S<strong>an</strong>ta Lucia Fndn., Rome, Italy; 2 INSERM U667, Paris, Fr<strong>an</strong>ce; 3 Clinica Neurologica, Univ.
Perugia, Perugia, Italy; 4 Ctr. of Excellence of Neurodegenerative Dis., Univ. of Mil<strong>an</strong>, Mil<strong>an</strong>,<br />
Italy; 5 Mol. <strong>an</strong>d Cell. Neurosci., Rockefeller Univ., New York, NY<br />
Abstract: A correct interplay between dopamine (DA) <strong>an</strong>d glutamate is essential <strong>for</strong><br />
corticostriatal synaptic plasticity <strong>an</strong>d motor activity. In experimental parkinsonism <strong>the</strong> complete<br />
depletion of striatal DA, mimicking adv<strong>an</strong>ced stages of Parkinson's disease (PD) induces a lack<br />
of synaptic plasticity at corticostriatal pathway, alterations of dendritic spines, <strong>an</strong>d ch<strong>an</strong>ges in<br />
glutamatergic signaling. All <strong>the</strong>se processes have been identified as critical mech<strong>an</strong>isms<br />
underlying <strong>the</strong> expression of motor symptoms following DA denervation. However, early PD<br />
stages are characterized by <strong>an</strong> incomplete DA level reduction. How this incomplete reduction in<br />
DA level affects striatal synaptic plasticity <strong>an</strong>d glutamatergic synapses is unknown. Here we<br />
present a model of early PD in which partial denervation, causing mild motor deficits, selectively<br />
affects NMDA-dependent Long Term Potentiation (LTP) but not Long Term Depression (LTD)<br />
<strong>an</strong>d dramatically alters NMDA receptor composition in <strong>the</strong> postsynaptic density (PSD). Our<br />
findings show that DA lowering influences corticostriatal synaptic plasticity depending upon <strong>the</strong><br />
level of depletion. Interestingly, <strong>the</strong> use of <strong>the</strong> TAT2A cell-permeable peptide, as <strong>an</strong> innovative<br />
<strong>the</strong>rapeutic strategy in early PD, rescues physiological NMDA receptor composition, synaptic<br />
plasticity <strong>an</strong>d motor behavior.<br />
Disclosures: B. Picconi, None; V. Bagetta, None; V. Paillé, None; V. Ghiglieri, None; C.<br />
Sgobio, None; I. Barone, None; M. Di Filippo, None; M. Viscomi, None; F. Gardoni,<br />
None; G. Bernardi, None; P. Greengard, None; M. Di Luca, None; P. Calabresi, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.4/K24<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: VIEP-BUAP-Hc-2008-<strong>2009</strong>.<br />
Title: The intramuscular administration of C-terminal fragment of tet<strong>an</strong>us toxin improves <strong>the</strong><br />
fine motor skills <strong>an</strong>d restored <strong>the</strong> catecholamines levels in striatum of hemiparkinsoni<strong>an</strong> rats<br />
Authors: *B. VENEGAS MENESES 1 , L. M. MENDIETA 1 , A. PATRICIO 1 , I. MARTÍNEZ 1 ,<br />
E. GARCÍA 2 , M. HERRANDO 3 , J. AGUILERA 3 , I. D. LIMÓN 1 ;<br />
1 Lab. de Neurofarmacologia, Benemerita Univ. Autónoma De Puebla, Puebla, Mexico; 2 Lab. de
Neuroinmunología, Inst. Nacional de Neurología y Neurocirugía "M<strong>an</strong>uel Velasco Suarez", D.F.,<br />
Mexico; 3 Bioquímica y Biología Mol., Univ. Autonoma de Barcelona, Barcelona, Spain<br />
Abstract: Recently, has been shown that in-vitro of <strong>the</strong> recombin<strong>an</strong>t C-terminal fragment of <strong>the</strong><br />
tet<strong>an</strong>us toxin (Hc-TeTx) had shown neuroprotective effects due to is retrogradely tr<strong>an</strong>sported <strong>an</strong>d<br />
bypass <strong>the</strong> blood-brain barrier (BBB) preventing neuronal death by apoptosis in cerebellar<br />
gr<strong>an</strong>ule neurons treated with 1-methyl-4-phenylpiridinium (Chaib-Oukadour et al., <strong>2009</strong>). The<br />
neuroprotective effect is due to activation of Trk receptors <strong>an</strong>d <strong>the</strong>ir signaling pathways.<br />
Moreover <strong>the</strong> first results in-vivo have shown <strong>the</strong> efficacy of local administration of Hc-TeTx on<br />
ameliorating behavior deficits <strong>an</strong>d improvement of dopaminergic system in MPP + -lesioned rats,<br />
however have not shown evidence of <strong>the</strong> intramuscular effect of Hc-TeTx in motor behaviors.<br />
The aim of this study was to evaluate <strong>the</strong> intramuscular administration of Hc-TeTx fragment in<br />
MPP + treated rats assessing fine motor skills in <strong>the</strong> staircase linking restored <strong>the</strong> catecholamine<br />
levels in striatum. Male Wistar rats (250-300g) were administered with 20 UI/Kg [2 µM i.m.] of<br />
Hc-TeTx three days previous to injection with MPP + 2µL [10µg/µL] in <strong>the</strong> left striatum. Ano<strong>the</strong>r<br />
group were injected only MPP + in <strong>the</strong> same brain area. Sixteen days were assessed <strong>the</strong> fine motor<br />
skills during fifteen days in staircase test <strong>an</strong>d finally <strong>the</strong> thirty days we measured <strong>the</strong> dopamine<br />
(DA) <strong>an</strong>d metabolites levels by HPLC with electrochemical detector in <strong>the</strong> striatum ipsilateral<br />
<strong>an</strong>d contralateral of lesion. <strong>When</strong> evaluated <strong>the</strong> rotation task <strong>the</strong> group Hc-TeTx + MPP + had<br />
been shown that decrease <strong>the</strong> ipsilateral turns respect to MPP + group. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>the</strong><br />
number of pellets taken with <strong>the</strong> contralateral <strong>for</strong>epaw increased a 43% in <strong>the</strong> Hc-TeTx + MPP +<br />
respect to MPP + group. Finally <strong>the</strong> DA levels restored a 53% <strong>an</strong>d DOPAC <strong>an</strong>d HVA levels<br />
increased a 70 y 42% respectively in ipsilateral striatum unch<strong>an</strong>ged in <strong>the</strong> contralateral striatum<br />
in <strong>the</strong> MPP + group. The present study clearly shows <strong>the</strong> effect intramuscular administration of<br />
Hc-TeTx in fine motor skills as a result of DA <strong>an</strong>d metabolites levels recovery in <strong>the</strong> ipsilateral<br />
striatum; <strong>the</strong>se results give evidence <strong>the</strong> new agent to reach CNS, without causing tissue damage.<br />
In general <strong>the</strong> Hc-TeTx shows a neuroprotective role <strong>an</strong>d modulator of <strong>the</strong> dopaminergic system<br />
because of existing reduction of DA neuron death induced by <strong>the</strong> specific toxin MPP + through of<br />
activation Trk receptor <strong>an</strong>d signaling pathways.<br />
Chaib-Oukadour et al., <strong>2009</strong>. Mol. Cell Neurosci. In press<br />
Disclosures: B. Venegas Meneses, None; L.M. Mendieta, None; A. Patricio, None; I.<br />
Martínez, None; E. García, None; M. Herr<strong>an</strong>do, None; J. Aguilera, None; I.D. Limón, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.5/K25
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: NIH Gr<strong>an</strong>t NS047198 (AKD)<br />
Title: Development of bivalent dopamine D2/D3 agonist as new generation <strong>the</strong>rapeutic agents<br />
<strong>for</strong> treatment of Parkinson’s disease<br />
Authors: B. GHOSH 1 , T. ANTONIO 2 , J. ZHEN 2 , A. K. DUTTA 1 , *M. E. REITH 2,3 ;<br />
1 Pharmaceut. Sci., Wayne State Univ., Detroit, MI; 2 Psychiatry, New York Univ. Schl Med.,<br />
New York, NY; 3 Pharmacol., New York Univ. Sch. of Med., New York, NY<br />
Abstract: Parkinson’s Disease (PD) is a progressive, chronic neurodegeneration disorder which<br />
is characterized by a gradual loss of dopaminergic neurons in <strong>the</strong> pars compacta of <strong>the</strong> subst<strong>an</strong>tia<br />
nigra. It is primarily a sporadic disorder although a rare subset of population ( 47) with preferential agonist affinity <strong>for</strong> D3 receptor <strong>an</strong>d also exhibited potent<br />
<strong>an</strong>tioxid<strong>an</strong>t activity. The lead molecule was evaluated in PD <strong>an</strong>imal models, unilaterally lesioned<br />
6-OHDA rat <strong>an</strong>d reserpinized rat models, to evaluate its efficacy in motor stimulation. Detailed<br />
structure activity relationship assessment along with in vitro <strong>an</strong>d in vivo studies will be<br />
presented.<br />
Disclosures: B. Ghosh, None; T. Antonio, None; J. Zhen, None; A.K. Dutta, None; M.E.<br />
Reith, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 532.6/K26<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: NIA PO1 AG023630<br />
Title: Oxidative stress mRNAch<strong>an</strong>ges in <strong>the</strong> subst<strong>an</strong>tia nigra of 3 month old methamphetaminetreated<br />
GDNF heterozygous versus wildtype mice<br />
Authors: *B. GO 1,2 , H. A. BOGER 2 , J. F. MCGINTY 2 ;<br />
1 Charleston, SC; 2 Med. Univ. of South Carolina, Charleston, SC<br />
Abstract: Dopaminergic neurons are believed to be particularly prone to oxidative stress due to<br />
<strong>the</strong>ir high rate of oxygen metabolism, low levels of <strong>an</strong>tioxid<strong>an</strong>ts, <strong>an</strong>d high iron content. An<br />
increase in <strong>the</strong> <strong>for</strong>mation of toxic hydroxyl radicals <strong>an</strong>d a decrease in <strong>the</strong> ability to detoxify <strong>the</strong>m<br />
are major risk factors in PD that may underlie <strong>the</strong> vulnerability of mitochondrial complex I to<br />
damage in SN DA neurons. For example, glutathione depletion in <strong>the</strong> SN of PD patients is<br />
accomp<strong>an</strong>ied by a decrease in catalase <strong>an</strong>d glutathione peroxidase activity that weakens a cell’s<br />
ability to counteract oxidative stress. Fur<strong>the</strong>rmore, oxidative stress has been implicated in <strong>the</strong><br />
neurotoxic damage associated with methamphetamine-induced toxicity. There<strong>for</strong>e, in this study,<br />
methamphetamine (METH HCl, 10 mg/kg, i.p.) or saline was injected into 2.5-month-old WT<br />
<strong>an</strong>d GDNF +/- mice every 2 hrs <strong>for</strong> 8 hrs. Two weeks post-injection (3 months of age), <strong>the</strong><br />
subst<strong>an</strong>tia nigra was dissected out, homogenized <strong>an</strong>d RNA extracted <strong>for</strong> <strong>an</strong>alysis using <strong>the</strong><br />
SuperArray RT2 Profiler PCR Array System Pathway-Focused Gene Expression Profiling<br />
technique <strong>for</strong> detection of markers associated with “Oxidative Stress <strong>an</strong>d Antioxid<strong>an</strong>t Defense”.<br />
Initial <strong>an</strong>alysis indicates that 3 month old GDNF +/- mice have lower mRNA expression of certain<br />
endogenous <strong>an</strong>ti-oxid<strong>an</strong>ts, such as extracellularly localized superoxide dismutase 3 (SOD-3),<br />
glutathione peroxidase 3, <strong>an</strong>d neuronal localized peroxiredoxin 3 compared to saline-treated WT<br />
mice. Two weeks after undergoing a toxic METH binge, GDNF +/- mice demonstrated <strong>an</strong> increase<br />
in endogenous <strong>an</strong>ti-oxid<strong>an</strong>t support by exhibiting a greater increase of mRNA expression of<br />
SOD-3, glutathione peroxidase 3, <strong>an</strong>d glial localized peroxiredoxin 6 compared to METH-treated<br />
WT mice. In addition, METH-treated GDNF +/- mice had increased mRNA expression of<br />
inducible nitric oxide synthase (iNOS) compared to METH-treated WT mice. These data suggest<br />
<strong>an</strong> import<strong>an</strong>t role of oxidative stress in <strong>the</strong> exacerbated DAergic damage observed in METHtreated<br />
GDNF +/- mice at 3 months of age. This work is supported by NIA PO1 AG023630.<br />
Disclosures: B. Go, None; H.A. Boger, None; J.F. McGinty, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.7/K27<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: CAPES/COFECUB<br />
Title: Behavioural alterations induced by intr<strong>an</strong>asal administration of MPTP, <strong>an</strong> experimental<br />
model of Parkinson´s disease, in mice with genetic deletion of <strong>the</strong> heparin binding growth factor<br />
Midkine<br />
Authors: *R. RAISMAN-VOZARI 1 , A. ROJAS-MAYORQUIN 1 , E. DELBEL 2 , R.<br />
MONGEAU 3 , L. LANFUMEY 3 , H. MURAMATSU 4 , J. COURTY 5 , R. PREDIGER 6 ;<br />
1 INSERM-UPMC CRICM-UMRS 975, Paris, Fr<strong>an</strong>ce; 2 FORP-University of SAO Paulo,<br />
Ribeirao Preto-Sao Paulo, Brazil; 3 INSERM, UMR 677, Paris, Fr<strong>an</strong>ce; 4 Aichi Gakuin Univ.,<br />
Aichi, Jap<strong>an</strong>; 5 UMR CNRS 7149, Creteil, Fr<strong>an</strong>ce; 6 UFSC, S<strong>an</strong>ta Catarina, Brazil<br />
Abstract: The “olfactory vector hypo<strong>the</strong>sis” postulates that Parkinson’s disease (PD) may be<br />
caused or catalyzed by agents that enter <strong>the</strong> brain via <strong>the</strong> olfactory mucosa. We have recently<br />
demonstrated that rats treated with intr<strong>an</strong>asal infusion of 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine<br />
(MPTP), a neurotoxin largely used to modeling PD in primates <strong>an</strong>d rodents,<br />
suffer <strong>from</strong> progressive signs of PD that are correlated with time-dependent degeneration in<br />
dopaminergic neurons. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, previous studies have suggested that heparin binding<br />
growth factors, such as Midkine (Mdk), might play <strong>an</strong> import<strong>an</strong>t role in nigrostriatal system<br />
development <strong>an</strong>d in <strong>the</strong> compensatory mech<strong>an</strong>isms that take place in PD. Here, we employed<br />
Mdk knockout mice (Mdk-Ko) to evaluate <strong>the</strong> relev<strong>an</strong>ce of such heparin binding growth factor in<br />
<strong>the</strong> olfactory, emotional, learning <strong>an</strong>d memory, <strong>an</strong>d motor deficits induced by a single intr<strong>an</strong>asal<br />
administration of MPTP (1 mg/nostril). Mimicking <strong>the</strong> clinical condition, wild type mice<br />
exhibited <strong>an</strong> early disruption in olfactory discrimination ability <strong>an</strong>d social recognition memory<br />
during <strong>the</strong> first two weeks after MPTP treatment. These responses were not due to locomotor<br />
impairments, since no behavioral alterations in <strong>the</strong> activity chambers were observed in <strong>the</strong> first<br />
post-treatment week. Interestingly, Mdk-Ko mice have a poorer per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> olfactory<br />
discrimination <strong>an</strong>d social recognition tests, but not in <strong>the</strong> elevated plus-maze, th<strong>an</strong> wild-type<br />
controls. Of high import<strong>an</strong>ce, selective locomotor impairments evaluated in activity chambers<br />
were observed only in Mdk-Ko mice at 3 weeks after MPTP infusion. The present results<br />
suggest, <strong>for</strong> <strong>the</strong> first time, <strong>the</strong> role of Mdk in olfactory <strong>an</strong>d short-term social memory processes<br />
in rodents. Moreover, our findings rein<strong>for</strong>ce <strong>the</strong> involvement of Mdk in compensatory<br />
mech<strong>an</strong>isms in PD, indicating that <strong>the</strong> genetic deletion of Mdk confers increased susceptibility to<br />
behavioural deficits induced by MPTP in mice.<br />
Disclosures: R. Raism<strong>an</strong>-Vozari, None; A. Rojas-Mayorquin, None; E. Delbel, None; R.<br />
Mongeau, None; L. L<strong>an</strong>fumey, None; H. Muramatsu, None; J. Courty, None; R. Prediger,<br />
None.
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.8/K28<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Title: Origin <strong>an</strong>d signific<strong>an</strong>ce of DA efflux in <strong>the</strong> subst<strong>an</strong>tia nigra pars reticulata in a rat model<br />
of L-DOPA-induced dyskinesia<br />
Authors: *H. S. LINDGREN 1 , D. R. ANDERSSON 2 , M. A. CENCI 1 , H. NISSBRANDT 2 ;<br />
1 2<br />
Basal G<strong>an</strong>glia pathophysiology, Lund, Sweden; <strong>the</strong> Sahlgrenska Acad., Dept. of Pharmacol.,<br />
Go<strong>the</strong>nburg, Sweden<br />
Abstract: L-DOPA remains <strong>the</strong> most effective treatment <strong>for</strong> Parkinson’s disease but its longterm<br />
use is hampered by severe side effects such as dyskinesia (abnormal involuntary<br />
movements, AIMs). The development of AIMs has been mainly attributed to neurochemical <strong>an</strong>d<br />
molecular ch<strong>an</strong>ges in <strong>the</strong> striatum. Recent studies have however reported pronounced<br />
electrophysiological <strong>an</strong>d cellular alterations in <strong>the</strong> subst<strong>an</strong>tia nigra pars reticulata (SNpr) in <strong>the</strong><br />
rat model of L-DOPA-induced dyskinesia. Here we have used intracerebral microdialysis in<br />
order to address <strong>the</strong> origin <strong>an</strong>d signific<strong>an</strong>ce of nigral DA efflux in <strong>the</strong> rat dyskinesia model. Rats<br />
with a 6-OHDA lesion were treated with L-DOPA <strong>for</strong> 14 days <strong>for</strong> <strong>an</strong> induction <strong>an</strong>d monitoring<br />
of axial, limb, <strong>an</strong>d orolingual AIMs. On day 14, <strong>the</strong> rats underwent dual probe impl<strong>an</strong>tation in<br />
<strong>the</strong> striatum <strong>an</strong>d SNpr, <strong>an</strong>d on day 16, <strong>the</strong> microdialysis experiment was per<strong>for</strong>med in freely<br />
moving <strong>an</strong>imals. Following a s.c. injection of L-DOPA, nigral extracellular DA levels showed a<br />
much larger increase in dyskinetic <strong>an</strong>imals compared to non-dyskinetic rats. This large DA surge<br />
was blunted by pre-treatment with 5-HT1A/B agonists <strong>an</strong>d/or TTX infusion, thus reflecting<br />
release <strong>from</strong> serotonin neurons. Blocking <strong>the</strong> conversion of L-DOPA to DA in <strong>the</strong> SNr by local<br />
infusion of benserazide had no effect on <strong>the</strong> expression of AIMs after L-DOPA-administration.<br />
In contrast, when applied to <strong>the</strong> striatum, benserazide infusion caused a 50% decrease in <strong>the</strong> L-<br />
DOPA-induced AIM scores. Reverse dialysis of L-DOPA in <strong>the</strong> SNr did not trigger <strong>the</strong><br />
expression of AIMs, although <strong>the</strong> induced extracellular DA levels were approximately 10-fold<br />
larger th<strong>an</strong> after a peripheral L-DOPA-injection.<br />
This study shows that treatment with L-DOPA induces a pronounced increase in nigral DA<br />
levels in <strong>an</strong>imals that exhibit AIMs, However, <strong>the</strong> reverse dialysis experiments show that nei<strong>the</strong>r<br />
local L-DOPA infusion nor a blockade of L-DOPA conversion in SNpr had <strong>an</strong>y <strong>an</strong> effect on <strong>the</strong><br />
expression of AIMs.. Studies with reverse dialysis of specific DA receptor agonists are ongoing.<br />
Disclosures: H.S. Lindgren, None; D.R. Andersson, None; M.A. Cenci, None; H.<br />
Nissbr<strong>an</strong>dt, None.
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.9/K29<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: NIH Gr<strong>an</strong>t R01 NS041674<br />
Title: The modulation of striatal aromatic l-amino acid decarboxylase by l-3,4dihydroxyphenylal<strong>an</strong>ine<br />
in a rodent model of Parkinson’s disease<br />
Authors: *J. M. KING 1 , G. MUTHIAN 2 , C. CHARLTON 2 ;<br />
1 Neurobio. <strong>an</strong>d Neurotoxicology, 2 Meharry Med. Col., Nashville, TN<br />
Abstract: Parkinson’s disease (PD) symptoms are initially relieved by <strong>the</strong> drug L-3,4dihydroxyphenylal<strong>an</strong>ine<br />
(L-DOPA), which improves <strong>the</strong> patients overall quality of life.<br />
However, after a number of years, L-DOPA <strong>the</strong>rapy begins to present motor side effects. The<br />
underlying mech<strong>an</strong>isms of L-DOPA-induced side effects have not been elucidated. Studies in<br />
<strong>an</strong>imal models of PD with L-DOPA induced involuntary movement have revealed large<br />
increases in <strong>the</strong> extra-cellular concentrations of DA in <strong>the</strong> striatum after peripheral<br />
administration of L-DOPA. Thus, excess DA may be a causative factor <strong>an</strong>d since L-aromatic<br />
amino acid decarboxylase (LAAD) is <strong>the</strong> rate-limiting enzyme that catalyzes <strong>the</strong> <strong>for</strong>mation of<br />
DA <strong>from</strong> L-DOPA in PD patients, we explore <strong>the</strong> modulatory role of L-dopa on LAAD, <strong>an</strong>d<br />
evaluate <strong>the</strong> ch<strong>an</strong>ges in <strong>the</strong> levels of DA. We considered whe<strong>the</strong>r DA production modulates<br />
LAAD through feedback inhibition that decreases <strong>the</strong> conversion of L-dopa to DA, leading to<br />
decreases in <strong>the</strong> efficacy of L-DOPA over time. For this project we prepared 1-methyl-4-phenyl-<br />
1,2,3,6-tetrahydropyridine (MPTP) PD disease mouse model <strong>an</strong>d treated <strong>the</strong>m with L-DOPA at<br />
100 mg/kg, twice daily <strong>for</strong> 14 days. We studied <strong>the</strong> locomotor activity of <strong>the</strong> mice using <strong>an</strong> open<br />
field test, Western blot assay <strong>for</strong> LAAD expression, <strong>an</strong>d also <strong>an</strong>alyzed LAAD activity <strong>an</strong>d DA<br />
<strong>an</strong>d its metabolites using High Per<strong>for</strong>m<strong>an</strong>ce Liquid Chromatography. Our studies found that <strong>the</strong><br />
sub-chronic administration of L-DOPA initially caused no difference in <strong>the</strong> expression of<br />
LAAD, when determined immediately after L-DOPA treatment. However, when LAAD was<br />
<strong>an</strong>alyzed 24 hrs after <strong>the</strong> last dose of L-dopa <strong>the</strong> expression <strong>an</strong>d activity of LAAD in <strong>the</strong> striatum<br />
was reduced by about 50%. The results also show that DA levels dropped signific<strong>an</strong>tly. Thus,<br />
when <strong>the</strong> ratio of DA to L-dopa (DA/L-dopa) is low, about 1 hr after L-dopa treatment, LAAD<br />
level is maintained, but when <strong>the</strong> ratio is high 24 hours after L-dopa <strong>the</strong> enzyme activity<br />
dropped. In a time-dependent study we also found that when <strong>the</strong> DA levels dropped signific<strong>an</strong>tly<br />
this led to a rise in <strong>the</strong> activity in LAAD over time.
These results suggest that L-DOPA initially drives <strong>the</strong> activity of LAAD <strong>the</strong>reby increasing <strong>the</strong><br />
production of DA. But, when DA levels decrease, this imbal<strong>an</strong>ce drives <strong>the</strong> catecholamine<br />
system to increase <strong>the</strong> activity of LAAD. This modulation of LAAD during L-DOPA <strong>the</strong>rapy<br />
may contribute to <strong>the</strong> motor complications seen during L-DOPA <strong>the</strong>rapy. These studies may<br />
show that <strong>the</strong> key side effects of L-dopa may be due to <strong>the</strong> disturb<strong>an</strong>ce of <strong>the</strong> homeostasis of <strong>the</strong><br />
catecholamine enzymes by <strong>the</strong> overload of L-DOPA.<br />
Disclosures: J.M. King, None; G. Muthi<strong>an</strong>, None; C. Charlton, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.10/K30<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: Kenneth Douglas Foundation (PMC)<br />
Title: An <strong>an</strong>giogenic inhibitor reduces dopamine neuron loss in <strong>an</strong> MPTP mouse model of<br />
Parkinson's disease<br />
Authors: *A. D. PATEL 1 , B. S. DESAI 1 , P. M. CARVEY 1,2 , B. HENDEY 1 ;<br />
1 Pharmacol., 2 Neurolog. Sci., Rush Univ., Chicago, IL<br />
Abstract: Introduction: Parkinson’s disease (PD) is a progressive neurodegenerative disorder,<br />
characterized by dopamine (DA) neuron loss in subst<strong>an</strong>tia nigra pars compacta (SNpc).<br />
Inflammatory mediators such as TNF-α <strong>an</strong>d VEGF are upregulated in PD <strong>an</strong>d also promote<br />
<strong>an</strong>giogenesis, <strong>the</strong> sprouting of new vessels <strong>from</strong> pre-existing vessels. In previous work using <strong>the</strong><br />
6-hydroxydopamine-rat model of PD, we have shown that areas exhibiting <strong>an</strong> increased<br />
expression of integrin β3, a marker <strong>for</strong> <strong>an</strong>giogenesis, co localize with blood brain barrier (BBB)<br />
disruption. Leaky <strong>an</strong>giogenic vessels may play <strong>an</strong> import<strong>an</strong>t role in disease progression by<br />
compromising BBB integrity <strong>an</strong>d allowing infiltration of toxic metabolites <strong>an</strong>d peripheral<br />
immune mediators into brain. Our group has also found evidence of <strong>an</strong>giogenesis in post-mortem<br />
hum<strong>an</strong> brain tissue of patients with PD, Alzheimer’s disease, incidental Lewy Body disease <strong>an</strong>d<br />
progressive supr<strong>an</strong>uclear palsy; suggesting <strong>an</strong>giogenesis may play a role in a variety of<br />
neurodegenerative disorders. Objective: Using <strong>an</strong> MPTP mouse model of PD, we tested whe<strong>the</strong>r<br />
<strong>the</strong> <strong>an</strong>giogenic inhibitor, cyclo Arg-Gly-Asp-D-Phe-Val (cyRGDfV), was effective in reducing<br />
BBB dysfunction <strong>an</strong>d preventing DA neuron loss. Methods: Mice were injected with MPTP<br />
(10mg/kg q 1hr x 4) or SALine. 24 hr later mice in each group were treated with SAL, cyRGDfV
(100µg/50µl) or cyRADfV (negative control) two-times/day <strong>for</strong> 3 consecutive days. Twenty-four<br />
hr following <strong>the</strong> last treatment, <strong>the</strong> <strong>an</strong>imals were assessed <strong>for</strong> FITC labeled albumin (F-LA)<br />
leakage into brain <strong>an</strong>d DA neuron loss [tyrosine hydroxylase immunoreactive (TH-ir) cell<br />
counts] in SNpc. Results: MPTP treatment resulted in a 32% reduction in TH-ir DA cell counts<br />
in SNpc compared to SAL treated mice [F(4,20)=17.032, p
improvement of dopaminergic system. The aim of this study was to evaluate <strong>the</strong> local<br />
administration effects of Hc-TeTx in left nigrostriatal pathways on motor behaviors, survival cell<br />
<strong>an</strong>d tyrosine hydroxylase immunoreactivity in 6-OHDA-treated rats. Male Wistar rats (200-250<br />
g) were unilateral injected with 2 µl (2µM) of Hc-TeTx previous to 6-OHDA 2 µl (8µg/µl) in<br />
left nigrostriatal pathways. O<strong>the</strong>r group were injected only with 6-OHDA in <strong>the</strong> same brain area.<br />
Then <strong>an</strong>imals were assessed by turn behaviour, cylinder <strong>an</strong>d stepping test. The viability cell in<br />
left <strong>an</strong>d right striatum of rat was examined by MTT assay <strong>an</strong>d <strong>the</strong> tyrosine hydroxylase<br />
immunoreactivity by ELISA striatum <strong>an</strong>d subst<strong>an</strong>tia nigra compacta. In turn behavior task Hc-<br />
TeTx+6-OHDA group displayed a decreased number of contralateral turns (88%), <strong>an</strong>d <strong>the</strong><br />
cylinder test was improved <strong>for</strong> both <strong>for</strong>elimb use asymmetry (71%) respect to control group. The<br />
Hc-TeTx+6-OHDA group had <strong>an</strong> improvement of contralateral limb asymmetry (43%) in <strong>the</strong><br />
stepping test respct to <strong>the</strong> same limb of 6-OHDA group. Finally was found that Hc-TeTx<br />
improved survival cell (36%) in left striatum <strong>an</strong>d increases tirosine hydroxylase<br />
immunoreactivity in <strong>the</strong> striatum (31%) <strong>an</strong>d SNpc (52%). The results clearly show that Hc-TeTx<br />
improves different motor behaviors <strong>an</strong>d regulates phosphorylation of tyrosine hydroxylase. The<br />
present study shows that <strong>the</strong> injection Hc-TeTx improves different motor behaviours <strong>an</strong>d<br />
increase tyrosine hydroxylase so strongly favor <strong>the</strong> hypo<strong>the</strong>sis of Hc-TeTx fragment enh<strong>an</strong>ce<br />
survival pathways that results in amelioration of dopaminergic system of rats with 6-OHDA.<br />
Chaib-Oukadour et al., <strong>2009</strong>. Mol. Cell. Neurosci. In press<br />
Disclosures: A. Patricio, None; V. Palafox, None; E. Bautista, None; L. Mendieta, None; B.<br />
Venegas, None; I. Martínez, None; M. Herr<strong>an</strong>do, None; J. Aguilera, None; I.D. Limón,<br />
None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.12/K32<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: FAPESP<br />
Title: A non-competitive NMDA receptor-ch<strong>an</strong>nel blocker into inferior colliculus c<strong>an</strong> modulate<br />
L-nitroarginine-induced catalepsy<br />
Authors: *L. L. MELO 1 , A. P. JACOPUCCI 1 , N. C. COIMBRA 2 ;<br />
1 Lab. Neuropsicofarmacologia, Univ. Sao Fr<strong>an</strong>cisco, Brag<strong>an</strong>ca Paulista, Brazil; 2 Dept. de<br />
Farmacologia, Univ. de Sao Paulo, Ribeirao Preto, Brazil
Abstract: The inferior colliculus (IC) is primarily involved in auditory in<strong>for</strong>mation processing<br />
but also integrates sensory in<strong>for</strong>mation of aversive nature. Electrical or chemical stimulation of<br />
this structure induces responses such as arousal, freezing <strong>an</strong>d escape responses that mimic fearful<br />
behavior elicited by environmental challenges. Much evidence suggests that <strong>the</strong> neural substrates<br />
responsible <strong>for</strong> defensive behavior in <strong>the</strong> IC c<strong>an</strong> also be regulated by excitatory amino acids<br />
since microinjections of NMDA into this structure induce defensive behaviors, characterized by<br />
running, rearing, <strong>an</strong>d jumping, that are prevented by AP7, a non-competitive NMDA receptorch<strong>an</strong>nel<br />
blocker. It has been shown that a nitric oxide (NO) donor injected into <strong>the</strong> IC induces<br />
flight reaction. This reaction was inhibited by AP7, suggesting that NO may induce defensive<br />
responses in this structure through glutamate-dependent mech<strong>an</strong>isms.<br />
Interestingly, it has been shown that acute ip administration of L G -nitro-arginine (L-NOARG), a<br />
nitric oxide synthase inhibitor, induces catalepsy in a dose-dependent m<strong>an</strong>ner. Linking <strong>the</strong>se two<br />
pieces of in<strong>for</strong>mation, this work examined whe<strong>the</strong>r <strong>the</strong> microinjection of <strong>the</strong> AP7, <strong>an</strong> NMDA<br />
<strong>an</strong>tagonist receptors into <strong>the</strong> IC is able to influence <strong>the</strong> catalepsy induced by systemic injections<br />
of L-NOARG. Each rat had a c<strong>an</strong>nula impl<strong>an</strong>ted in <strong>the</strong> IC <strong>an</strong>d received microinjection of AP7<br />
(10 or 20 nmol/0.5 microliter) or saline into <strong>the</strong> IC prior to systemic administration of L-<br />
NOARG (90 mg/kg). Immediately after that <strong>the</strong>y were placed in <strong>an</strong> open-field where three<br />
catalepsy evaluations were recorded during 10 minutes. The results showed that IC<br />
microinjection of AP7 signific<strong>an</strong>tly reversed <strong>the</strong> catalepsy elicited by systemic injections of Lnitroarginine.<br />
Based on <strong>the</strong>se findings, it is suggested that fear-induced reactions mediated by<br />
excitatory amino acids neurotr<strong>an</strong>smission in <strong>the</strong> IC may play a key-role in <strong>the</strong> L-NOARG -<br />
induced catalepsy.<br />
Disclosures: L.L. Melo, None; A.P. Jacopucci, None; N.C. Coimbra, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.13/L1<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: Swedish Research Council<br />
MJ Fox Foundation <strong>for</strong> Parkinson's Research<br />
EU FP7-ITN Neuromodel
Title: Behavioural <strong>an</strong>d molecular responses to L-DOPA in mice injected with 6hydroxydopamine<br />
in <strong>the</strong> striatum or in <strong>the</strong> medial <strong>for</strong>ebrain bundle<br />
Authors: V. FRANCARDO 1 , A. RECCHIA 1 , *A. M. CENCI 2,1 ;<br />
1 2<br />
Exptl. Med. Sci., Biomed. Ctr. (BMC F11), 22184 Lund, Sweden; Wallenberg Neurosci Ctr.,<br />
223 62 Lund, Sweden<br />
Abstract: Unilateral lesions of <strong>the</strong> nigrostriatal dopamine (DA) pathway with 6hydroxydopamine<br />
(6-OHDA) c<strong>an</strong> be used to obtain a model of Parkinson´s Disease in <strong>the</strong><br />
mouse. Toxin injections c<strong>an</strong> be per<strong>for</strong>med ei<strong>the</strong>r at <strong>the</strong> origin or in <strong>the</strong> terminal field of <strong>the</strong> DA<br />
projections. The adv<strong>an</strong>tages <strong>an</strong>d disadv<strong>an</strong>tages of different lesion procedures have not been<br />
systematically compared. In <strong>the</strong> present study we examine two types of unilateral 6-OHDA<br />
lesions in <strong>the</strong> mouse <strong>for</strong> <strong>the</strong>ir ability to induce stable motor deficits <strong>an</strong>d a supersensitive<br />
molecular <strong>an</strong>d behavioural response to L-DOPA.<br />
Three µg/µl free-base 6-OHDA was injected at two striatal coordinates (2 µl per site) or in <strong>the</strong><br />
medial <strong>for</strong>ebrain bundle (“MFB”; 1µl). Spont<strong>an</strong>eous rotation, horizontal activity, <strong>an</strong>d <strong>for</strong>elimb<br />
use asymmetry were assessed at 2 weeks post-lesion. Animals were treated with L-DOPA <strong>for</strong> 12<br />
days (3 <strong>an</strong>d 6 mg/kg/day <strong>for</strong> 4 <strong>an</strong>d 8 days respectively, combined with 12 mg/kg benserazide),<br />
during which ratings of abnormal involuntary movements (AIMs) were carried out every o<strong>the</strong>r<br />
day. The 6-OHDA-induced denervation pattern was determined by tyrosine hydroxylase (TH)<br />
immunohystochemistry. Molecular supersensitivity to L-DOPA was assessed by<br />
immunohistochemical staining <strong>for</strong> FosB/∆FosB on striatal sections. Both of <strong>the</strong> lesion<br />
procedures examined produced marked <strong>for</strong>elimb use asymmetry in <strong>the</strong> cylinder test (20-30%<br />
contralateral limb use) <strong>an</strong>d spont<strong>an</strong>eous rotational bias in <strong>the</strong> direction ipsilateral to <strong>the</strong> lesion. L-<br />
DOPA treatment produced maximally severe AIMs in all <strong>the</strong> mice with MFB lesions. Among <strong>the</strong><br />
mice with striatal lesions, 65% did not exhibit dyskinesia, whereas 35% developed AIMs during<br />
<strong>the</strong> treatment, which, however reached only 50% of <strong>the</strong> severity scores measured in <strong>the</strong> MFB<br />
lesion group.<br />
L-DOPA-induced AIM score showed linear positive correlations both with <strong>the</strong> levels of<br />
FosB/∆FosB immunoreactivity <strong>an</strong>d with <strong>the</strong> number of TH-positive cell bodies in <strong>the</strong> striatum.<br />
Fur<strong>the</strong>r biochemical assays are in progress. The results thus far indicate that 6-OHDA injection<br />
in <strong>the</strong> MFB is <strong>the</strong> procedure of choice to obtain a mouse model of evenly distributed DA<br />
denervation <strong>an</strong>d maximally severe supersensitivity to L-DOPA.<br />
Disclosures: V. Fr<strong>an</strong>cardo, None; A. Recchia, None; A.M. Cenci, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 532.14/L2<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: VIEP/BUAP-Hc-2008-<strong>2009</strong><br />
Title: Neuroprotection by tet<strong>an</strong>us toxin HC fragment in rat models of Parkinson’s disease<br />
Authors: *L. MARTINEZ MENDIETA 1 , E. BAUTISTA 2 , V. PALAFOX 3 , I. MARTÍNEZ 3 ,<br />
M. HERRANDO 4 , J. AGUILERA 4 , I. D. LIMÓN 3 ;<br />
1<br />
Dept Pharmacy, Neuropharmacol, Benemerita Univ. Autonoma de Puebla, Puebla, Mexico;<br />
2 3 4<br />
Pharm., Benemérita Univ. Autónoma de Puebla, Puebla, Mexico; Univ. Autónoma de<br />
Barcelona, Barcelona, Spain<br />
Abstract: The non-toxic carboxy-terminal domain of tet<strong>an</strong>us toxin heavy-chain (HC-TeTx)<br />
elicits neuroprotection of cerebelar gr<strong>an</strong>ule neurons against apototic death induced by ei<strong>the</strong>r,<br />
potassium withdrawal <strong>an</strong>d 1-methyl-4-phenlpyridinium (MPP + ) intoxication. Previous results<br />
demonstrated that HC-TeTx stimulates tyrosine phosphorylation of Trk neurotrophin receptors,<br />
<strong>an</strong>d results in activation of PI3-K/Akt <strong>an</strong>d p21ras/ERK mediated pathways. These pathways are<br />
involved in cell survival mediated by Akt <strong>an</strong>d BAD phosphorylation, sequestering BAD by Bcl-<br />
XL dimerization, cytochrome c stabilisation, <strong>an</strong>d blockade of procaspase-3 activation (Chaïb-<br />
Oukadour et al., J Neurochem 2004, <strong>an</strong>d Mol Cel Neurosc <strong>2009</strong>). We have previously shown that<br />
intracr<strong>an</strong>ially injected recombin<strong>an</strong>t protein HC-TeTx protects nigrostriatal dopaminergic<br />
pathways <strong>an</strong>d improves motor behavior in rat’s hemi-parkinsonism models MPP + <strong>an</strong>d 6hydroxydopamine<br />
(6-OHDA); however, <strong>the</strong> motor behavior improvement with peripheral HC-<br />
TeTx application have not yet proved. Since it has been amply demonstrated that HC-TeTx<br />
fragment binds to neuromuscular endings <strong>an</strong>d is retroaxonally tr<strong>an</strong>sported to SNC, in <strong>the</strong> present<br />
study we have investigated <strong>the</strong> effects of intramuscular application of HC-TeTx fragment in<br />
motor asymmetry <strong>an</strong>d in <strong>the</strong> nigrostriatal dopaminergic system in rat’s hemi-parkinsonism 6-<br />
OHDA model. Adult male Wistar rats were intramuscularly treated with <strong>the</strong> recombin<strong>an</strong>t HC-<br />
TeTx (20 ul, i.m.) at 0 µM to 2 µM concentration, three days be<strong>for</strong>e dopaminergic denervation<br />
with 6-OHDA (2 µL, 8 µg/µl) into left striatum. Motor function was evaluated by turning<br />
behavior, stepping test <strong>an</strong>d <strong>the</strong> cylinder test. The motor improvement functions were extending<br />
during 4 weeks in all HC-TeTx groups obtaining <strong>the</strong> maximum improvement with 2 µM<br />
concentration of Hc-TeTx. In motor behavior tests, improvements close to 90%, 30% <strong>an</strong>d 45%<br />
were found in turning behaviour, stepping test <strong>an</strong>d <strong>the</strong> cylinder test, respectively, in HC-TeTx/6-<br />
OHDA treated-<strong>an</strong>imals respect to 6-OHDA group. After motor behaviour tests, encephalic<br />
structures were dissected <strong>an</strong>d tyrosine hydroxilase <strong>from</strong> <strong>the</strong> subst<strong>an</strong>tia nigra pars compcta<br />
(SNpc) <strong>an</strong>d striatum was immunohistochemically assessed, obtaining signific<strong>an</strong>t increases in<br />
positive immunostaining <strong>from</strong> SNpc in HC-TeTx/6-OHDA treated-<strong>an</strong>imals respect to 6-OHDA<br />
group. These results encourage exploring <strong>the</strong> <strong>the</strong>rapeutic potential of non-toxic tet<strong>an</strong>us toxin<br />
derivatives in neurodegenerative diseases.<br />
Disclosures: L. Martinez Mendieta, None; E. Bautista, None; V. Palafox, None; I. Martínez,<br />
None; M. Herr<strong>an</strong>do, None; J. Aguilera, None; I.D. Limón, None.
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.15/L3<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: Center <strong>for</strong> Development <strong>an</strong>d Behavioral Neuroscience at Binghamton University<br />
NIH-NINDS gr<strong>an</strong>t NS059600<br />
Title: 5-HT1A receptor stimulation reduces L-DOPA-induced striatal glutamate efflux in <strong>the</strong><br />
hemiparkinsoni<strong>an</strong> rat<br />
Authors: *K. B. DUPRE, K. L. ESKOW JAUNARAJS, T. BUTTON, C. Y. OSTOCK, C.<br />
BISHOP;<br />
Binghamton Univ., Binghamton, NY<br />
Abstract: Chronic dopamine (DA) replacement <strong>the</strong>rapy with L-DOPA has profound effects on<br />
<strong>the</strong> corticostriatal glutamate system. Upon DA depletion <strong>an</strong>d subsequent L-DOPA treatment,<br />
augmentation of striatal glutamate levels <strong>an</strong>d increased expression of glutamatergic receptors<br />
have been postulated to result in L-DOPA-induced dyskinesia (LID). Convergent evidence has<br />
indicated that serotonin 5-HT1A receptor (5-HT1AR) agonists reduce LID, but <strong>the</strong>ir <strong>an</strong>tidyskinetic<br />
mech<strong>an</strong>ism of action is not fully understood. Interestingly, 5-HT1AR stimulation has<br />
been shown to reduce striatal glutamate levels in DA-depleted rats. Taken as a whole, 5-HT1AR<br />
agonists may be diminishing LID in part through attenuation of L-DOPA-induced striatal<br />
glutamate efflux. In order to test this, rats with unilateral medial <strong>for</strong>ebrain bundle DA lesions <strong>an</strong>d<br />
striatal microdialysis c<strong>an</strong>nulae were primed with L-DOPA (12 mg/kg + benserazide, 15 mg/kg,<br />
sc) until abnormal involuntary movements (AIMs) stabilized. On test days, rats were treated<br />
with: Vehicle (0.9% NaCl) + Vehicle (0.9% NaCl, 0.1% ascorbic acid), Vehicle (0.9% NaCl) +<br />
L-DOPA (12 mg/kg, sc), or <strong>the</strong> full 5-HT1AR agonist ±8-OH-DPAT (1.0 mg/kg, sc) + L-DOPA<br />
(12 mg/kg, sc). Striatal sample fractions were collected every 20 min <strong>for</strong> 2 h <strong>for</strong> <strong>an</strong>alysis of<br />
glutamate levels <strong>an</strong>d AIMs were observed every 10 min during this time. While L-DOPA<br />
induced AIMs <strong>an</strong>d enh<strong>an</strong>ced striatal glutamate levels, pretreatment with ±8-OH-DPAT<br />
concomit<strong>an</strong>tly diminished AIMs expression <strong>an</strong>d L-DOPA-related striatal glutamate efflux. These<br />
novel findings indicate a potential mech<strong>an</strong>ism of action of 5-HT1AR agonists in <strong>the</strong>ir ability to<br />
reduce LID with implications <strong>for</strong> <strong>the</strong> improved treatment of Parkinson’s disease.
Disclosures: K.B. Dupre, None; K.L. Eskow Jaunarajs, None; T. Button, None; C.Y.<br />
Ostock, None; C. Bishop, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.16/L4<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: Center <strong>for</strong> Development <strong>an</strong>d Behavioral Neuroscience at Binghamton University (CB)<br />
NIH-NINDS Gr<strong>an</strong>t NS059600 (CB)<br />
Title: 5-HT1A receptor stimulation within <strong>the</strong> primary motor cortex reduces L-DOPA-induced<br />
dyskinesias in <strong>the</strong> hemiparkinsoni<strong>an</strong> rat<br />
Authors: *C. OSTOCK, K. L. ESKOW, K. B. DUPRE, H. WALTERS, C. BISHOP;<br />
Psychology, Binghamton Univ., Binghamton, NY<br />
Abstract: The most common treatment <strong>for</strong> Parkinson’s Disease (PD) is dopamine (DA)<br />
replacement <strong>the</strong>rapy with L-DOPA. However, chronic L-DOPA treatment often leads to <strong>the</strong><br />
expression of debilitating motor side effects know as L-DOPA-induced dyskinesias (LID). While<br />
serotonin 5-HT1A receptor (5-HT1AR) agonists have been shown to reduce <strong>the</strong> expression of<br />
LID by normalizing activity in multiple basal g<strong>an</strong>glia sites, recent evidence suggests putative 5-<br />
HT1AR within <strong>the</strong> primary motor cortex (M1) may also contribute. In order to test this,<br />
unilaterally DA depleted rats with M1 c<strong>an</strong>nulae were primed with L-DOPA (12 mg/kg, +<br />
benserazide, 15mg/kg, sc) <strong>for</strong> 7 days <strong>an</strong>d were tested <strong>for</strong> abnormal involuntary movements<br />
(AIMS) <strong>an</strong>d rotations on <strong>the</strong> last day of priming. On test days, primed rats reaching criterion<br />
(AIMs score > 20) received unilateral microinfusions of: Vehicle (0.9% NaCl), <strong>the</strong> full 5-<br />
HT1AR agonist ±8-OH-DPAT (1 or 10 µg), <strong>the</strong> 5-HT1AR <strong>an</strong>tagonist WAY100635 (5 µg), or<br />
combined WAY100635 (5 µg) + ±8-OH-DPAT (10 µg) into 1 of 2 different locations within M1<br />
(<strong>an</strong>terior or posterior) followed by systemic treatment with L-DOPA (12mg/kg, + benserazide,<br />
15 mg/kg) <strong>an</strong>d were rated <strong>for</strong> AIMs. Microinfusions of ±8-OH-DPAT into M1 dose dependently<br />
reduced <strong>the</strong> expression of AIMs, however no regional differences were found. These effects<br />
were reversed with <strong>the</strong> coadministration of WAY100635. The current results suggest that<br />
cortical 5-HT1AR stimulation contributes to <strong>the</strong> <strong>an</strong>tidyskinetic effects of 5-HT1AR agonists<br />
indicating <strong>the</strong> ubiquitous effects of <strong>the</strong>se compounds within <strong>the</strong> PD brain.
Disclosures: C. Ostock, None; K.L. Eskow, None; K.B. Dupre, None; H. Walters, None; C.<br />
Bishop, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.17/L5<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: NIAAA<br />
NIEHS<br />
Title: NADPH oxidase (NOX) contributes to systemic LPS-induced microglial activation <strong>an</strong>d<br />
neurodegeneration<br />
Authors: *L. QIN 1 , Y. X. LIU 1,2 , M. L. BLOCK 2 , J. S. HONG 2 , F. T. CREWS 1 ;<br />
1 Bowles Ctr. Alcohol Studies, Univ. North Carolina, Sch. Med., Chapel Hill, NC; 2 Lab. of<br />
Pharmacol. <strong>an</strong>d Chem., NIH/NIEHS, Rtp, NC<br />
Abstract: Systemic lipopolysaccharide (LPS) induces a persistent increase in brain TNFα <strong>an</strong>d<br />
oxidative stress that in time leads to neurodegeneration. The purpose of this study was to<br />
investigate <strong>the</strong> role of NADPH oxidase (NOX) in LPS-induced microglial activation <strong>an</strong>d<br />
neurotoxicity. Treatment of C57BL/6 mice with LPS (single i.p. injection, 5mg/kg, 24h) resulted<br />
in a 12.1 fold increase in NOX subunit gp91 phox mRNA, 1.57 fold increase in cytosolic NOX<br />
subunit p67 phox mRNA <strong>an</strong>d 1.28 fold increase in Iba1 (microglial marker) mRNA in brain<br />
compared to saline controls (rtPCR). Immunohistochemical <strong>an</strong>alysis indicated increased<br />
gp91+IR <strong>an</strong>d p67+IR cells 24 hours after LPS. Production of O2 − <strong>an</strong>d O2 − -derived oxid<strong>an</strong>ts was<br />
assessed by hydroethidine histochemistry. Peripherial LPS signific<strong>an</strong>tly increased <strong>the</strong> production<br />
of O2 - <strong>an</strong>d O2 - -derived oxid<strong>an</strong>ts in a time-dependent m<strong>an</strong>ner. Confocal microscopy shows that<br />
gp91 phox +IR cells are triple-localized with O2 - <strong>an</strong>d Iba1 or with O2 - <strong>an</strong>d TH (a marker <strong>for</strong><br />
dopaminergic neurons), indicating NOX activation <strong>an</strong>d superoxide production occur in microglia<br />
<strong>an</strong>d DA neurons. Conversely, no gp91 phox +IR cells or O2 - co-localized with astrocyte marker<br />
glial fibrillary acidic protein (GFAP). To investigate <strong>the</strong> role of NOX we compared LPS<br />
responses in NOX gp91-deficient (NOX gp91-/- ) mice <strong>an</strong>d wild type C57BL/6 mice. Microglia in<br />
brain sections <strong>from</strong> NOX +/+ C57BL/6 mice become activated as indicated by increased cell size,<br />
irregular shape <strong>an</strong>d intensified Iba1 immunoreactivity 3 hours after LPS injection. In NOX gp91-/-<br />
mice microglia show reduced markers of microglial activation. Our previous studies indicated
loss of DA neurons 10 months after LPS prompting experiments in NOX gp91-/- mice. NOX gp91-/-<br />
mice show signific<strong>an</strong>tly less LPS-induced loss of nigral DA neurons compared to NOX +/+<br />
C57BL/6 mice. These results suggest that NOX contributes to persistent microglial activation<br />
<strong>an</strong>d neurodegeneration following LPS (Supported by NIAAA <strong>an</strong>d NIEHS).<br />
Disclosures: L. Qin, None; Y.X. Liu, None; M.L. Block, None; J.S. Hong, None; F.T. Crews,<br />
None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.18/L6<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: NIH Gr<strong>an</strong>t 5U54NS041071<br />
NIH Gr<strong>an</strong>t 9T32NS061201<br />
Title: Selective 5-HT2A receptor <strong>an</strong>tagonists decrease motor deficits in MPTP mouse model of<br />
Parkinson's disease<br />
Authors: M. C. FERGUSON, T. NAYYAR, *T. A. ANSAH;<br />
Meharry Med. Col., Nashville, TN<br />
Abstract: Parkinson’s disease is neurological disease associated with a decrease in dopamine in<br />
<strong>the</strong> striatum due to <strong>the</strong> degeneration of dopamine producing neurons in <strong>the</strong> subst<strong>an</strong>tia nigra. The<br />
disease is characterized by rigidity, bradykinesia, tremors, <strong>an</strong>d stooped posture. The method of<br />
treatment <strong>for</strong> Parkinson’s disease is dopamine replacement <strong>the</strong>rapy with 3, 4dihydroxyphenylal<strong>an</strong>ine<br />
(L-dopa). The L-dopa <strong>the</strong>rapy is effective <strong>for</strong> initial treatment of <strong>the</strong><br />
disease, but with continued usage L-dopa <strong>the</strong>rapy causes motor fluctuations <strong>an</strong>d dyskinesia. As a<br />
result, <strong>the</strong>re exists <strong>the</strong> need to identify non-dopaminergic drug targets. Anatomical studies have<br />
identified 5-HT2A receptors in <strong>the</strong> striatum. Our goal is to determine if 5-HT2A receptor<br />
<strong>an</strong>tagonists acting on cortico-striatal terminals may restore motor function in 1-methyl-4-phenyl-<br />
1, 2, 3, 6-tetraphydropyridine (MPTP) model of Parkinsonism. In order to evaluate <strong>the</strong> effects of<br />
5-HT2A receptor <strong>an</strong>tagonists on <strong>the</strong> MPTP model of Parkinsonism, <strong>the</strong> model was characterized<br />
to determine motor deficits <strong>an</strong>d neurochemical ch<strong>an</strong>ges. The motor function of <strong>the</strong> MPTP treated<br />
mice were assessed by various methods including beam traversal, <strong>an</strong>d gait <strong>an</strong>alysis, 3 weeks after<br />
vehicle or MPTP treatment (four intraperitoneal injections of 20 mg/kg MPTP at two hour
intervals). The MPTP treated mice exhibited deficits in beam traversal <strong>an</strong>d alterations in gait.<br />
Intraperitoneal administration of a mixed 5-HT2A/2C receptor <strong>an</strong>tagonist <strong>an</strong>d a selective 5-HT2A<br />
receptor <strong>an</strong>tagonist improved motor deficits produced by MPTP. The results obtained thus<br />
suggest that 5-HT2A receptor <strong>an</strong>tagonists may be of potential benefit in treating motor deficits in<br />
Parkinson’s disease.<br />
Disclosures: M.C. Ferguson, None; T. Nayyar, None; T.A. Ansah, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.19/L7<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: NIH Gr<strong>an</strong>t NS053912<br />
National Parkinson Foundation<br />
Title: A novel biochemical assay to detect intracellular α-synuclein aggregation<br />
Authors: *D. C. BUTLER 1,2 , S. M. LYNCH 1,2 , A. MESSER 1,2 ;<br />
1 2<br />
Wadsworth Ctr., New York State Dept. of Hlth., Alb<strong>an</strong>y, NY; Biomed. Sci., Univ. at Alb<strong>an</strong>y,<br />
Alb<strong>an</strong>y, NY<br />
Abstract: Since α-synuclein (α-syn) is a major component of Lewy bodies in Parkinson’s<br />
disease (PD) brains, misfolded α-synuclein is <strong>an</strong> attractive target <strong>for</strong> <strong>the</strong>rapeutic intervention. αsyn<br />
is a natively unstructured 140 amino acid (14 kDa) cytosolic protein, containing <strong>an</strong><br />
amphipathic N-terminal region <strong>an</strong>d acidic C-terminal region that fl<strong>an</strong>k a hydrophobic core, <strong>the</strong><br />
non-beta-amyloid component (NAC). The NAC has been identified as <strong>the</strong> critical region <strong>for</strong> asyn<br />
misfolding. Missense mutations A30P, A53T, E46K characteristic of familial PD result in<br />
impaired cellular degradation <strong>an</strong>d show <strong>an</strong> increased tendency to aggregate into toxic<br />
intermediates. Previously, we have shown that intrabodies targeted against <strong>the</strong> NAC region or<br />
monomeric α-syn reduce detergent insoluble α-syn, syn-A53T/synphillin aggregates, <strong>an</strong>d cellular<br />
toxicity. Here, we describe a simple method <strong>for</strong> qualitative <strong>an</strong>d qu<strong>an</strong>titative characterization of<br />
sodium dodecyl sulfate (SDS) insoluble aggregate species <strong>from</strong> mammali<strong>an</strong> cells tr<strong>an</strong>siently<br />
tr<strong>an</strong>sfected with full length <strong>an</strong>d familial <strong>for</strong>ms of α-syn. We were able to <strong>an</strong>alyze SDS insoluble<br />
aggregate species of α-syn by SDS agarose gel electrophoresis (SDSAGE). Import<strong>an</strong>tly, <strong>the</strong>se<br />
insoluble species were detected in <strong>the</strong> absence of visible aggregates or <strong>the</strong> co-aggregating
enh<strong>an</strong>cer synphillin. We are currently using this assay to screen intrabodies against α-syn, <strong>an</strong>d it<br />
may be applied to o<strong>the</strong>r <strong>the</strong>rapeutics <strong>for</strong> Parkinson’s disease as well brain tissue.<br />
Disclosures: D.C. Butler, None; S.M. Lynch, None; A. Messer, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.20/L8<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: Cognitive Deficits <strong>an</strong>d Mood Disorders in PD gr<strong>an</strong>t <strong>from</strong> Michael J. Fox Foundation<br />
(to EVG)<br />
NIH Gr<strong>an</strong>t NS45117 (to EVG)<br />
NIH Gr<strong>an</strong>t MH073159 (to RDB)<br />
Title: A novel mouse model of Parkinson’s disease with dementia: Striatal dopamine-depleted,<br />
choline tr<strong>an</strong>sporter (CHT) heterozygous mice<br />
Authors: *L. ZURKOVSKY 1 , E. R. BYCHKOV 1 , R. D. BLAKELY 1,2 , E. V. GUREVICH 1 ;<br />
1 Pharmacol., 2 Psychiatry, V<strong>an</strong>derbilt Univ. Med. Ctr., Nashville, TN<br />
Abstract: Dementia in Parkinson’s Disease (PDD) occurs in about 40% of Parkinson’s patients.<br />
In contrast to Alzheimer’s dementia, deficits in memory are observed on tests of recall but not<br />
recognition, <strong>an</strong>d are generally milder th<strong>an</strong> in Alzheimer’s patients. However, in PD subjects,<br />
per<strong>for</strong>m<strong>an</strong>ce on tasks targeting executive functioning is consistently impaired. Since postmortem<br />
studies reveal lower levels of cholinergic markers in PDD compared to Parkinson’s<br />
disease without dementia, we hypo<strong>the</strong>size that a combined loss of dopamine (DA) <strong>an</strong>d<br />
acetylcholine (ACh) is a critical factor in PDD. C57Bl/6 mice, heterozygous <strong>for</strong> <strong>the</strong> high-affinity,<br />
hemicholinium-3 sensitive choline tr<strong>an</strong>sporter (CHT +/-), exhibit reduced levels of ACh in <strong>the</strong><br />
striatum <strong>an</strong>d cortex compared to wild-type (WT) littermates, but display no differences in<br />
measures of <strong>an</strong>xiety, spont<strong>an</strong>eous locomotion, or spatial memory. We sought to evaluate whe<strong>the</strong>r<br />
combined DA-ACh depletion could generate features of PDD. CHT+/- mice <strong>an</strong>d WT littermates<br />
were given bilateral infusions of 6-hydroxydopamine (6-OHDA; 2.0 µg in 1.0 µl) or vehicle into<br />
<strong>the</strong> striatum. Following a one-week recovery, mice were trained on attention set shifting or<br />
recognition tasks. An attention set-shifting task was used to challenge executive functioning as
this task requires <strong>an</strong>imals to self-generate a pl<strong>an</strong> to find food rewards that are hidden under<br />
scented media, ch<strong>an</strong>ge that pl<strong>an</strong> when necessary, <strong>an</strong>d create a set rule by which <strong>the</strong> food reward<br />
is consistently found. Trials to criterion <strong>an</strong>d number of errors serve as dependent variables.<br />
Object <strong>an</strong>d spatial recognition memory are indicated by relative time spent exploring two<br />
objects: one familiar <strong>an</strong>d in a familiar location, <strong>the</strong> o<strong>the</strong>r novel (object) or in a novel location<br />
(spatial). Memory was measured with 5- <strong>an</strong>d 30-minute delays between sample <strong>an</strong>d test sessions.<br />
Our initial findings reveal impaired attention set shifting in CHT+/- mice lesioned with 6-OHDA<br />
as compared to vehicle-treated WT <strong>an</strong>imals or 6-OHDA-treated WT <strong>an</strong>imals. Specifically, 6-<br />
OHDA-treated CHT+/- mice showed impaired ability to self-generate a pl<strong>an</strong> <strong>an</strong>d create a set, but<br />
were not impaired in flexibly ch<strong>an</strong>ging a pl<strong>an</strong> when necessary. Recognition scores showed no<br />
effect of genotype or lesion on memory when tested with a novel or spatially reconfigured<br />
object. These findings display similarities with <strong>the</strong> cognitive profile of PDD patients suggesting<br />
that <strong>the</strong> DA-depleted CHT+/- mice may represent <strong>an</strong> <strong>an</strong>imal model of PDD. Fur<strong>the</strong>rmore, <strong>the</strong><br />
results support a role <strong>for</strong> cholinergic depletion in <strong>the</strong> disease.<br />
Disclosures: L. Zurkovsky, None; E.R. Bychkov, None; R.D. Blakely, MH073159, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); E.V. Gurevich, Cognitive Deficits <strong>an</strong>d Mood Disorders in PD gr<strong>an</strong>t<br />
<strong>from</strong> Michael J. Fox Foundation, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); NS45117, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received).<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.21/L9<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: CREST, Jap<strong>an</strong><br />
Title: Endoplasmic reticulum stress sensor ATF6α plays a critical role in <strong>the</strong> protection <strong>from</strong> 1methyl-4-phenyl-1,<br />
2, 3, 6-tetrahydropyridine-induced dopaminergic neuronal death<br />
Authors: *N. EGAWA 1 , K. YAMAMOTO 2 , H. INOUE 1 , K. NISHI 3 , K. MORI 2 , R.<br />
TAKAHASHI 1 ;<br />
1 Dept. of Neurology, Grad. Sch. of Medicine, Kyoto Univ., Kyoto, Jap<strong>an</strong>; 2 Dept. of Biophysics,
Grad. Sch. of Science, Kyoto Univ., Kyoto, Jap<strong>an</strong>; 3 Tokyo Metropolit<strong>an</strong> Inst. <strong>for</strong> Neurosci. 2-6<br />
Musashidai, Fuchu, Tokyo, Tokyo, Jap<strong>an</strong><br />
Abstract: Oxidative stress <strong>an</strong>d endoplasmic reticulum (ER) stress are thought to contribute to<br />
<strong>the</strong> pathogenesis of neurodegenerative disease including Parkinson’s disease. However, <strong>the</strong><br />
cause-effect relationship between <strong>the</strong>se stresses are poorly understood. ATF6α is <strong>an</strong> membr<strong>an</strong>ebound<br />
tr<strong>an</strong>scription factor activated by ER stress induced by protein misfolding in <strong>the</strong> ER <strong>an</strong>d<br />
functions as a critical regulator of ER quality control in mammali<strong>an</strong> cells. Here we demonstrate<br />
that 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin<br />
producing oxidative stress, induces more prominent ubiquitin-immunopositive inclusion<br />
<strong>for</strong>mation <strong>an</strong>d dopaminergic neuronal loss in mice deficient in ATF6α th<strong>an</strong> in wild-type mice. In<br />
response to MPTP treatment phosphorylation of p38 Mitogen-Activated Protein Kinase<br />
(p38MAPK) was enh<strong>an</strong>ced accomp<strong>an</strong>ied by upregulation of ATF6α <strong>an</strong>d ER chaperones in<br />
dopaminergic neurons. Cultured cell experiments revealed that hydrogen peroxide (H2O2)induced<br />
oxidative stress promotes phosphorylation of p38MAPK, enh<strong>an</strong>ces <strong>the</strong> interaction of<br />
phosphorylated p38MAPK with ATF6α, leading to tr<strong>an</strong>scriptional upregulation of ER<br />
chaperones <strong>an</strong>d ERAD components presumably through phosphorylation-induced activation of<br />
ATF6α. Taken toge<strong>the</strong>r, our study indicates that ATF6α plays a critical role in <strong>the</strong> protection of<br />
<strong>the</strong> dopaminergic neurons <strong>from</strong> MPTP through its p38MAPK-mediated activation.<br />
Disclosures: N. Egawa, CREST, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); K. yamamoto, CREST, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); H. inoue, CREST, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); K. nishi, None;<br />
K. Mori, CREST, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); R. takahashi, CREST, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received).<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.22/L10<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Support: CINN Gr<strong>an</strong>t
Title: Behavioral <strong>an</strong>d histological <strong>an</strong>alyses of hum<strong>an</strong> umbilical cord blood <strong>an</strong>d embryonic stem<br />
cells tr<strong>an</strong>spl<strong>an</strong>tation in a rodent model of Parkinson’s disease<br />
Authors: L. P. KELLY 1 , *M. B. NEWMAN 1 , A. P. SMITH 2 , R. A. E. BAKAY 1 ;<br />
1 Depts.NeuroSurg & Pharmacol, 2 Dept.NeuroSurg, Rush Univ. Med. Ctr., Chicago, IL<br />
Abstract: We previously reported <strong>the</strong> first study indicating hum<strong>an</strong> umbilical cord blood (hUCB)<br />
cells, both <strong>the</strong> heterogeneous mononuclear fraction (MNF) <strong>an</strong>d neural-induced CD-133<br />
(nCD133) stem cells (a subclass of hUCB cells), yield signific<strong>an</strong>t behavioral improvements over<br />
neural-induced hum<strong>an</strong> embryonic stem cells (nhESC) in Parkinson’s Disease. In comparison to<br />
hESCs, hUCBs are a heterogeneous population rich in haematopoietic stem <strong>an</strong>d progenitor cells,<br />
lack ethical implications, c<strong>an</strong> be collected easily <strong>an</strong>d noninvasively, are more immune immature<br />
th<strong>an</strong> o<strong>the</strong>r adult stem cells, <strong>an</strong>d have over two decades of history in treating various<br />
nonmalign<strong>an</strong>t <strong>an</strong>d malign<strong>an</strong>t diseases.<br />
We provide fur<strong>the</strong>r immunohistological data on our study that determined <strong>the</strong> viability <strong>an</strong>d<br />
potential of nhESCs, MNF <strong>an</strong>d nCD133s to repair, replace, or regenerate <strong>the</strong> loss of dopamine<br />
(DA) in <strong>the</strong> striatum of Parkinsoni<strong>an</strong>-like rats. Rats (n=28) were lesioned twice in <strong>the</strong> right<br />
medial <strong>for</strong>ebrain bundle with 6-hydroxydopamine (6-OHDA) or sham lesioned (n=8). Baseline<br />
rotational behavioral testing was per<strong>for</strong>med 1-2 days be<strong>for</strong>e lesion surgery <strong>an</strong>d 3 weeks after <strong>for</strong><br />
inclusion criteria. In this model of PD, at least 60% of DA neurons in <strong>the</strong> SNpc are lost, with<br />
~80% depletion of DA levels in <strong>the</strong> striatum, <strong>an</strong>d full PD-like motor deficits are observed by 3<br />
weeks post lesion. Unilateral cell tr<strong>an</strong>spl<strong>an</strong>tations were per<strong>for</strong>med into <strong>the</strong> ipsilateral striatum<br />
with ei<strong>the</strong>r nhESCs, MNF, or nCD133s. In each group of rats, <strong>the</strong> brains, spleens, bone marrow<br />
<strong>an</strong>d peripheral blood were taken <strong>for</strong> immunohistochemical dependent measures. Tissue sections<br />
were stained <strong>for</strong> tyrosine hydroxylase (TH), neuronal nuclei (NeuN), hum<strong>an</strong> nuclei (HuN), or<br />
double-labeled <strong>for</strong> NeuN/HuN <strong>an</strong>d TH/HuN <strong>for</strong> cell counts.<br />
The specific aim of our study was to determine whe<strong>the</strong>r <strong>the</strong> nCD133 stem cells could yield<br />
behavioral improvements that have been previously reported <strong>for</strong> nhESC tr<strong>an</strong>spl<strong>an</strong>tations. The<br />
most fundamental finding was that both <strong>the</strong> MNF <strong>an</strong>d nCD133s produced signific<strong>an</strong>t<br />
improvements in <strong>the</strong> rotational, swing, <strong>an</strong>d <strong>for</strong>elimb behavioral tests to nearly complete recovery<br />
at 1 <strong>an</strong>d 2 months. The nhESC stem cells, however, only showed signific<strong>an</strong>t improvements in <strong>the</strong><br />
swing <strong>an</strong>d <strong>for</strong>elimb behavioral tests at 1 <strong>an</strong>d 2 months. In addition, histologically, <strong>the</strong><br />
tr<strong>an</strong>spl<strong>an</strong>ted hUCB cells appeared to be more numerous <strong>an</strong>d widely distributed th<strong>an</strong> <strong>the</strong> nhESCs.<br />
Interestingly, both hUCB groups were distributed bilaterally <strong>an</strong>d heavily localized throughout<br />
blood vessels. We are now determining whe<strong>the</strong>r <strong>the</strong> cells migrate outside <strong>the</strong> CNS.<br />
Disclosures: L.P. Kelly, None; M.B. Newm<strong>an</strong>, None; A.P. Smith, None; R.A.E. Bakay,<br />
None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.23/L11<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment<br />
Title: DNMT 1 is expressed in neurons of Parkinson disease brains<br />
Authors: *O. KAUT 1 , M. MAJORES 2 , U. WUELLNER 2 ;<br />
1 Bonn, Germ<strong>an</strong>y; 2 Univ. of Bonn, Bonn, Germ<strong>an</strong>y<br />
Abstract: Objective: To evaluate <strong>the</strong> protein expression of <strong>the</strong> DNA methyltr<strong>an</strong>sferase (DNMT<br />
1) of Parkinson’s disease (PD) patients <strong>an</strong>d of healthy individuals, we used<br />
immunohistochemistry (IHC) <strong>for</strong> slices of cortex <strong>an</strong>d subst<strong>an</strong>tia nigra.<br />
Background: DNA (cytosine-5) methyltr<strong>an</strong>sferases are enzymes that catalyse <strong>the</strong> methylation of<br />
<strong>the</strong> 5´ position of <strong>the</strong> cytosine ring expressed in promoter GpG isl<strong>an</strong>ds. The methylation of<br />
promoter regions is associated with gene silencing. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d hypomethylation c<strong>an</strong><br />
induce gene activation, possibly due to a reduced expression of methyltr<strong>an</strong>sferases. It has<br />
previously been shown by Pieper et al. that CpG isl<strong>an</strong>ds of <strong>the</strong> tumor necrosis factor-alpha (TNFalpha)<br />
promoter are hypomethylated in Parkinson diseased brains. This may induce <strong>an</strong> activation<br />
of <strong>the</strong> TNF-alpha promoter. We hypo<strong>the</strong>size that this hypomethylation is based on a reduced<br />
protein expression of DNMT 1.<br />
Methods: To test this hypo<strong>the</strong>sis we per<strong>for</strong>med immunohistochemistry of paraffin embedded<br />
brain slices of cortex <strong>an</strong>d subst<strong>an</strong>tia nigra with a polyclonal hum<strong>an</strong> DNMT 1 <strong>an</strong>tibody. Each<br />
group (Parkinson’s disease, controls) consisted of 5 individuals.<br />
Results: Cortical neurons of PD patients, as well as of healthy individuals showed a moderate<br />
immunoreactivity. No difference between both groups was seen. The protein was predomin<strong>an</strong>tly<br />
localized in <strong>the</strong> cytoplasmatic compartment. No specific signals were detectable in <strong>the</strong> pars<br />
compacta of <strong>the</strong> subst<strong>an</strong>tia nigra. Conclusions: Thus a reduction or misregulation of DNMT 1<br />
protein expression seems not be involved in PD.<br />
Disclosures: O. Kaut, None; M. Majores, None; U. Wuellner, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.24/L12<br />
Topic: C.03.f. Mech<strong>an</strong>isms <strong>an</strong>d degeneration treatment
Support: The Picower Foundation<br />
Title: Exploration of GATA-2 as a <strong>the</strong>rapeutic target in Parkinson's disease<br />
Authors: *M. P. HOROWITZ 1,2,4 , E. H. BRESNICK 5 , J. GREENAMYRE 3,6 ;<br />
1 Univ. Pittsburgh, Pittsburgh, PA; 2 Med. Scientist Training Program, 3 Pittsburgh Inst. <strong>for</strong><br />
Neurodegenerative Dis., Univ. of Pittsburgh Sch. of Med., Pittsburgh, PA; 4 Ctr. <strong>for</strong> Neurosci.<br />
Univ. of Pittsburgh, Pittsburgh, PA; 5 Pharmacol., Univ. of Wisconsin, Madison, Madison, WI;<br />
6 Dept. of Neurol., Univ. of Pittsburgh Med. Ctr., Pittsburgh, PA<br />
Abstract: Parkinson’s disease (PD) is a currently incurable chronic movement disorder with<br />
degeneration of neurons in <strong>the</strong> subst<strong>an</strong>tia nigra (SN) <strong>an</strong>d elsewhere. Alpha-synuclein <strong>an</strong>d iron<br />
pathologically accumulate in <strong>the</strong> parkinsoni<strong>an</strong> SN in hum<strong>an</strong> PD <strong>an</strong>d in <strong>the</strong> rotenone model of PD<br />
<strong>for</strong> unknown reasons, <strong>an</strong>d both are known to be cytotoxic when present in excess. Halting <strong>the</strong><br />
accumulation of <strong>the</strong>se subst<strong>an</strong>ces may <strong>the</strong>re<strong>for</strong>e represent a valuable <strong>the</strong>rapeutic approach <strong>for</strong><br />
PD. However, key regulators of <strong>the</strong>se processes have not been identified. Recently, our<br />
laboratory discovered a tr<strong>an</strong>sferrin/tr<strong>an</strong>sferrin receptor 2 (TfR2)-mediated mech<strong>an</strong>ism <strong>for</strong><br />
cellular iron uptake unique to dopamine neurons within <strong>the</strong> SN. Interestingly, TfR2 levels are<br />
increased in <strong>the</strong>se cells in <strong>the</strong> rotenone rat, allowing increased cellular iron loading to occur. The<br />
regulation of TfR2 is poorly understood. GATA tr<strong>an</strong>scription factors were recently shown to<br />
coordinately <strong>an</strong>d positively regulate <strong>the</strong> expression of alpha-synuclein <strong>an</strong>d 3 heme biosyn<strong>the</strong>sis<br />
genes. Import<strong>an</strong>tly, <strong>the</strong> GATA iso<strong>for</strong>m, GATA-2, is present in hum<strong>an</strong> SN homogenates,<br />
supporting <strong>the</strong> possibility that aberr<strong>an</strong>t GATA-2 activity may contribute to PD pathogenesis by<br />
up-regulating alpha-synuclein <strong>an</strong>d possibly o<strong>the</strong>r disease-related genes. Given <strong>the</strong> connection<br />
between alpha-synuclein <strong>an</strong>d iron-related genes, we explored whe<strong>the</strong>r TfR2 may also be<br />
regulated by GATA tr<strong>an</strong>scription factors. TfR2 contains two GATA elements in its promoter,<br />
<strong>an</strong>d preliminary studies have revealed that GATA-2 binds <strong>the</strong> TfR2 promoter <strong>an</strong>d up-regulates<br />
TfR2 tr<strong>an</strong>scription in mouse embryonic stem cells. We have localized GATA-2 protein to<br />
dopamine neurons in <strong>the</strong> rat SN by immunohistochemistry, <strong>an</strong>d preliminary studies suggest that<br />
it is relatively up-regulated in degenerating neurons as <strong>the</strong>y lose <strong>the</strong>ir phenotypic markers in <strong>the</strong><br />
rotenone model. Fur<strong>the</strong>r, preliminary experiments indicate that silencing of GATA2 in <strong>the</strong><br />
hum<strong>an</strong> dopaminergic neuronal cell line, SH-SY5Y, down-regulates TfR2 expression. We are<br />
now combining in vitro GATA-2 silencing <strong>an</strong>d chromatin immunoprecipitation experiments with<br />
in vivo examination of GATA-2 regulation in <strong>the</strong> rotenone rat SN to explore <strong>the</strong> potential role of<br />
GATA-2 in PD pathogenesis. If GATA-2 coordinately <strong>an</strong>d positively regulates both alphasynuclein<br />
<strong>an</strong>d TfR2, <strong>the</strong>n GATA-2 may be a useful <strong>the</strong>rapeutic target to simult<strong>an</strong>eously mitigate<br />
<strong>the</strong> toxic effects of alpha-synuclein <strong>an</strong>d iron in PD.<br />
Disclosures: M.P. Horowitz, None; E.H. Bresnick, None; J. Greenamyre, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.25/L13<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: Edmonds bequest <strong>for</strong> PD research<br />
Title: L-DOPA-induced dyskinesia priming is reversed by calcium-permeable AMPA receptor<br />
blockade in <strong>the</strong> 6-OHDA-lesioned rat model of Parkinson’s disease<br />
Authors: *P. RAVENSCROFT 1 , C. KOBYLECKI 2 , A. R. CROSSMAN 2 ;<br />
2 Fac. of Life Sci., 1 Univ. of M<strong>an</strong>chester, M<strong>an</strong>chester, United Kingdom<br />
Abstract: L-DOPA-induced dyskinesia (LID) is a common complication of dopamine<br />
replacement <strong>the</strong>rapy <strong>for</strong> Parkinson’s disease (PD). Overactivity of striatal α-amino-3-hydroxy-5methyl-4-isoxazolepropionic<br />
acid (AMPA) receptors has been implicated in LID<br />
pathophysiology, <strong>an</strong>d acute treatment with AMPA receptor (AMPAR) <strong>an</strong>tagonists shows<br />
efficacy in non-hum<strong>an</strong> primate models of PD <strong>an</strong>d LID. However, <strong>the</strong> effects of chronic treatment<br />
have not been assessed. The objective of this study was to compare <strong>the</strong> effects of acute <strong>an</strong>d<br />
chronic treatment with selective AMPAR <strong>an</strong>tagonists in <strong>the</strong> 6-hydroxydopamine (6-OHDA)lesioned<br />
rat model of LID.<br />
In <strong>the</strong> first part of our study, unilaterally 6-OHDA-lesioned rats previously primed with L-<br />
DOPA/benserazide (6/15 mg/kg, i.p.) <strong>for</strong> 21 days <strong>an</strong>d showing abnormal involuntary movements<br />
(AIMs) received acute treatment with <strong>the</strong> non-competitive AMPAR <strong>an</strong>tagonist GYKI 52466 (1,<br />
5, 10 mg/kg, i.p.) <strong>an</strong>d <strong>the</strong> Ca 2+ -permeable AMPAR <strong>an</strong>tagonist IEM 1460 (1, 3 mg/kg, i.p.). In <strong>the</strong><br />
second part, 6-OHDA-lesioned rats previously primed with L-DOPA/benserazide <strong>for</strong> 21 days<br />
received chronic treatment <strong>for</strong> seven days with GYKI 52466 (5 mg/kg), IEM 1460 (3 mg/kg) or<br />
vehicle, toge<strong>the</strong>r with L-DOPA. The effects of treatment on AIMs <strong>an</strong>d rotarod per<strong>for</strong>m<strong>an</strong>ce were<br />
assessed. The effect on priming <strong>for</strong> dyskinesia was assessed with <strong>an</strong> L-DOPA challenge 24 hours<br />
after <strong>the</strong> end of <strong>the</strong> chronic drug treatment period.<br />
Acute treatment with both GYKI 52466 5 mg/kg (p
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.26/L14<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Support: NS 047452 (ARW)<br />
Parkinson's Disease Foundation<br />
Title: Inhibition of soluble gu<strong>an</strong>ylyl cyclase reverses electrophysiological <strong>an</strong>d behavioral<br />
abnormalities associated with experimental parkinsonism<br />
Authors: *D. PARK 1 , S. SAMMUT 1 , E. SUNU 1 , M. PARK 3 , R. SOBHANI 4 , S. BLUME 1 , K.<br />
TSENG 1 , A. WEST 2 ;<br />
2 1 3<br />
Neurosci., Rosalind Fr<strong>an</strong>klin Univ., North Chicago, IL; Washington Univ., St. Louis, MO;<br />
4<br />
Lake Forest Col., Lake Forest, IL<br />
Abstract: The soluble gu<strong>an</strong>ylyl cyclase (sGC)-cGMP signaling cascade is <strong>an</strong> import<strong>an</strong>t, yet<br />
understudied, pathway which potently modulates corticostriatal tr<strong>an</strong>smission. Striatal mediumsized<br />
spiny neurons (MSNs) contain high levels of sGC, cGMP, <strong>an</strong>d cGMP-dependent protein<br />
kinases. In <strong>the</strong> dopamine (DA)-depleted striatum, neuroadaptations in cGMP syn<strong>the</strong>sis <strong>an</strong>d<br />
metabolism occur which may contribute to <strong>the</strong> enduring ch<strong>an</strong>ges in neuronal excitability <strong>an</strong>d<br />
locomotor activity observed in parkinsoni<strong>an</strong> <strong>an</strong>imals. In <strong>the</strong> current study we have examined <strong>the</strong><br />
utility of systemic administration of <strong>the</strong> selective sGC inhibitor [1H-[1,2,4] oxadiazolo-[4,3a]quinoxalin-1-one]<br />
(ODQ) <strong>for</strong> reversing electrophysiological <strong>an</strong>d behavioral correlates of<br />
experimental parkinsonism induced following unilateral 6-hydroxydopamine infusion into <strong>the</strong><br />
medial <strong>for</strong>ebrain bundle. Local field potentials (LFPs) were recorded in <strong>the</strong> motor cortex <strong>an</strong>d<br />
striatum simult<strong>an</strong>eously with striatal single-units <strong>an</strong>d neuronal synchrony was assessed by<br />
<strong>an</strong>alyzing <strong>the</strong> temporal relationship between <strong>the</strong>se measures. A strong trend towards <strong>an</strong> increase<br />
in <strong>the</strong> peak frequency of LFPs (within <strong>the</strong> delta b<strong>an</strong>d) was observed in <strong>the</strong> cortex of DA-depleted<br />
rats. Analysis of striatal recordings showed that measures of LFPs <strong>an</strong>d neuronal synchrony were<br />
not different across sham-operated <strong>an</strong>d 6-hydroxydopamine lesioned groups. However, DAdepleted<br />
rats exhibited robust elevations in spont<strong>an</strong>eous firing activity as compared to shamlesioned<br />
controls. Systemic administration of ODQ did not affect <strong>the</strong> peak frequency of LFPs<br />
recorded in <strong>the</strong> cortex or striatum of sham-operated or DA-depleted rats, but did decrease <strong>the</strong><br />
strength of neuronal synchrony observed between <strong>the</strong>se regions. Interestingly, <strong>the</strong> effect of ODQ<br />
administration on neuronal synchrony recorded in DA-depleted rats was associated with <strong>an</strong><br />
earlier onset time <strong>an</strong>d longer duration as compared to <strong>the</strong> time course of drug effects observed in<br />
sham-lesioned rats. Fur<strong>the</strong>rmore, elevations in spont<strong>an</strong>eous firing of striatal neurons were
tr<strong>an</strong>siently reversed in DA-depleted rats following systemic administration of ODQ. The effects<br />
of ODQ on electrophysiological measures appeared to be behaviorally relev<strong>an</strong>t as <strong>the</strong> same<br />
treatment tr<strong>an</strong>siently attenuated <strong>the</strong> reduction in <strong>for</strong>elimb use observed in DA-depleted rats.<br />
Toge<strong>the</strong>r, <strong>the</strong>se findings indicate that <strong>the</strong> increase in neuronal excitability observed in DAdepleted<br />
<strong>an</strong>imals is partially mediated or exacerbated by activation of <strong>the</strong> sGC-cGMP signaling<br />
pathway. Thus, pharmacological attenuation of sGC-cGMP signaling holds considerable promise<br />
as a novel, non-dopaminergic treatment of parkinsoni<strong>an</strong> symptoms.<br />
Disclosures: D. Park, None; S. Sammut, None; E. Sunu, None; M. Park, None; R. Sobh<strong>an</strong>i,<br />
None; S. Blume, None; K. Tseng, None; A. West, None.<br />
Poster<br />
532. Parkinson's Disease: Rat <strong>an</strong>d Mice Toxins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 532.27/L15<br />
Topic: C.03.e. Rat <strong>an</strong>d mouse toxin <strong>an</strong>d behavior models<br />
Title: Partial, bilateral 6-hydroxydopamine lesions to <strong>the</strong> dorsolateral striatum contribute to<br />
extinction failure in acquisition of <strong>an</strong> oper<strong>an</strong>tly conditioned visual discrimination task in rat<br />
Authors: *A. L. EAGLE 1 , J. OH 2 ;<br />
2 Psychology, 1 Central Michig<strong>an</strong> Univ., Mount Pleas<strong>an</strong>t, MI<br />
Abstract: Current <strong>an</strong>imal models of Parkinson’s disease (PD) using 6-hydroxydopamine (6-<br />
OHDA) surgical lesions have revealed that cognitive <strong>an</strong>d motor dysfunction arises <strong>from</strong> <strong>the</strong><br />
denervation of nigrostriatal dopaminergic neurons. Recent studies indicate that dopamine within<br />
<strong>the</strong> striatum may play a modulatory role in neural tr<strong>an</strong>smission associated with corticostriatal<br />
input into <strong>the</strong> basal g<strong>an</strong>glia in cognitive processes. Early stage PD has been associated with<br />
specific cognitive deficits, including deficits in attentional set-shifts. Attentional set-shifts have<br />
been modeled using oper<strong>an</strong>t conditioning paradigms of stimulus discrimination in nigrostriatal<br />
dopamine denervated rats by injuring striatum. Previous research has shown conflicting results<br />
with regards to <strong>the</strong> acquisition of a discrimination task by rats rendered Parkinsoni<strong>an</strong> by<br />
nigrostriatal injury. The current project investigated <strong>the</strong> effects of partial, bilateral 6hydroxydopamine<br />
(6-OHDA; 5.5 µg on each side) lesions to <strong>the</strong> dorsolateral striatum on<br />
acquisition of a visual discrimination oper<strong>an</strong>t task in rats. Sham-lesioned <strong>an</strong>d 6-OHDA-lesioned<br />
<strong>an</strong>imals were tested on a two-component multiple VR15 (variable ratio 15), extinction schedule<br />
associated with a visual light stimulus <strong>for</strong> 7 to 16 post-surgery days. Results showed that 6-<br />
OHDA lesioned <strong>an</strong>imals had signific<strong>an</strong>tly lower level of discrimination th<strong>an</strong> sham-lesioned
<strong>an</strong>imals, starting at post-surgical day 14. Fur<strong>the</strong>r <strong>an</strong>alysis of discrimination data revealed that <strong>the</strong><br />
deficit in discrimination may be due to extinction failure of oper<strong>an</strong>tly conditioned behavior.<br />
These findings could indicate that <strong>the</strong> basal g<strong>an</strong>glia extrapyramidal pathways compromised in<br />
PD may contribute to deficits in attentional processes or perseveration purported to be linked to<br />
behavioral extinction. These results are consistent with recent reports implicating evidence of<br />
visual neglect <strong>an</strong>d response perseveration both in clinical <strong>an</strong>d <strong>an</strong>imal models of PD. These<br />
findings provide new insight into basal g<strong>an</strong>glia mech<strong>an</strong>isms associated with discrimination<br />
conditioning <strong>an</strong>d attentional processes in PD, <strong>an</strong>d could accelerate development of novel<br />
approaches to diagnose as well as to <strong>the</strong> improved m<strong>an</strong>agement of motor as well as non-motor<br />
symptoms of PD.<br />
Disclosures: A.L. Eagle, None; J. Oh, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.1/L16<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: Gr<strong>an</strong>ts-in-aid <strong>from</strong> <strong>the</strong> Ministry of Education, Science, Culture <strong>an</strong>d Sport of Jap<strong>an</strong><br />
The Program <strong>for</strong> Promotion of Fundamental Studies in Health Sciences of <strong>the</strong> National<br />
Institute of Biomedical Innovation (NIBIO) in Jap<strong>an</strong><br />
Title: Effects of MM-1 <strong>an</strong>d molecular chaperones on <strong>for</strong>mation of polyglutamine aggregation<br />
Authors: *H. ARIGA, E. TASHIRO, M. MIYAZAWA, H. KITAURA, S. M. IGUCHI-<br />
ARIGA;<br />
Grad. Pharm. Hokkaido Univ., Sapporo, Jap<strong>an</strong><br />
Abstract: MM-1, a c-Myc-binding protein, is a tumor suppressor <strong>an</strong>d suppressesactivities of cell<br />
growth <strong>an</strong>d tr<strong>an</strong>s<strong>for</strong>mation catalyzed by c-myc/ras. MM-1 is also known to be Prefoldin5, a<br />
subunit of Prefoldin. Prefoldin regulates <strong>the</strong> folding of nascent polypeptide chain in cooperation<br />
with Hsp70/Hsp40 <strong>an</strong>d Tric/CCT. Since it has been reported that molecular chaperones,<br />
including Hsp70, Hsp40 <strong>an</strong>d Tric/CCT, affect <strong>for</strong>mation of polyglutamine aggregation, we<br />
investigated a role of MM-1 in process of polyQ <strong>for</strong>mation. Aggregation <strong>an</strong>d oligomers of<br />
EGFP-polyQ72 were found to be increased in MM-1 knockdown Neuro2A cells by using siRNA
<strong>an</strong>d by Filter-trap assays <strong>an</strong>d FCS (Fluorescence Correlation Spectroscopy) were used. These<br />
results indicate that Prefoldin affects <strong>the</strong> <strong>for</strong>mation of polyQ aggregations.<br />
Disclosures: H. Ariga, None; E. Tashiro, None; M. Miyazawa, None; H. Kitaura,<br />
None; S.M. Iguchi-ariga, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.2/L17<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: Huntington's Disease <strong>Society</strong> of America<br />
Novartis Foundation<br />
EPFL<br />
Title: In silico cell type-specific <strong>an</strong>alysis of tr<strong>an</strong>scriptional dysregulation in Huntington’s disease<br />
Authors: *A. KUHN, R. LUTHI-CARTER;<br />
Lab. of Functional Neurogenomics, EPFL, Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d<br />
Abstract: Microarray-based gene expression profiling is a powerful method to measure<br />
tr<strong>an</strong>scriptional ch<strong>an</strong>ges occuring in <strong>the</strong> course of <strong>an</strong> import<strong>an</strong>t biological process, such as a<br />
disease. The detected ch<strong>an</strong>ges define <strong>the</strong> molecular pathology of <strong>the</strong> disease <strong>an</strong>d c<strong>an</strong> in<strong>for</strong>m our<br />
underst<strong>an</strong>ding of disease etiology. However, a disease condition often results in ch<strong>an</strong>ges of tissue<br />
composition, confounding <strong>the</strong> <strong>an</strong>alysis of molecular expression ch<strong>an</strong>ges. There<strong>for</strong>e, when<br />
per<strong>for</strong>ming a comparison of gene expression levels in normal <strong>an</strong>d diseased tissue samples, we<br />
might detect ch<strong>an</strong>ges that are <strong>the</strong> mere consequence of ch<strong>an</strong>ges in <strong>the</strong> proportions of various cell<br />
populations. Possible experimental solutions to identify ch<strong>an</strong>ges within specific cell populations<br />
include tissue microdissection <strong>an</strong>d <strong>the</strong> use of fluorescence-activated cell sorting (FACS) <strong>for</strong><br />
inst<strong>an</strong>ce. Here, we propose a statistical method to address this issue <strong>an</strong>d detect cell populationspecific<br />
ch<strong>an</strong>ges in gene expression in silico.<br />
The development of this method was motivated by our study of Huntington’s disease (HD). HD<br />
is <strong>an</strong> autosomal domin<strong>an</strong>t disease associated with dramatic ch<strong>an</strong>ges in motor, cognitive <strong>an</strong>d/or<br />
psychiatric per<strong>for</strong>m<strong>an</strong>ce, <strong>an</strong>d eventually, death. A striking feature of HD pathology is a loss of<br />
neurons in <strong>the</strong> caudate nucleus of <strong>the</strong> striatum, as well as <strong>the</strong> proliferation of glial cells. In
addition, tr<strong>an</strong>scriptional dysregulation has been shown to occur early in <strong>the</strong> course of <strong>the</strong> disease<br />
<strong>an</strong>d has been proposed to participate in disease etiology. We thus aimed at detecting ch<strong>an</strong>ges<br />
occurring specifically in <strong>the</strong> neuronal population which reflect <strong>the</strong> etiology of <strong>the</strong>ir degeneration.<br />
Using our previous microarray dataset obtained <strong>from</strong> post-mortem caudate samples of hum<strong>an</strong><br />
HD patients <strong>an</strong>d controls, we developed <strong>an</strong>d applied a statistical method to estimate <strong>the</strong><br />
contribution of each cell population to <strong>the</strong> total expression of each gene (i.e. measured <strong>from</strong><br />
homogenate samples), as well as gene expression ch<strong>an</strong>ges specific to each population.<br />
Interestingly, this method allowed us to identify genes upregulated in neuron which went<br />
undetected in a conventional comparison of diseased versus normal samples because <strong>the</strong><br />
decreased number of neurons in HD compensated <strong>for</strong> <strong>the</strong> gene’s specific upregulation. Fur<strong>the</strong>r<br />
experimental investigation of such newly detected gene expression ch<strong>an</strong>ges is currently ongoing<br />
in our laboratory.<br />
The proposed <strong>an</strong>alysis method is widely applicable <strong>an</strong>d could be useful in addressing m<strong>an</strong>y<br />
biological problems where tr<strong>an</strong>scriptional regulation is investigated in <strong>the</strong> context of histological<br />
ch<strong>an</strong>ges or variation, such as in tumor biology or development.<br />
Disclosures: A. Kuhn, None; R. Luthi-Carter, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.3/L18<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Disruption of <strong>the</strong> endoplasmic reticulum by polyglutamine exp<strong>an</strong>sion induces Apaf-1dependent,<br />
but apoptosome-independent, activation of caspase-7<br />
Authors: *M. UEDA, S. LI, M. ITOH, Y. HIDA, Y. HAYAKAWA-YANO, Y. SUZUKI, K.<br />
OHTA, E. OHTA, A. MIZUNO, T. NAKAGAWA;<br />
Gifu Univ. Grad Sch. Med., Gifu, Jap<strong>an</strong><br />
Abstract: We discovered <strong>the</strong> novel processes of caspase activation initiated at <strong>the</strong> endoplasmic<br />
reticulum (ER), which was involved in polyglutamine exp<strong>an</strong>sion induced cell death.<br />
Apoptosis is import<strong>an</strong>t <strong>for</strong> normal tissue homeostasis, development <strong>an</strong>d its dysregulation causes<br />
diseases including neurodegenerative diseases, such as Huntington’s disease. Caspases play <strong>an</strong><br />
essential role in apoptosis, which is classified into initiator caspases (such as caspase-9) <strong>an</strong>d<br />
effecter caspases (such as caspase-7). Caspase-9 is activated by <strong>the</strong> <strong>for</strong>mation of apoptosome,<br />
consisting of Apaf-1 <strong>an</strong>d cytochrome c released <strong>from</strong> mitochondria, which is regulated by
members of <strong>the</strong> Bcl-2 family. In addition to mitochondria, <strong>the</strong> ER c<strong>an</strong> be a site <strong>for</strong> initiation of<br />
apoptosis in neurodegenerative diseases. However, little is known about <strong>the</strong> mech<strong>an</strong>ism of<br />
caspase activation at <strong>the</strong> ER.<br />
We found that <strong>the</strong> ER membr<strong>an</strong>e was distorted in a mouse model of Huntington’s disease, R6/2<br />
HD exon 1 tr<strong>an</strong>sgenic mice. Morphological <strong>an</strong>alysis of neurons using electron microscopy (EM)<br />
indicated that <strong>the</strong> luminal space of <strong>the</strong> rough ER in <strong>the</strong> neurons of R6/2 mice was wider th<strong>an</strong> that<br />
in wild-type mice. An irregularity of <strong>the</strong> rough ER <strong>an</strong>d nuclear membr<strong>an</strong>e was observed in R6/2<br />
mice, which was exacerbated during <strong>the</strong> progressing stage of <strong>the</strong> phenotype, leading to activation<br />
of caspase-7 in <strong>the</strong> striatum. Immunocytochemical <strong>an</strong>alysis showed that <strong>the</strong> ER was distorted in<br />
HEK293T cells after tr<strong>an</strong>sfection of 82-repeated polyglutamine (polyQ82), which was prevented<br />
by Congo red, indicating that polyglutamine oligomer may affect <strong>the</strong> structure of <strong>the</strong> ER. In<br />
HEK293T cells expressing polyQ82, caspase-7 was activated, which was inhibited by <strong>the</strong> ERlocalizing<br />
Bcl-xL (ER-Bcl-xL) generated by <strong>the</strong> fusion of <strong>the</strong> tr<strong>an</strong>smembr<strong>an</strong>e domain of cb5 <strong>an</strong>d<br />
murine Bcl-xL lacking its C-terminal region. Similarly, caspase-7 activation in ER stress <strong>an</strong>d<br />
ER-targeting Bax induced cell death was prevented by ER-Bcl-xL. Analysis of knockdown of<br />
caspase-9 <strong>an</strong>d Apaf-1 indicated that Apaf-1, but not caspase-9, was required <strong>for</strong> caspase-7<br />
activation in ER-targeting Bax induced cell death. These results suggested that polyglutamine<br />
exp<strong>an</strong>sion affects <strong>the</strong> ER membr<strong>an</strong>e, leading to activation of caspase-7 dependently of Apaf-1.<br />
Thus, it is import<strong>an</strong>t to explore <strong>the</strong> mech<strong>an</strong>ism of caspase activation initiated at <strong>the</strong> ER, leading<br />
to develop <strong>the</strong> <strong>the</strong>rapeutic interventions of polyglutamine exp<strong>an</strong>sion diseases, such as<br />
Huntington’s disease.<br />
Disclosures: M. Ueda, None; S. Li, None; M. Itoh, None; Y. Hida, None; Y. Hayakawa-<br />
Y<strong>an</strong>o, None; Y. Suzuki, None; K. Ohta, None; E. Ohta, None; A. Mizuno, None; T.<br />
Nakagawa, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.4/L19<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NS041744<br />
Title: Effect of PPARγ activation on mitochondrial morphology <strong>an</strong>d function in Huntington’s<br />
disease cell model
Authors: *Y. JIN, R. A. QUINTANILLA, G. V. W. JOHNSON;<br />
Univ. Rochester, Rochester, NY<br />
Abstract: Huntington’s disease (HD) is <strong>an</strong> autosomal domin<strong>an</strong>t neurodegenerative disease<br />
caused by abnormal exp<strong>an</strong>sion of CAG repeats in <strong>the</strong> huntingtin (htt) gene. Initially <strong>the</strong> striatum,<br />
<strong>an</strong>d later <strong>the</strong> cortex undergo neurodegeneration in HD. Although <strong>the</strong> causative mutation in HD<br />
was uncovered decades ago, it is still unclear how mut<strong>an</strong>t htt leads to pathological progression.<br />
Recent studies suggest <strong>the</strong> involvement of mitochondrial impairment in HD. In addition, it is<br />
becoming increasingly apparent that mut<strong>an</strong>t htt results in tr<strong>an</strong>scriptional dysregulation, which in<br />
turn impairs mitochondrial function. Peroxisome proliferator-activated receptor γ (PPARγ)<br />
coactivator-1α (PGC-1α), a master player <strong>for</strong> mitochondrial biogenesis, has been suggested to<br />
associate with pathological mech<strong>an</strong>isms in HD patients <strong>an</strong>d mouse HD models. PPARγ also<br />
plays <strong>an</strong> import<strong>an</strong>t role in mitochondrial biogenesis, as well as fatty acid oxidation <strong>an</strong>d its<br />
beneficial effect has been shown in several models of neurological disease. Our previous data<br />
demonstrate that <strong>the</strong> expression of PPARγ <strong>an</strong>d its activity are signific<strong>an</strong>tly downregulated in<br />
immortalized striatal cells expressing mut<strong>an</strong>t htt, compared with cells expressing wild type htt.<br />
Treatment with rosiglitazone, a PPARγ agonist signific<strong>an</strong>tly attenuates thapsigargin-induced<br />
mitochondrial potential loss <strong>an</strong>d cell death in striatal cells expressing mut<strong>an</strong>t htt, suggesting that<br />
mitochondrial impairment may be at least in part due to dysregulation of PPARγ <strong>an</strong>d be rescued<br />
by its activation or upregulation. We are currently investigating how upregulation or activation<br />
of PPARγ modulates mitochondrial <strong>for</strong>m <strong>an</strong>d function in HD cell models, <strong>an</strong>d <strong>the</strong> mech<strong>an</strong>isms<br />
by which PPARγ activation protects against cell death.<br />
Disclosures: Y. Jin, None; R.A. Quint<strong>an</strong>illa, None; G.V.W. Johnson, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.5/L20<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Development of a cell based assay to monitor mut<strong>an</strong>t huntingtin protein clear<strong>an</strong>ce by<br />
autophagy<br />
Authors: *J. R. MAZZULLI 1 , H. JEONG 1,2 , P. ISAKSON 2 , A. SIMONSEN 2 , A.<br />
YAMAMOTO 3 , D. KRAINC 1 ;<br />
1 Dept Neurol, Massachusetts Gen. Hosp. (MIND), Charlestown, MA; 2 Ctr. <strong>for</strong> C<strong>an</strong>cer
Biomedicine <strong>an</strong>d Dept. of Biochem., Univ. of Oslo <strong>an</strong>d The Norwegi<strong>an</strong> Radium Hosp., Oslo,<br />
Norway; 3 Neurol., Col. of Physici<strong>an</strong> <strong>an</strong>d Surgeons, Columbia Univ., New York, NY<br />
Abstract: The expression <strong>an</strong>d accumulation of polyglutamine exp<strong>an</strong>ded mut<strong>an</strong>t huntingtin (Htt)<br />
protein plays a key role in <strong>the</strong> pathogenesis of Huntington’s disease (HD). Enh<strong>an</strong>cing <strong>the</strong><br />
degradation of mut<strong>an</strong>t Htt protein is expected to delay or prevent disease onset. We previously<br />
demonstrated that mut<strong>an</strong>t Htt protein degradation is accelerated upon acetylation at lysine 444 by<br />
creb binding protein, leading to neuroprotection. Acetylation was found to enh<strong>an</strong>ce mut<strong>an</strong>t Htt<br />
clear<strong>an</strong>ce through <strong>an</strong> autophagic pathway. Here, we developed a cell-based assay to monitor <strong>the</strong><br />
autophagosomal clear<strong>an</strong>ce of mut<strong>an</strong>t Htt protein. Stable tr<strong>an</strong>sfected HeLa cell lines were<br />
established which express mut<strong>an</strong>t Htt-cherry fusion protein <strong>an</strong>d LC3-YFP, a well established<br />
marker of autophagosomes. Co-localization of Htt-cherry with LC3-YFP was used to monitor<br />
trafficking of mut<strong>an</strong>t Htt into autophagosomes. This assay is utilized to fur<strong>the</strong>r examine cellular<br />
pathways that enh<strong>an</strong>ce Htt protein acetylation <strong>an</strong>d <strong>the</strong> co-localization of mut<strong>an</strong>t Htt protein with<br />
LC3. In addition, <strong>the</strong> clear<strong>an</strong>ce rate of mut<strong>an</strong>t Htt protein will be established in <strong>the</strong> presence of<br />
compounds that promote Htt acetylation. These studies provide <strong>an</strong> assay to monitor mut<strong>an</strong>t Htt<br />
protein clear<strong>an</strong>ce <strong>an</strong>d identify c<strong>an</strong>didate compounds that promote autophagic degradation of <strong>the</strong><br />
mut<strong>an</strong>t protein.<br />
Disclosures: J.R. Mazzulli, None; H. Jeong, None; P. Isakson, None; A. Simonsen, None; A.<br />
Yamamoto, None; D. Krainc, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.6/L21<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Characterization of tr<strong>an</strong>sglutaminase-2 inhibitors with multiple pharmacological profiles:<br />
Working towards a <strong>the</strong>rapeutic agent <strong>for</strong> <strong>the</strong> treatment of Huntington’s disease<br />
Authors: *D. M. MACDONALD 1 , S. SCHAERTL 2 , A. SCHEEL 2 , G. SCHMIEDEL 2 , J.<br />
REINSHAGEN 2 , S. COURTNEY 3 , R. MARSTON 3 , J. PALFREY 3 , M. PRIME 3 , M. TAYLOR 3 ,<br />
C. YARNOLD 3 , J. WITYAK 1 ;<br />
1 Drug Discovery, CHDI M<strong>an</strong>agement, Inc., Los Angeles, CA; 2 Evotec, AG, Hamburg, Germ<strong>an</strong>y;<br />
3 Evotec, Ltd, Abingdon, United Kingdom
Abstract: Tissue tr<strong>an</strong>sglutaminase 2 (TG2; TGM2; hum<strong>an</strong> Gene ID# 7052) is a multi-functional<br />
protein primarily known <strong>for</strong> its calcium-dependent enzymatic activity of crosslinking proteins<br />
via <strong>an</strong> isopeptide bond <strong>for</strong>mation between glutamine <strong>an</strong>d lysine residues, <strong>an</strong>d has also been<br />
shown to act as a GTPase <strong>an</strong>d ATPase. Increased TG2 expression <strong>an</strong>d tr<strong>an</strong>sglutaminase<br />
enzymatic activity have been reported in samples derived <strong>from</strong> Huntington’s disease (HD)<br />
patients <strong>an</strong>d rodent models. Import<strong>an</strong>tly, genetic deletion of TG2 in two different HD mouse<br />
models, R6/1 <strong>an</strong>d R6/2, results in improved phenotypes including a reduction in neuronal death<br />
<strong>an</strong>d prolonged survival. Thus, we are interested in <strong>the</strong> discovery of potent <strong>an</strong>d selective TG2<br />
inhibitors <strong>for</strong> proof-of-concept experiments <strong>an</strong>d eventual clinical development as <strong>an</strong> HD<br />
modifying agent. To that end we have evaluated several TG2 inhibitors with distinct mech<strong>an</strong>istic<br />
actions. A set of biochemical <strong>an</strong>d cellular assays evaluating potency, efficacy, <strong>an</strong>d selectivity<br />
have been optimized to identify potential validating lig<strong>an</strong>ds <strong>for</strong> in vivo efficacy trials. We will<br />
present profiles of covalent peptidic inhibitors, dipeptide Michael acceptors, <strong>an</strong>d small molecule<br />
TG2 tr<strong>an</strong>sglutaminase inhibitors that display distinct modes of action, but do not in all cases<br />
show target specificity, nor signs of cellular activity, which are requisite starting points <strong>for</strong> lead<br />
compound optimization. This assay plat<strong>for</strong>m will enable <strong>the</strong> fur<strong>the</strong>r development of lead-like<br />
molecules to pharmacologically validate TG2 as a <strong>the</strong>rapeutic target <strong>for</strong> Huntington’s disease<br />
using in vivo efficacy studies in murine models, <strong>an</strong>d ultimately in <strong>the</strong> clinic.<br />
Disclosures: D.M. Macdonald, None; S. Schaertl, None; A. Scheel, None; G. Schmiedel,<br />
None; J. Reinshagen, None; S. Courtney, None; R. Marston, None; J. Palfrey, None; M.<br />
Prime, None; M. Taylor, None; C. Yarnold, None; J. Wityak, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.7/L22<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NINDS<br />
Taube-Koret Center <strong>for</strong> Huntington's Disease Research<br />
Hillbloom Foundation Predoctoral Fellowship<br />
NIH-NIGMS-UCSF Medical Scientist Training Program
Title: Pathogenic mech<strong>an</strong>ism <strong>an</strong>d small molecule inhibition of a mut<strong>an</strong>t huntingtin con<strong>for</strong>mer<br />
that predicts striatal neurodegeneration<br />
Authors: *J. M. MILLER 1,2,3 , I. KRATTER 1,2 , L. QUINTI 4 , M. GLICKSMAN 4 , R. STEIN 4 , S.<br />
FINKBEINER 1,2,3 ;<br />
1 UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 2 Gladstone Inst. of Neurolog. Dis., S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 3 Taube-<br />
Koret Ctr. <strong>for</strong> Huntington's Dis. Res., S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 4 LDDN, Harvard Ctr. <strong>for</strong><br />
Neurodegeneration <strong>an</strong>d Repair, Cambridge, MA<br />
Abstract: The mut<strong>an</strong>t version of <strong>the</strong> protein responsible <strong>for</strong> Huntington’s disease, huntingtin,<br />
contains <strong>an</strong> abnormally long polyglutamine stretch that causes huntingtin to fold into multiple<br />
monomeric, oligomeric, <strong>an</strong>d larger aggregated structures. Which of <strong>the</strong>se m<strong>an</strong>y putative species<br />
of mut<strong>an</strong>t huntingtin is toxic has been long debated. We previously demonstrated that <strong>the</strong><br />
monoclonal <strong>an</strong>tibody 3B5H10 recognizes a species of huntingtin that strongly predicts death in<br />
<strong>the</strong> type of neurons that are preferentially affected in Huntington’s disease. Here, we pursue <strong>the</strong><br />
hypo<strong>the</strong>sis that <strong>the</strong> con<strong>for</strong>mation of huntingtin recognized by 3B5H10 may mediate pathogenesis<br />
by mimicking protein interaction structural motifs displayed by o<strong>the</strong>r proteins in <strong>the</strong> neuron. This<br />
mimicry may promote abnormal, toxic interactions between mut<strong>an</strong>t huntingtin <strong>an</strong>d cellular<br />
protein partners. We pl<strong>an</strong> on using protein array technology, immunoprecipitation, <strong>an</strong>d coimmunoprecipitation<br />
to identify which of <strong>the</strong> more th<strong>an</strong> 400 known binding partners of<br />
huntingtin may be mediating pathogenesis with <strong>the</strong> 3B5H10 con<strong>for</strong>mer. In parallel, we are also<br />
screening <strong>for</strong> small molecules which c<strong>an</strong> disrupt <strong>the</strong> con<strong>for</strong>mation of mut<strong>an</strong>t huntingtin<br />
recognized by 3B5H10. Hits <strong>from</strong> this screen will be assayed <strong>for</strong> <strong>the</strong>ir ability to alleviate mut<strong>an</strong>t<br />
huntingtin toxicity in a primary striatal neuron model of HD. Thus, our work aims to i)<br />
underst<strong>an</strong>d how a con<strong>for</strong>mation of mut<strong>an</strong>t huntingtin known to predict neurodegeneration<br />
mediates pathogenesis <strong>an</strong>d ii) <strong>the</strong>rapeutically decrease <strong>the</strong> predictive con<strong>for</strong>mation’s abund<strong>an</strong>ce<br />
through small molecules.<br />
Disclosures: J.M. Miller, None; I. Kratter, None; L. Quinti, None; M. Glicksm<strong>an</strong>, None; R.<br />
Stein, None; S. Finkbeiner, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.8/L23<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms
Title: Investigation of <strong>the</strong> crosstalk between neurons <strong>an</strong>d glia upon inflammation in<br />
Huntington’s disease<br />
Authors: *H.-Y. HSIAO 1,2 , Y. CHERN 2 ;<br />
1 2<br />
Natl. Y<strong>an</strong>g-Ming Univ., Taipei, Taiw<strong>an</strong>; Inst. of Biomed. Science, Academia Sinica, Taipei,<br />
Taiw<strong>an</strong><br />
Abstract: Huntington’s disease (HD) is <strong>an</strong> autosomal domin<strong>an</strong>t inherited disease caused by<br />
expended CAG repeat in <strong>the</strong> exon 1 of huntingtin gene (Htt), <strong>an</strong>d preferentially affects <strong>the</strong><br />
functions of <strong>the</strong> striatum <strong>an</strong>d cortex in <strong>the</strong> brain. A major hallmark of HD is <strong>the</strong> accumulation of<br />
mut<strong>an</strong>t Htt as aggregates in <strong>the</strong> nuclear in neurons <strong>an</strong>d astrocytes. It has been reported that mHtt<br />
suppresses <strong>the</strong> production of a chemokine (CCL5/RANTES) in R6/2 astrocytes which<br />
contributes to <strong>the</strong> neuronal dysfunction in HD. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, accumulated evidence<br />
demonstrated that microglial activation correlates with <strong>the</strong> severity of HD. Altered immune<br />
response in HD patients has also been reported. However, <strong>the</strong> cellular <strong>an</strong>d molecular<br />
mech<strong>an</strong>isms remain unclear. In <strong>the</strong> present study, we showed that primary cortical neurons<br />
harvested <strong>from</strong> a tr<strong>an</strong>sgenic HD mouse model (R6/2) developed axonal varicosities starting <strong>from</strong><br />
DIV 7. No varicosity was observed in wildtype neurons under <strong>the</strong> same culture condition.<br />
Immunocytochemical staining revealed that intensive signals of ubiquitin <strong>an</strong>d activated caspase 3<br />
were localized to axonal varicosities in HD neurons, indicating potential neuronal degeneration.<br />
Using a neurons-astrocyte coculture system, we fur<strong>the</strong>r demonstrated that HD neurons are more<br />
susceptible to <strong>the</strong> damage caused by inflammation, <strong>an</strong>d that astrocytes play a critical role in<br />
neuronal survival under inflammation.<br />
Disclosures: H. Hsiao, None; Y. Chern, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.9/L24<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Impaired AMPA receptor trafficking <strong>an</strong>d function by mut<strong>an</strong>t huntingtin<br />
Authors: *M. MANDAL, E. YUEN, J. WEI, Z. YAN;<br />
Univ. Buffalo, Buffalo, NY
Abstract: Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by <strong>the</strong><br />
polyglutamine exp<strong>an</strong>sion (polyQ) of huntingtin protein (htt). Several htt-interacting proteins<br />
implicated in intracellular tr<strong>an</strong>sport have been identified, one of which is huntingtin-associated<br />
protein 1 (HAP1). HAP1 interacts more tightly with polyQ-htt th<strong>an</strong> wild-type htt, <strong>an</strong>d may act as<br />
a key mediator of pathological alterations in membr<strong>an</strong>e trafficking by mut<strong>an</strong>t htt. In this study,<br />
we examined <strong>the</strong> impact of abnormal interaction between polyQ-htt/HAP1 on <strong>the</strong> trafficking of<br />
AMPA receptors, <strong>the</strong> major player mediating excitatory synaptic tr<strong>an</strong>smission in CNS. We found<br />
that knockdown of HAP1 or expression of polyQ-htt reduced <strong>the</strong> frequency <strong>an</strong>d amplitude of<br />
AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) in cultured cortical<br />
neurons. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, overexpression of wild-type htt increased mEPSC. Moreover,<br />
suppressing <strong>the</strong> kinesin microtubule motor, KIF5, occluded <strong>the</strong> effect of polyQ-htt on mEPSC.<br />
Disruption of <strong>the</strong> GluR2/KIF5/microtubule complex was also found in a tr<strong>an</strong>sgenic mouse model<br />
of HD, N171-82Q, which expresses <strong>an</strong> 82Q-containing N-terminal fragment of htt. Toge<strong>the</strong>r,<br />
<strong>the</strong>se data suggest that AMPAR trafficking is impaired by mut<strong>an</strong>t htt, presumably due to <strong>the</strong><br />
interference of KIF5-mediated microtubule-based tr<strong>an</strong>sport of AMPA receptors. The diminished<br />
strength of excitatory synaptic tr<strong>an</strong>smission in HD conditions could contribute to <strong>the</strong> loss of <strong>the</strong><br />
cortical output that is import<strong>an</strong>t <strong>for</strong> movement control.<br />
Disclosures: M. M<strong>an</strong>dal, None; E. Yuen, None; J. Wei, None; Z. Y<strong>an</strong>, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.10/L25<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: MRC<br />
Title: A HAP1-KIF5 trafficking complex directs tr<strong>an</strong>sport of GABAA receptors to synapses <strong>an</strong>d<br />
is inhibited by mut<strong>an</strong>t huntingtin<br />
Authors: *A. E. TWELVETREES 1 , E. Y. YUEN 2 , L. ARANCIBIA-CARCAMO 1 , A. F.<br />
MACASKILL 1 , M. J. LUMB 1 , Z. YAN 2 , J. T. KITTLER 1 ;<br />
1 Neuroscience, Physiol. <strong>an</strong>d Pharmacol., Univ. Col. London, London, United Kingdom; 2 Dept.<br />
of Physiol. <strong>an</strong>d Biophysics, State Univ. of New York at Buffalo, Buffalo, NY<br />
Abstract: Regulating <strong>the</strong> number of gamma-aminobutyric acid type A receptors (GABAA<br />
receptors) at <strong>the</strong> post synapse is essential <strong>for</strong> controlling <strong>the</strong> strength of neuronal inhibition. In
addition, dysregulation of synaptic GABAA receptor number may also contribute to pathological<br />
alterations in neuronal activity, leading to disrupted in<strong>for</strong>mation processing <strong>an</strong>d excitotoxicity.<br />
Microtubule mediated tr<strong>an</strong>sport plays <strong>an</strong> import<strong>an</strong>t role in regulating <strong>the</strong> trafficking of postsynaptic<br />
components, but <strong>the</strong> microtubule motor proteins import<strong>an</strong>t <strong>for</strong> <strong>the</strong> synaptic delivery of<br />
GABAA receptors are unspecified. Here we show that <strong>the</strong> Huntingtin Associated Protein 1<br />
(HAP1) links kinesin KIF5 microtubule motor proteins to GABAARs. This direct interaction<br />
between <strong>the</strong> KIF5 motors <strong>an</strong>d HAP1, links GABAA receptors to <strong>an</strong>terograde microtubule<br />
tr<strong>an</strong>sport to regulate inhibitory synaptic tr<strong>an</strong>smission. Disrupting <strong>the</strong> KIF5/HAP1/GABAA<br />
receptor complex, using domin<strong>an</strong>t negative constructs, RNAi <strong>an</strong>d function blocking <strong>an</strong>tibodies,<br />
reduces <strong>the</strong> level of surface GABAA receptor expression, <strong>the</strong> number of GABAA receptors at<br />
inhibitory synapses <strong>an</strong>d <strong>the</strong> strength of synaptic inhibition by reducing <strong>the</strong> rate of synaptic<br />
delivery of GABAA receptors <strong>from</strong> <strong>an</strong> internal pool.<br />
In Huntington’s disease, which is caused by a polyglutamine exp<strong>an</strong>sion in <strong>the</strong> protein huntingtin,<br />
toxic alterations of huntingtin function disrupt <strong>the</strong> normal cell biological role of HAP1. We<br />
demonstrate that huntingtin c<strong>an</strong> also be found complexed with GABAA receptors <strong>an</strong>d that mut<strong>an</strong>t<br />
polyQ-huntingtin disrupts inhibitory synaptic responses <strong>an</strong>d <strong>the</strong> normal KIF5/HAP1-dependent<br />
trafficking of GABAA receptors to synapses. Our results demonstrate that KIF5/HAP1 dependent<br />
GABAA receptor trafficking is critical <strong>for</strong> <strong>the</strong> mainten<strong>an</strong>ce of inhibitory synaptic tr<strong>an</strong>smission<br />
<strong>an</strong>d disruption of this trafficking by mut<strong>an</strong>t polyQ-huntingtin results in compromised neuronal<br />
inhibition in HD.<br />
Disclosures: A.E. Twelvetrees, None; E.Y. Yuen, None; L. Ar<strong>an</strong>cibia-Carcamo, None; A.F.<br />
MacAskill, None; M.J. Lumb, None; Z. Y<strong>an</strong>, None; J.T. Kittler, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.11/L26<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t P20RR020171<br />
NIH Gr<strong>an</strong>t K01MH067123<br />
Title: Huntingtin regulates RE1-silencing tr<strong>an</strong>scription factor/neuron-restrictive silencer factor<br />
(REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting<br />
LIM domain protein (RILP) <strong>an</strong>d dynactin p150 Glued
Authors: *M. SHIMOJO;<br />
Univ. Kentucky, Coll Med., Lexington, KY<br />
Abstract: Huntington’s disease is a choreic-psychiatric neurodegenerative disease caused by a<br />
mutation in <strong>the</strong> huntingtin protein. The pathogenic mech<strong>an</strong>isms induced by mut<strong>an</strong>t huntingtin are<br />
not clearly understood. Huntingtin has been reported to regulate <strong>the</strong> nuclear tr<strong>an</strong>slocation of <strong>the</strong><br />
tr<strong>an</strong>scriptional repressor RE1-silencing tr<strong>an</strong>scription factor/neuron-restrictive silencer factor<br />
(REST/NRSF). The interaction between huntingtin <strong>an</strong>d REST/NRSF was reported to retain<br />
REST/NRSF in <strong>the</strong> cytosol, <strong>the</strong>reby preventing REST/NRSF target gene repression. The<br />
REST/NRSF-Interacting LIM domain Protein (RILP) is a binding protein of REST/NRSF. RILP<br />
has also been shown to regulate REST/NRSF nuclear tr<strong>an</strong>slocation. There<strong>for</strong>e, we were<br />
prompted to address <strong>the</strong> question of how two distinct proteins could have <strong>the</strong> same function. To<br />
more fully underst<strong>an</strong>d <strong>the</strong> function, yeast two-hybrid screen with C-terminal fragment of RILP<br />
was per<strong>for</strong>med to look <strong>for</strong> <strong>an</strong> interaction between huntingtin <strong>an</strong>d RILP. As a result, dynactin<br />
p150 Glued was identified as <strong>an</strong> interacting protein. Coimmunoprecipitation <strong>an</strong>alysis of proteins in<br />
vitro expressed in a reticulocyte lysate system showed <strong>an</strong> interaction between REST/NRSF <strong>an</strong>d<br />
RILP as well as between RILP <strong>an</strong>d dynactin p150 Glued . Coimmunoprecitation <strong>an</strong>alysis fur<strong>the</strong>r<br />
showed a complex containing RILP, dynactin p150 Glued , <strong>an</strong>d huntingtin. Huntingtin did not<br />
interact directly with ei<strong>the</strong>r REST/NRSF or RILP, but did interact with dynactin p150 Glued . The<br />
N-terminal fragment of wild-type huntingtin did not affect <strong>the</strong> interaction between dynactin<br />
p150 Glued <strong>an</strong>d RILP; however, mut<strong>an</strong>t huntingtin weakened this interaction. We fur<strong>the</strong>r showed<br />
that HAP1 (huntingtin-associated protein-1) prevents this complex <strong>from</strong> tr<strong>an</strong>slocating<br />
REST/NRSF to <strong>the</strong> nucleus. It was suggested that HAP1 affects <strong>the</strong> interaction of mut<strong>an</strong>t<br />
huntingtin with <strong>the</strong> complex <strong>an</strong>d leads to a weakening of <strong>the</strong> interaction of RILP with <strong>the</strong><br />
complex. However, HAP1 did not affect this interaction in <strong>the</strong> presence of wild-type huntingtin.<br />
Thus, <strong>the</strong>se studies show that <strong>the</strong> RILP-dynactin p150 Glued -Htt is complex is involved in <strong>the</strong><br />
tr<strong>an</strong>slocation of REST/NRSF into <strong>the</strong> nucleus. Fur<strong>the</strong>rmore HAP1 when present in <strong>the</strong> complex<br />
prevents REST/NRSF cellular localization in neurons. This study provides new insights into <strong>the</strong><br />
role of RILP, huntingtin, dynactin p150 Glued , <strong>an</strong>d HAP1 in <strong>the</strong> regulation of neuron-specific gene<br />
expression by REST/NRSF. This study is supported, in whole or in part, by NIH P20RR020171<br />
<strong>from</strong> <strong>the</strong> National Center <strong>for</strong> Research Resources <strong>an</strong>d K01MH067123 <strong>from</strong> <strong>the</strong> National Institute<br />
of Mental Health.<br />
Disclosures: M. Shimojo, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.12/L27
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: KOSEF Gr<strong>an</strong>t R01-2007-000-20135-0<br />
KIMST Gr<strong>an</strong>t 2007-000-000-6069<br />
KOSEF Gr<strong>an</strong>t <strong>2009</strong>0070560<br />
KOSEF Gr<strong>an</strong>t <strong>2009</strong>0070562<br />
Title: GABAB receptor agonist altered ubiquitin proteasome system function in Huntington's<br />
disease model<br />
Authors: *W. KIM, H. SEO;<br />
Mol & Life Sci., H<strong>an</strong>y<strong>an</strong>g Univ., Ans<strong>an</strong>, Republic of Korea<br />
Abstract: Huntington's disease (HD) is <strong>an</strong> autosomal neurodegenerative disease, which shows<br />
<strong>the</strong> selective degeneration of <strong>the</strong> medium sized spiny neurons (MSN) in <strong>the</strong> striatum. The<br />
selective vulnerability of <strong>the</strong>se GABAergic MSN cells c<strong>an</strong> be explained by <strong>the</strong> abnormal protein<br />
accumulation, excytotoxicity, mitochondrial dysfunction, failure of trophic control, etc. In this<br />
study, we addressed to determine <strong>the</strong> effects of <strong>the</strong> stimulation of GABAergic neuron on <strong>the</strong><br />
protein degradation mech<strong>an</strong>ism in <strong>the</strong> normal or pathological GABAergic neurons in vitro. First,<br />
to study whe<strong>the</strong>r GABAergic stimulation alter <strong>the</strong> proteasome activities in <strong>the</strong> HD, we<br />
administered GABAB receptor agonist, baclofen, to <strong>the</strong> wild-type or mut<strong>an</strong>t huntingtin<br />
expressing striatal neurons. Interestingly, chymotrypsin-like proteasome activities signific<strong>an</strong>tly<br />
increased in <strong>the</strong> mut<strong>an</strong>t huntingtin expressing striatal neurons after GABAB receptor agonist<br />
treatment. Second, we administered proteasome inhibitor, MG132, to <strong>the</strong> wild-type or mut<strong>an</strong>t<br />
huntingtin expressing striatal neurons, to determine whe<strong>the</strong>r proteasome is directly related to <strong>the</strong><br />
control of <strong>the</strong> cellular function of GABAB receptor protein. Proteasome inhibition by MG132<br />
decreased <strong>the</strong> protein expression levels of GABAB receptor in this experiment. Third, we also<br />
determined <strong>the</strong> ch<strong>an</strong>ges of cell viability after GABAB receptor agonist treatment. These data<br />
suggest that <strong>the</strong> activities of <strong>the</strong> cellular ubiquitin proteasome <strong>an</strong>d functional GABAergic<br />
neurons are directly related to each o<strong>the</strong>r.<br />
Disclosures: W. Kim, None; H. Seo, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 533.13/L28<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NeRF Gr<strong>an</strong>t to L.G.<br />
EU FP7 gr<strong>an</strong>t 222943 "NeuroStemCell "<br />
Title: Doublecortin-like kinase 3 (DCLK3), a novel striatum-enriched species, is a modulator of<br />
mut<strong>an</strong>t huntingtin in vivo<br />
Authors: L. GALVAN 1 , M.-C. GAILLARD 2 , M. DE CHALDÉE 2 , G. AUREGAN 1 , N.<br />
DUFOUR 1 , M. GUILLERMIER 1 , D. HOUITTE 1 , F. PETIT 1 , C. MALGORN 1 , G. LIOT 3 , S.<br />
HUMBERT 3 , J. ELALOUF 2 , N. DÉGLON 1 , *E. P. BROUILLET 1 ;<br />
1 CEA, MIRCen, URA CEA-CNRS 2210, Fontenay-aux-Roses, Fr<strong>an</strong>ce; 2 CEA-IBITec-<br />
S/SBIGeM, Saclay, Fr<strong>an</strong>ce; 3 Inst. Curie, UMR 146, CNRS, Orsay, Fr<strong>an</strong>ce<br />
Abstract: Huntington's disease (HD) is a neurodegenerative disorder caused by <strong>an</strong> abnormal<br />
CAG repeat exp<strong>an</strong>sion coding <strong>for</strong> <strong>an</strong> exp<strong>an</strong>ded polyglutamine tract in <strong>the</strong> protein "huntingtin"<br />
(Htt). Although this mut<strong>an</strong>t Htt (mHtt) is expressed ubiquitously throughout <strong>the</strong> brain, <strong>the</strong><br />
striatum is found preferentially affected. One hypo<strong>the</strong>sis to explain this particular vulnerability is<br />
that striatal neurons express a particular set of proteins that make <strong>the</strong>m highly vulnerable to<br />
mHtt. In order to fur<strong>the</strong>r examine this hypo<strong>the</strong>sis, we carried out a tr<strong>an</strong>scriptome <strong>an</strong>alysis of<br />
different brain territories <strong>an</strong>d identified more th<strong>an</strong> 100 molecular markers i.e. tr<strong>an</strong>scripts that are<br />
highly enriched in <strong>the</strong> mouse striatum. We recently focused our interest on a subset of striatalenriched<br />
tr<strong>an</strong>scripts of poorly characterized tr<strong>an</strong>scripts.<br />
We here report <strong>the</strong> study of one of <strong>the</strong>se markers, <strong>the</strong> CAMKII family-related kinase DCLK3.<br />
We found that DCLK3 is mainly expressed in <strong>the</strong> adult striatum in rodent with low level of<br />
expression in <strong>the</strong> newborn <strong>an</strong>d striatal primary cultures. Reduced mRNA levels of DCLK3 were<br />
found in <strong>the</strong> striatum of tr<strong>an</strong>sgenic mouse models of HD. We thus studied <strong>the</strong> effect of DCLK3<br />
overexpression <strong>an</strong>d knock-down in a mouse model of HD using lentiviral vectors coding <strong>for</strong> a Nterminal<br />
fragment of mHtt. DCLK3 <strong>an</strong>d its related siRNA were delivered using lentiviral<br />
vectors. Striatal degeneration produced by mHtt was characterized using immunohistochemistry<br />
of DARPP32, Cytochrome oxidase <strong>an</strong>d ubiquitin followed by qu<strong>an</strong>titative histological<br />
evaluation. Results showed that lenti-siRNA targeting DCLK3 increased mHtt toxicity when<br />
compared to <strong>the</strong> control. On <strong>the</strong> contrary, overexpression of DCLK3 reduced <strong>the</strong> striatal lesions<br />
produced by mHtt in vivo. DCLK3 also decreased <strong>the</strong> number <strong>an</strong>d size of ubiquitin-containing<br />
nuclear inclusions. Current experiments are examining <strong>the</strong> mech<strong>an</strong>isms that could underlie <strong>the</strong><br />
neuroprotective effect of DCLK3 in striatal neurons. The present study suggests that DCLK3 is a<br />
potential modifier of <strong>the</strong> disease <strong>an</strong>d might be considered in HD <strong>the</strong>rapy to slow disease<br />
progression.<br />
Disclosures: L. Galv<strong>an</strong>, None; M. Gaillard, None; M. de Chaldée, None; G. Aureg<strong>an</strong>,<br />
None; N. Dufour, None; M. Guillermier, None; D. Houitte, None; F. Petit, None; C.<br />
Malgorn, None; G. Liot, None; S. Humbert, None; J. Elalouf, None; N. Déglon, None; E.P.<br />
Brouillet, None.
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.14/L29<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: CHDI<br />
HDSA<br />
NINDS<br />
Title: Mapping novel huntingtin phosphorylation sites<br />
Authors: *E. WATKIN 1 , T. RATOVITSKI 1 , R. O’MEALLY 2 , R. N. COLE 2 , F. SAUDOU 4 , S.<br />
HUMBERT 4 , C. A. ROSS 1,3 ;<br />
1 Psychiatry, 2 Mass Spectrometry <strong>an</strong>d Proteomics Facility, 3 Depts of Neurol. <strong>an</strong>d Neurosci., Johns<br />
Hopkins Sch. of Med., Baltimore, MD; 4 Ctr. Universitaire, Orsay, Fr<strong>an</strong>ce<br />
Abstract: Huntington’s disease (HD) is <strong>an</strong> inherited autosomal domin<strong>an</strong>t disease caused by<br />
exp<strong>an</strong>sion of <strong>the</strong> polyglutamine (polyQ) region of <strong>the</strong> huntingtin (Htt) protein. Modification of<br />
Htt by phosphorylation may play <strong>an</strong> import<strong>an</strong>t role in HD by affecting <strong>the</strong> toxicity of mut<strong>an</strong>t<br />
polyQ-Htt; Htt phosphorylation at S421 by Akt <strong>an</strong>d SGK kinases has been shown to be<br />
neuroprotective, to reduce accumulation of Htt fragments, <strong>an</strong>d to restore <strong>an</strong>terograde <strong>an</strong>d<br />
retrograde tr<strong>an</strong>sport in neurons. We have used mass spectrometry to map several novel<br />
phosphorylation sites. HEK 293 cells were co-tr<strong>an</strong>sfected with truncated (N511) Htt fragments<br />
containing 52Q repeats <strong>an</strong>d SGK. Htt proteins were purified using Flag affinity chromatography<br />
<strong>an</strong>d subjected to mass spectrometric <strong>an</strong>alysis. C<strong>an</strong>didate sites include S120, T127, T259, <strong>an</strong>d<br />
T488. We are developing phospho-specific <strong>an</strong>tibodies to two of those sites. We are also<br />
generating al<strong>an</strong>ine or aspartate substitutions <strong>for</strong> functional studies. Using <strong>the</strong>se tools, we are<br />
examining <strong>the</strong> effects of Htt phosphorylation at specific sites on cleavage, aggregation, <strong>an</strong>d<br />
toxicity. The results of <strong>the</strong>se studies could lead to new <strong>the</strong>rapeutic targets, as well as a deeper<br />
underst<strong>an</strong>ding of disease toxicity.<br />
Disclosures: E. Watkin, None; T. Ratovitski, None; R. O’Meally, None; R.N. Cole, None; F.<br />
Saudou, None; S. Humbert, None; C.A. Ross, None.
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.15/L30<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: HDF gr<strong>an</strong>t<br />
TCP<br />
Government of Navarra<br />
Title: Altered synaptic composition <strong>an</strong>d trafficking of NMDA receptors in early stages of<br />
Huntington´s disease<br />
Authors: S. MARCO 1 , J. TORRES-PERAZA 2 , A. GIRALT 2 , L. S. KALTENBACH 3 , R. K.<br />
GRAHAM 4,5 , M. R. HAYDEN 4,5 , D. C. LO 3 , J. ALBERCH 2 , *I. PEREZ-OTANO 6,1 ;<br />
1 Neurosciences, Ctr. <strong>for</strong> Applied Med. Res. (CIMA) <strong>an</strong>d Univ. of Navarra, Pamplona, Spain;<br />
2 Cell. Biology, Immunol. <strong>an</strong>d Neurosci., Univ. of Barcelona, Barcelona, Spain; 3 Ctr. <strong>for</strong> Drug<br />
Discovery, Duke Univ., Durham, NC; 4 Ctr. <strong>for</strong> Mol. Med. <strong>an</strong>d Therapeutics, Dept. of Med.<br />
Genet., 5 Child <strong>an</strong>d Family Res. Inst., Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada;<br />
6 FIMA, Pamplona, Spain<br />
Abstract: Recent studies implicate selective alterations in NMDA receptors (NMDARs) in <strong>the</strong><br />
cell-specific death observed in models of Huntington’s disease expressing mutated huntingtin<br />
(htt). Yet how <strong>the</strong> mutations are linked to alterations in NMDAR function remains unclear. We<br />
have recently discovered that <strong>the</strong> htt-interacting protein PACSIN1/syndapin1 behaves as <strong>an</strong><br />
endocytic adaptor <strong>for</strong> NMDARs by targeting <strong>the</strong> NMDAR subunit NR3A (Nature Neuroscience,<br />
9, 611, 2006; Hum Mol Genet. 11, 2547, 2002). Because <strong>the</strong> interaction of PACSIN1 with htt is<br />
repeat-length dependent <strong>an</strong>d enh<strong>an</strong>ced with mut<strong>an</strong>t htt, we hypo<strong>the</strong>sized that mut<strong>an</strong>t htt disrupts<br />
NMDAR endocytic trafficking by altering <strong>the</strong> subcellular location of PACSIN1, leading to<br />
enh<strong>an</strong>ced NMDAR surface expression <strong>an</strong>d synaptic dysfunction. Our results show that, indeed,<br />
N-terminal fragments of mut<strong>an</strong>t htt tr<strong>an</strong>sfected into striatal neurons sequester PACSIN1 <strong>an</strong>d<br />
o<strong>the</strong>r endocytic proteins such as dynamin 1 into aggregates, limiting <strong>the</strong>ir dendritic availability.<br />
These defects in dendritic localization are accomp<strong>an</strong>ied by accumulation of NR3A <strong>an</strong>d NR1containing<br />
NMDARs at <strong>the</strong> plasma membr<strong>an</strong>e without concomit<strong>an</strong>t alterations in general<br />
endocytic capacity or surface expression of o<strong>the</strong>r glutamate receptors. Consistent with our in<br />
vitro data, R6/1 mice expressing exon1 as well as YAC128 mice expressing full-length mut<strong>an</strong>t<br />
htt (with 115 <strong>an</strong>d 128 CAG repeats, respectively) display reduced PACSIN1 <strong>an</strong>d increased<br />
NR3A levels in striatal synaptic plasma membr<strong>an</strong>es. Protein ch<strong>an</strong>ges are observed early, at times
coinciding with <strong>the</strong> first appear<strong>an</strong>ce of motor symptoms. Fur<strong>the</strong>r, increased levels of NR3A: 1)<br />
occur in medium-sized spiny neurons, <strong>the</strong> neuronal population most affected in HD, <strong>an</strong>d 2) could<br />
contribute to neuronal degeneration because increasing levels of NR3A in striatal slices by<br />
biolistic tr<strong>an</strong>sfection potentiates cell death. Current studies are directed to map <strong>the</strong> trafficking<br />
pathways involved, explain <strong>the</strong> sources of specificity, <strong>an</strong>d investigate its causative/contributory<br />
role to HD neuronal degeneration <strong>an</strong>d synaptic deficits.<br />
Disclosures: S. Marco, None; J. Torres-Peraza, None; A. Giralt, None; L.S. Kaltenbach,<br />
None; R.K. Graham, None; M.R. Hayden, None; D.C. Lo, None; J. Alberch, None; I. Perez-<br />
Ot<strong>an</strong>o, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.16/L31<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NIH NS038144-08<br />
NINDS 16375<br />
Title: Caspase 6 <strong>an</strong>d downstream huntingtin cleavage events in Huntington disease pathogenesis<br />
Authors: E. WALDRON 1 , *T. RATOVITSKI 2 , E. CHIGHLADZE 1 , A. T. TEBBENKAMP 3 ,<br />
D. R. BORCHELT 3 , C. A. ROSS 1 ;<br />
1 2 3<br />
Johns Hopkins Univ., Baltimore, MD; JHU, BALTIMORE, MD; McKnight Brain Institute,<br />
Univ. of Florida, Gainesville, FL<br />
Abstract: Huntington disease (HD) is <strong>an</strong> autosomal domin<strong>an</strong>t disease, which belongs to a family<br />
of inherited neurodegenerative disorders caused by exp<strong>an</strong>sion of polyglutamine (polyQ) tracts in<br />
<strong>the</strong> associated disease proteins. Strong evidence exists supporting a role <strong>for</strong> huntingtin<br />
proteolysis in disease pathogenesis. Particular interest has been paid to <strong>the</strong> caspase 6 derived<br />
fragment of huntingtin- N586. While failure in <strong>the</strong> generation of this fragment is sufficient to<br />
rescue <strong>the</strong> phenotype, it is not yet known whe<strong>the</strong>r cleavage products downstream of N586 are<br />
also a contributing <strong>for</strong>ce in disease progression. We found that proteolysis of huntingtin at<br />
position 167 (generating <strong>the</strong> cp-2 N-terminal fragment) is associated with toxicity in neuronal<br />
HT22 cell. Deletion of <strong>the</strong> cp-2 sites results in <strong>an</strong> increase in huntingtin aggregation. In addition,<br />
exp<strong>an</strong>ded huntingtin devoid of cp-2 site seems to acquire altered con<strong>for</strong>mation with different
iochemical properties. To fur<strong>the</strong>r validate <strong>the</strong> downstream cleavage events of caspase 6generated<br />
fragment of huntingtin, we also generated a new mouse model expressing <strong>the</strong> N586-<br />
82Q truncated huntingtin. These mice produce high levels of <strong>the</strong> tr<strong>an</strong>sgene <strong>an</strong>d <strong>for</strong>m huntingtin<br />
aggregates (based on SDS-PAGE <strong>an</strong>alysis). We continue to monitor <strong>the</strong> <strong>an</strong>imals <strong>for</strong> signs of<br />
phenotypes similar to o<strong>the</strong>r HD mouse models. To investigate <strong>the</strong> contribution of <strong>the</strong> cp-2<br />
fragment towards N586 toxicity in vivo, development of <strong>an</strong>o<strong>the</strong>r N586-82Q mouse model with<br />
<strong>the</strong> cp-2 site deleted is also on-going. The events leading up to N586 proteolysis are also under<br />
study through <strong>the</strong> determination of caspase 6 interacting proteins. We have per<strong>for</strong>med a yeast<br />
two hybrid screen using caspase 6 made inactive as bait, <strong>an</strong>d are assessing <strong>the</strong> influence of<br />
selected caspase 6-interacting proteins on <strong>the</strong> cleavage of a longer huntingtin fragment (N1176)<br />
by caspase 6 into N586. We will also address <strong>the</strong> effect of such proteins over cleavage into<br />
N167/cp-2. Underst<strong>an</strong>ding what regulates <strong>the</strong> cleavage of htt by caspase 6 will not only provide<br />
us with new <strong>the</strong>rapeutic c<strong>an</strong>didates but also a more insightful view towards huntingtin<br />
function(s).<br />
Disclosures: E. Waldron, None; T. Ratovitski, None; E. Chighladze, None; A.T.<br />
Tebbenkamp, None; D.R. Borchelt, None; C.A. Ross, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.17/L32<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Functional roles of AMP-activated protein kinase in Huntington’s disease<br />
Authors: *T. C. JU 1,2,3 , H.-M. CHEN 3 , Y. CHERN 3 ;<br />
1 Inst. of Biomed. Sciences|910003215|0, Taipei, Taiw<strong>an</strong>; 2 Inst. of Neuroscience, Natl. Y<strong>an</strong>g<br />
Ming Univ., Taipei, Taiw<strong>an</strong>; 3 Div. of Neuroscience, Inst. of Biomed. Sciences, Academia Sinica,<br />
Taipei, Taiw<strong>an</strong><br />
Abstract: Abstract:<br />
Huntington’s disease (HD) is <strong>an</strong> autosomal domin<strong>an</strong>t neurological disorder that evokes<br />
progressive cognitive, motor, <strong>an</strong>d psychiatric dysfunction. It is caused by CAG trinucleotide<br />
exp<strong>an</strong>sion in <strong>the</strong> exon 1 of Huntingtin (Htt) gene. The accumulated evidence on HD <strong>from</strong> hum<strong>an</strong><br />
patients <strong>an</strong>d <strong>an</strong>imal models suggests <strong>the</strong> involvement of energy metabolism, excitotoxicity, <strong>an</strong>d<br />
oxidative stress in HD pathogenesis. The common pathways linking Htt mutation with energy<br />
deficiency, oxidative damage, <strong>an</strong>d cell death in HD remain largely unknown. We reported earlier
that a metabolic sensor 5’AMP-activated protein kinase (AMPK) was overactivated in a<br />
tr<strong>an</strong>sgenic mouse model of HD (R6/2). AMPK is a downstream mediator of m<strong>an</strong>y different types<br />
of stress. It is a heterotrimeric kinase consisting of a catalytic (α) <strong>an</strong>d two regulatory subunits (β<br />
<strong>an</strong>d γ), <strong>an</strong>d c<strong>an</strong> be activated by CaMKK <strong>an</strong>d LKB1 via phosphorylation at <strong>the</strong> threonine residue<br />
172 of AMPKα. In addition to suppressing metabolic enzymes (e.g., acetyl-CoA carboxylase)<br />
<strong>an</strong>d reducing ATP consumption upon stress, AMPK also phosphorylates a wide variety of<br />
proteins with different functions. In <strong>the</strong> present study, we characterized <strong>the</strong> dysregulated energy<br />
homeostasis <strong>an</strong>d elevated oxidative stress in HD using R6/2 mice <strong>an</strong>d three striatal progenitor<br />
cell lines (ST14A; STHdh Q7 , STHdh Q109 ). We demonstrated that expression of mHtt selectively<br />
phosphorylated <strong>an</strong>d activated AMPK in striatal cells. In addition, treatments with <strong>an</strong> agonist<br />
(CGS21680) of <strong>the</strong> A2A adenosine receptor (A2A receptor) normalized <strong>the</strong> overactivated AMPK<br />
by mHtt. Since activation of <strong>the</strong> A2A receptor stimulates Gsα protein <strong>an</strong>d enh<strong>an</strong>ces <strong>the</strong> cAMP<br />
production, our findings shed new light on <strong>the</strong> protective effects of cAMP-elevating reagents in<br />
HD.<br />
Disclosures: T.C. Ju, None; H. Chen, None; Y. Chern, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.18/L33<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t NS058792<br />
CHDI Foundation<br />
Title: Hypoacetylation <strong>an</strong>d hypermethylation as central <strong>an</strong>d peripheral biomarkers in<br />
Huntington's disease<br />
Authors: *K. M. SMITH 1,2 , B. B. HUNT 2 , D. J. AMANTE 2 , J. P. MOODY 2 , K. A.<br />
CORMIER 1 , J. KIM 2 , S. M. HERSCH 3 , R. J. FERRANTE 1,2 ;<br />
1 2 3<br />
Bed<strong>for</strong>d VA Med. Ctr., Bed<strong>for</strong>d, MA; Neurol., Boston Univ. Sch. Med., Boston, MA; Neurol.,<br />
Massachusetts Gen. Hosp., Boston, MA<br />
Abstract: Huntington's disease (HD) is caused by <strong>an</strong> exp<strong>an</strong>ded trinucleotide CAG repeat in <strong>the</strong><br />
gene coding <strong>for</strong> <strong>the</strong> protein huntingtin. Although <strong>the</strong> underlying biochemical alterations <strong>an</strong>d <strong>the</strong><br />
mech<strong>an</strong>isms of neuronal degeneration remain unknown in HD, recent findings have showed that
aberr<strong>an</strong>t chromatin remodeling is a central feature in <strong>the</strong> pathology of HD. Epigenetic<br />
modification in <strong>the</strong> <strong>for</strong>m of marked hypoacetylation with concomit<strong>an</strong>t hypermethylation is a<br />
consistent finding observed in HD patients <strong>an</strong>d tr<strong>an</strong>sgenic HD mice. Peripheral biomarkers are<br />
urgently needed <strong>for</strong> diagnosis, disease progression, <strong>an</strong>d <strong>for</strong> potential disease-modifying <strong>the</strong>rapies<br />
that are being developed <strong>an</strong>d evaluated in clinical trials, especially at <strong>the</strong> preclinical stage. While<br />
profiling objective biomarker measurements of HD has proven difficult, <strong>the</strong> mut<strong>an</strong>t huntingtin<br />
protein is expressed ubiquitously throughout <strong>the</strong> body <strong>an</strong>d may cause detectable ch<strong>an</strong>ges in<br />
biochemistry <strong>an</strong>ywhere. We examined complimentary processes of acetylation <strong>an</strong>d methylation<br />
measurements in <strong>the</strong> brain <strong>an</strong>d blood <strong>from</strong> HD subjects <strong>an</strong>d HD mice. Parallel studies in<br />
tr<strong>an</strong>sgenic R6/2 <strong>an</strong>d 140 CAG knock-in mice <strong>an</strong>d early m<strong>an</strong>ifest disease HD patients showed a<br />
corresponding hypoacetylation of H4 <strong>an</strong>d hypermethylation of dimethyl-histone H3, lysine 9, in<br />
both brain <strong>an</strong>d blood buffy coat samples using Western <strong>an</strong>d dot blot <strong>an</strong>alyses. Interestingly brain<br />
<strong>an</strong>d blood samples <strong>from</strong> prem<strong>an</strong>ifest mice showed a signific<strong>an</strong>t reduction in acetylation <strong>an</strong>d<br />
increased methylation. These findings suggest that markers of altered nucleosomal dynamics<br />
may be useful as peripheral biomarkers of disease. The correlation of hypoacetylation <strong>an</strong>d<br />
hypermethylation as disease biomarkers in both mouse <strong>an</strong>d m<strong>an</strong> fur<strong>the</strong>r validate <strong>the</strong> murine<br />
models <strong>an</strong>d may provide a powerful me<strong>an</strong>s to assess <strong>the</strong>rapeutic treatments to hum<strong>an</strong>s<br />
experiencing HD <strong>an</strong>d predict <strong>the</strong> potential magnitude of benefits in <strong>the</strong>se patients.<br />
Disclosures: K.M. Smith, None; B.B. Hunt, None; D.J. Am<strong>an</strong>te, None; J.P. Moody,<br />
None; K.A. Cormier, None; J. Kim, None; S.M. Hersch, None; R.J. Ferr<strong>an</strong>te, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.19/L34<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: Down-regulation of brain type creatine kinase contributes to neuron dysfunction in<br />
Huntington’s disease<br />
Authors: *Y.-S. LIN 1,2 , H.-M. CHEN 1 , C.-P. CHANG 1 , Y. CHERN 1 ;<br />
1 2<br />
Inst. of Biomed. Sci., Academia Sinica, Taipei, Taiw<strong>an</strong>; Inst. of Neurosci., Natl. Y<strong>an</strong>g-Ming<br />
Univ., Taipei, Taiw<strong>an</strong><br />
Abstract: Huntington’s disease (HD) is caused by expended CAG repeat in <strong>the</strong> exon 1 of<br />
huntingtin gene, <strong>an</strong>d preferentially affects <strong>the</strong> functions of <strong>the</strong> striatum <strong>an</strong>d cortex in <strong>the</strong> brain.<br />
Recent studies indicate that energy metabolism is impaired in HD, but <strong>the</strong> mech<strong>an</strong>isms remain
unclear. Using 2D gel electrophoresis, we found that brain type creatine kinase (CKB) was<br />
down-regulated in <strong>the</strong> striatum of a tr<strong>an</strong>sgenic HD mouse model (R6/2). The major function of<br />
CKB is known to regenerate ATP through regulating <strong>the</strong> levels of creatine <strong>an</strong>d phosphor-creatine<br />
at specific energy consuming sites. By using a qu<strong>an</strong>titative RT-PCR <strong>an</strong>alysis <strong>an</strong>d <strong>the</strong> promoter<br />
assay, we found that <strong>the</strong> tr<strong>an</strong>script level of CKB was suppressed by polyglutamine-exp<strong>an</strong>ded<br />
mut<strong>an</strong>t huntingtin (htt109Q). Immunocytochemical staining revealed that CKB existed in both<br />
soma <strong>an</strong>d neuronal processes. Interestingly, expression of CKB protein in <strong>the</strong> neuronal processes<br />
of R6/2 mice was greatly reduced when compared to those of age-controlled wildtype mice.<br />
Exogenous expression of CKB rescued <strong>the</strong> mut<strong>an</strong>t Htt-induced defects including cell survival,<br />
Htt aggregate, <strong>an</strong>d damaged neurite outgrowth. Our data suggest that downregulation of CKB<br />
contributes to neuronal dysfunction in HD. Strategies aimed at increasing <strong>the</strong> expression or<br />
activity of CKB may lead to beneficial effects on <strong>the</strong> progression of HD.<br />
Disclosures: Y. Lin, None; H. Chen, None; C. Ch<strong>an</strong>g, None; Y. Chern, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.20/L35<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NIH NINDS NS053679<br />
NIH NINDS16375<br />
Title: Purification <strong>an</strong>d characterization of mut<strong>an</strong>t huntingtin exon-1 expressed in a mammali<strong>an</strong><br />
system -- Identification of multiple intermediate species<br />
Authors: *X. FENG 1 , T. RATOVITSKI 1 , Y. PORAT 1 , J. MILLER 2 , P. H. PATTERSON 3 , S.<br />
FINKBEINER 2 , C. A. ROSS 1 , M. A. POIRIER 1 ;<br />
1 Psychiatry <strong>an</strong>d Behavior Sci., Johns Hopkins Univ., Baltimore, MD; 2 Gladstone Inst. of<br />
Neurolog. Dis., Univ. of Cali<strong>for</strong>nia, S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 3 Div. of Biol., Caltech,<br />
Pasadena, CA<br />
Abstract: Huntington’s disease is a neurodegenerative disorder characterized by neuronal<br />
degeneration <strong>an</strong>d inclusions containing N-terminal fragments of mut<strong>an</strong>t huntingtin (htt).<br />
Polyglutamine aggregation is a complex multi-step process, involving <strong>the</strong> assembly of multiple<br />
intermediates prior to <strong>the</strong> <strong>for</strong>mation of inclusions. Recent studies have suggested that soluble htt
oligomeric or protofibrillar <strong>for</strong>ms may be more toxic th<strong>an</strong> late-stage fibrils. Previously, we<br />
expressed recombin<strong>an</strong>t mut<strong>an</strong>t htt exon-1 fragment in bacteria. We monitored aggregation over<br />
time <strong>an</strong>d showed that globular <strong>an</strong>d protofibrillar species comprised of beta-structure participate<br />
in <strong>the</strong> genesis of mature fibrils. In <strong>the</strong> present study, we have established a mammali<strong>an</strong> system<br />
<strong>for</strong> expression of GST-tagged htt exon-1 in HEK293 cells. Our preliminary aggregation data<br />
indicate that recombin<strong>an</strong>t mut<strong>an</strong>t htt exon-1 produced in a mammali<strong>an</strong> system aggregates more<br />
slowly th<strong>an</strong> <strong>the</strong> protein generated in bacteria, possibly resulting <strong>from</strong> post-tr<strong>an</strong>slational<br />
modifications. Numerous soluble oligomeric species <strong>for</strong>med throughout <strong>the</strong> aggregation process<br />
were observed using both SDS-PAGE <strong>an</strong>d Blue Native-PAGE, a new type of native gel <strong>an</strong>alysis.<br />
Import<strong>an</strong>tly, <strong>the</strong>se species were not previously detected using <strong>the</strong>se methods with <strong>the</strong> comparable<br />
protein generated in bacteria, <strong>an</strong>d could represent a toxic <strong>for</strong>m of mut<strong>an</strong>t Htt. Future experiments<br />
will be aimed at isolating mammali<strong>an</strong> htt exon-1 monomer <strong>an</strong>d aggregation species <strong>for</strong> fur<strong>the</strong>r<br />
characterization <strong>an</strong>d determination of <strong>the</strong>ir potential effect on cytotoxicity.<br />
Disclosures: X. Feng, None; T. Ratovitski, None; Y. Porat, None; J. Miller, None; P.H.<br />
Patterson, None; S. Finkbeiner, None; C.A. Ross, None; M.A. Poirier, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.21/L36<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t K99-ES016352 (PGM)<br />
A gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Picower Foundation (JTG)<br />
Title: Abnormal interaction of mut<strong>an</strong>t huntingtin exon 1 with <strong>the</strong> mitochondrial malate-aspartate<br />
shuttle leads to bio-energetic defects in Huntington’s disease<br />
Authors: *P. MASTROBERARDINO 1 , A. L. ORR 3 , O. VERGUN 1 , X. HU 1 , M.<br />
JAYARAMAN 2 , X.-J. LI 3 , R. WETZEL 2 , J. GREENAMYRE 1 ;<br />
1 Dept. of Neurology, Pittsburgh Inst. <strong>for</strong> Neurodegenerative Dis., 2 Dept. of Structural Biology,<br />
Pittsburgh Inst. <strong>for</strong> Neurodegenerative Dis., Univ. Pittsburgh, Pittsburgh, PA; 3 Dept. of Hum<strong>an</strong><br />
Genet., Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Huntington’s disease (HD) is a domin<strong>an</strong>t, genetic disorder associated with a<br />
polyglutamine (polyQ) exp<strong>an</strong>sion in <strong>the</strong> huntingtin (htt) protein. Among <strong>the</strong> diverse molecular
factors participating in <strong>the</strong> initiation of <strong>the</strong> disease, abnormal protein-protein interactions<br />
occurring by virtue of <strong>the</strong> exp<strong>an</strong>ded stretch of polyQ in mut<strong>an</strong>t htt (mu-htt) may be critical.<br />
Several independent studies have demonstrated that mu-htt is associated with mitochondria <strong>an</strong>d<br />
that this interaction is paralleled by bioenergetic defects <strong>an</strong>d <strong>an</strong>omalies in calcium h<strong>an</strong>dling.<br />
Despite this evidence, our underst<strong>an</strong>ding of <strong>the</strong> abnormal molecular interactions that lead to <strong>the</strong><br />
observed defects in mitochondrial physiology is rudimentary. In this study, we sought to identify<br />
<strong>the</strong> mitochondrial protein partners of mu-htt to better elucidate <strong>the</strong> mech<strong>an</strong>istic details underlying<br />
pathogenesis. Our results show that binding of mu-htt exon 1 to mitochondria depends upon <strong>the</strong><br />
length of <strong>the</strong> polyQ tract as well as <strong>the</strong> mitochondrial energization state. Mu-htt c<strong>an</strong> be copurified<br />
- <strong>an</strong>d hence interacts - with <strong>the</strong> mitochondrial iso<strong>for</strong>m of aspartate aminotr<strong>an</strong>sferase<br />
(mtAAT). This enzyme is a component of <strong>the</strong> malate-aspartate shuttle which, in addition to its<br />
role in providing substrates <strong>for</strong> intermediary metabolism, contributes to glutamate scavenging,<br />
<strong>the</strong>reby preventing excitotoxicity. We found that mtAAT enzymatic activity is impaired when<br />
isolated mitochondria or permeabilized neurons are incubated with monomeric mu-htt exon 1.<br />
MtAAT activity is also decreased in HD tr<strong>an</strong>sgenic mouse brain, even though its expression<br />
levels remain unch<strong>an</strong>ged. More import<strong>an</strong>tly, decreased AAT activity has been well described in<br />
hum<strong>an</strong> HD cases <strong>an</strong>d <strong>an</strong> inhibitor of <strong>the</strong> malate-aspartate shuttle - amino-oxyacetic acid - has<br />
previously been proposed as a toxin model of HD. In conclusion, our results provide novel<br />
mech<strong>an</strong>istic insights into <strong>the</strong> bioenergetic <strong>an</strong>d metabolic defects in HD <strong>an</strong>d provide <strong>an</strong> import<strong>an</strong>t<br />
link between polyQ-mediated mitochondrial dysfunction <strong>an</strong>d glutamate induced excitotoxicity.<br />
Disclosures: P. Mastroberardino, None; A.L. Orr, None; O. Vergun, None; X. Hu, None; M.<br />
Jayaram<strong>an</strong>, None; X. Li, None; R. Wetzel, None; J. Greenamyre, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.22/L37<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Title: A gene <strong>an</strong>tisense to huntingtin may modify huntingtin toxicity<br />
Authors: *W. CHUNG, L. YU, C. ZHANG, C. A. ROSS, D. D. RUDNICKI, R. L.<br />
MARGOLIS;<br />
Psychiatry, Johns Hopkins Univ., Baltimore, MD<br />
Abstract: While ample evidence supports a major role <strong>for</strong> polyglutamine toxicity in<br />
Huntington’s disease (HD), <strong>the</strong> complete expl<strong>an</strong>ation <strong>for</strong> HD pathogenesis, including selective
neurodegeneration in <strong>the</strong> striatum <strong>an</strong>d cortex, remains elusive. A number of lines of investigation<br />
have suggested a pathological role of <strong>an</strong>tisense tr<strong>an</strong>script present in <strong>the</strong> loci of repeat exp<strong>an</strong>sion<br />
disorders such as DM1, SCA8, Fragile X, <strong>an</strong>d FXTAS. Based on <strong>the</strong>se studies, we hypo<strong>the</strong>sized<br />
that functional tr<strong>an</strong>scripts are produced <strong>an</strong>tisense to huntingtin. Consistent with this hypo<strong>the</strong>sis,<br />
we identified <strong>an</strong>d mapped a previously unreported gene located on <strong>the</strong> opposite str<strong>an</strong>d to<br />
huntingtin, tentatively termed <strong>an</strong>ti-sense HD (ASHD), which overlaps with <strong>the</strong> promoter <strong>an</strong>d<br />
exon 1 of huntingtin. One ASHD splice vari<strong>an</strong>t, ASHD1, is of particular interest because it<br />
includes <strong>the</strong> CAG/CTG repeat that exp<strong>an</strong>ds to cause HD. Using a reporter assay, we determined<br />
that 1) ASHD1 has a functional promoter, 2) repeat exp<strong>an</strong>sion decreases ASHD1 expression, <strong>an</strong>d<br />
3) <strong>the</strong> CTCF site fl<strong>an</strong>king ASHD1 modulates ASHD1 expression. Consistent with <strong>the</strong>se<br />
observations, ASHD1 levels are decreased in RNA extracted <strong>from</strong> post-mortem HD brain<br />
compared to control brain. Fur<strong>the</strong>r, when overexpressed in cell models, ASHD1 decreases<br />
huntingtin expression. These preliminary data suggest a model in which ASHD1 suppresion of<br />
huntingtin expression is lifted in <strong>the</strong> setting of repeat exp<strong>an</strong>sion, potentially leading to increased<br />
levels of mut<strong>an</strong>t huntingtin <strong>an</strong>d greater neurotoxicity.<br />
Disclosures: W. Chung, None; L. Yu, None; C. Zh<strong>an</strong>g, None; C.A. Ross, None; D.D.<br />
Rudnicki, None; R.L. Margolis, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.23/L38<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: NSF Gr<strong>an</strong>t 0548733<br />
NIH DE017102, 5P20RR017699<br />
Title: TRPC1 protects neuroblastoma cells against 3-nitropropionic acid via ERK <strong>an</strong>d CREB<br />
activation<br />
Authors: *S. SELVARAJ 1 , B. B. SINGH 2 ;<br />
1 2<br />
biochemistry <strong>an</strong>d molecular biology, Biochem. <strong>an</strong>d molecular biology, Univ. of North Dakota,<br />
Gr<strong>an</strong>d Forks, ND<br />
Abstract: TRPC ch<strong>an</strong>nels have wide variety of physiological function including cell survival<br />
<strong>an</strong>d proliferation. Here we report that <strong>the</strong> member of TRPC subfamily, TRPC1 protected hum<strong>an</strong>
neuroblastoma cells (SH-SY5Y) against 3-nitropropionic acid (3-NPA), a mitochondrial toxin<br />
that induces a clinical <strong>an</strong>d pathological phenotype similar to Huntington disease. In vitro studies<br />
using SH-SY5Y showed that 3-NPA treatment (4mM <strong>for</strong> 12h) down regulated <strong>the</strong> TRPC1<br />
protein level. Moreover, blocking TRPC1 ch<strong>an</strong>nel by SKF96365 or down regulating TRPC1 by<br />
shRNA increases susceptibility to 3-NPA. By contrast, TRPC1 overexpression signific<strong>an</strong>tly<br />
attenuated 3-NPA -induced pro-apoptotic markers, including Bax tr<strong>an</strong>slocation to mitochondria,<br />
cytochrome c release, <strong>an</strong>d caspase-9 <strong>an</strong>d caspase-3 activation. Also TRPC1 overexpression in 3-<br />
NPA treated cells resulted in a persistent activation of extracellular signal-regulated kinase 1/2<br />
(ERK1/2), <strong>an</strong>d phosphorylation <strong>an</strong>d nuclear localization of CREB (pCREB), a downstream<br />
molecule of ERK. TRPC1 <strong>an</strong>tagonist SKF96365 signific<strong>an</strong>tly attenuated TRPC1-mediated<br />
activation of ERK <strong>an</strong>d CREB phosphorylation. Interestingly, ERK1/2 phosphorylation<br />
(pERK1/2) was found to be critical <strong>for</strong> mediating TRPC1 induced neuroprotection since<br />
inhibitors of mitogen-activated protein kinase kinases 1 <strong>an</strong>d 2 (MEK1/2) <strong>an</strong>d ERK1/2 blocked<br />
<strong>the</strong> pCREB levels <strong>an</strong>d TRPC1-mediated cell protection. Overall, <strong>the</strong>se studies clearly<br />
demonstrate a novel role of TRPC1 in <strong>the</strong> protection of SH-SY5Y cells against 3-NPA by<br />
regulating <strong>the</strong> activity of several downstream effectors, such as ERK <strong>an</strong>d <strong>the</strong> tr<strong>an</strong>scription factors<br />
CREB.<br />
Disclosures: S. Selvaraj, None; B.B. Singh, Associate Professor, A. Employment (full or parttime);<br />
Principle Investigator, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.24/M1<br />
Topic: C.04.a. Huntington's disease: Mech<strong>an</strong>isms<br />
Support: CHDI<br />
Title: Dopaminergic signaling <strong>an</strong>d striatal neurodegeneration in Huntington’s disease<br />
Authors: H. WANG, *X. CHEN, T.-S. TANG, Y. LI, I. BEZPROZVANNY;<br />
UT Southwestern Med. Ctr., Dallas, TX<br />
Abstract: Huntington’s disease (HD) is a neurodegenerative disorder caused by polyglutamine<br />
(polyQ) exp<strong>an</strong>sion in Huntingtin protein (Htt). PolyQ exp<strong>an</strong>sion in Htt exp causes selective<br />
degeneration of striatal medium spiny neurons (MSN) in HD patients. A central question in <strong>the</strong>
study of HD is how this polyQ-exp<strong>an</strong>sion of Htt leads to <strong>the</strong> neurodegeneration of MSN. Our<br />
previous studies with <strong>the</strong> yeast artificial chromosome (YAC128) tr<strong>an</strong>sgenic HD mouse model<br />
suggest that dopamine signaling pathway plays <strong>an</strong> import<strong>an</strong>t role in HD pathogenesis <strong>an</strong>d that<br />
<strong>an</strong>tagonists of dopamine pathway such as tetrabenazine (TBZ) or dopamine receptors blockers<br />
may have a <strong>the</strong>rapeutic potential <strong>for</strong> treatment of HD (T<strong>an</strong>g at al, 2007, J. Neuroscience, 27:7899<br />
-7910).<br />
To fur<strong>the</strong>r investigate <strong>an</strong> import<strong>an</strong>ce of dopamine signaling in HD pathogenesis repeated TBZ<br />
trial in YAC128 mice starting TBZ treatment early (2 months of age, “presymptomatic”) <strong>an</strong>d late<br />
(6 months of age, “symptomatic”). The trial was continued until <strong>the</strong> mice were 12 months of age.<br />
The results of behavioral <strong>an</strong>d neuropathological assessment will be presented.<br />
Because of severe side-effects of TBZ in patients, additional dopamine <strong>an</strong>tagonists should be<br />
considered <strong>for</strong> treatment of HD. In our experiments we evaluated potential neuroprotective<br />
effects of clinically relev<strong>an</strong>t D1 <strong>an</strong>tagonists in experiments with YAC128 MSN cultures. These<br />
data will be presented.<br />
Our results will provide fur<strong>the</strong>r in<strong>for</strong>mation about <strong>an</strong> import<strong>an</strong>ce of dopamine pathway <strong>for</strong><br />
neurodegeneration in HD <strong>an</strong>d help to define <strong>the</strong>rapeutic potential of TBZ <strong>an</strong>d D1 <strong>an</strong>tagonists <strong>for</strong><br />
HD treatment at “presymptomatic” <strong>an</strong>d “symptomatic” stages of <strong>the</strong> disease.<br />
Disclosures: H. W<strong>an</strong>g, None; X. Chen, CHDI, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); University of<br />
Texas Southwestern Medical Center at Dallas, A. Employment (full or part-time); T. T<strong>an</strong>g,<br />
None; Y. Li, None; I. Bezprozv<strong>an</strong>ny, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.25/M2<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Support: NINDS NS045195 (CLJ)<br />
Title: Vascular endo<strong>the</strong>lial growth factor is concentrated at <strong>the</strong> neuromuscular junction<br />
Authors: *J. E. POORT 1 , L. QI 2 , S. M. BREEDLOVE 2 , C. L. JORDAN 2 ;<br />
2 Neurosci., 1 Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Deficits in vascular endo<strong>the</strong>lial growth factor (VEGF) levels are implicated in<br />
motoneuron disease, including amyotrophic lateral sclerosis (ALS) <strong>an</strong>d spinal bulbar muscular
atrophy (Neurosci Res. 62:71; Biochim Biophys Acta 1762:1109). VEGF levels are reduced in<br />
several mouse models of motoneuron disease, whereas mice lacking <strong>the</strong> hypoxia-response<br />
element in <strong>the</strong> VEGF promoter show a motoneuron disease phenotype. Moreover, exogenously<br />
supplied VEGF slows disease progression in ALS rat <strong>an</strong>d mouse models <strong>an</strong>d protects cultured<br />
motoneurons <strong>from</strong> cell death during exposure to various apoptotic inducers. Given that<br />
motoneurons express VEGF receptors <strong>an</strong>d skeletal muscles express VEGF itself, muscle-derived<br />
VEGF may act directly on motoneurons to protect <strong>the</strong>m. Localization studies indicate that<br />
skeletal muscle fibers express VEGF (FASEB 20: E921). Interestingly, VEGF immunoreactivity<br />
appears in discrete patches at <strong>the</strong> perimeter of fibers. This patchy distribution, along with <strong>the</strong> fact<br />
that VEGF has a heparin-binding domain, suggests that VEGF could be concentrated at <strong>the</strong><br />
neuromuscular junction (NMJ). To explore this possibility, <strong>the</strong> extensor digitorum longus (EDL)<br />
<strong>from</strong> adult wildtype C57BL/6J male mice was harvested, frozen in liquid N2 <strong>an</strong>d cross-sectioned<br />
at 10 µm using a cryostat. Sections were co-stained <strong>for</strong> VEGF <strong>an</strong>d acetylcholine receptors<br />
(AChRs) using VEGF <strong>an</strong>tisera <strong>from</strong> goat or mouse <strong>an</strong>d fluorescently-tagged α-bungarotoxin. As<br />
expected, we find VEGF immunoreactivity localized to blood vessels, but also in nuclei of<br />
muscle fibers <strong>an</strong>d o<strong>the</strong>r cells in skeletal muscle. Import<strong>an</strong>tly, we find VEGF immunoreactivity in<br />
discrete patches at <strong>the</strong> periphery of muscle fibers that precisely align with <strong>the</strong> postsynaptic<br />
junction as marked by a high density of AChRs. This result suggests that VEGF is concentrated<br />
at <strong>the</strong> NMJ <strong>an</strong>d raises <strong>the</strong> possibility that depletion of muscle-derived VEGF at <strong>the</strong> NMJ<br />
promotes motoneuron disease by depriving motor nerve terminals of <strong>an</strong> essential neurotrophic<br />
factor.<br />
Disclosures: J.E. Poort, None; L. Qi, None; S.M. Breedlove, None; C.L. Jord<strong>an</strong>, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.26/M3<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Support: NIH NS045195 (CLJ)<br />
NS055746 (APL)<br />
NS054517 (JAJ)<br />
1F32AR055848-02. (MQK)
Title: Androgen receptor <strong>an</strong>tagonist increases lifesp<strong>an</strong> <strong>an</strong>d motor function in mouse models of<br />
SBMA<br />
Authors: *S. M. TROXELL 1 , J. A. JOHANSEN 1 , M. Q. KEMP 1 , A. P. LIEBERMAN 2 , S. M.<br />
BREEDLOVE 1 , C. L. JORDAN 1 ;<br />
1 Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI; 2 Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Spinal Bulbar Muscular Atrophy (SBMA) is <strong>an</strong> <strong>an</strong>drogen-dependent,<br />
neurodegenerative disease causing progressive loss of muscle strength only in males. SBMA is<br />
caused by a CAG repeat mutation in <strong>the</strong> <strong>an</strong>drogen receptor (AR) gene, resulting in <strong>an</strong> exp<strong>an</strong>ded<br />
polyglutamine (polyQ) tract in <strong>the</strong> protein. We study <strong>the</strong> progression of this disease in two<br />
different mouse models of SBMA: a tr<strong>an</strong>sgenic (TG) mouse expressing a wildtype AR<br />
exclusively in skeletal muscle fibers, <strong>an</strong>d a knock-in (KI) mouse model that expresses a<br />
hum<strong>an</strong>ized AR disease allele containing 113 CAGs. Previous studies in our lab have shown that<br />
<strong>the</strong> AR <strong>an</strong>tagonist flutamide blocks <strong>an</strong>drogen-induced disease in TG females. The goal of this<br />
study was to see if flutamide could also rescue symptomatic males <strong>from</strong> disease in <strong>the</strong>se two<br />
different SBMA mouse models. Both TG <strong>an</strong>d KI males were approximately 90 days old when<br />
flutamide treatment beg<strong>an</strong>. Treatment consisted of time-release 100 mg flutamide pellets (<strong>from</strong><br />
Innovative Research of America) designed to release approximately 5mg flutamide/day <strong>for</strong> 21<br />
days. We find that TG males given flutamide show a signific<strong>an</strong>t improvement in grip strength<br />
compared to control-treated TG males by day 7 of treatment. We also see a drastic increase in<br />
lifesp<strong>an</strong> in response to flutamide in KI males, with a 40% increase in survival by day 21 of<br />
treatment. These data suggest that flutamide may be <strong>an</strong> effective <strong>the</strong>rapeutic <strong>for</strong> hum<strong>an</strong> SBMA.<br />
Because AR acts in muscles to cause disease in our myogenic model of SBMA, blocking AR<br />
action selectively in muscle may be sufficient to rescue SBMA patients <strong>from</strong> disease.<br />
Disclosures: S.M. Troxell, None; J.A. Joh<strong>an</strong>sen, None; M.Q. Kemp, None; A.P. Lieberm<strong>an</strong>,<br />
None; S.M. Breedlove, None; C.L. Jord<strong>an</strong>, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.27/M4<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Support: NINDS NS045195
Title: Slow twitch muscles are more severely affected th<strong>an</strong> fast in a myogenic mouse model of<br />
SBMA<br />
Authors: *K. OKI 1 , S. BREEDLOVE 1 , R. W. WISEMAN 2 , C. L. JORDAN 1 ;<br />
1 Neurosci. Program, 2 Dept. of Physiol., Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Our laboratory has produced a line of tr<strong>an</strong>sgenic (tg) mice that overexpress <strong>the</strong> rat<br />
wildtype <strong>an</strong>drogen receptor selectively in skeletal muscle fibers. Unexpectedly, such mice<br />
exhibit <strong>an</strong> <strong>an</strong>drogen-dependent disease phenotype suggestive of spinal bulbar muscular atrophy<br />
(SBMA; Monks et al., 2007, PNAS, 104(46):18259-64). Only tg males show a loss of motor<br />
function, but treating tg females with testosterone (T) rapidly induces disease symptoms. Tests of<br />
muscle strength based on <strong>for</strong>elimb grip strength <strong>an</strong>d ability to h<strong>an</strong>g <strong>from</strong> a wire grid indicate that<br />
affected mice have progressive muscle weakness. Results <strong>from</strong> previous experiments indicate<br />
that <strong>the</strong> isolated fast twitch extensor digitorum longus (EDL) muscle <strong>from</strong> diseased, T-treated tg<br />
female mice produces much less contractile <strong>for</strong>ce th<strong>an</strong> <strong>the</strong> EDL <strong>from</strong> non-diseased controls<br />
(vehicle- <strong>an</strong>d T-treated wildtype <strong>an</strong>d vehicle-treated tg female mice), despite <strong>the</strong> muscles <strong>from</strong><br />
diseased <strong>an</strong>d healthy mice being of comparable size (Oki et al., 2008, SfN Abstract<br />
745.20/W25). Given that fast twitch muscles appear more susceptible to disease th<strong>an</strong> slow twitch<br />
muscles in some models of motoneuron disease, we set out to examine whe<strong>the</strong>r <strong>the</strong> soleus<br />
(SOL), composed largely of slow-oxidative fibers, is also compromised by disease in our SBMA<br />
model. Isolated SOLs were superfused in mammali<strong>an</strong> Ringer’s (25C) <strong>an</strong>d maintained at resting<br />
length. Muscles were stimulated directly to induce single twitch <strong>an</strong>d tet<strong>an</strong>ic contractions (30-40<br />
volts, 1 sec duration). We find that <strong>the</strong> SOL muscle is more severely affected th<strong>an</strong> <strong>the</strong> EDL by<br />
disease. Whereas <strong>the</strong> EDL <strong>from</strong> diseased mice produces about half <strong>the</strong> amount of tension<br />
compared to controls, <strong>the</strong> diseased SOL produces 5-7 fold less tension th<strong>an</strong> controls, based on<br />
both twitch <strong>an</strong>d tet<strong>an</strong>ic tensions. This profound loss in contractile strength of <strong>the</strong> SOL <strong>from</strong><br />
diseased mice c<strong>an</strong>not be explained by a loss in muscle mass, since <strong>the</strong> deficit persists even when<br />
contractile <strong>for</strong>ce is normalized <strong>for</strong> muscle weight. We conclude that slow-oxidative fibers are<br />
more severely affected th<strong>an</strong> fast-glycolytic fibers in this SBMA model, which suggests that slow<br />
fibers may also be more profoundly affected by SBMA in hum<strong>an</strong>s. The observed motor deficits<br />
in diseased mice are likely a result of contractile deficits in both fiber types. Possible<br />
mech<strong>an</strong>isms underlying <strong>the</strong>se contractile deficits include altered calcium h<strong>an</strong>dling <strong>an</strong>d/or<br />
metabolic deficits which future experiments will examine directly.<br />
Disclosures: K. Oki, None; S. Breedlove, None; R.W. Wisem<strong>an</strong>, None; C.L. Jord<strong>an</strong>, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 533.28/M5<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Support: NIH NS-045195<br />
NIH NS-28421<br />
NIH NRSA 1F32AR055848-01<br />
Title: Live imaging of endosomal trafficking deficits in sciatic nerves of a myogenic mouse<br />
model of SBMA<br />
Authors: *M. KEMP, J. E. POORT, R. M. BAQRI, S. M. BREEDLOVE, K. E. MILLER, C. L.<br />
JORDAN;<br />
Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Spinal bulbar muscular atrophy (SBMA) is a hereditary neurodegenerative disease<br />
caused by <strong>an</strong> exp<strong>an</strong>sion of a trinucleotide CAG repeat encoding <strong>the</strong> polyglutamine tract in <strong>the</strong><br />
<strong>an</strong>drogen receptor (AR) gene. We developed a tr<strong>an</strong>sgenic (tg) mouse model that mimics SBMA<br />
by over-expressing <strong>the</strong> wild-type (wt) rat AR selectively in skeletal muscle fibers. Like o<strong>the</strong>r<br />
mouse models of SBMA, our tg mice show <strong>an</strong> <strong>an</strong>drogen-dependent loss of motor function. We<br />
have shown that such deficits in motor function are accomp<strong>an</strong>ied by signific<strong>an</strong>t delays in <strong>the</strong><br />
retrograde tr<strong>an</strong>sport of cholera toxin-horseradish peroxidase (CT-HRP) by spinal motoneurons<br />
suggesting that endosomal trafficking in motor axons may be impaired. To investigate this<br />
possibility, we have developed a technique that allows us to monitor trafficking endosomes in<br />
real time in living axons of mouse sciatic nerves using time-lapse video capture. Endosomes are<br />
visualized using CT-Alexa Fluor 488. From time-lapse movies, kymographs are made that<br />
display in two dimensions trafficking endosomes moving along <strong>the</strong> length of <strong>an</strong> axon as a<br />
function of time. From such kymographs, we c<strong>an</strong> derive estimates of <strong>the</strong> number of moving<br />
endosomes (flux) <strong>an</strong>d <strong>the</strong> rate at which <strong>the</strong>y move (velocity), providing insight into whe<strong>the</strong>r<br />
trafficking, dye uptake or both account <strong>for</strong> why diseased mice show a slower accumulation of<br />
CT-HRP in <strong>the</strong>ir motoneurons. We find that T-treated tg females with profound motor<br />
dysfunction show a signific<strong>an</strong>t (35%) reduction in endosomal flux, but not velocity, compared to<br />
control-treated tg females with normal motor function. These data suggest that delayed<br />
accumulation of CT-HRP in motoneuronal cell bodies may be caused by impaired dye uptake,<br />
suggesting that dysregulation at <strong>the</strong> motor nerve terminal, involving impaired endocytosis <strong>an</strong>d/or<br />
terminal retraction, may critically mediate <strong>the</strong> loss of motor function in SBMA. Moreover, given<br />
that in our model <strong>the</strong> disease-causing gene is expressed only in muscle fibers, <strong>the</strong>se data also<br />
show that signals em<strong>an</strong>ating <strong>from</strong> muscle regulate motoneuronal function.<br />
Disclosures: M. Kemp, None; J.E. Poort, None; R.M. Baqri, None; S.M. Breedlove,<br />
None; K.E. Miller, None; C.L. Jord<strong>an</strong>, None.
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.29/M6<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Title: Unusual distribution of ApoE genotypes in spinal <strong>an</strong>d bulbar muscular atrophy<br />
Authors: R. SCHNEIDER 1 , A. KÖHLER 1 , C. ACHMÜLLER 2 , *S. M. BOESCH 3 , W.<br />
LÖSCHER 2 ;<br />
1 Inst. of Biochem. <strong>an</strong>d Ctr. <strong>for</strong> Mol. Biosci., Leopold Fr<strong>an</strong>zens Univ., Innsbruck, Austria;<br />
2 Neurol., 3 Med. Univ. Innsbruck, Innsbruck, Austria<br />
Abstract: Apolipoprotein E (APOE) is a carrier protein <strong>an</strong>d directly involved in <strong>the</strong> uptake <strong>an</strong>d<br />
distribution of plasma lipids <strong>an</strong>d cholesterol <strong>an</strong>d thus essential <strong>for</strong> normal lipoprotein<br />
metabolism. The polymorphic ApoE gene is characterized by three major alleles, ApoE2, ApoE3<br />
<strong>an</strong>d ApoE4, which differ by base substitutions in two codons, resulting in amino acid<br />
replacements in position 112 (Cys to Arg) <strong>an</strong>d 158 (Arg to Cys). ApoE2 (Cys112, Cys158) shows<br />
less th<strong>an</strong> 2% of receptor binding activity <strong>an</strong>d is linked to lower serum cholesterol <strong>an</strong>d higher<br />
ApoE concentration. ApoE4 (Arg112, Arg158) is associated with high serum cholesterol <strong>an</strong>d low<br />
ApoE concentrations <strong>an</strong>d has been implicated in a<strong>the</strong>rosclerosis, impaired cognitive function,<br />
reduced neurite outgrowth <strong>an</strong>d Alzheimer's disease.<br />
Spinal <strong>an</strong>d bulbar muscular atrophy (SBMA) is a late-onset, X-linked recessive inherited<br />
progressive disease of lower motor neurons that is caused by a polyglutamine repeat exp<strong>an</strong>sion<br />
in <strong>the</strong> <strong>an</strong>drogen receptor (AR). The key symptoms of SBMA are progressive weakness <strong>an</strong>d<br />
atrophy of bulbar, facial <strong>an</strong>d limb muscles due to neurodegeneration especially in <strong>the</strong> <strong>an</strong>terior<br />
horns of <strong>the</strong> spinal cord <strong>an</strong>d in <strong>the</strong> bulbar region of <strong>the</strong> brainstem. There is evidence that<br />
<strong>an</strong>drogen increases <strong>the</strong> severity of clinical symptoms in SBMA. X-linked inherit<strong>an</strong>ce of <strong>the</strong> AR<br />
<strong>an</strong>d <strong>an</strong>drogen concentrations in males cause a strong gender dependency, with only males being<br />
fully affected.<br />
Since SBMA phenotype is <strong>an</strong>drogen-modulated <strong>an</strong>d may <strong>the</strong>re<strong>for</strong>e be cholesterol-dependent we<br />
hypo<strong>the</strong>sized that <strong>the</strong> ApoE genotype may possibly be disease modifying. To investigate,<br />
whe<strong>the</strong>r <strong>the</strong>re is a deviation <strong>from</strong> <strong>the</strong> usual ApoE genotype distribution, 12 unrelated male<br />
patients with genetically confirmed SBMA were <strong>an</strong>alysed <strong>for</strong> <strong>the</strong>ir ApoE genotype by PCRrestriction<br />
fragment length polymorphism <strong>an</strong>alysis.<br />
We found a striking enh<strong>an</strong>cement of <strong>the</strong> heterozygous E2/E3 combination frequency in SBMA<br />
patients. The E2/E3 combination was found in 33.3% SBMA patients compared to only 11%<br />
reported in normal individuals (p=0,015). Underlying mech<strong>an</strong>isms that possibly involve <strong>the</strong><br />
cholesterol-<strong>an</strong>drogen axis of <strong>the</strong> mo<strong>the</strong>r <strong>an</strong>d/or <strong>the</strong> affected offspring have to be elucidated in<br />
future large multicenter studies.
Disclosures: R. Schneider, None; A. Köhler, None; C. Achmüller, None; S.M. Boesch,<br />
None; W. Löscher, None.<br />
Poster<br />
533. Huntington's Disease <strong>an</strong>d SBMA: Molecular <strong>an</strong>d Cellular Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 533.30/M7<br />
Topic: C.04.d. Repeat exp<strong>an</strong>sion diseases: Non-Huntington’s disease<br />
Support: MDA Gr<strong>an</strong>t 92333<br />
NINDS intramural funding<br />
Title: B2 modifies polyglutamine <strong>an</strong>drogen receptor toxicity in cell <strong>an</strong>d fly models of SBMA<br />
Authors: *M. PENNUTO 1,2 , I. PALAZZOLO 3 , N. NEDELSKY 4 , G. HARMISON 3 , J. P.<br />
TAYLOR 4 , K. H. FISCHBECK 3 ;<br />
1 Dept Neurol, Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA; 2 Neurosci. <strong>an</strong>d Brain Technol., Itali<strong>an</strong> Inst.<br />
of Technol., Genoa, Italy; 3 NINDS, NIH, Be<strong>the</strong>sda, MD; 4 Neurol., St Jude Children's Hosp.,<br />
Memphis, TN<br />
Abstract: Spinal <strong>an</strong>d bulbar muscular atrophy (SBMA) is <strong>an</strong> inherited neurodegenerative<br />
disorder caused by polyglutamine exp<strong>an</strong>sion in <strong>the</strong> gene encoding <strong>the</strong> <strong>an</strong>drogen receptor.<br />
Exp<strong>an</strong>sion of polyglutamine is <strong>the</strong> cause of eight o<strong>the</strong>r neurodegenerative diseases, including<br />
Huntington’s disease. Exp<strong>an</strong>sion of <strong>the</strong> polyglutamine tract confers a toxic gain of function to<br />
<strong>the</strong> bearing protein, which <strong>for</strong> <strong>an</strong>drogen receptor is lig<strong>an</strong>d-dependent. In <strong>the</strong> absence of its<br />
lig<strong>an</strong>d, testosterone or its derivative dihydrotestosterone, <strong>the</strong> <strong>an</strong>drogen receptor is located in <strong>the</strong><br />
cytoplasm in <strong>an</strong> inactive state. Upon lig<strong>an</strong>d binding, <strong>the</strong> <strong>an</strong>drogen receptor tr<strong>an</strong>slocates to <strong>the</strong><br />
nucleus <strong>an</strong>d becomes highly toxic to motor neurons. B2 has been previously described to<br />
decrease <strong>the</strong> toxicity of polyglutamine huntingtin in cells. In this study we <strong>an</strong>alyzed <strong>the</strong> effects of<br />
B2 in SBMA. We found that in motor neuron-derived SBMA cells B2 reduces <strong>the</strong> caspase 3<br />
activation induced by polyglutamine <strong>an</strong>drogen receptor. In flies, B2 attenuates <strong>the</strong> lig<strong>an</strong>ddependent<br />
neurodegeneration induced by mut<strong>an</strong>t <strong>an</strong>drogen receptor when expressed in <strong>the</strong><br />
ommatidia. Interestingly, we found that B2 affects tr<strong>an</strong>sactivation of mut<strong>an</strong>t <strong>an</strong>drogen receptor<br />
<strong>an</strong>d reduces <strong>the</strong> ability of <strong>the</strong> mut<strong>an</strong>t protein to bind lig<strong>an</strong>d. Our findings show <strong>for</strong> <strong>the</strong> first time<br />
<strong>the</strong> effect of B2 on polyglutamine toxicity in vivo <strong>an</strong>d suggest <strong>the</strong> use of B2 as <strong>the</strong>rapy <strong>for</strong><br />
SBMA.
Disclosures: M. Pennuto, None; I. Palazzolo, None; N. Nedelsky, None; G. Harmison,<br />
None; J.P. Taylor, None; K.H. Fischbeck, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.1/M8<br />
Topic: C.11.p. Neuroinflammation: HIV <strong>an</strong>d infections<br />
Support: NIH Gr<strong>an</strong>t MH071151<br />
NIH Gr<strong>an</strong>t MH01717<br />
NIH Gr<strong>an</strong>t T32MH018273<br />
Title: Peripheral macrophage infection is associated with <strong>the</strong> development of simi<strong>an</strong><br />
immunodeficiency virus encephalitis in pigtailed macaques<br />
Authors: *S. J. BISSEL, G. WANG, D. BONNEH-BARKAY, A. STARKEY, M. F.<br />
STAUFFER, J. NYAUNDI, M. MURPHEY-CORB, C. A. WILEY;<br />
Univ. Pittsburgh, Pittsburgh, PA<br />
Abstract: The brains of individuals with lentiviral-associated encephalitis contain <strong>an</strong> abund<strong>an</strong>ce<br />
of infected <strong>an</strong>d activated macrophages. It has been hypo<strong>the</strong>sized that encephalitis develops when<br />
increased numbers of infected monocytes traffic into <strong>the</strong> central nervous system (CNS) during<br />
<strong>the</strong> end stages of immunosuppression. The relationships between <strong>the</strong> infection of brain <strong>an</strong>d<br />
systemic macrophages during <strong>the</strong> development of lentiviral encephalitis are unknown. We<br />
examined <strong>the</strong> extent of macrophage infection in pigtailed macaques that did or did not develop<br />
simi<strong>an</strong> immunodeficiency virus encephalitis (SIVE). At necropsy, macaques with SIVE had<br />
more infected macrophages in peripheral org<strong>an</strong>s, most notable in <strong>the</strong> small bowel. Duodenal<br />
biopsies <strong>from</strong> infected macaques have been followed every 2 weeks post-infection to determine<br />
whe<strong>the</strong>r systemic macrophage infection mirrors CNS infection as determined by CSF viral load.<br />
Biopsy samples also give <strong>an</strong> opportunity to examine <strong>the</strong> immune response to macrophage<br />
infection. In <strong>the</strong> CNS, T cells <strong>an</strong>d NK cells with cytotoxic potential were more abund<strong>an</strong>t in<br />
macaques with SIVE; however, T-cell <strong>an</strong>d NK-cell infiltration in SIVE <strong>an</strong>d hum<strong>an</strong><br />
immunodeficiency virus encephalitis was more modest th<strong>an</strong> that observed in classical acute<br />
herpes simplex virus encephalitis. These findings support <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> development of<br />
lentiviral encephalitis occurs in hosts that are unable to control macrophage infection.
Disclosures: S.J. Bissel, None; G. W<strong>an</strong>g, None; D. Bonneh-Barkay, None; A. Starkey,<br />
None; M.F. Stauffer, None; J. Nyaundi, None; M. Murphey-Corb, None; C.A. Wiley, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.2/M9<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Title: TDP-43 proteinopathy in centenari<strong>an</strong>s<br />
Authors: *T. IWASE 1 , Y. HASHIZUME 2 , K. OJIKA 3 ;<br />
1 Dept. of Neurol., Nagoya City Koseiin Med. Welfare Ctr., Nagoya, Jap<strong>an</strong>; 2 Neuropathology,<br />
Inst. <strong>for</strong> Med. Sci. of Aging, Aichi Med. Univ., Nagakute-cho, Aichi-gun, Jap<strong>an</strong>; 3 Neurol.,<br />
Nagoya City Univ., Nagoya, Jap<strong>an</strong><br />
Abstract: Tr<strong>an</strong>sactive response (TAR)-DNA-binding protein 43 (TDP-43) was first identified as<br />
a major component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) <strong>an</strong>d<br />
frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). The presence of<br />
abnormal aggregates of phosphorylated <strong>an</strong>d ubiquitinated TDP-43 has also been found<br />
accumulated in o<strong>the</strong>r neurodegenerative diseases including Pick disease, Alzheimer’s disease,<br />
Lewy body disease, <strong>an</strong>d corticobasal degeneration. In this study, we investigated <strong>the</strong> TDP-43positive<br />
structures in centenari<strong>an</strong>s. The autopsied brains <strong>an</strong>d spinal cords <strong>from</strong> 42 centenari<strong>an</strong>s<br />
were studied. In all cases, neuropathological diagnosis was made with st<strong>an</strong>dard stainings of<br />
hematoxylin-eosin, Klüber-Barrera <strong>an</strong>d Gallyas-Braak, <strong>an</strong>d α-synuclein, phosphorylated tau<br />
(AT8) <strong>an</strong>d amyloid-β immunostainings. No ALS or FTLD-U case was found. We per<strong>for</strong>med<br />
TDP-43 <strong>an</strong>d ubiquitin immunostaining of <strong>the</strong> hippocampal dentate gyrus, <strong>an</strong>d found<br />
intracytoplasmic inclusions in 10 cases, yielding <strong>an</strong> overall prevalence of 23.8%. TDP-43positive<br />
inclusions were also ubiquitin-positive. For 10 cases with TDP-43-positive inclusions in<br />
<strong>the</strong> hippocampus, we fur<strong>the</strong>r studied <strong>the</strong> spinal cords, <strong>an</strong>d found TDP-43-positive structures in<br />
only one case. Among 10 cases with TDP-43-positive inclusions, all 8 demented cases were<br />
complicated with o<strong>the</strong>r neurological diseases including Alzheimer’s disease, argyrophilic grain<br />
disease, Lewy body disease, Binsw<strong>an</strong>ger’s subcortical encephalopathy, <strong>an</strong>d senile dementia of<br />
<strong>the</strong> neurofibrillary t<strong>an</strong>gle type. Interestingly, two cases solely with TDP-43-positive inclusions<br />
were not demented. Our findings indicate that <strong>the</strong>re is a high prevalence of TDP-43-positive<br />
structures with limited distribution in centenari<strong>an</strong>s. A small number of TDP-43 positive<br />
structures may be found even in normal aging hum<strong>an</strong> brains.
Disclosures: T. Iwase, None; Y. Hashizume, None; K. Ojika, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.3/M10<br />
Topic: C.04.i. Prion diseases<br />
Support: NIH Gr<strong>an</strong>t NS 044209<br />
Title: Prion protein knock-out mice show altered iron metabolism: A functional role <strong>for</strong> prion<br />
protein in iron uptake <strong>an</strong>d tr<strong>an</strong>sport<br />
Authors: *N. SINGH, A. SINGH, X. LUO;<br />
Dept Pathology, Case Western Reserve Univ., Clevel<strong>an</strong>d, OH<br />
Abstract: Prion protein (PrP) is a ubiquitously expressed cell surface glycoprotein linked to <strong>the</strong><br />
plasma membr<strong>an</strong>e by a glycosylphosphatidylinositol <strong>an</strong>chor. Although several lines of evidence<br />
support <strong>the</strong> obligate role of PrP in <strong>an</strong>imal <strong>an</strong>d hum<strong>an</strong> prion disorders, relatively less is known<br />
about <strong>the</strong> normal physiological function of this evolutionarily conserved protein. In a recent<br />
report we demonstrated that PrP mediates iron uptake <strong>an</strong>d tr<strong>an</strong>sport in hum<strong>an</strong> neuroblastoma<br />
cells (Plos One), <strong>an</strong>d aggregation of PrP to <strong>the</strong> disease causing PrP-scrapie (PrP Sc ) <strong>for</strong>m induces<br />
imbal<strong>an</strong>ce of iron homeostasis in prion disease affected hum<strong>an</strong>, hamster <strong>an</strong>d mouse brains (Plos<br />
Pathogens). We have now extended <strong>the</strong>se studies to mouse models to underst<strong>an</strong>d <strong>the</strong> role of PrP<br />
in systemic iron homeostasis. Using tr<strong>an</strong>sgenic mice lacking PrP expression (PrP KO ) <strong>an</strong>d wild<br />
type (Wt) controls, we demonstrate that absence of PrP induces a phenotype of systemic iron<br />
deficiency in PrP KO mice as reflected by various hematological parameters such as lower levels<br />
of serum iron, tr<strong>an</strong>sferrin iron binding capacity (TIBC), iron saturation of tr<strong>an</strong>sferrin, ferritin,<br />
<strong>an</strong>d mild reticulocytosis in <strong>the</strong> peripheral blood relative to Wt controls. A similar phenotype of<br />
iron deficiency is noted in major org<strong>an</strong>s such as <strong>the</strong> liver, spleen, <strong>an</strong>d brain of PrP KO mice as<br />
indicated by increased expression of iron uptake proteins tr<strong>an</strong>sferrin (Tf) <strong>an</strong>d tr<strong>an</strong>sferrin receptor<br />
(TfR), <strong>an</strong>d decreased expression of iron storage protein ferritin relative to Wt controls. The<br />
cerebellar purkinje cell layer of PrP KO brains shows a similar up regulation of TfR, reflecting a<br />
phenotype of neuronal iron deficiency. Introduction of radiolabeled iron ( 59 FeCl3) by gastric<br />
gavage reveals inefficient tr<strong>an</strong>sport of 59 Fe <strong>from</strong> <strong>the</strong> duodenum to <strong>the</strong> blood stream, <strong>an</strong> early<br />
abortive spike of erythropoiesis in <strong>the</strong> long bones <strong>an</strong>d spleen, <strong>an</strong>d eventual decreased 59 Fe<br />
content of red blood cells <strong>an</strong>d all major org<strong>an</strong>s of PrP KO mice relative to Wt controls. The<br />
underlying cause of iron deficiency in PrP KO mice is impaired tr<strong>an</strong>sport of iron <strong>from</strong> <strong>the</strong>
duodenal epi<strong>the</strong>lium to <strong>the</strong> blood stream, <strong>an</strong>d a similar defect in <strong>the</strong> uptake of iron by <strong>the</strong><br />
parenchymal cells of various org<strong>an</strong>s <strong>an</strong>d hematopoietic precursor cells. The iron deficient<br />
phenotype of PrP KO mice is rescued by introducing PrP on <strong>the</strong> PrP KO background, indicating a<br />
functional role <strong>for</strong> PrP in iron uptake <strong>an</strong>d tr<strong>an</strong>sport. These results uncover a novel physiological<br />
function of PrP in systemic <strong>an</strong>d brain iron metabolism that could have signific<strong>an</strong>t implications<br />
<strong>for</strong> prion disease pathogenesis due to <strong>the</strong> highly toxic nature of iron, <strong>an</strong>d could account <strong>for</strong> <strong>the</strong><br />
reported imbal<strong>an</strong>ce of iron homeostasis in prion disease affected hum<strong>an</strong> <strong>an</strong>d <strong>an</strong>imal brains.<br />
Disclosures: N. Singh , None; A. Singh, None; X. Luo, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.4/M11<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: Je<strong>an</strong> <strong>an</strong>d Pape Adams Foundation<br />
<strong>the</strong> J<strong>an</strong>e B. Barsumi<strong>an</strong> Trust Fund<br />
Title: Increase oxidative stress <strong>an</strong>d ch<strong>an</strong>ges in <strong>the</strong> iron m<strong>an</strong>agement protein expression in brains<br />
of mice carrying <strong>the</strong> H67D vari<strong>an</strong>t of <strong>the</strong> HFE gene<br />
Authors: *W. NANDAR 1 , E. NEELY 1 , Z. SIMMONS 2 , J. R. CONNOR 1 ;<br />
1 Dept. of Neurosurg., The Pennsylv<strong>an</strong>ia State University, , M. S. Hershey Med. Ctr., Hershey,<br />
PA; 2 Dept. of Neurol., The Pennsylv<strong>an</strong>ia State University, M. S. Hershey Med. Ctr., Hershey,<br />
PA<br />
Abstract: Iron overload <strong>an</strong>d increased oxidative stress have been observed in several<br />
neurodegenerative diseases such as Alzheimer’s disease, Amyotrophic lateral sclerosis <strong>an</strong>d<br />
Parkinson’s disease. One of <strong>the</strong> mech<strong>an</strong>isms that c<strong>an</strong> lead to iron accumulation is a mutation in<br />
hemochromatosis (HFE) gene, which plays a role in tr<strong>an</strong>sferrin- mediated iron uptake. HFE gene<br />
mutations have been proposed as genetic modifiers <strong>for</strong> risk of neurodegenerative disorders. The<br />
most common mutation in HFE gene, H63D, has been shown to influence cellular iron<br />
homeostasis. Neuroblastoma (SH-SY5Y) cell lines carrying a H63D mutation have increased<br />
oxidative stress, glutamate secretion <strong>an</strong>d inflammation. Since both genetic <strong>an</strong>d environmental<br />
factors c<strong>an</strong> influence iron homeostasis <strong>the</strong> question of whe<strong>the</strong>r a H63D mutation is associated<br />
with <strong>an</strong> increased risk of neurodegenerative disorders still needs to be clarified. Thus, <strong>an</strong> in vivo
model is import<strong>an</strong>t to evaluate neurological consequences of a H63D mutation under controlled<br />
environmental conditions. The mouse model <strong>for</strong> H63D mutation used in <strong>the</strong> present study was<br />
generated by intercrossing heterozygous H67D mice (mouse homologous to H63D in hum<strong>an</strong>),<br />
which were produced using a site-directed mutagenesis <strong>an</strong>d germ-line chimeras. Although<br />
juvenile H67D knock-in mice displayed increased hepatic iron levels, <strong>the</strong> neurological<br />
characterization of H67D homozygous mice has not been reported. At 6 months of age, <strong>the</strong><br />
brains <strong>from</strong> wild type <strong>an</strong>d homozygous H67D mice were harvested <strong>an</strong>d <strong>the</strong> expression of<br />
divalent metal tr<strong>an</strong>sporter-1 (DMT-1) was determined by Westernblot <strong>an</strong>alysis. The brains of<br />
wild type <strong>an</strong>d H67D mice were not different macroscopically; however, total brain iron level<br />
tended to be higher in H67D mice compared to <strong>the</strong> wild type. DMT-1 expression was<br />
downregulated in <strong>the</strong> brains of H67D mice compared to <strong>the</strong> wild type. Immunohistochemical<br />
<strong>an</strong>alysis also showed that DMT-1 immunoreactivity was decreased in <strong>the</strong> motor cortex, striatum<br />
<strong>an</strong>d cerebellum of H67D mice. These findings suggested <strong>an</strong> increased brain iron level in H67D<br />
knock-in mice. We previously reported abnormal glutmatergic secretion <strong>an</strong>d release in <strong>the</strong> H63D<br />
cells so we determined <strong>the</strong> expression of <strong>the</strong> cystine glutamate <strong>an</strong>tiporter, xCT, which is<br />
associated with glutamate release <strong>an</strong>d oxidative stress. Increased xCT <strong>an</strong>tiporter level was<br />
observed in <strong>the</strong> brains of H67D compared to wild type mice. Although evaluation of o<strong>the</strong>r iron<br />
m<strong>an</strong>agement proteins in H67D knock-in mice is still in progress, a H67D knock-in mouse<br />
appears to be a potential <strong>an</strong>imal model to determine <strong>the</strong> neurological consequences of a H63D<br />
mutation <strong>an</strong>d its effect on <strong>the</strong> pathogenesis of neurodegenerative diseases.<br />
Disclosures: W. N<strong>an</strong>dar, None; E. Neely, None; Z. Simmons, None; J.R. Connor, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.5/M12<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: CIHR Fellow ship<br />
National Multiple Sclerosis <strong>Society</strong><br />
Title: Nlrp3 gene plays critical role in <strong>the</strong> development of experimental allergic<br />
encephalomyelitis by mediating TH1 <strong>an</strong>d TH17 responses
Authors: *D. GRIS 1 , Z. YE 1 , H. WEN 1 , S. D. MILLER 2 , J. P. Y. TING 1 ;<br />
1 2<br />
Micrology immunology, LCCC UNC, Chapel Hill, NC; Microbiology Immunol., Northwest<br />
Univ. Med. Sch., Chicago, IL<br />
Abstract: Multiple sclerosis is a devastating pathology that develops as a consequence of<br />
disregulation of immune response. The interplay between innate <strong>an</strong>d adaptive immune system is<br />
believed to be <strong>an</strong> origin of this pathology. Downstream cytokines such as interleukin (IL) IL-<br />
1beta <strong>an</strong>d IL-18 play <strong>the</strong> crucial role in <strong>the</strong> development of neuroinflammatory diseases. The<br />
regulation of secretion of <strong>the</strong>se cytokines relies on inflammasome, a protein complex that<br />
activates caspase-1, which cleaves IL-1beta <strong>an</strong>d IL-18. Nlrp3 is one of <strong>the</strong> inflammasome<br />
proteins that plays critical role in <strong>the</strong> regulation of <strong>the</strong> innate <strong>an</strong>d adaptive immune system. We<br />
used mouse model of multiple sclerosis, experimental autoimmune encephalitis (EAE), to<br />
investigate <strong>the</strong> role of Nlrp3 protein in <strong>the</strong> development of EAE. Nlrp3-/- had delayed course <strong>an</strong>d<br />
reduced severity of disease. We observed improved histology in <strong>the</strong> spinal cords with reduced<br />
destruction of myelin <strong>an</strong>d reduced astrogliosis that were evident <strong>from</strong> immunohistochemical<br />
staining <strong>an</strong>d western blotting <strong>an</strong>alysis. Activated splenocytes <strong>from</strong> Nlrp3-/- EAE mice produced<br />
3 fold less interferon-gamma (INF-gamma) <strong>an</strong>d 2 fold less IL-17. We also observed signific<strong>an</strong>t<br />
reduction of INF-gamma <strong>an</strong>d IL-17 in spinal cord homogenates in Nlrp3-/- mice. We<br />
demonstrated that Nlrp3 protein plays a critical role in <strong>the</strong> induction of <strong>the</strong> EAE in mice.<br />
Disclosures: D. Gris, None; Z. Ye, None; H. Wen, None; S.D. Miller, None; J.P.Y. Ting,<br />
None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.6/M13<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: CASE Educational <strong>an</strong>d Research Support Fund<br />
Title: Mech<strong>an</strong>isms underlying cerebellar Purkinje cell death in <strong>the</strong> shaker mut<strong>an</strong>t rat<br />
Authors: N. S. EREKAT, *D. L. TOLBERT;<br />
St Louis Univ. Sch. Med., St. Louis, MO<br />
Abstract: In shaker mut<strong>an</strong>t rats hereditary degeneration of at risk cerebellar Purkinje cells (PCs)<br />
occurs between 7 <strong>an</strong>d 14 postnatal weeks of age in restricted <strong>an</strong>terior <strong>an</strong>d posterior vermal
compartments. Secure PCs in a flocculonodular survival compartment always survive. We<br />
hypo<strong>the</strong>size that hereditary shaker mut<strong>an</strong>t PC death may be due to activation of multiple death<br />
pathways. In this study we sought to determine if at risk PCs die by apoptosis. We first<br />
investigated DNA fragmentation characteristic of apoptosis using terminal deoxynucleotidyl<br />
tr<strong>an</strong>sferase dUTP nick end labeling (TUNEL). Subst<strong>an</strong>tial numbers of at risk PCs nuclei were<br />
first labeled <strong>for</strong> TUNEL at 7 weeks of age in <strong>the</strong> <strong>an</strong>terior degeneration compartment <strong>an</strong>d at 9<br />
weeks in <strong>the</strong> posterior compartment. This is spatially <strong>an</strong>d temporally congruous with <strong>the</strong> pattern<br />
of calbindin immunolabeling of degenerating PCs that is characterized by dendritic atrophy,<br />
shrinkage of PC cell bodies <strong>an</strong>d <strong>the</strong> appear<strong>an</strong>ce of PC axonal torpedoes. Purkinje neurons were<br />
<strong>the</strong> only cerebellar cells labeled <strong>for</strong> TUNEL in <strong>the</strong> shaker mut<strong>an</strong>t. Using immunofluorescence we<br />
<strong>the</strong>n investigated if apoptosis of at risk PCs occurred by activated caspase 3 expression. Active<br />
caspase-3 expression was observed in a few at risk PCs as early as 2-3 weeks of age. Starting,<br />
however, at 5 weeks of age <strong>the</strong> numbers of caspase 3-immunoreactive at risk PCs increased<br />
dramatically in <strong>the</strong> <strong>an</strong>terior degeneration compartment. This was followed at 8 weeks of age with<br />
m<strong>an</strong>y at risk PCs in <strong>the</strong> posterior compartment expressing activated caspase-3. TUNEL labeling<br />
<strong>an</strong>d active caspase-3 expression was not observed in <strong>the</strong> cerebellar cortex after 13-14 weeks of<br />
age. Secure PCs in <strong>the</strong> flocculonodular survival compartment were never positive <strong>for</strong> TUNEL or<br />
immunoreactive <strong>for</strong> active caspase-3. Collectively, <strong>the</strong>se observations indicate <strong>the</strong> onset of<br />
hereditary PC death occurs 2-3 weeks earlier th<strong>an</strong> DNA fragmentation <strong>an</strong>d overt degeneration of<br />
at risk PCs. It <strong>the</strong>re<strong>for</strong>e appears that apoptosis takes place in at risk PCs in <strong>the</strong> shaker mut<strong>an</strong>t rat.<br />
We have o<strong>the</strong>r findings that shaker mut<strong>an</strong>t PCs also die as <strong>the</strong> result of autophagic mech<strong>an</strong>isms.<br />
Disclosures: N.S. Erekat, None; D.L. Tolbert, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.7/M14<br />
Topic: C.04.j. Dystonia<br />
Support: CIHR MOP-42505<br />
Title: Frequency-dependent effects of microstimulation in <strong>the</strong> globus pallidus of dystonia<br />
patients<br />
Authors: L. D. LIU 1 , N. T. HOLZAPFEL 2 , J. O. DOSTROVSKY 1 , M. HODAIE 3 , A.<br />
LOZANO 3 , *W. D. HUTCHISON 3 ;
1 2 3<br />
Physiol., Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada; Div. Neurosurg., Toronto Western Hosp.,<br />
Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Deep Brain Stimulation (DBS) in <strong>the</strong> globus pallidus interna (GPi) has been shown to<br />
improve <strong>the</strong> sustained muscle contractions <strong>an</strong>d repetitive, twisting movements known as<br />
dystonia. Clinical studies show that DBS at frequencies above 60Hz produces <strong>the</strong>rapeutic effects<br />
while stimulation at 10-30 Hz has no effect or may exacerbate motor symptoms. We recently<br />
found that short trains of high frequency stimulation (HFS) enh<strong>an</strong>ce <strong>the</strong> amplitude of a positive<br />
field evoked potential (fEP) suggesting <strong>an</strong> involvement of activity-dependent synaptic plasticity<br />
in GPi. The aim of <strong>the</strong> study was twofold; first to examine <strong>the</strong> frequency-dependent response of<br />
focal microstimulation on GPi neuronal activity <strong>an</strong>d second, to examine <strong>the</strong> effects of synaptic<br />
plasticity on <strong>the</strong> frequency-dependent response in order to model <strong>the</strong> effects of DBS in GPi.<br />
Stimulation trains of 50 pulses at 100 uA intensity <strong>an</strong>d various frequencies (1, 2, 5, 10, 20, 30, 50<br />
<strong>an</strong>d 100 Hz) were delivered via one electrode while monitoring <strong>the</strong> effects on cell firing <strong>an</strong>d fEPs<br />
via a second electrode ~ 1 mm away within <strong>the</strong> GPi of 7 dystonia patients. Spikes were template<br />
matched, <strong>an</strong>d stimulus artefacts rejected. Average firing rates were calculated at baseline <strong>an</strong>d<br />
during <strong>the</strong> stimulation period (n = 10). The fEP amplitudes to <strong>the</strong> first pulse in <strong>the</strong> train, <strong>the</strong><br />
average amplitudes <strong>for</strong> all pulses at a given frequency, <strong>an</strong>d fiber volleys (n=4) were measured.<br />
The results were normalized to baseline <strong>an</strong>d plotted against stimulation frequency be<strong>for</strong>e <strong>an</strong>d up<br />
to 1 min after HFS (4 X 2 s, 10s apart). ANOVA tested main effects of frequency <strong>an</strong>d HFS <strong>an</strong>d<br />
<strong>the</strong>ir interaction. Be<strong>for</strong>e HFS, <strong>the</strong> average fEP amplitude increased at frequencies of 10 to 30 Hz,<br />
whereas <strong>the</strong> average firing rate decreased, suggesting synaptic release of <strong>an</strong> inhibitory<br />
tr<strong>an</strong>smitter, likely GABA. Above 50 Hz <strong>the</strong> firing rate was virtually silenced but <strong>the</strong> fEPs<br />
persisted albeit at a much reduced amplitude. Following <strong>the</strong> HFS trains, fEPs at low frequencies<br />
were enh<strong>an</strong>ced but <strong>the</strong> peak in <strong>the</strong> 20 - 30 Hz r<strong>an</strong>ge was lower, suggesting DBS reduces synaptic<br />
reson<strong>an</strong>ce at this frequency. In contrast, fiber volleys were stable but showed a tr<strong>an</strong>sient 20%<br />
decrease in amplitude <strong>an</strong>d prolonged latency at 100 Hz. The brief duration <strong>an</strong>d short fixed<br />
latency of a few spikes seen near <strong>the</strong> peak of <strong>the</strong> fEP suggest <strong>an</strong>tidromic axonal activation or<br />
direct activation of dendrites. The <strong>the</strong>rapeutic effects of DBS may be mediated by m<strong>an</strong>y factors<br />
including inhibitory neurotr<strong>an</strong>smitter release, excitation of axons <strong>an</strong>d dendrites, <strong>an</strong>d synaptic<br />
plasticity. The data support <strong>the</strong> hypo<strong>the</strong>sis that DBS in GPi works by reduction of neuronal firing<br />
rate, disruption of firing patterns <strong>an</strong>d imposing a high frequency in <strong>the</strong> local field potential.<br />
Disclosures: L.D. Liu, None; N.T. Holzapfel, None; J.O. Dostrovsky, None; M. Hodaie,<br />
None; A. Loz<strong>an</strong>o, None; W.D. Hutchison, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 534.8/M15<br />
Topic: C.04.i. Prion diseases<br />
Title: Impaired autophagy in prion protein-deficient Purkinje cells<br />
Authors: *Y. J. BAILLY, N. J. GRANT;<br />
CNRS UPR3212 INCI, Strasbourg, Fr<strong>an</strong>ce<br />
Abstract: The ectopic expression of <strong>the</strong> prion protein homologue Doppel (Dpl) in brain neurons<br />
causes progressive cerebellar Purkinje cell death in prion protein-deficient Ngsk mice (NP0/0).<br />
The neurotoxicity caused by Dpl involves Bax-dependent apoptotic pathways as well as o<strong>the</strong>r yet<br />
to be characterized cell death mech<strong>an</strong>isms in <strong>the</strong> NP0/0 Purkinje cells. These neurons display<br />
increased amounts of several autophagy-related molecules such as <strong>the</strong> scrapie-responsive gene<br />
one (Scrg1), LC3B-II <strong>an</strong>d p62 without showing <strong>an</strong>y ch<strong>an</strong>ges in mRNA expression. In addition,<br />
autolysosomes accumulate in all neuronal compartments including axon terminals. This suggests<br />
that Dpl toxicity impairs <strong>the</strong> autophagic flux, which may trigger apoptosis in <strong>the</strong>se neurons,<br />
similar to <strong>the</strong> way neurodegeneration is thought to occur in Alzheimer <strong>an</strong>d prion diseases.<br />
Purkinje cells feature early axonal <strong>an</strong>d presynaptic autophagy in both NP0/0 <strong>an</strong>d GluRdelta2Lc<br />
mut<strong>an</strong>ts, but no signs of autophagic flux impairment are evident in GluRdelta2Lc Lurcher<br />
suggesting that different pathogenic stimuli (i.e., Dpl versus GluRdelta2Lc) trigger different cell<br />
death modalities involving autophagy <strong>an</strong>d apoptosis in <strong>the</strong> same type of neuron. The interplay<br />
between <strong>the</strong>se multiple pathways of programmed cell death needs to be fur<strong>the</strong>r investigated in<br />
cellular <strong>an</strong>d <strong>an</strong>imal models of neurodegenerative diseases in order to develop new <strong>the</strong>rapies of<br />
neurodegenerative diseases.<br />
Disclosures: Y.J. Bailly, None; N.J. Gr<strong>an</strong>t, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.9/M16<br />
Topic: C.04.i. Prion diseases<br />
Support: NIH NINDS (R01 NS046037)<br />
Brain Research Foundation
Pioneer Fund<br />
Title: Induction of autophagy by T182A mut<strong>an</strong>t prion protein<br />
Authors: C. J. CORTES 1 , *J. A. MASTRIANNI 2 ;<br />
1 Cell Physiol., 2 Univ. Chicago, Chicago, IL<br />
Abstract: Background: Prion diseases result <strong>from</strong> <strong>the</strong> accumulation of misfolded prion protein<br />
(PrP) in <strong>the</strong> central nervous system. In recent years, several neurodegenerative-related<br />
proteinopathies have been shown to employ autophagy in <strong>the</strong> removal of aggregated proteins;<br />
however, its role in prion disease has not been established.<br />
Objective: To study <strong>the</strong> potential involvement of autophagy in <strong>the</strong> intracellular degradation of<br />
<strong>the</strong> disease-linked T182A PrP mut<strong>an</strong>t, known to exhibit aberr<strong>an</strong>t trafficking .<br />
Results: Confocal immunofluorescence microscopy showed Mut-PrP incompletely colocalized<br />
with Golgi markers, did not label <strong>the</strong> plasma membr<strong>an</strong>e (PM), but it did accumulate within ER<br />
<strong>an</strong>d was detected in lysosomes. GFP-tagged Mut-PrP signific<strong>an</strong>tly colocalized with LysoTracker<br />
Red in live cells, <strong>an</strong>d lysosomal inhibitors increased steady state levels of Mut <strong>an</strong>d wid-type<br />
(WT) PrP. This persistent co-localization of Mut-PrP with lysosomes in <strong>the</strong> absence of PM<br />
localization suggested bulk intracellular tr<strong>an</strong>sport, such as autophagy. In support of this, EM<br />
<strong>an</strong>alysis revealed <strong>an</strong> accumulation of autophagic structures in Mut-PrP expressing cells. Mut-PrP<br />
also colocalized signific<strong>an</strong>tly with <strong>the</strong> autophagy marker LC3. Compared with stably expressing<br />
WT PrPells, those stably expressing Mut-PrP displayed signific<strong>an</strong>tly higher levels of LC3<br />
(Atg8), beclin (Atg6), LAMP-1, phospho-mTOR, <strong>an</strong>d a higher LC3II/LC3I ratio, all classic<br />
hallmarks of autophagy induction. Inhibition of autophagy by 3-MAproduced a concentrationdependent<br />
increase in <strong>the</strong> level of Mut-PrP, whereas activation of autophagy with rapamycin,<br />
reduced <strong>the</strong> levels. In addition, cellular fractionation studies show that treatment with 3-MA<br />
shifted Mut, but not WT, PrP <strong>from</strong> <strong>the</strong> lysosomal fractions into heavier fractions, which were<br />
enriched withER markers, suggesting ER retention of aggregated PrP. Finally, delivery of GFPtagged<br />
Mut-PrP to Lysotracker positive compartments was signific<strong>an</strong>tly reduced in <strong>an</strong> autophagy<br />
deficient (ATG5-/-) cell line.<br />
Discussion: These results suggest a role <strong>for</strong> autophagy in <strong>the</strong> intracellular tr<strong>an</strong>sport of misfolded<br />
PrP to lysosomes. Based on recent evidence that ER stress c<strong>an</strong> trigger autophagy, we hypo<strong>the</strong>size<br />
that ER accumulation of misfolded/aggregated Mut-PrP stimulates autophagy, leading to its<br />
packaging <strong>an</strong>d tr<strong>an</strong>sport to lysosomes <strong>for</strong> elimination.<br />
Disclosures: C.J. Cortes, None; J.A. Mastri<strong>an</strong>ni, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 534.10/M17<br />
Topic: C.04.i. Prion diseases<br />
Support: GIS ‘Infections a prions’ Gr<strong>an</strong>t F127<br />
Europe<strong>an</strong> Commission FP6 programme Gr<strong>an</strong>t Food-CT-2004-506579 (Network of<br />
Excellence ‘NeuroPrion’)<br />
Title: Loss of cerebellar gr<strong>an</strong>ule neurons <strong>an</strong>d prion protein (PrP) deposition pattern in sporadic<br />
Creutzfeldt-Jakob disease. Investigation of <strong>the</strong> three PrP codon 129 genotypes<br />
Authors: *B. A. FAUCHEUX 1 , J.-P. BRANDEL 2 , E. MORAIN 3 , V. DIOURON 3 , D.<br />
SALOMON 2 , V. SAZDOVITCH 3 , N. PRIVAT 3 , J.-L. LAPLANCHE 4 , J.-J. HAUW 5 , S. HAIK 5 ;<br />
1 AP-HP, Lab. De Neuropathologie, CRICM, INSERM Umrs975, Paris, Fr<strong>an</strong>ce; 2 AP-HP,<br />
CRICM, INSERM Umrs975, Paris, Fr<strong>an</strong>ce; 3 AP-HP, Lab. Neuropathol, CRICM, INSERM<br />
Umrs975, Paris, Fr<strong>an</strong>ce; 4 AP-HP, Biochem. <strong>an</strong>d Molec Biol Dept, Lariboisiere Hosp., Paris,<br />
Fr<strong>an</strong>ce; 5 AP-HP, Lab. Neuropathol, CRICM, INSERM Umrs975, Salpetriere Hosp., Paris,<br />
Fr<strong>an</strong>ce<br />
Abstract: Prion diseases are rare <strong>an</strong>d fatal neurodegenerative disorders also called tr<strong>an</strong>smissible<br />
spongi<strong>for</strong>m encephalopathies. They c<strong>an</strong> occur as sporadic, genetic or infectious.The hum<strong>an</strong> prion<br />
diseases include Creutzfeldt-Jakob disease (CJD), Gerstm<strong>an</strong>n-Sträussler-Scheinker (GSS)<br />
syndrome, <strong>an</strong>d fatal familial insomnia (FFI). Symptoms <strong>an</strong>d neuropathology vary between <strong>the</strong><br />
diseases, with patients afflicted with sporadic CJD developing dementia <strong>an</strong>d focal neurological<br />
signs. The diversity of sCJD clinical symptoms has been related to <strong>the</strong> protease resist<strong>an</strong>t<br />
iso<strong>for</strong>ms of PrP (type 1 <strong>an</strong>d 2 prions) <strong>an</strong>d <strong>the</strong> Met/Val polymorphism at codon 129 of <strong>the</strong> prion<br />
protein gene on chromosome 20. Neuronal death is a central component of <strong>the</strong> neuropathology<br />
but <strong>the</strong> mech<strong>an</strong>isms involved in neurodegeneration are still poorly understood. The toxicity of<br />
disease-related prion protein (PrPSc) has been proposed to play a central role. However, whe<strong>the</strong>r<br />
neuronal loss correlates with PrP deposition remains <strong>an</strong> open question. We investigated neuronal<br />
loss in <strong>the</strong> gr<strong>an</strong>ule layer of <strong>the</strong> cerebellum which is associated with ataxia <strong>an</strong>d examined <strong>the</strong><br />
statistical relationships with <strong>the</strong> pattern of PrP accumulation in 100 sCJD cases (35 Met/Met 129<br />
homozygous; 34 Val/Val 129 homozygous; 31 Met/Val 129 heterozygous) <strong>an</strong>d 31 control cases.<br />
Neuronal loss <strong>an</strong>d <strong>the</strong> density of focal (5- to 50-µm wide) PrP deposits were estimated with a<br />
computer-assisted image <strong>an</strong>alysis system. Precise qu<strong>an</strong>tifications of neurons <strong>an</strong>d scores of<br />
abnormal PrP accumulation showed correlations between neuronal loss <strong>an</strong>d <strong>the</strong> type of abnormal<br />
PrP deposition. Pathological PrP deposits of <strong>the</strong> punctate type (“synaptic type”) were associated<br />
with neuronal loss. Fur<strong>the</strong>rmore, our data show that <strong>the</strong> number of large deposits were not<br />
associated with neuronal loss in sCJD-VV <strong>an</strong>d sCJD-MV cases with occurrence of PrPSc type 2,<br />
<strong>an</strong>d unexpectedly <strong>the</strong>re was a trend <strong>for</strong> a better neuronal survival when focal deposits were more<br />
numerous. Our results support <strong>the</strong> hypo<strong>the</strong>sis of putative pathogenic roles <strong>for</strong> disease-related PrP<br />
through neurotoxic mech<strong>an</strong>isms altering cellular homeostasis <strong>an</strong>d ending in neurodegeneration<br />
within <strong>the</strong> hum<strong>an</strong> brain. These findings are in accord<strong>an</strong>ce with <strong>the</strong> view that punctate deposits<br />
may point out <strong>the</strong> <strong>an</strong>atomical location of neuronal death process <strong>an</strong>d suggest, at <strong>the</strong> histological<br />
level, that large aggregates may have a protective role against <strong>the</strong> neurotoxic process.
Disclosures: B.A. Faucheux, None; J. Br<strong>an</strong>del, None; E. Morain, None; V. Diouron,<br />
None; D. Salomon, None; V. Sazdovitch, None; N. Privat, None; J. Lapl<strong>an</strong>che, None; J.<br />
Hauw, None; S. Haik, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.11/M18<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: Fondecyt Nº 108 0396<br />
Title: Longitudinal studies of Tax <strong>an</strong>d Foxp3+ in regulatory T-cells in patients with spastic<br />
paraparesis linked to HTLV-I<br />
Authors: *J. F. PUENTE 1 , E. RAMIREZ 2 , L. CARTIER 3 , Y. TANAKA 4 , Y. TANAKA 5 ;<br />
1 Biochem. <strong>an</strong>d Mol. Biol., Univ. Chile, Fac Chem. & Pharmaceut. Sci., S<strong>an</strong>tiago, Chile;<br />
2 Program of Virology, Fac. of Medicine; Virology, ISP, 3 Neurolog. Sciences, Fac. of Med.,<br />
Univ. of Chile, S<strong>an</strong>tiago, Chile; 4 Div. of Immunol., 5 Div. of Immunology; Fac. of Med., Univ. of<br />
Ryukyus, Nishihara, Jap<strong>an</strong><br />
Abstract: HTLV-I virus is <strong>the</strong> ethiological agent of <strong>the</strong> chronic myelopathy known as tropical<br />
spastic paraparesis (HAM/TSP). The virus inserts in T lymphocytes, mainly in <strong>the</strong> regulatory<br />
ones (Treg), but not infect neurons. This neurologic disease is characterized by <strong>the</strong> degeneration<br />
of <strong>the</strong> long axons of <strong>the</strong> corticospinal tracts. The viral protein Tax seems to be <strong>the</strong> main viral<br />
component associated with <strong>the</strong> progression of <strong>the</strong> neurological disease, however <strong>the</strong> mech<strong>an</strong>ism<br />
is still not known. Some evidences point to that alteration in immunoregulation could play <strong>an</strong><br />
import<strong>an</strong>t role in <strong>the</strong> disease development due to <strong>an</strong> imbal<strong>an</strong>ce in Treg levels associated with <strong>the</strong><br />
presence of <strong>the</strong> Tax protein. The lack of longitudinal studies makes difficult to find a relation<br />
between Tax in Treg in <strong>the</strong> same HAM/TSP patient. There<strong>for</strong>e, we have initiated a longitudinal<br />
study working with PBMC (peripheral blood mononuclear cells) of HAM/TSP patients (who<br />
signed <strong>the</strong> in<strong>for</strong>med consent according to <strong>the</strong> protocol of work approved by <strong>the</strong> Ethical<br />
Institutional Committee) to evaluate <strong>an</strong>d correlate ch<strong>an</strong>ges in Tax, both at mRNA <strong>an</strong>d protein<br />
levels, with Foxp3+ also at levels of mRNA <strong>an</strong>d protein. We additionally identified <strong>the</strong> presence<br />
of Tax in Treg cells (CD4+CD25+ high ) isolated by immunomagnetic methods. Levels of mRNA<br />
were followed by real time PCR, <strong>an</strong>d those of protein by flow cytometry using fluorescent<br />
markers <strong>for</strong> CD4+, Treg (Foxp3+) <strong>an</strong>d Tax. The results indicate <strong>an</strong> inverse relation between Tax<br />
<strong>an</strong>d Foxp3+ levels, both at mRNA <strong>an</strong>d protein levels. Treg cells have <strong>the</strong> capacity of suppressing
<strong>the</strong> immune response mediated by T-effector cells, reducing inflammatory processes. This<br />
immunosupression is produced by <strong>the</strong> tr<strong>an</strong>scriptional factor Foxp3+, <strong>the</strong> reduction of <strong>the</strong> levels<br />
of this protein in HAM/TSP indicates that Treg cells would have loss its immunoregulatory<br />
function.<br />
Disclosures: J.F. Puente, None; E. Ramirez, None; L. Cartier, None; Y. T<strong>an</strong>aka, None; Y.<br />
T<strong>an</strong>aka, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.12/M19<br />
Topic: C.04.i. Prion diseases<br />
Support: FAPESP<br />
HHMI<br />
Title: Control of protein syn<strong>the</strong>sis by cellular <strong>an</strong>d scrapie prion<br />
Authors: *G. HAJJ 1 , M. ROFFE 2 , F. H. BERALDO 3 , R. ROST 4 , M. NUNZIANTE 4 , C.<br />
BACH 4 , G. MANCINI 1 , I. VORBERG 4 , B. CASTILHO 2 , V. MARTINS 1 ;<br />
1 Cell. & Mol. Biol Lab., Ludwig Inst. C<strong>an</strong>cer Res., Sao Paulo, Brazil; 2 Univ. Federal de São<br />
Paulo, São Paulo, Brazil; 3 Robarts Res. Insitute, London, ON, C<strong>an</strong>ada; 4 Tech. Univ. of Munich,<br />
Munich, Germ<strong>an</strong>y<br />
Abstract: Tr<strong>an</strong>smissible spongi<strong>for</strong>m encephalopathies (TSE) are fatal neurodegenerative<br />
diseases caused by <strong>the</strong> abnormal folding of <strong>the</strong> cellular prion (PrP C ) into its abnormal iso<strong>for</strong>m<br />
(PrP Sc ). PrP C has m<strong>an</strong>y described cellular functions, among <strong>the</strong>m <strong>the</strong> control of apoptosis, cell<br />
proliferation <strong>an</strong>d differentiation. The Stress Inducible Protein 1 (STI1) is a co-chaperone that<br />
interacts with Hsp70/90 that is secreted by astrocytes <strong>an</strong>d tumor cells. Interestingly, STI1<br />
interacts with PrP C promoting neuritogenesis <strong>an</strong>d neuroprotection, suggesting that STI1 may act<br />
as new neurotrophic factor. In this work we describe that <strong>the</strong> interaction of PrP C with<br />
STI1stimulates protein syn<strong>the</strong>sis in primary cultured hippocampal neurons in a PI3K-Akt-mTOR<br />
<strong>an</strong>d ERK-MNK pathways dependent m<strong>an</strong>ner. This STI1 effect is specific of its PrPc interaction<br />
since no signaling was observed in PrP C knockout neurons. STI1 deleted <strong>for</strong> <strong>the</strong> PrP c binding site<br />
on STI1 also has no effect. Import<strong>an</strong>tly, neuritogenesis induced by STI1 is dependent on <strong>the</strong><br />
activation of both signaling cascades, whereas neuroprotection mediated by STI1 requires only
mTOR activation. Since STI1 has import<strong>an</strong>t effects in normal neuronal cells, we decided to<br />
investigate its effects in Scrapie infected cells. In a neuronal cell line (N2a) persistently infected<br />
with prions <strong>the</strong> addition of STI1 was able to increase protein syn<strong>the</strong>sis, but not to <strong>the</strong> levels<br />
observed in non-infected cells. The same effect was also observed in primary hippocampal<br />
cultures acutely infected with PrP Sc . We found that prion infection increased <strong>the</strong> phosphorylation<br />
of eIF2α, which is a known rate limiting step <strong>for</strong> tr<strong>an</strong>slation initiation <strong>an</strong>d that this effect is at<br />
least mediated by <strong>the</strong> activation of PKR, one of <strong>the</strong> four known mammali<strong>an</strong> eIF2α kinases. In<br />
conclusion, we demonstrated that STI1 induces protein syn<strong>the</strong>sis through its interaction with<br />
PrP C in non-pathological conditions, suggesting a neurotrophic role <strong>for</strong> this interaction.<br />
Import<strong>an</strong>tly, STI1 effects are abrogated by prion infection, which could be <strong>an</strong> import<strong>an</strong>t cause of<br />
<strong>the</strong> pathology, explaining alterations such as dementia.<br />
Disclosures: G. Hajj, None; M. Roffe, None; F.H. Beraldo, None; R. Rost, None; M.<br />
Nunzi<strong>an</strong>te, None; C. Bach, None; G. M<strong>an</strong>cini, None; I. Vorberg, None; B. Castilho, None; V.<br />
Martins, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.13/M20<br />
Topic: C.11.p. Neuroinflammation: HIV <strong>an</strong>d infections<br />
Support: NIH Gr<strong>an</strong>t NS064872-01 to ANC<br />
Title: Neuropathology induced by HIV-Tat protein associated with lasting impairment in<br />
learning <strong>an</strong>d memory<br />
Authors: *A. N. CAREY 1 , E. I. SYPEK 1 , J. P. MCLAUGHLIN 1,2 ;<br />
1 2<br />
Psychology, Nor<strong>the</strong>astern Univ., Boston, MA; Torrey Pines Inst. <strong>for</strong> Mol. Studies, Port St.<br />
Lucie, FL<br />
Abstract: Recent studies suggest that HIV-dementia may be attributable to <strong>the</strong> neurotoxic <strong>an</strong>d<br />
neuromodulatory actions of <strong>the</strong> HIV-accessory protein, Tat. We hypo<strong>the</strong>size that<br />
neurodegeneration <strong>an</strong>d neuronal dysfunction arising <strong>from</strong> Tat expression alone is sufficient to<br />
mediate behavioral disorders associated with HIV infection in <strong>the</strong> brain. Using <strong>the</strong> GT-tg bigenic<br />
mouse with brain-selective expression of Tat protein induced with doxycycline (Dox) treatments,<br />
we evaluated <strong>the</strong> effect of Tat in behavioral assays of learning <strong>an</strong>d memory per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d<br />
histology to characterize neurodegeneration. Uninduced GT-tg <strong>an</strong>d Dox-treated C57Bl/6 mice
spent more time exploring a novel object ra<strong>the</strong>r th<strong>an</strong> a familiar object in <strong>the</strong> test trial of a novel<br />
object recognition (NOR) assay. In contrast, mice expressing Tat did not spend more time<br />
exploring <strong>the</strong> novel object, suggesting a deficit in novel object recognition. <strong>When</strong> mice were<br />
induced to express Tat <strong>for</strong> 3, 5, <strong>an</strong>d 7 days (100 mg/kg Dox, i.p.), <strong>the</strong> mice displayed signific<strong>an</strong>t<br />
deficits in NOR when tested within 48 hours after induction. Also, mice induced <strong>for</strong> 7 days with<br />
50 <strong>an</strong>d 100, but not 25 mg/kg Dox also demonstrated deficits in learning <strong>an</strong>d memory when<br />
tested within 48 hours of induction. <strong>When</strong> separate groups of mice were induced <strong>an</strong>d tested one<br />
week later, mice induced with 100, 50, <strong>an</strong>d 25 mg/kg DOX demonstrated deficits in learning <strong>an</strong>d<br />
memory. These data suggest that <strong>the</strong> Tat-induced deficit in NOR <strong>an</strong>d learning <strong>an</strong>d memory<br />
per<strong>for</strong>m<strong>an</strong>ce were evident beyond <strong>the</strong> period when Tat was actively being produced. Moreover,<br />
<strong>the</strong>se data suggest that with smaller qu<strong>an</strong>tities of Tat expression, behavioral deficits that may not<br />
be immediately apparent c<strong>an</strong> develop over time. The effects of Tat protein expression on spatial<br />
learning <strong>an</strong>d memory will also be discussed. Fur<strong>the</strong>rmore, brains of Tat induced mice were<br />
examined with histology to confirm <strong>the</strong> severity <strong>an</strong>d type of degeneration associated with <strong>the</strong>se<br />
behavioral deficits. Preliminary findings detailing cell death <strong>an</strong>d degeneration, reactive<br />
astrocytes, microglial activation, <strong>an</strong>d oxidative stress during examination of <strong>the</strong> CA1 <strong>an</strong>d CA3<br />
regions of <strong>the</strong> hippocampus <strong>an</strong>d perirhinal cortex will be discussed. Given <strong>the</strong> concerns over <strong>the</strong><br />
effectiveness of prolonged <strong>an</strong>ti-retroviral <strong>the</strong>rapies at preventing brain dysfunction, research<br />
along <strong>the</strong>se lines is intended to identify targets <strong>for</strong> <strong>the</strong> development of novel NeuroAIDS<br />
treatment <strong>the</strong>rapies.<br />
Disclosures: A.N. Carey, None; E.I. Sypek, None; J.P. McLaughlin, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.14/M21<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: HHS gr<strong>an</strong>ts: DA026174 (IM)<br />
1U01MH083545 (NNTC)<br />
UO1-AI-35004 (WISH)<br />
Title: Brain-derived neurotrophic factor levels are reduced in <strong>the</strong> brain <strong>an</strong>d blood of HIV-1<br />
positive subjects
Authors: *A. BACHIS 1 , V. AVDOSHINA 1 , C. LIU 2 , M. A. YOUNG 2 , I. MOCCHETTI 1 ;<br />
1 Neurosci., 2 Med., Georgetown Univ., Washington, DC<br />
Abstract: Progressive <strong>an</strong>d irreversible loss of specific neuronal cell populations of <strong>the</strong> central<br />
nervous system (CNS) is commonly seen in individuals with Hum<strong>an</strong> Immunodeficiency Virus-1<br />
(HIV) associated dementia (HAD). Evidence is accumulating that apoptosis is a crucial cellular<br />
event responsible <strong>for</strong> <strong>the</strong> dysfunction <strong>an</strong>d death of neurons in this disease. However, despite<br />
numerous adv<strong>an</strong>ces in our underst<strong>an</strong>ding of HAD, <strong>the</strong> underlying causes of neurological<br />
symptoms <strong>an</strong>d pathological alterations observed in <strong>the</strong>se individuals are poorly understood. The<br />
HIV-1 envelope protein gp120 has been shown to decrease neurotrophic support in <strong>the</strong> rat brain.<br />
Indeed, it reduces <strong>the</strong> levels of brain-derived neurotrophic factor (BDNF) in <strong>the</strong> striatum <strong>an</strong>d<br />
glial-cell derived neurotrophic factor GDNF in <strong>the</strong> subst<strong>an</strong>tia nigra (SN). We <strong>the</strong>re<strong>for</strong>e tested <strong>the</strong><br />
hypo<strong>the</strong>sis that HIV-1 infection lowers <strong>the</strong> levels of BDNF <strong>an</strong>d GDNF in hum<strong>an</strong> subjects. BDNF<br />
<strong>an</strong>d GDNF levels were measured by ELISA in five different brain areas of HAD subjects <strong>an</strong>d<br />
controls (HIV positive with no dementia) <strong>from</strong> <strong>the</strong> National NeuroAIDS Tissue Consortium<br />
(NNTC). We found lower levels of BDNF <strong>an</strong>d GDNF in <strong>the</strong> striatum <strong>an</strong>d SN, respectively in<br />
HAD subjects when compared to controls. Our results indicate that HIV causes a widespread<br />
reduction in neurotrophic support, which could contribute to <strong>the</strong> neuronal loss observed in HAD.<br />
To provide in<strong>for</strong>mation as to whe<strong>the</strong>r BDNF c<strong>an</strong> be used as a biomarker <strong>for</strong> HAD, we have<br />
examined BDNF levels in <strong>the</strong> blood of 174 cases of women with or at risk of HIV infection<br />
enrolled in <strong>the</strong> Women’s Interagency HIV Study (WIHS). Serum BDNF levels were decreased in<br />
HIV positive individuals, fur<strong>the</strong>r suggesting that HIV infection lowers <strong>the</strong> levels of BDNF.<br />
Disclosures: A. Bachis, None; V. Avdoshina, None; C. Liu, None; M.A. Young, None; I.<br />
Mocchetti, None.<br />
Poster<br />
534. Neurodegeneration: Tr<strong>an</strong>smissable <strong>an</strong>d Infectious Forms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 534.15/M22<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: NIH Gr<strong>an</strong>t DA013137<br />
NIH Gr<strong>an</strong>t HD043680<br />
Title: Assessing biomarkers of dopamine availability in HIV-1 tr<strong>an</strong>sgenic rats, a model of HIV<br />
pediatric encephalopathy
Authors: *K. M. WEBB 1 , M. AKSENOV 2 , C. F. MACTUTUS 2 , R. M. BOOZE 2 ;<br />
1 Columbia, SC; 2 Univ. of South Carolina, Columbia, SC<br />
Abstract: Highly Active AntiRetroviral Therapy (HAART) has signific<strong>an</strong>tly extended longevity<br />
<strong>for</strong> HIV-infected children. However, neurologic impairments associated with HIV infection,<br />
referred to as pediatric encephalopathy, c<strong>an</strong> develop despite HAART <strong>an</strong>d may have a subst<strong>an</strong>tial<br />
impact on quality of life. The neurologic ch<strong>an</strong>ges associated with pediatric encephalopathy may<br />
include difficulties with expressive l<strong>an</strong>guage, motor function <strong>an</strong>d attention/focus abilities, each<br />
of which is signific<strong>an</strong>tly modulated by dopamine neurotr<strong>an</strong>smission. In this project we have<br />
evaluated <strong>an</strong> <strong>an</strong>imal model, <strong>the</strong> HIV-1 tr<strong>an</strong>sgenic rat, <strong>for</strong> basic biomarkers indicative of<br />
dopamine availability including tyrosine hydroxylase (TH). We isolated total protein <strong>an</strong>d RNA<br />
<strong>from</strong> <strong>the</strong> midbrain of 3 male HIV tr<strong>an</strong>sgenic rats at 21 days old <strong>for</strong> western blotting <strong>an</strong>d Real-<br />
Time PCR. Thus far our findings indicate <strong>an</strong> increase in total <strong>an</strong>d phosphorylated TH protein<br />
expression with no ch<strong>an</strong>ge in TH mRNA levels. Initial results examining dopamine tr<strong>an</strong>sporter<br />
(DAT) mRNA levels suggest that <strong>the</strong>re is less DAT message in HIV tr<strong>an</strong>sgenic <strong>an</strong>imals. These<br />
findings may suggest a decrease in dopamine (DA) availability via a decrease in catecholamine<br />
syn<strong>the</strong>sis, <strong>an</strong>d <strong>the</strong> increase we have observed in total <strong>an</strong>d phosphorylated TH may be a<br />
compensatory mech<strong>an</strong>ism <strong>for</strong> maintaining DA availability. The HIV tr<strong>an</strong>sgenic rats possess a<br />
viral plasmid that does not contain <strong>the</strong> genes Gag or Pol, so <strong>the</strong>se <strong>an</strong>imals may be considered a<br />
model of HIV infection with <strong>an</strong>tiretroviral <strong>the</strong>rapy as <strong>the</strong> products of Gag <strong>an</strong>d Pol are critical <strong>for</strong><br />
virion production. These findings present interesting implications <strong>for</strong> <strong>the</strong> possible dopaminergic<br />
alterations that may be affecting neurologic function in HIV-infected children on <strong>an</strong>tiretroviral<br />
<strong>the</strong>rapy.<br />
Disclosures: K.M. Webb, None; M. Aksenov, None; C.F. Mactutus, None; R.M. Booze,<br />
None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.1/M23<br />
Topic: C.05.a. Molecular studies<br />
Support: RR-000163<br />
Title: Effects of age <strong>an</strong>d gender in gene expression in <strong>the</strong> rhesus hippocampus
Authors: *S. G. KOHAMA 1 , S. G. KRYGER 1 , K. SORWELL 1 , B. PARK 2 , V.<br />
GARYFALLOU 1 , K. SONMEZ 2 , H. F. URBANSKI 1 ;<br />
1 Oregon Natl. Primate Res. Ctr., Beaverton, OR; 2 Oregon Hlth. <strong>an</strong>d Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: The hippocampus has been shown to play a vital role in aspects of learning <strong>an</strong>d<br />
memory, as well as in spatial navigation. Because of <strong>the</strong> clinical relev<strong>an</strong>ce of potential sex <strong>an</strong>d<br />
aging-regulated effects in this region, we have conducted a cross-sectional aging study of <strong>the</strong><br />
hippocampus <strong>from</strong> male <strong>an</strong>d female rhesus macaques. In order to get a comprehensive view of<br />
potential ch<strong>an</strong>ges, we used high-throughput microarray technology to globally examine gene<br />
regulation. RNA was extracted <strong>from</strong> <strong>the</strong> hippocampus of adult (6-11 yrs), middle-aged (15-20<br />
yrs) <strong>an</strong>d old (24-31 yrs) male <strong>an</strong>d female rhesus monkeys; n=8/age group. After RNA<br />
qu<strong>an</strong>tification <strong>an</strong>d quality assessment, samples were processed <strong>for</strong> <strong>an</strong>alysis on rhesus-specific,<br />
Affymetrix GeneChips. Each hippocampal sample was run on its own individual chip <strong>an</strong>d preprocessing<br />
<strong>an</strong>d normalization of <strong>the</strong> data was per<strong>for</strong>med using <strong>the</strong> Robust Multi-Chip Analysis<br />
(RMA), <strong>for</strong> low level <strong>an</strong>alysis of <strong>the</strong> study. Expression levels of all tr<strong>an</strong>scripts were subjected to<br />
a two-way ANOVA, to look <strong>for</strong> signific<strong>an</strong>t (p
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: GlaxoSmithKline (UK) <strong>an</strong>d <strong>the</strong> Industrial Development Authority (Irel<strong>an</strong>d)<br />
Health Research Board (Irel<strong>an</strong>d)<br />
Wellcome Trust<br />
Title: Acute <strong>an</strong>d chronic rosiglitazone administration enh<strong>an</strong>ce hippocampal-associated learning,<br />
synaptic plasticity <strong>an</strong>d place cell activity in middle-aged rats<br />
Authors: *V. HOK 1 , B. WANG 1 , A. DELLA CHIESA 1 , C. K. CALLAGHAN 1 , S. BARLOW 1 ,<br />
M. TSANOV 1 , R. BECHARA 1 , E. A. IRVING 2 , D. J. VIRLEY 2 , N. UPTON 2 , S. M. O'MARA 1 ;<br />
1 2<br />
Trinity Col. Dublin, Dublin, Irel<strong>an</strong>d; Neurosciences CEDD, GlaxoSmithKline, Harlow, Essex,<br />
United Kingdom<br />
Abstract: Currently two thiazolidinediones, Rosiglitazone (ROSI) <strong>an</strong>d Pioglitazone, are U.S.<br />
Food <strong>an</strong>d Drug Administration-approved <strong>for</strong> treatment of type 2 diabetes. As <strong>an</strong> <strong>an</strong>ti-diabetic<br />
agent, ROSI binds <strong>an</strong>d activates peroxisome proliferator-activator receptor gamma (PPAR-g),<br />
<strong>an</strong>d alters <strong>the</strong> expression of genes involved in glucose uptake <strong>an</strong>d disposal, as well as lipid<br />
metabolism. Recent clinical studies suggest that PPAR-g agonists may offer a new avenue of<br />
treatment <strong>for</strong> Alzheimer's Disease, as insulin abnormalities may promote pathophysiological<br />
processes observed in patients with neurodegenerative disorders, <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e underlie<br />
disruptions of normal memory functioning.<br />
Here, we investigate <strong>the</strong> <strong>the</strong>rapeutic potential of ROSI <strong>for</strong> cognitive deficits evident in middleaged<br />
(MA) rats in which a pattern of reliable age-related neurocognitive decline may be detected.<br />
We have found that acute ROSI treatment (one dose; 3mg/kg; p.o.) enh<strong>an</strong>ces initial acquisition in<br />
<strong>the</strong> water-plus maze (a modified version of <strong>the</strong> Morris water maze task), relative to untreated<br />
controls; fur<strong>the</strong>rmore, we have also found that chronic ROSI treatment enh<strong>an</strong>ces subsequent<br />
per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> same task.<br />
In a separate study, we found that <strong>the</strong> ratio of population spike amplitude over fEPSP slope of<br />
ROSI-aged group reached <strong>the</strong> level of young <strong>an</strong>imals, whereas <strong>the</strong> same parameter was<br />
signific<strong>an</strong>tly lower <strong>for</strong> <strong>the</strong> control-aged group. Our data suggest that acute ROSI administration<br />
potentiates <strong>the</strong> probability of fEPSPs to cause firing of action potentials in aged <strong>an</strong>imals.<br />
Finally, we have found that <strong>the</strong> localized firing pattern of place cells in hippocampal area CA1 is<br />
signific<strong>an</strong>tly improved by chronic ROSI administration. Place fields spatial coherence <strong>an</strong>d<br />
in<strong>for</strong>mation density increased while <strong>the</strong> average firing activity remained unch<strong>an</strong>ged, suggesting<br />
that hippocampal place cells modulations were not due to a non-specific ch<strong>an</strong>ge in <strong>the</strong> basic<br />
physiological properties.<br />
Overall, <strong>the</strong>se results imply that, in a preclinical model at least, both acute <strong>an</strong>d chronic treatment<br />
with ROSI c<strong>an</strong> reverse age-related deficits in neurocognitive function. ROSI may <strong>the</strong>re<strong>for</strong>e offer<br />
a treatment to reverse or halt age-related decline in cognitive function.<br />
Disclosures: V. Hok, None; B. W<strong>an</strong>g, None; A. Della Chiesa, None; C.K. Callagh<strong>an</strong>,<br />
None; S. Barlow, None; M. Ts<strong>an</strong>ov, None; R. Bechara, None; E.A. Irving, None; D.J. Virley,<br />
None; N. Upton, None; S.M. O'Mara, None.
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.3/M25<br />
Topic: C.05.c. Oxidative stress<br />
Support: NIH-AG028072<br />
NIH-RR00163<br />
Title: Mitochondrial DNA deletions <strong>an</strong>d differential mitochondrial DNA content in rhesus<br />
monkeys: Implications <strong>for</strong> aging<br />
Authors: *P. MAO, P. GALLAGHER, S. NEDUNGADI, S. G. KOHAMA, B. FERGUSON, P.<br />
H. REDDY;<br />
Oregon Natl. Primate Res. Ctr., Oregon Hlth. & Sci. Univ., Beaverton, OR<br />
Abstract: BACKGROUND<br />
The mitochondrial <strong>the</strong>ory of aging proposes that defects in mitochondrial DNA (mtDNA) result<br />
in increased oxidative damage, decreased cellular energy, cell death, <strong>an</strong>d senescence. The<br />
purpose of this study was to determine <strong>the</strong> relationship between mtDNA deletions/mtDNA<br />
content <strong>an</strong>d <strong>the</strong> effects of aging in <strong>the</strong> rhesus macaques.<br />
METHODS<br />
Using rhesus macaques as a model org<strong>an</strong>ism, a protocol was established to assess <strong>the</strong><br />
relationship among mtDNA deletions, mtDNA content, <strong>an</strong>d aging. To assess <strong>the</strong> mtDNA<br />
deletions <strong>an</strong>d content, we used 2 sets of oligonucleotide primers <strong>an</strong>d amplified <strong>the</strong> rhesus<br />
macaque mitochondrial genome: set 1, 8 kb DNA covering a common 5 Kb deletion; <strong>an</strong>d set 2,<br />
<strong>the</strong> entire 16.5 kb. We studied a total of 66 DNA samples: 39 were prepared <strong>from</strong> a peripheral<br />
buffy-coat <strong>an</strong>d 27 were prepared <strong>from</strong> occipital cortex tissues <strong>from</strong> <strong>the</strong> brains of young (5 to 12<br />
years) <strong>an</strong>d aged (13 to 31 years) rhesus monkeys. mtDNA content <strong>an</strong>d mtDNA deletions were<br />
evaluated by visualizing PCR products, <strong>an</strong>d <strong>the</strong> densitometry <strong>an</strong>alysis of PCR products. The<br />
mtDNA data were assessed using a permutation test to identify differences in mDNA of different<br />
monkey groups (e.g., young <strong>an</strong>d aged, male <strong>an</strong>d female). A permutation test provided a nonparametric<br />
comparison groups.<br />
RESULTS<br />
We found 5 Kb mtDNA deletions in all 27 (100%) samples prepared <strong>from</strong> <strong>the</strong> occipital cortex of<br />
young <strong>an</strong>d aged monkeys, <strong>an</strong>d 5 Kb mtDNA deletions in 17 of 39 (43.6%) samples <strong>from</strong> <strong>the</strong><br />
buffy-coat DNA in young <strong>an</strong>d aged monkeys. In <strong>the</strong> younger monkey group, we found 5 Kb
deletions in 7 of 17 (41%), <strong>an</strong>d in <strong>the</strong> aged group, we found 5 kb deletions in 12 of 22 (54%),<br />
suggesting that mtDNA deletions were increased with age. Our combined densitometry <strong>an</strong>alysis<br />
of mtDNA content (brain DNA+buffy-coat DNA) revealed a signific<strong>an</strong>t increase in mtDNA in<br />
aged monkeys (P
Abstract: Oxidative stress is associated with <strong>the</strong> aging process, a risk factor <strong>for</strong><br />
neurodegenerative diseases, <strong>an</strong>d decreased by reduced energy intake. Oxidative modifications to<br />
cells c<strong>an</strong> affect protein function, <strong>an</strong>d <strong>the</strong> sulfur-containing amino acid residues, including<br />
methionine, are particularly susceptible to oxidation. A methionine sulfoxide (MetO) c<strong>an</strong> be<br />
enzymatically reduced by <strong>the</strong> methionine sulfoxide reductase (Msr) system. The Msr system is<br />
comprised of MsrA <strong>an</strong>d MsrB enzymes that reduce S-MetO <strong>an</strong>d R-MetO, respectively.<br />
Previously, we have shown MsrA -/- mice exhibit lower locomotor activity <strong>an</strong>d altered brain<br />
dopamine levels as function of age. Previous studies have demonstrated that a caloric restriction<br />
(CR) diet enh<strong>an</strong>ces <strong>an</strong>tioxid<strong>an</strong>t defense <strong>an</strong>d reduces <strong>the</strong> action of reactive oxygen species.<br />
Additionally, dopamine (DA) signaling was shown to increase in rodents undergoing CR. Here<br />
we compare <strong>the</strong> MsrA -/- <strong>an</strong>d control mice (WT) locomotor behavior <strong>an</strong>d DA levels with a CR<br />
diet starting at 8 months of age <strong>an</strong>d ending at 17 months of age. The MsrA -/- mice did not have<br />
<strong>an</strong>y signific<strong>an</strong>t difference in total dist<strong>an</strong>ce traveled when compared to WT at 17 months of age.<br />
In contrast, our previous report showed decreased locomotor activity in <strong>the</strong> MsrA -/- mice at 12<br />
months of age <strong>an</strong>d older when fed ad libitum. After completion of <strong>the</strong> CR diet, DA levels were<br />
comparable to control mice at 12 <strong>an</strong>d 17 months of age. This differs <strong>from</strong> <strong>the</strong> abnormal DA<br />
levels previously observed in MsrA -/- mice fed ad libitum. Thus, <strong>the</strong> CR diet had a neutralization<br />
effect on MsrA ablation, possibly via <strong>an</strong> increase of <strong>an</strong>tioxid<strong>an</strong>t capability. Supportive evidence<br />
<strong>for</strong> <strong>the</strong> involvement of reduced energy intake on reduction of MetO levels is shown by <strong>the</strong><br />
relatively lower levels of MetO in MsrA -/- mice fed a CR diet, which was identified by our novel<br />
<strong>an</strong>ti-MetO <strong>an</strong>tibodies. The MsrA -/- mice have <strong>an</strong> average life-sp<strong>an</strong> of 14 months. In comparison<br />
to MsrA -/- mice that were fed ad libitum, all <strong>the</strong> MsrA -/- mice fed CR diet have survived to age of<br />
17 months, be<strong>for</strong>e <strong>the</strong>y were euth<strong>an</strong>ized. This phenomenon suggests that MsrA -/- mice average<br />
life sp<strong>an</strong> may increase under CR diet. In summary, it is suggested that caloric restriction protects<br />
against <strong>the</strong> accumulation of <strong>the</strong> MetO in proteins.<br />
Disclosures: D.B. Oien, None; G.L. Osterhaus, None; S.C. Fowler, None; J. Moskovitz,<br />
None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.5/M27<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: NIH Gr<strong>an</strong>t R01-NS037527<br />
NIH Gr<strong>an</strong>t R01-AG23686
Keck Foundation<br />
NIH Gr<strong>an</strong>t K02-AA13430<br />
NIH Gr<strong>an</strong>t P01-DK68229<br />
NIH NIH Gr<strong>an</strong>t NCRR/GCRC M01-RR00125<br />
Howard Hughes Medical Institute<br />
Title: Altered brain energy metabolism in healthy aging assessed in vivo by 13C <strong>an</strong>d 1H MRS:<br />
Evidence <strong>for</strong> mitochondrial dysfunction<br />
Authors: F. BOUMEZBEUR 1,2 , G. F. MASON 1 , R. A. DE GRAAF 1 , *K. L. BEHAR 1 , G. W.<br />
CLINE 1 , G. I. SHULMAN 1,3 , D. L. ROTHMAN 1 , K. F. PETERSEN 1 ;<br />
1 Yale Univ. Sch. Med., New Haven, CT; 2 Neurospin, I2BM, CEA, Gif-sur-Yvette, Fr<strong>an</strong>ce;<br />
3 Howard Hughes Med. Inst., New Haven, CT<br />
Abstract: Decline in brain function is characteristic of healthy aging but little is known about<br />
<strong>the</strong> biological basis of <strong>the</strong>se ch<strong>an</strong>ges. To determine <strong>the</strong> nature of <strong>the</strong> alterations in brain<br />
metabolism with healthy aging, we combined 13C Magnetic Reson<strong>an</strong>ce Spectroscopy (MRS)<br />
with infusions of [1-13C]glucose <strong>an</strong>d [2-13C]acetate to characterize qu<strong>an</strong>titatively <strong>the</strong> neuronal<br />
<strong>an</strong>d astroglial TCA cycle rates (VTCAn,VTCAg), as well as <strong>the</strong> glutamate-glutamine cycle rate<br />
(Vcyc) in healthy elderly <strong>an</strong>d young volunteers. 13C MRS data were acquired at 4Tesla <strong>from</strong> a<br />
100 mL volume of occipital-parietal lobe of healthy young (3F/5M, age, 26±7 yrs; Body-Mass<br />
Index (BMI), 23 ± 4 kg/m2) <strong>an</strong>d elderly (2F/5M; age, 76±8 yrs; BMI 24 ± 3 kg/m2) volunteers,<br />
be<strong>for</strong>e <strong>an</strong>d during intravenous infusions of [1-13C]glucose or [2-13C]acetate. Brain metabolite<br />
concentrations were assessed using 1H MRS. 13C <strong>an</strong>d 1H spectra were fitted with a Linear<br />
Combination Model algorithm (LCModel 6.1) with simulated basis sets. A neuron-astrocyte<br />
metabolic model was fitted to <strong>the</strong> combined data sets of 13C labeling time courses <strong>for</strong> glutamate<br />
(Glu)-C4,C3,C2 <strong>an</strong>d glutamine (Gln)-C4,C3,C2 <strong>from</strong> <strong>the</strong> [1-13C]glucose <strong>an</strong>d [2-13C]acetate<br />
infusions yielding values <strong>for</strong> Vcyc, VTCAn <strong>an</strong>d VTCAg. We found that compared with young<br />
subjects, <strong>the</strong> brain of elderly subjects showed decreased neuronal oxidative metabolism<br />
(VTCAn, -28%, p=0.006) <strong>an</strong>d glutamate/glutamine neurotr<strong>an</strong>smitter cycling (Vcyc, -24%),<br />
whereas glial oxidative metabolism was increased (VTCAg, +30%, p=0.005) (me<strong>an</strong>, Bonferroni<br />
test). The aging associated ch<strong>an</strong>ges in neuronal <strong>an</strong>d glial metabolic rates correlated with<br />
decreased levels of <strong>the</strong> neuronal markers, N-acetylaspartate (-10%) <strong>an</strong>d glutamate (-14%), <strong>an</strong>d<br />
increased levels of <strong>the</strong> glial marker, myo-inositol (+25%). Based on <strong>the</strong> absence of reports of<br />
signific<strong>an</strong>t morphological ch<strong>an</strong>ges in <strong>the</strong> measured brain region, our findings indicate that<br />
healthy aging is associated with reduced cortical neuronal mitochondrial energy production <strong>an</strong>d<br />
glutamatergic neurotr<strong>an</strong>smission.<br />
Disclosures: F. Boumezbeur, None; G.F. Mason, None; R.A. de Graaf, None; K.L. Behar,<br />
None; G.W. Cline, None; G.I. Shulm<strong>an</strong>, None; D.L. Rothm<strong>an</strong>, None; K.F. Petersen, None.
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.6/M28<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Title: Ch<strong>an</strong>ges in mast cell distribution over time<br />
Authors: *Q. MACH 1 , R. RAHEMTULLA 2 , K. R. GEORGE 2 , M. A. PERSINGER 2 ;<br />
2 Behavioural Neurosci., 1 Laurenti<strong>an</strong> Univ., Sudbury, ON, C<strong>an</strong>ada<br />
Abstract: Mast cell gr<strong>an</strong>ules are filled with a myriad of bioactive <strong>an</strong>d psychoactive compounds.<br />
Deriving <strong>from</strong> bone marrow stem cells, <strong>the</strong>se cells migrate across <strong>the</strong> org<strong>an</strong>ism, localized<br />
primarily in connective tissue. The distribution of mast cells has only been acutely described <strong>an</strong>d<br />
never in a systematic m<strong>an</strong>ner. Following <strong>the</strong> initial localization <strong>an</strong>d distribution; it has been<br />
postulated that fur<strong>the</strong>r migration c<strong>an</strong> occur, resulting in a distributional ch<strong>an</strong>ge over time<br />
throughout <strong>the</strong> body. Brain mast cells are of critical import<strong>an</strong>ce in development. Using littermates<br />
sacrificed at different ages we counted <strong>an</strong>d assessed degree of degr<strong>an</strong>ulation on a fourpoint<br />
scale, of brain <strong>an</strong>d thyroid mast cells in order to develop <strong>an</strong> underst<strong>an</strong>ding of mast cell<br />
distribution at different developmental states. Counts were fur<strong>the</strong>r divided by leptomenengies<br />
versus parenchyma in <strong>the</strong> brain <strong>an</strong>d by <strong>the</strong> four regions of <strong>the</strong> thyroid. The addition to <strong>the</strong> field of<br />
basic <strong>an</strong>atomy has profound implications <strong>for</strong> <strong>the</strong> underst<strong>an</strong>ding of <strong>the</strong> role of mast cells in <strong>the</strong><br />
neurophysiology of development <strong>an</strong>d aging.<br />
Disclosures: Q. Mach, None; R. Rahemtulla, None; K.R. George, None; M.A. Persinger,<br />
None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.7/M29<br />
Topic: C.05.a. Molecular studies
Support: NIGMS 1SC3GM086323<br />
CTSC GRANT #UL1-RR024996<br />
RR03037 to Hunter College<br />
PSC-CUNY 61853-00 39<br />
Title: Overexpressing prosbeta5 of proteasome in Drosophila nervous system with eGFP U as a<br />
dynamic reporter in vivo<br />
Authors: *W. S. WONG 1,2 , M. SANTOS 1 , M. JANSON 1 , V. VERNANCE 1 , M.<br />
FIGUEIREDO-PEREIRA 1 , T. SCHMIDT-GLENEWINKEL 1 ;<br />
1 Biol. Sci., Hunter Col. - CUNY, New York, NY; 2 Biol., Grad. Ctr. - CUNY, New York, NY<br />
Abstract: M<strong>an</strong>y neurodegenerative disorders such as Alzheimer’s, Parkinson’s <strong>an</strong>d Huntington’s<br />
diseases are characterized by intracellular proteinaceous inclusions composed of ubiquitin<br />
aggregates. If neurons c<strong>an</strong> not remove <strong>the</strong>se inclusions via <strong>the</strong> Ubiquitin Proteasome Pathway<br />
(UPP), <strong>the</strong> protein excess will impede metabolic processes, leading to cellular toxicity. If <strong>the</strong><br />
neuronal impairment continues, eventually induction of cell death occurs via apoptosis,<br />
lysosomal autophagy or necrosis. This is <strong>the</strong> cellular basis <strong>for</strong> <strong>the</strong> neurological pathology of<br />
memory loss, cognitive disability <strong>an</strong>d motor dysfunction. The molecular mech<strong>an</strong>ism, however,<br />
remains unknown. We hypo<strong>the</strong>size that <strong>an</strong> import<strong>an</strong>t risk factor to neurodegeneration is a<br />
reduction of functional protein degradation controlled by <strong>the</strong> UPP.<br />
In order to establish cause-<strong>an</strong>d-effect relationships of UPP with aging <strong>an</strong>d neurodegeneration, we<br />
are establishing a Drosophila model which overexpresses <strong>the</strong> dβ5 subunit of <strong>the</strong> proteasome in a<br />
GAL4-UAS context with <strong>the</strong> ELAV promoter <strong>for</strong> control of spatial expression to <strong>the</strong> central <strong>an</strong>d<br />
peripheral nervous systems. We expect that increased proteasome function will increase<br />
longevity. Fur<strong>the</strong>r, we are establishing a Drosophila model with <strong>an</strong> indirect reporter <strong>for</strong><br />
proteasome activity by modifying <strong>the</strong> pGreen Pelic<strong>an</strong> (gift <strong>from</strong> Dr. Posakony, UCSD) by<br />
attaching <strong>the</strong> CL1 degron (ACKNWFSSLSHFVIHL) to <strong>the</strong> carboxyl-terminal part of enh<strong>an</strong>ced<br />
green fluorescent protein (EGFP) which shortens its half-life, resulting in <strong>an</strong> undetectable EGFP U<br />
within cells with normal proteasome activity.<br />
Disclosures: W.S. Wong, None; M. S<strong>an</strong>tos, None; M. J<strong>an</strong>son, None; V. Vern<strong>an</strong>ce, None; M.<br />
Figueiredo-Pereira, None; T. Schmidt-Glenewinkel, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 535.8/M30<br />
Topic: C.05.c. Oxidative stress<br />
Title: Protein malnourishment induces neurodegenerative ch<strong>an</strong>ges which phenocopy age<br />
associated neurodegeration in <strong>the</strong> motor neurons of Wistar rats<br />
Authors: S. K. GUPTA 1 , *S. PANGULURI 2 ;<br />
1 Zoology, Kurukshetra Univ., Kurukshetra, India; 2 Univ. Louisville, Louisville, KY<br />
Abstract: Protein malnourishment (PM) is one of <strong>the</strong> key health problem in <strong>the</strong><br />
third world countries. Although <strong>the</strong> ill effects of protein<br />
malnourishment on brain development are well documented but not m<strong>an</strong>y<br />
such studies have been done to evaluate <strong>the</strong> effects of PM on neuronal<br />
integrity of <strong>the</strong> young <strong>an</strong>d adults. There<strong>for</strong>e we chronically protein<br />
malnourished three months old male wistar rats <strong>for</strong> three <strong>an</strong>d six<br />
months <strong>an</strong>d found that compared to age matched controls, PM induces two<br />
fold higher accumulation of lipofuscin, which is considered as one of<br />
<strong>the</strong> hallmark of aged neurons (age pigment) in <strong>the</strong> motor neurons. The<br />
increased lipofuceniogenesis was also accomp<strong>an</strong>ied by enh<strong>an</strong>ced lipid<br />
peroxidative damage neurodegenerative ch<strong>an</strong>ges in <strong>the</strong> PM <strong>an</strong>imals. The<br />
PM group rats also showed signific<strong>an</strong>t increased oxygen consumption<br />
levels. These observations clearly indicate that protein malnourishment<br />
probably accelerated lipofuscin <strong>for</strong>mation by making cell more prone to<br />
free radical attack as more targets were available due to cell<br />
degenerative ch<strong>an</strong>ges. Thus it enh<strong>an</strong>ces aging process by accelerating <strong>the</strong><br />
<strong>for</strong>mation <strong>an</strong>d accumulation of lipofuscin.<br />
Disclosures: S.K. Gupta, None; S. P<strong>an</strong>guluri, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.9/M31<br />
Topic: C.05.a. Molecular studies<br />
Support: NIGMS 1SC3GM086323
NIH CTSC GRANT #UL1-RR024996<br />
NIH RR03037<br />
PSC-CUNY 61853-00 39<br />
Title: The effect of AIRAP on modulating proteasome activity in <strong>the</strong> nervous system of<br />
Drosophila<br />
Authors: S. TEEGALA 1 , M. JANSEN 1 , C.-H. YEH 1 , J. GAO 1 , W. WONG 1 , M.<br />
FIGUEIREDO-PEREIRA 1 , *T. SCHMIDT-GLENEWINKEL 2 ;<br />
1 Biol. Sci., 2 Hunter Col. of CUNY, New York, NY<br />
Abstract: Proteolysis is <strong>an</strong> import<strong>an</strong>t cellular event involving tightly regulated removal of<br />
unw<strong>an</strong>ted proteins <strong>an</strong>d retention of those that are essential. Although it is currently accepted that<br />
protein degradation declines with age <strong>the</strong> mech<strong>an</strong>isms responsible <strong>for</strong> this decline are not yet<br />
delineated. In a variety of neurological disorders such as Alzheimer’s disease, Parkinson’s<br />
disease, Huntington’s disease <strong>an</strong>d amyotrophic lateral sclerosis, aggregates of ubiquitinated<br />
proteins are detected in neuronal inclusions in selective sets of neurons. The mech<strong>an</strong>isms leading<br />
to <strong>the</strong> <strong>for</strong>mation of such abnormal aggregates <strong>an</strong>d <strong>the</strong>ir role in <strong>the</strong> progression of<br />
neurodegeneration is unknown. The function of ubiquitin/proteasome pathway, responsible <strong>for</strong><br />
<strong>the</strong> bulk of intracellular protein degradation, declines with age. One potentially import<strong>an</strong>t factor<br />
in this decline might be <strong>the</strong> decrease in ATP levels as a function of age. It has been reported that<br />
AIRAP (Arsenite-inducible RNA-associated protein) stabilizes dramatically proteasome activity<br />
in <strong>the</strong> absence of ATP. In our studies we have investigated if inducible overexpression of AIRAP<br />
protein in Drosophila will stabilize <strong>the</strong> 26S proteasome <strong>an</strong>d prevents <strong>the</strong> age-dependent decline<br />
in proteasome activity in Drosophila.<br />
Disclosures: S. Teegala, None; M. J<strong>an</strong>sen, None; C. Yeh, None; J. Gao, None; W. Wong,<br />
None; M. Figueiredo-Pereira, None; T. Schmidt-Glenewinkel, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.10/M32<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: Center <strong>for</strong> Alzheimer's Disease <strong>an</strong>d Related Disorders (CADRD)
Title: 13C isotopic incorporation studies reveal alteration in glutamate-glutamine cycling <strong>the</strong> <strong>the</strong><br />
hippocampus of aged Fischer 344 rats<br />
Authors: *J. M. EBERSOLE 1,2,3 , D. J. KANAK 2,3 , A. E. CORBIN 3 , T. EID 5 , G. M. ROSE 3,4 ,<br />
P. R. PATRYLO 2,3,4 ;<br />
1 Sou<strong>the</strong>rn Illinois Univ, Carbondale, Carbondale, IL; 2 Dept. of Physiol., 3 Ctr. <strong>for</strong> Integrated Res.<br />
in Cognitive <strong>an</strong>d Neural Sci., 4 Dept. of Anat., SIUC, Carbondale, IL; 5 Dept. of Intrnl. Med., Yale<br />
Univ., New Haven, CT<br />
Abstract: Hippocampal function declines with age. Hippocampal function is dependent on fast<br />
synaptic glutamatergic <strong>an</strong>d GABAergic tr<strong>an</strong>smission, which, in turn, requires adequate syn<strong>the</strong>sis<br />
of releasable glutamate <strong>an</strong>d GABA. In this study we beg<strong>an</strong> to test <strong>the</strong> hypo<strong>the</strong>sis that alterations<br />
in glutamate-glutamine cycling (Glu-Gln), <strong>the</strong> primary process underlying fast amino acid<br />
neurotr<strong>an</strong>smitter production, could contribute to aging-related hippocampal dysfunction.<br />
Hippocampal slices <strong>from</strong> young adult (Y, 3-6 months, n = 4) <strong>an</strong>d aged (A, > 24 months, n = 3)<br />
male Fischer 344 rats were incubated <strong>for</strong> two hours in artificial cerebral spinal fluid (aCSF)<br />
containing 10 mM 13C-labeled glucose. 13C isotopic enrichments of Glu <strong>an</strong>d Gln were <strong>the</strong>n<br />
assessed in <strong>the</strong> dentate gyrus (DG) <strong>an</strong>d CA1 by t<strong>an</strong>dem mass spectrometry. Percent isotopic<br />
enrichment of Glu was signific<strong>an</strong>tly decreased in both regions in slices <strong>from</strong> aged <strong>an</strong>imals. (DG;<br />
Y = 42.8 ± 0.8, A = 33.4 ± 1.4; p < 0.05: CA1; Y = 37.1 ± 1.4, A = 30.8 ± 2.2; p
Program#/Poster#: 535.11/M33<br />
Topic: C.05.a. Molecular studies<br />
Support: Fondecyt 1090353<br />
VRAID-Puente08 Pontificia Universidad Católica de Chile<br />
Title: Microglial activation in aging <strong>an</strong>d its modulation by TGFβ-Smad3 signaling <strong>an</strong>d pasive<br />
immune-<strong>the</strong>rapy in a murine model of Alzheimer disease<br />
Authors: *J. E. TICHAUER, R. VON BERNHARDI;<br />
P. Univ. Católica de Chile, S<strong>an</strong>tiago, Chile<br />
Abstract: Microglia are <strong>the</strong> main immune effectors of <strong>the</strong> brain. In aging, <strong>the</strong>re is <strong>an</strong> increased<br />
inflammatory environment <strong>an</strong>d glial cell activation. We have shown that astrocytes modulate<br />
microglial cytotoxicity through <strong>the</strong> secretion of <strong>the</strong> <strong>an</strong>ti inflammatory cytokine TGFβ. There is<br />
evidence that TGFβ is increased in AD but <strong>the</strong> expression of Smad3, its main pathway effector,<br />
is reduced. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d immuno<strong>the</strong>rapy appears to modulate microglial activation but its<br />
mech<strong>an</strong>isms has not been elucidated. Aim: To study <strong>the</strong> effect of <strong>the</strong> TGFβ-Smad3 pathway in<br />
microglial activation <strong>an</strong>d <strong>the</strong> effect of immuno<strong>the</strong>rapy on TGFβ expression in aged <strong>an</strong>imals.<br />
Results: We evaluated nitrite secretion, scavenger receptor expression <strong>an</strong>d phagocytosis in<br />
microglial cultures exposed to various stimuli with & without TGFβ <strong>an</strong>d different pathway<br />
inhibitors. 1 µg/ml LPS induced a 4.1±0.5-fold increase on nitrite secretion, which was down<br />
regulated (0.82±0.05-fold) by 2ng/ml TGFβ. ERK <strong>an</strong>d p38 inhibitors by <strong>the</strong>mselves decreased<br />
LPS-induced nitrite secretion, <strong>an</strong>d TGFβ potentiated <strong>the</strong> decrease. TGFβ increased SR-A<br />
expression by 2.4±0.3-fold <strong>an</strong>d Aβ phagocytosis by 51%, effects that were prevented by Smad3<br />
inhibition. We also determined in 2- <strong>an</strong>d 12-month old APP/PS1 mice treated with 0.5 mg/kg<br />
LPS <strong>an</strong>d with <strong>an</strong>tibodies against Aβ, <strong>the</strong> expression of Smad3 <strong>an</strong>d TGFβ in <strong>the</strong> hippocampus <strong>an</strong>d<br />
<strong>the</strong> effect of Smad3 inhibition on phagocytosis by adult microglia. We observed that LPS<br />
induced <strong>an</strong> increase in Smad3 protein levels (1.58±0.18-fold) in 2 month- but not in 12 monthold<br />
mice hippocampus or in immuno<strong>the</strong>rapy-treated mice. Fur<strong>the</strong>rmore, active TGFβ was<br />
decreased in LPS <strong>an</strong>d immunized WT <strong>an</strong>d APP/PS1 mice. Microglial cultures <strong>from</strong> WT <strong>an</strong>d<br />
APP/PS1 mice treated with LPS increased Aβ-uptake when obtained <strong>from</strong> 2 month- but not <strong>from</strong><br />
12 month-old mice. Treatment with 2ng/ml TGFβ increased microglial Aβ-uptake by 72% in 2<br />
month WT <strong>an</strong>d 57% in APP/PS1. No potentiation was observed <strong>for</strong> 12 month-old mice.<br />
Increased phagocytosis was abolished by Smad3 inhibition. Chronic immune <strong>the</strong>rapy increased<br />
proliferation rate but not phagocytosis in microglial cultures <strong>from</strong> 12 moth-old mice. At <strong>the</strong> brain<br />
level, we observed that chronic LPS increased MHCII(+) cells <strong>an</strong>d plaque area in APP/PS1 mice<br />
whereas immuno<strong>the</strong>rapy decreased MHCII(+) cells <strong>an</strong>d plaque area. Conclusion: TGFβ-Smad3<br />
pathway is relev<strong>an</strong>t <strong>for</strong> microglial cells regulation. It is affected by aging <strong>an</strong>d inflammatory<br />
condition. However, <strong>the</strong> immunomodulatory effect of immunization doesn’t appear to be<br />
mediated by TGFβ.<br />
Disclosures: J.E. Tichauer, None; R. von Bernhardi, None.
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.12/M34<br />
Topic: C.05.c. Oxidative stress<br />
Support: NIH [NIGMS 1SC3GM086323 <strong>an</strong>d CTSC GRANT #UL1-RR024996]<br />
RR03037<br />
PSC-CUNY 61853-00 39<br />
Title: Proteasome function <strong>an</strong>d mitochondrial deficiency in Drosophila<br />
Authors: *J. GAO 1 , M. JANSEN 2 , C.-H. YEH 2 , M. FIGUEIREDO-PEREIRA 2 , T. SCHMIDT-<br />
GLENEWINKEL 2 ;<br />
1 CUNY GC, New York, NY; 2 CUNY GC <strong>an</strong>d Hunter Col., New York, NY<br />
Abstract: Age-related neurodegenerative diseases share a similar morphological feature, <strong>the</strong><br />
intracellular deposition of aggregated <strong>an</strong>d ubiquitinated proteins in affected neurons of <strong>the</strong><br />
central nervous system. Even in normal aging, <strong>the</strong> build-up of aggregated <strong>an</strong>d ubiquitinated<br />
proteins is one of <strong>the</strong> most apparent characteristics. Most of <strong>the</strong> proteins that accumulate under<br />
<strong>the</strong>se conditions depend on <strong>the</strong> ubiquitin/proteasome pathway <strong>for</strong> <strong>the</strong>ir degradation. The inability<br />
to break down ubiquitinated proteins might be due to proteasome impairment known to be<br />
associated with aging, mitochondrial dysfunction <strong>an</strong>d conditions such as oxidative stress. This<br />
observation supports our hypo<strong>the</strong>sis, which is that proteasome impairment is <strong>an</strong> import<strong>an</strong>t risk<br />
factor in neurodegeneration <strong>an</strong>d aging. We have observed a steep decline in proteasome activity<br />
<strong>an</strong>d ATP concentration in flies between <strong>the</strong> ages of 43 to 47 days (Vernace et al. (2007) FASEB<br />
J. 21(11):2672-82). Proteasome activity is ATP-depended. Mitochondria is <strong>the</strong> ATP syn<strong>the</strong>sis<br />
factory. We have <strong>an</strong>alyzed <strong>the</strong> time course of ch<strong>an</strong>ge in ATP levels, proteasome activity <strong>an</strong>d<br />
mitochondrial activity in several longevity mut<strong>an</strong>ts of Drosophila as well as wild-type flies under<br />
caloric restrictions. Using tr<strong>an</strong>sgenic methods we will fur<strong>the</strong>r study <strong>the</strong> aging impact on<br />
proteasome dysfunction <strong>an</strong>d mitochondria impairment.<br />
Disclosures: J. Gao, None; M. J<strong>an</strong>sen, None; C. Yeh, None; M. Figueiredo-Pereira,<br />
None; T. Schmidt-Glenewinkel, None.
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.13/M35<br />
Topic: C.05.c. Oxidative stress<br />
Support: NIH<br />
Title: Lipid peroxidation in Alzheimer’s disease plaques<br />
Authors: H. SCHALL 1 , J. FAWVER 1 , J. ROBERTS 1 , E. FANG 1 , G. ELLIS 1 , B. DESUTTER 1 ,<br />
D. PAPPALARDO 1 , E. ROLTSCH 4 , L. HOLCUMB 2 , D. ZIMMER 4 , T. GUMIENNY 5 , *I. V.<br />
MURRAY 3 ;<br />
2 3 1<br />
Psychiatry <strong>an</strong>d Behavioral Sci., Dept NeXT, Texas A&M Hlth. Sci. Ctr., College Station, TX;<br />
4 5<br />
Vet. Pathobiology, Texas A&M, Vet. Sch., College Station, TX; Mol. <strong>an</strong>d Cell. Med.,<br />
TAMHSC, College Station, TX<br />
Abstract: Lipid peroxidation is a well-documented pathological feature of Alzheimer’s disease<br />
(AD). We sought to determine if this peroxidation is intimately associated with amyloid plaques,<br />
as a first step in <strong>the</strong> demonstration of a feed-<strong>for</strong>ward mech<strong>an</strong>ism where Aβ increases lipid<br />
peroxidation, which <strong>the</strong>n augments amyloidogenesis. We visualized lipid peroxidation using two<br />
independent methods, biocytin hydrazide tagging of aldehydes <strong>an</strong>d ketones <strong>an</strong>d specific<br />
<strong>an</strong>tibodies to lipid peroxidation adducts of 4-hydroxy-2-nonenal (HNE). There was robust lipid<br />
peroxidation of amyloid plaques of <strong>the</strong> PSAPP Alzheimer’s disease mouse model at 5 <strong>an</strong>d 12<br />
months of age. Since Aβ, <strong>the</strong> protein misfolded in amyloid plaques, has been implicated directly<br />
in this lipid oxidation <strong>an</strong>d is potentially modified in vivo, we used syn<strong>the</strong>tic Aβ-HNE to develop<br />
methods to purify such adducts. Aβ is <strong>an</strong> ideal model peptide <strong>for</strong> this purpose, as it is likely<br />
modified in vivo, lipid modification augments Aβ misfolding in vitro <strong>an</strong>d, as <strong>an</strong> aggregation<br />
prone protein, is difficult to identify/isolate by traditional proteomic methods. Using Aβ-HNE,<br />
we sought to identify drugs which would prevent such modification <strong>an</strong>d misfolding, albeit in <strong>the</strong><br />
test-tube. Of <strong>the</strong> hydrazide compounds tested, two had <strong>the</strong> unique ability to both prevent HNE<br />
mediated misfolding of Aβ, <strong>an</strong>d unfold HNE-Aβ. Finally, preliminary experiments indicate that<br />
oxidative damage augments Aβ misfolding in a C. eleg<strong>an</strong>s AD model. Thus, we provide<br />
evidence <strong>for</strong> a possible feed-<strong>for</strong>ward mech<strong>an</strong>ism where Aβ augments lipid peroxidation, which<br />
<strong>the</strong>n results in Aβ modification <strong>an</strong>d misfolding. Additionally we identified drugs that c<strong>an</strong><br />
potentially prevent such lipid peroxidation <strong>an</strong>d Aβ misfolding in Alzheimer’s disease.<br />
Disclosures: H. Schall, None; J. Fawver, None; J. Roberts, None; E. F<strong>an</strong>g, None; G. Ellis,<br />
None; B. DeSutter, None; D. Pappalardo, None; E. Roltsch, None; L. Holcumb, None; D.<br />
Zimmer, None; T. Gumienny, patent process, E. Ownership Interest (stock, stock options,
patent or o<strong>the</strong>r intellectual property); I.V. Murray, NIH, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
patent process, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.14/M36<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: Evelyn F. McKnight Brain Research Gr<strong>an</strong>t to TCF<br />
NIH gr<strong>an</strong>ts MH059891 to TCF<br />
NIH gr<strong>an</strong>ts AG014979 to TCF<br />
J. Crayton Pruitt Family Biomedical Engineering Department Startup Funds to BKO<br />
Pruitt Scholarship to RBS<br />
Title: Biomarkers predict successful versus unsuccessful aging in rats<br />
Authors: *B. K. ORMEROD 1 , R. B. SPEISMAN 1 , A. KUMAR 2 , T. C. FOSTER 2 ;<br />
1 2<br />
Dept Biomed Engin., J. Crayton Pruitt Family, Gainesville, FL; Neurosci., McKnight Brain<br />
Inst., Gainesville, FL<br />
Abstract: Chronological age does not predict cognitive success across senescence, which c<strong>an</strong><br />
vary <strong>from</strong> “successful” with minimum impairment to “unsuccessful” with signific<strong>an</strong>t impairment<br />
despite no identifiable pathology. We have shown previously that senescent rats c<strong>an</strong> be<br />
characterized as memory unimpaired (MU) <strong>an</strong>d memory impaired (MI) using <strong>the</strong> spatial water<br />
maze <strong>an</strong>d inhibitory avoid<strong>an</strong>ce tasks, which exhibit a good degree of concord<strong>an</strong>ce in <strong>the</strong>ir<br />
sensitivity to age-related memory impairments. Interestingly, senescent rats that are categorized<br />
as MI begin to show impaired cognition in middle age. We take adv<strong>an</strong>tage of this observation to<br />
investigate whe<strong>the</strong>r ei<strong>the</strong>r hypothalamic-pituitary-adrenal axis or inflammatory biomarkers <strong>for</strong><br />
“unsuccessful aging” emerge in middle-aged rats. To test this hypo<strong>the</strong>sis young (8 mo), middle<br />
aged (14 mo), <strong>an</strong>d aged (20 mo) male Fischer 344 rats were trained <strong>an</strong>d tested on <strong>the</strong> spatial<br />
water maze task <strong>an</strong>d <strong>the</strong>n in <strong>an</strong> inhibitory avoid<strong>an</strong>ce task, where some <strong>an</strong>imals received a mild<br />
foot shock during training <strong>an</strong>d some acted as no shock controls. Based upon <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce in
<strong>the</strong>se tasks, <strong>the</strong> rats were described as memory unimpaired (MU) or memory impaired (MI) <strong>an</strong>d<br />
<strong>the</strong>n sacrificed so that blood <strong>an</strong>d hippocampal/cortical tissue samples could be collected.<br />
Analytes in blood serum <strong>an</strong>d protein harvested <strong>from</strong> brain tissue samples were qu<strong>an</strong>tified using a<br />
multiplex ELISA strategy that permits <strong>the</strong> detection of up to 23 pro- <strong>an</strong>d <strong>an</strong>ti-inflammatory<br />
chemokines/cytokines or 3 stress hormones simult<strong>an</strong>eously. Our preliminary results show that<br />
circulating levels of <strong>the</strong> homeostatic cytokine eotaxin <strong>an</strong>d proinflammatory cytokine RANTES<br />
tended to increase with age (p=0.086 <strong>an</strong>d 0.078 respectively). Interestingly,circulating levels of<br />
eotaxin, RANTES, leptin, Interleukin-4, <strong>an</strong>d growth related oncogene (GRO-KC) were lower in<br />
<strong>an</strong>imals that received shock during inhibitory avoid<strong>an</strong>ce training. Within <strong>the</strong> cortex <strong>an</strong>d<br />
hippocampus, multiple pro-inflammatory cytokines including interleukin-6 (IL-6), tumor<br />
necrosis factor-α (TNF-α), <strong>an</strong>d interleukin-1β (IL-1β), <strong>an</strong>d interestingly interleukin-17 (IL-17),<br />
interleukin- 4 (IL-4), leptin <strong>an</strong>d monocyte chemoattract<strong>an</strong>t protein-1 (MCP-1) initially increased<br />
with age but <strong>the</strong>n decreased in old age. Corticosterone levels increased across age, but <strong>the</strong> age x<br />
shock interaction was signific<strong>an</strong>t in that shock-induced increases in hippocampal (<strong>an</strong>d perhaps<br />
serum) corticosterone levels decreased with age (p < 0.05). Our preliminary data have revealed<br />
interesting c<strong>an</strong>didate biomarkers of aging that we are currently validating against per<strong>for</strong>m<strong>an</strong>ce in<br />
behavioral tasks.<br />
Disclosures: B.K. Ormerod, None; R.B. Speism<strong>an</strong>, None; A. Kumar, None; T.C. Foster,<br />
None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.15/M37<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: GlaxoSmithKline (UK) <strong>an</strong>d <strong>the</strong> industrial Development Authority (Irel<strong>an</strong>d)<br />
Title: 5HT6 receptor <strong>an</strong>tagonist SB742457 recovers <strong>an</strong> age related deficit in working memory<br />
but not in spatial memory in <strong>the</strong> rat<br />
Authors: *C. K. CALLAGHAN 1 , B. WAN WANG 1 , M. MALONEY 1 , H. MILTON 1 , A.<br />
DELLA CHIESA 1 , V. HOK 1 , D. J. VIRLEY 2 , N. UPTON 2 , S. M. O'MARA 1 ;<br />
1 2<br />
Inst. of Neurosci., Trinity Col., Dublin, Irel<strong>an</strong>d; GlaxoSmithKline, Neurosciences CEDD,<br />
Harlow, Essex, United Kingdom
Abstract: The serotonergic system is fast becoming a new objective in development of<br />
treatments targeting <strong>the</strong> symptoms of Alzheimer’s disease (AD). The serotonergic system has<br />
been linked to cognition, depression, psychosis <strong>an</strong>d aggression, all of which decline or are<br />
elevated in this progressive disease. Studies have also found serotonergic denervation within <strong>the</strong><br />
AD brain, <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e regulation of this system appears <strong>an</strong> attractive potential <strong>the</strong>rapeutic<br />
treatment of neurodegenerative disorders.<br />
Serotonin (5-HT) plays <strong>an</strong> import<strong>an</strong>t role in cognition through activation of <strong>the</strong> serotonergic<br />
receptors. The 5-HT6 receptor is one of <strong>the</strong> most recent additions to <strong>the</strong> 5-HT receptor family. It<br />
is widely distributed within <strong>the</strong> CNS, including, striatum, nucleus accumbens, hippocampal<br />
<strong>for</strong>mation, cerebral cortex <strong>an</strong>d amygdala. This study examined <strong>the</strong> effects of chronic systemic<br />
administration of <strong>the</strong> 5-HT6 <strong>an</strong>tagonist SB742457 in <strong>the</strong> delayed non-match to sample (DNMS)<br />
task <strong>an</strong>d in <strong>the</strong> water-plus maze (WPM) task.<br />
The oper<strong>an</strong>t DNMS task is designed to simulate <strong>an</strong>d measure working memory in <strong>the</strong> rodent, <strong>an</strong>d<br />
is closely associated with activity in <strong>the</strong> hippocampal <strong>for</strong>mation (HF). The WPM task, a<br />
modified version of <strong>the</strong> Morris Water Maze, is aimed at targeting spatial learning <strong>an</strong>d memory,<br />
<strong>an</strong>d because of <strong>the</strong> possibility of using a motor strategy as well as spatial strategy to per<strong>for</strong>m <strong>the</strong><br />
task is associated with activity in HF <strong>an</strong>d striatum. It has been shown that <strong>the</strong>re are age-related<br />
delay-dependent deficits in per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong> DNMS task, as well as also age-related deficits in<br />
<strong>the</strong> WPM task; thus <strong>the</strong>se tasks may serve as <strong>an</strong> appropriate model to <strong>an</strong>alyze <strong>the</strong> effects of<br />
SB742457 on cognition in <strong>an</strong> aged population.<br />
Following a six week training protocol in <strong>the</strong> DNMS task, both young (3month) <strong>an</strong>d middle age<br />
(12-14month) rats received chronic treatment (3mg/day SB742457). This treatment resulted in a<br />
recovery of <strong>the</strong> age-related deficit in <strong>the</strong> middle aged group, sufficient to match <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce<br />
of <strong>the</strong> young control group. In <strong>the</strong> WPM task <strong>an</strong>imals received treatment (3mg/day SB742457)<br />
24hr be<strong>for</strong>e commencing training <strong>an</strong>d <strong>for</strong> <strong>the</strong> duration of <strong>the</strong> experiment. By contrast, we found<br />
no ch<strong>an</strong>ge in <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of young or middle age <strong>an</strong>imals in this task.<br />
These results suggest that regulation of <strong>the</strong> serotonergic system enh<strong>an</strong>ces working memory, but<br />
may not enh<strong>an</strong>ce spatial memory in <strong>the</strong> rat, suggesting that <strong>the</strong> 5-HT6 <strong>an</strong>tagonist may be of<br />
benefit in treating mnemonic aspects of neurodegenerative diseases.<br />
Disclosures: C.K. Callagh<strong>an</strong>, None; B. W<strong>an</strong> W<strong>an</strong>g, None; M. Maloney, None; H. Milton,<br />
None; A. Della Chiesa, None; V. Hok, None; D.J. Virley, None; N. Upton, None; S.M.<br />
O'Mara, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.16/M38
Topic: F.02.i. Learning <strong>an</strong>d memory: Physiology <strong>an</strong>d imaging<br />
Support: NIA Gr<strong>an</strong>t P01 AG11370<br />
Title: Altered basal cerebrovascular perfusion <strong>an</strong>d perfusion response to hypercapnia examined<br />
using FAIR <strong>an</strong>d BOLD MRI predict age-related cognitive impairment in rodents<br />
Authors: *M. C. MITSCHELEN 1 , P. GARTEISER 2 , B. A. CARNES 1 , J. A. FARLEY 1 , S.<br />
DOBLAS 2 , J. H. DEMOE 2 , J. P. WARRINGTON 1 , H. YAN 1 , M. M. NICOLLE 3 , R.<br />
TOWNER 2 , W. E. SONNTAG 1 ;<br />
1 Geriatric Med., Univ. of Oklahoma Hlth. Sci. Ctr., Oklahoma City, OK; 2 Adv<strong>an</strong>ced Magnetic<br />
Reson<strong>an</strong>ce Ctr., Oklahoma Med. Res. Fndn., Oklahoma City, OK; 3 Intrnl. Med., Wake Forest<br />
Univ. Hlth. Sci., Winston-Salem, NC<br />
Abstract: Although <strong>the</strong> etiology of cognitive impairment is poorly understood, reduced cerebral<br />
blood flow is implicated in multiple types of cognitive impairment <strong>an</strong>d dementias in hum<strong>an</strong>s. In<br />
this study we address whe<strong>the</strong>r baseline or <strong>the</strong> hypercapnia-induced increase in perfusion is<br />
altered with age <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>se measures are predictive of cognitive status in rodents. Adult<br />
(9 m) <strong>an</strong>d aged (26 m) F344xBN male rats were evaluated using a hippocampally-dependent<br />
spatial task in a Morris Water Maze <strong>an</strong>d aged rats were classified as memory impaired or intact<br />
based on per<strong>for</strong>m<strong>an</strong>ce comparisons with adult rats. FAIR <strong>an</strong>d BOLD MRI <strong>an</strong>alyses were<br />
undertaken using a 7T MRI, examining <strong>an</strong> axial slice including dorsal hippocampus <strong>an</strong>d<br />
retrospleenal cortex. Animals were <strong>an</strong>es<strong>the</strong>tized with isoflur<strong>an</strong>e using air or oxygen as a carrier<br />
gas, <strong>an</strong>d perfusion was assessed by FAIR <strong>an</strong>alysis be<strong>for</strong>e <strong>an</strong>d after 10% CO2. The tr<strong>an</strong>sition<br />
period between CO2 concentrations was examined with BOLD. FAIR <strong>an</strong>alysis did not reveal<br />
signific<strong>an</strong>t differences in basal perfusion between adult <strong>an</strong>d aged rats, but separation of aged<br />
<strong>an</strong>imals into intact <strong>an</strong>d impaired categories revealed increased perfusion in dorsal hippocampus<br />
of impaired compared to intact <strong>an</strong>imals (p
Disclosures: M.C. Mitschelen, None; P. Garteiser, None; B.A. Carnes, None; J.A. Farley,<br />
None; S. Doblas, None; J.H. DeMoe, None; J.P. Warrington, None; H. Y<strong>an</strong>, None; M.M.<br />
Nicolle, None; R. Towner, None; W.E. Sonntag, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.17/N1<br />
Topic: C.05.c. Oxidative stress<br />
Support: NIH Gr<strong>an</strong>t DK047320<br />
NIH Gr<strong>an</strong>t G12RR003061<br />
NIH Gr<strong>an</strong>t NS40302<br />
Title: The neuroprotective functions of selenoprotein M <strong>an</strong>d selenoprotein I<br />
Authors: *M. A. REEVES, F. P. BELLINGER, M. J. BERRY;<br />
CMB, Univ. Hawaii M<strong>an</strong>oa, JABSOM, Honolulu, HI<br />
Abstract: Cumulative oxidative damage is strongly implicated in neurological <strong>an</strong>d<br />
neurodegenerative diseases. The brain has multiple defense mech<strong>an</strong>isms against oxidative<br />
damage. Among <strong>the</strong>se defenses are selenoproteins, which contain <strong>the</strong> trace element selenium<br />
incorporated as selenocysteine, <strong>the</strong> 21st amino acid. Some members of <strong>the</strong> selenoprotein family,<br />
including <strong>the</strong> glutathione peroxidases, have well-characterized <strong>an</strong>tioxid<strong>an</strong>t activities, but <strong>the</strong><br />
functions of m<strong>an</strong>y of <strong>the</strong> 25 hum<strong>an</strong> selenoproteins are unknown. We are studying <strong>the</strong> functions<br />
of selenoprotein M (SelM) <strong>an</strong>d selenoprotein I (SelI). Our results confirm that SelM plays <strong>an</strong><br />
import<strong>an</strong>t role in protecting <strong>the</strong> brain against oxidative damage as well as potentially functioning<br />
in calcium regulation. Additionally SelM protects against toxicity caused by aggregated amyloid<br />
beta protein, a hallmark of Alzheimer’s disease, <strong>an</strong>d <strong>from</strong> methamphetamine exposure. SelI is a<br />
mammali<strong>an</strong> <strong>for</strong>m of eth<strong>an</strong>olamine phosphotr<strong>an</strong>sferase involved in production of phosphatadyl<br />
eth<strong>an</strong>olamine, <strong>an</strong>d may be import<strong>an</strong>t in neuronal growth <strong>an</strong>d phospholipid signaling pathways.<br />
SelI overexpression in neuronal <strong>an</strong>d glial cell lines reduced levels of reactive oxygen species <strong>an</strong>d<br />
apoptotic cell death. In addition cells overexpressing SelI had altered cytosolic calcium levels<br />
<strong>an</strong>d calcium responses to oxidative stress. These results suggest that SelM <strong>an</strong>d SelI are<br />
neuroprotective, although <strong>the</strong> mech<strong>an</strong>isms remain to be elucidated. A greater underst<strong>an</strong>ding of
<strong>the</strong>se, <strong>an</strong>d o<strong>the</strong>r selenoproteins may provide future <strong>the</strong>rapeutic applications which could be<br />
beneficial to our aging populations.<br />
Disclosures: M.A. Reeves, None; F.P. Bellinger, None; M.J. Berry, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.18/N2<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: Hart<strong>for</strong>d/AFAR Beeson AAAE8665<br />
Title: Histone deacetylase inhibitors preferentially target <strong>the</strong> dentate gyrus: Relev<strong>an</strong>ce to<br />
cognitive aging<br />
Authors: *S. T. JONES, A. S. BENDER, A. MUHAMMAD, F. HUA, S. A. SMALL;<br />
Taub Inst., Columbia Univ. Med. Ctr., New York, NY<br />
Abstract: Background: A growing number of studies suggest that within <strong>the</strong> hippocampal<br />
<strong>for</strong>mation, <strong>the</strong> dentate gyrus (DG) is differentially affected by aging. Recent molecular studies<br />
have implicated DG-selective defects in histone acetylation. Accordingly, we used mouse<br />
functional magnetic reson<strong>an</strong>ce imaging (fMRI) to determine which hippocampal subregion<br />
benefits most <strong>from</strong> <strong>the</strong> histone deacetylase inhibitors (HDACi) suberoy<strong>an</strong>ilide hydroxamic acid<br />
(SAHA) <strong>an</strong>d sodium butyrate (SB).<br />
Methods: A high-resolution, cross-species vari<strong>an</strong>t of fMRI that relies on cerebral blood volume<br />
(CBV) was used, which has previously documented age-related DG dysfunction in primates <strong>an</strong>d<br />
rodents (Small et al, PNAS 2004; 18:7181 & Moreno et al, Archives of Neurology, 2007;<br />
64:1467). As previously described (Moreno et al, NMR in Biomedicine, 2006; 19:535), relative<br />
CBV maps were generated using a 9.4 tesla dedicated mouse sc<strong>an</strong>ner (AVANCE 400WB<br />
spectrometer, Bruker NMR, Inc., Billerica, MA), generating T2-weighted images (TR/TEeff=<br />
2000ms/70ms; 16; FOV=19.6mm; acquisition matrix = 256 x 256; 8 slices; slice<br />
thickness=0.6mm, slice gap=0.1mm; NEX=28) be<strong>for</strong>e <strong>an</strong>d after intra-peritoneal (IP)<br />
administration of gadolinium. C57/Black6 mice were used in all studies. For <strong>the</strong> SAHA<br />
experiment, 10 mice were administered drug IP <strong>for</strong> 3 weeks (50 mg/kg in saline/dimethyl<br />
sulfoxide), <strong>an</strong>d 10 mice were administered vehicle control (saline/dimethyl sulfoxide). For <strong>the</strong><br />
SB experiment, 10 mice consumed drug per os (PO) <strong>for</strong> 4 weeks (5 g/kg in drinking water) <strong>an</strong>d<br />
10 mice consumed equivalent amounts of water without drug. On average all mice consumed 4
ml water/day.<br />
Results: A multivariate ANOVA was used in which rCBV <strong>from</strong> <strong>the</strong> five hippocampal<br />
subregions were included as <strong>the</strong> dependent variables (entorhinal cortex, DG, CA3, CA1, <strong>an</strong>d <strong>the</strong><br />
subiculum) <strong>an</strong>d group (drug vs. control) was included as <strong>the</strong> fixed factor. Although a mild drug<br />
effect was detected in o<strong>the</strong>r hippocampal subregions, <strong>the</strong> strongest <strong>an</strong>d domin<strong>an</strong>t effect was<br />
observed in <strong>the</strong> DG <strong>for</strong> both SAHA (F=9.58, p=0.006) <strong>an</strong>d SB (F=6.48, p=0.021). A direct<br />
comparison between SAHA <strong>an</strong>d SB revealed no between-drug difference.<br />
Conclusions: Interpreted in <strong>the</strong> context of previous <strong>an</strong>atomical <strong>an</strong>d molecular findings related to<br />
age-related hippocampal dysfunction, <strong>the</strong>se results suggest that behavioral or pharmacological<br />
interventions that increase histone acetylation may ameliorate cognitive aging.<br />
Disclosures: S.T. Jones, None; A.S. Bender, None; A. Muhammad, None; F. Hua,<br />
None; S.A. Small, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.19/N3<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: NIH Gr<strong>an</strong>t AG017542<br />
Title: Epigenetic regulation via histone acetylation reverses aging-associated decline in<br />
neurotrophin expression <strong>an</strong>d hippocampal synaptic plasticity<br />
Authors: Y. ZENG, X. YIN, M. TAN, J. B. WATSON, *C.-W. XIE;<br />
Psychiatry, UCLA Sch. Med., Los Angeles, CA<br />
Abstract: Chromatin modification via histone acetylation is known to enh<strong>an</strong>ce synaptic<br />
plasticity <strong>an</strong>d memory. The cellular basis <strong>for</strong> this enh<strong>an</strong>cement <strong>an</strong>d its potential ch<strong>an</strong>ges in<br />
cognitive disorders have not been fully understood. We examined <strong>the</strong> impact of aging on this<br />
epigenetic regulatory mech<strong>an</strong>ism <strong>an</strong>d result<strong>an</strong>t ch<strong>an</strong>ges in hippocampal synaptic plasticity,<br />
neurotrophin expression, <strong>an</strong>d regulation of several plasticity-related proteins. Hippocampal longterm<br />
potentiation (LTP) was determined in brain slices derived <strong>from</strong> young (1-2 months old) <strong>an</strong>d<br />
aged (22-23 months old) Fischer 344 rats. Western blot <strong>an</strong>alysis was used to measure agedependent<br />
<strong>an</strong>d drug-induced ch<strong>an</strong>ges in protein expression. A high frequency stimulation (HFS)<br />
to <strong>the</strong> Schaffer collateral-CA1 pathway induced long-lasting increases in evoked synaptic<br />
responses in young but not aged slices (144+1.2% vs. 105+3.0% at 60 min post-HFS, p
Preincubation of slices <strong>for</strong> 3 hr with trichostatin (TSA), a histone deacetylase inhibitor, dosedependently<br />
enh<strong>an</strong>ced LTP in both young <strong>an</strong>d aged slices (164+2.4% <strong>an</strong>d 146+2.7%,<br />
respectively, with 2 µM TSA, P
Abstract: Neurodegenerative disorders, including Alzheimer’s (AD), Parkinson’s (PD),<br />
Huntington’s <strong>an</strong>d prion diseases, are characterized by a selective loss of neurons in specific<br />
regions of <strong>the</strong> brain. The result is often a disruption to motor, sensory or cognitive systems,<br />
resulting in severe disability of <strong>the</strong> patient. A hallmark of <strong>the</strong> diseases involves protein<br />
misfolding <strong>an</strong>d aggregation, resulting in inclusion bodies <strong>an</strong>d o<strong>the</strong>r aggregates within cells. In<br />
general, <strong>the</strong>se inclusions consist of insoluble, unfolded, partially ubiquitinated polypeptides that<br />
fail to be targeted <strong>an</strong>d degraded by <strong>the</strong> 26S proteasome. Production of intracellular protein<br />
aggregates in cells profoundly impairs <strong>the</strong> functional capacity of <strong>the</strong> ubiquitin-proteasome<br />
system (UPS), a major intracellular proteolytic pathway. A dysfunctional UPS may <strong>the</strong>n cause<br />
proteins that are normally turned over by this pathway to aggregate <strong>an</strong>d <strong>for</strong>m inclusions. We<br />
hypo<strong>the</strong>size that dysfunction in <strong>the</strong> UPS pathway causes <strong>the</strong> accumulation of ubiquitin-protein<br />
aggregates. To investigate this hypo<strong>the</strong>sis, we developed a Drosophila model with a “tunable”<br />
proteasome by modulating <strong>the</strong> expression of one of its catalytic subunits, <strong>the</strong> dβ5 subunit,<br />
through double-str<strong>an</strong>ded RNA interference (RNAi). Expression of <strong>the</strong> dβ5 RNAi is controlled by<br />
a RU486 inducible act5C promoters. Reduction of dβ5 subunit expression was caused by RU486<br />
administration. The loss of dβ5 function caused <strong>an</strong> early onset of proteasome activity disruption<br />
<strong>an</strong>d a age-related accumulation of ubiquitinated proteins. Disruption of proteasome activity<br />
shortens lifesp<strong>an</strong> of flies <strong>an</strong>d enh<strong>an</strong>ced sensitivity to oxidative stress. Our data demonstrate that<br />
age-related proteasome dysfunction could be <strong>the</strong> major factor <strong>for</strong> age associated ubiquitinated<br />
protein accumulation with a subsequent effect on <strong>the</strong> lifesp<strong>an</strong>. We will fur<strong>the</strong>r investigate how<br />
reduced proteasome activity affects <strong>the</strong> nervous system of Drosophila.<br />
Disclosures: C. Yeh, None; M. J<strong>an</strong>sen, None; J. Gao, None; W. Wong, None; M. Figueiredo-<br />
Pereira, None; T. Schmidt-Glenewinkel, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.21/N5<br />
Topic: C.11.g. Neuroprotective mech<strong>an</strong>isms: Oxidative stress<br />
Support: VA R&D<br />
AHA<br />
Title: Resveratrol modulates NAD + metabolism through AMP-activated protein kinase
Authors: *C. C. ALANO 1 , H. LU 2 , S. WON KIM 2 ;<br />
1 2<br />
NEUROLOGY, VAMC/UCSF, SAN FRANCISCO, CA; NEUROLOGY, VAMC/UCSF, S<strong>an</strong><br />
fr<strong>an</strong>cisco, CA<br />
Abstract: Resveratrol (RSV) is a polyphenol shown to have neuroprotective effects, <strong>an</strong>d is<br />
classically associated with activation of SIRT1. However, o<strong>the</strong>rs have shown that its actions<br />
c<strong>an</strong>not be exclusively associated with SIRT1 activation. We have shown that prevention of NAD<br />
depletion ei<strong>the</strong>r through 1) inhibition of NAD catabolic enzymes, or 2) direct NAD application,<br />
promoted neuronal recovery after injury. Here we show that RSV increased NAD levels in cells<br />
through activation of AMP-activated protein kinase (AMPK) independent of SIRT1 activation.<br />
In cultured mouse cortical neurons <strong>an</strong>d astrocytes, RSV treatment led to increased AMPK<br />
phosphorylation <strong>an</strong>d activation. The AMPK activator 5-Amino-4-imidazolecarboxamide riboside<br />
(AICAR) increased AMPK phosphorylation <strong>an</strong>d activation. AMPK phosphorylation <strong>an</strong>d<br />
activation with ei<strong>the</strong>r RSV or AICAR were not dependent on calcium. The AMPK inhibitor,<br />
Compound C (CC) did not prevent AMPK phosphorylation with RSV or AICAR, but CC<br />
treatment decreased AMPK activity. These were not prevented by sirt1 inhibition with sirtinol.<br />
RSV treatment increased total NAD levels, <strong>an</strong>d increased <strong>the</strong> NAD/NADH ratio in cells. RSV<br />
treatment also increased mitochondrial protein levels. These ch<strong>an</strong>ges were reversed by inhibition<br />
with CC. RSV treatment partially recovered NAD levels after PARP-1 activation, <strong>an</strong>d protected<br />
neurons against excitotoxic injury. RSV did not inhibit PARP-1 activity. These findings indicate<br />
that activation of AMPK by resveratrol could affect energy homeostasis, <strong>an</strong>d suggest a<br />
potentially novel pathway of resveratrol protection through 1) modulation of NAD + metabolism,<br />
<strong>an</strong>d 2) promotion of mitochondrial biogenesis.<br />
Disclosures: C.C. Al<strong>an</strong>o, None; H. Lu, None; S. Won Kim, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.22/N6<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: Hartwell Foundation Individual Research Award (A.A.P.)<br />
NIH Director's Pioneer Award (S.L.M)<br />
Title: The role of hippocampal neurogenesis in age-related impairments in learning <strong>an</strong>d memory
Authors: *G. L. BECKER 1 , P. J. HUNTINGON 1 , J. ZHONG 1 , J. H. HSIEH 2 , S. L.<br />
MCKNIGHT 3 , A. A. PIEPER 1,3 ;<br />
1 Psychiatry, 2 Mol. Biol. <strong>an</strong>d Green Ctr. <strong>for</strong> Reproductive Biol. Sci., 3 Biochem., The Univ. of<br />
Texas Southwestern Med. Ctr., Dallas, TX<br />
Abstract: Age-related decline in cognitive functioning is associated with diminished<br />
hippocampal volume <strong>an</strong>d repressed hippocampal neurogenesis. It is proposed that pharmacologic<br />
agents that augment postnatal hippocampal neurogenesis might help preserve <strong>the</strong> morphological<br />
<strong>an</strong>d functional integrity of <strong>the</strong> aging hippocampus, <strong>an</strong>d thus prevent or ameliorate hippocampalrelated<br />
cognitive deficits commonly seen in aging. We are testing this hypo<strong>the</strong>sis with a novel,<br />
non-toxic, pro-neurogenic small molecule that readily crosses <strong>the</strong> blood brain barrier. We<br />
identified this pharmacologic agent through a large-scale in vivo screening process <strong>for</strong> small,<br />
drug-like molecules that promote hippocampal neurogenesis in living rodents. This molecule<br />
effectively restored hippocampal neurogenesis, as well as overall morphology <strong>an</strong>d<br />
electrophysiologic functioning, in a mouse in which postnatal hippocampal neurogenesis was<br />
virtually abolished (Neuronal PAS Domain Protein 3 - deficient mice). We have fur<strong>the</strong>r shown<br />
that this molecule quadrupled <strong>the</strong> amount of hippocampal neurogenesis in aged 18 month old<br />
Fisher 344 rats, <strong>an</strong>d also augmented <strong>the</strong> extension of neuritic processes in <strong>the</strong>se newborn cells.<br />
We are now evaluating <strong>the</strong> efficacy of long term (4 months), daily administration of this<br />
molecule to 18 month old Fisher 344 rats to evaluate <strong>the</strong> efficacy of augmenting hippocampal<br />
neurogenesis <strong>an</strong>d <strong>the</strong> effect on learning <strong>an</strong>d memory as assessed by per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> Morris<br />
water maze <strong>an</strong>d novel object recognition tasks. Histologic measures, including<br />
bromodeoxyuridine <strong>an</strong>d doublecortin immunostaining, as well as Golgi-Cox <strong>an</strong>alysis, were used<br />
to characterize <strong>the</strong> effect of prolonged stimulation of hippocampal neurogenesis on <strong>the</strong> <strong>an</strong>atomy<br />
of <strong>the</strong> aging hippocampus. If augmenting hippocampal neurogenesis proves to ameliorate agerelated<br />
decline in hippocampal <strong>an</strong>atomy <strong>an</strong>d function, this work may provide a basis <strong>for</strong> <strong>the</strong><br />
development of new <strong>the</strong>rapeutic options <strong>for</strong> treating patients with age-related cognitive decline.<br />
Disclosures: G.L. Becker, None; P.J. Huntingon, None; J. Zhong, None; J.H. Hsieh,<br />
None; S.L. McKnight, None; A.A. Pieper, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.23/N7<br />
Topic: C.05.c. Oxidative stress<br />
Title: Neuroprotective <strong>an</strong>d <strong>an</strong>ti-ageing effect of bacosides
Authors: *M. RASTOGI 1 , R. P. OJHA 1 , B. P. DEVI 1 , G. RAJAMANICKAM 1 , A.<br />
AGRAWAL 2 , G. DUBEY 1 ;<br />
1 SASTRA Univ., Th<strong>an</strong>javur, India; 2 BHU, Var<strong>an</strong>asi, India<br />
Abstract: Ageing represents <strong>the</strong> major risk factor <strong>for</strong> plethora of age-related neurodegenerative<br />
disorders. Despite, recent adv<strong>an</strong>cement in underst<strong>an</strong>ding <strong>the</strong> molecular mech<strong>an</strong>isms of ageing,<br />
<strong>the</strong>rapeutic interventions that could <strong>for</strong>estall or reverse its deleterious effects are limited.<br />
The present study aims to investigate <strong>the</strong> neuroprotective effect of bacosides, isolated <strong>from</strong><br />
Bacopa monnieri (L.), <strong>an</strong> Ayurvedic drug in brain of aged mice. Bacosides (300mg/kg) was<br />
orally administered to C57Bl/6 mice (6 <strong>an</strong>d 18 months) <strong>for</strong> three months. A signific<strong>an</strong>t increase<br />
in TBARS content with concomit<strong>an</strong>t decrease in GSH, SOD <strong>an</strong>d GPx activity were observed in<br />
<strong>the</strong> aged brain. Fur<strong>the</strong>r, mitochondrial complex I <strong>an</strong>d IV activity were found to be downregulated<br />
which leads to <strong>the</strong> depletion of ATP content in <strong>the</strong> ageing brain. However,<br />
accumulation of lipofuscin, was elevated with ageing.<br />
Treatment with bacosides signific<strong>an</strong>tly modulates <strong>the</strong> endogenous <strong>an</strong>tioxid<strong>an</strong>t marker enzymes<br />
activity to normal level. Moreover, up-regulation of mitochondrial complex activity along with<br />
ATP content was also observed. Intra-neuronal accumulation of lipofuscin was also found to be<br />
markedly reduced on oral administration of bacosides. The present study suggest that bacosides<br />
exerts neuroprotective effect by attenuating oxidative stress markers, mitochondrial complex<br />
activity <strong>an</strong>d lipofuscin aggregation. Thus, bacosides may act as a potential <strong>an</strong>ti-ageing agent <strong>an</strong>d<br />
may be used in <strong>the</strong> treatment <strong>an</strong>d/or m<strong>an</strong>agement of age -related neurodegenerative disorders.<br />
Disclosures: M. Rastogi, None; R.P. Ojha, None; B.P. Devi, None; G. Rajam<strong>an</strong>ickam,<br />
None; A. Agrawal, None; G. Dubey, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.24/N8<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: IRP of <strong>the</strong> NIH, National Institute on Aging<br />
Title: In vivo amyloid deposition detected by [11C]PIB PET <strong>an</strong>d neuropathology in nondemented<br />
older adults<br />
Authors: *J. SOJKOVA 1 , D. IACONO 2 , Y. ZHOU 2 , W. YE 2 , M. A. KRAUT 2 , L.<br />
FERRUCCI 1 , C. A. MATHIS 3 , W. E. KLUNK 3 , D. F. WONG 2 , J. C. TRONCOSO 2 , S. M.
RESNICK 1 ;<br />
1 Natl. Inst. On Aging/NIH, Baltimore, MD; 2 Johns Hopkins Med. Institutions, Baltimore, MD;<br />
3 Univ. of Pittsburgh, Pittsburgh, PA<br />
Abstract: Although up to 30% of non-demented older adults have elevated levels of β-amyloid,<br />
<strong>the</strong> relationship between in vivo imaging <strong>an</strong>d postmortem assessment of β-amyloid load remains<br />
unclear.<br />
Methods:<br />
Six non-demented particip<strong>an</strong>ts of <strong>the</strong> Neuroimaging Substudy of <strong>the</strong> Baltimore Longitudinal<br />
Study of Aging (NI-BLSA) were prospectively followed by [11C]PIB PET imaging <strong>an</strong>d<br />
neuropsychological testing <strong>an</strong>d evaluated postmortem a me<strong>an</strong> of 1.5 (SD 0.9) years after <strong>the</strong>ir<br />
last [11C]PIB study. Me<strong>an</strong> cortical DVR (cDVR) <strong>an</strong>d parametric images reflecting <strong>the</strong><br />
distribution volume ratio (DVR), were generated <strong>from</strong> [11C]PIB dynamic PET using SRTM-<br />
LRSC. Neuropathology evaluation included CERAD rating <strong>an</strong>d assessment of amyloid<br />
<strong>an</strong>giopathy. The associations between <strong>an</strong>temortem [11C] PIB retention <strong>an</strong>d neuropathological<br />
findings were investigated in <strong>the</strong> context of cognitive status.<br />
Results:<br />
On postmortem evaluation, 1/6 had frequent, 3/6 particip<strong>an</strong>ts had moderate, 1/6 sparse, <strong>an</strong>d 1/6<br />
no detectable neuritic plaques. Of <strong>the</strong> two particip<strong>an</strong>ts with higher in vivo amyloid load (cDVR ><br />
1.4), one individual became demented be<strong>for</strong>e <strong>the</strong> third [11C] PIB PET, <strong>an</strong>d this individual had a<br />
moderate number of neuritic plaques on postmortem evaluation. The o<strong>the</strong>r individual with<br />
cDVR>1.4 had sparse neuritic plaques <strong>an</strong>d amyloid <strong>an</strong>giopathy on postmortem evaluation. Three<br />
of <strong>the</strong> 4 particip<strong>an</strong>ts with lower in vivo amyloid load (cDVR < 1.2) had moderate numbers of<br />
neuritic plaques. 3/6 individuals (1 of whom had cDVR < 1.2) were found to have amyloid<br />
<strong>an</strong>giopathy on postmortem evaluation.<br />
Discussion:<br />
While in vivo imaging <strong>an</strong>d neuropathology were concord<strong>an</strong>t with respect to β-amyloid load in<br />
<strong>the</strong> non-demented older adult who progressed to dementia in some non-demented adults<br />
differences were observed between amyloid load detected in vivo by imaging <strong>an</strong>d CERAD<br />
ratings of amyloid plaques. More detailed investigations are needed to provide better<br />
underst<strong>an</strong>ding of <strong>the</strong> relationship between imaging <strong>an</strong>d neuropathological measures of β-amyloid<br />
load in non-demented older adults.<br />
Disclosures: J. Sojkova, None; D. Iacono, None; Y. Zhou, None; W. Ye, None; M.A. Kraut,<br />
None; L. Ferrucci, None; C.A. Mathis, GE Healthcare holds a license agreement with <strong>the</strong> U. of<br />
Pittsburgh based on <strong>the</strong> PIB technology. I am co-inventors of PIB <strong>an</strong>d, as such, have a fin<strong>an</strong>cial<br />
interest in this license agreement., O<strong>the</strong>r; W.E. Klunk, GE Healthcare holds a license agreement<br />
with <strong>the</strong> U. of Pittsburgh based on <strong>the</strong> PIB technology. I am co-inventors of PIB <strong>an</strong>d, as such,<br />
have a fin<strong>an</strong>cial interest in this license agreement., O<strong>the</strong>r; D.F. Wong, None; J.C. Troncoso,<br />
None; S.M. Resnick, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.25/N9<br />
Topic: C.05.b. Physiological <strong>an</strong>d molecular correlates<br />
Support: NIH NR010827<br />
NIH NS042861<br />
Athinoula A. Martinos Center <strong>for</strong> Biomedical Imaging<br />
NIH P41RR14075<br />
Title: The association between white matter integrity <strong>an</strong>d cortical thickness in younger <strong>an</strong>d older<br />
adults<br />
Authors: *D. H. SALAT 1 , S. Y. LEE 2 , H. D. ROSAS 2 ;<br />
1 MGH-NMR Ctr., Charlestown, MA; 2 Neurol., Massachusetts Gen. Hospital/Athinoula A.<br />
Martinos Ctr. <strong>for</strong> Biomed. Imaging, Charlestown, MA<br />
Abstract: It is now established that <strong>the</strong> brain undergoes complex ch<strong>an</strong>ges in gray <strong>an</strong>d white<br />
matter tissue integrity with adv<strong>an</strong>cing age. Little in<strong>for</strong>mation exists; however, about whe<strong>the</strong>r<br />
ch<strong>an</strong>ges in gray <strong>an</strong>d white matter are driven by common mech<strong>an</strong>isms or whe<strong>the</strong>r <strong>the</strong>se different<br />
tissue classes exhibit a differential <strong>an</strong>d independent decline with aging. We examined how white<br />
matter integrity measured by diffusion tensor imaging (DTI) is associated with cortical thickness<br />
in a population of 150 individuals across <strong>the</strong> adult age-sp<strong>an</strong>. Cortical thickness regionally<br />
declined with increasing age as described in previous work. Regional patterns of white matter<br />
loss with aging were dependent on <strong>the</strong> specific DTI metric examined. For example, whereas<br />
ch<strong>an</strong>ges in <strong>the</strong> fractional <strong>an</strong>isotropy were spread across a wide r<strong>an</strong>ge of <strong>an</strong>terior brain regions,<br />
<strong>the</strong> axial diffusivity was most affected in periventricular regions <strong>an</strong>d in <strong>the</strong> <strong>an</strong>terior limb of <strong>the</strong><br />
internal capsule <strong>an</strong>d radial diffusivity was strongly affected in prefrontal white matter. There<br />
were associations between cortical thickness <strong>an</strong>d whole brain diffusion measures; however, <strong>the</strong>se<br />
associations were regionally selective within <strong>the</strong> cortex. Statistically controlling <strong>for</strong> age in this<br />
<strong>an</strong>alysis resulted in a small number of remaining regional associations. Axial diffusivity showed<br />
a strong relationship with cortical thickness after controlling <strong>for</strong> age <strong>an</strong>d was increased in<br />
individuals with increased cortical thickness. Regionally, <strong>the</strong>re were associations between DTI<br />
FA in <strong>the</strong> <strong>an</strong>terior corpus callosum, but not posterior corpus callosum. These associations were<br />
minimal after statistically controlling <strong>for</strong> age. The associations between DTI measures <strong>an</strong>d<br />
cortical thickness were highly dependent on <strong>the</strong> procedure utilized <strong>for</strong> extracting <strong>the</strong> DTI<br />
measures, <strong>an</strong>d thus, it is possible that optimal procedures <strong>for</strong> examining such associations are yet<br />
to be elucidated. Never<strong>the</strong>less, <strong>the</strong>se results demonstrate that <strong>the</strong>re is a complex relationship<br />
between alterations in gray <strong>an</strong>d white matter tissue structure with aging.
Disclosures: D.H. Salat, None; S.Y. Lee, None; H.D. Rosas, None.<br />
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.26/N10<br />
Topic: C.05.c. Oxidative stress<br />
Support: NIH Gr<strong>an</strong>t PO1AG02219-28<br />
Title: Regulation of brain energy metabolism as influenced by diabetes in oldest-old dementia<br />
cases<br />
Authors: *W. ZHAO, X. QIAN, V. HAROUTUNIAN, G. M. PASINETTI;<br />
Psychiatry, Mount Sinai Sch. of Med., New York, NY<br />
Abstract: Recent evidence suggests that <strong>the</strong> neuropathological features of dementia in <strong>the</strong><br />
oldest-old were not <strong>the</strong> same as those of cognitively impaired younger-old persons. There is also<br />
evidence suggesting that reductions in cerebral metabolism sufficient to impair cognition in<br />
normal individuals also occur in Alzheimer’s disease (AD). Previous data <strong>from</strong> our lab indicates<br />
that diabetes may have some impact in AD. There<strong>for</strong>e, we tested whe<strong>the</strong>r impairments in<br />
tricarboxylic acid (TCA) cycle enzymes of mitochondria correlate with diabetes <strong>an</strong>d/or CDR<br />
between two different age groups: young-old (60-85 y.o.) <strong>an</strong>d oldest-old (86-106 y.o.). Postmortem<br />
brains samples <strong>from</strong> patients with autopsy-confirmed AD <strong>an</strong>d clinical dementia ratings<br />
(CDRs) be<strong>for</strong>e death were used in this study. We observed a faster decline in <strong>the</strong> expression<br />
level of <strong>the</strong> alpha-ketoglutarate dehydrogenase complex <strong>an</strong>d malate dehydrogenase in <strong>the</strong> brain<br />
samples (parahippocampal cortex area, BM36) of oldest-old diabetic subjects th<strong>an</strong> <strong>the</strong> nondiabetic<br />
subjects in <strong>the</strong> same age group. This study suggests that improvement of TCA cycle<br />
metabolism might benefit AD patients, especially those affected by diabetes. Fur<strong>the</strong>r<br />
investigations of mitochondrial oxidative profile <strong>an</strong>d TCA cycle enzyme activities are ongoing in<br />
<strong>the</strong> lab.<br />
Disclosures: W. Zhao, None; X. Qi<strong>an</strong>, None; V. Haroutuni<strong>an</strong>, None; G.M. Pasinetti, None.<br />
Poster
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.27/N11<br />
Topic: C.05.a. Molecular studies<br />
Title: Snapin-dynein-mediated coordination of tr<strong>an</strong>sport <strong>an</strong>d membr<strong>an</strong>e trafficking in regulating<br />
neuronal autophagy-lysosomal function<br />
Authors: *Q. CAI, L. LU, J.-H. TIAN, Y.-B. ZHU, Z.-H. SHENG;<br />
Synaptic Function Section, NINDS, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: Lysosomes are dynamic org<strong>an</strong>elles that receive <strong>an</strong>d degrade macromolecules <strong>an</strong>d<br />
org<strong>an</strong>elles <strong>from</strong> secretory, endocytic, <strong>an</strong>d autophagic pathways. Although some features of late<br />
endocytic trafficking have been described, regulation of this process in neurons remains to be<br />
elucidated. Here, we report <strong>an</strong> essential role <strong>for</strong> Snapin in regulating neuronal autophagylysosomal<br />
function by coordinating dynein-driven retrograde tr<strong>an</strong>sport <strong>an</strong>d late endocytic<br />
membr<strong>an</strong>e trafficking. First, deletion of <strong>the</strong> snapin gene in mice results in impaired late<br />
endocytic tr<strong>an</strong>sport <strong>an</strong>d aberr<strong>an</strong>t focal accumulation of late endosomes along neuronal processes,<br />
a phenotype that c<strong>an</strong> be efficiently rescued by reintroducing <strong>the</strong> snapin gene into mut<strong>an</strong>t neurons.<br />
Second, Snapin is relatively enriched in <strong>the</strong> late endocytic compartments. We show that Snapin<br />
stabilizes dynein motor complex attached to late endocytic org<strong>an</strong>elles. Third, snapin deficiency<br />
in mouse embryonic fibroblasts (MEFs) signific<strong>an</strong>tly results in impaired degradation of<br />
internalized EGF <strong>an</strong>d BSA-gold, retention of internalized Dextr<strong>an</strong> in late endosomes ra<strong>the</strong>r th<strong>an</strong><br />
delivery to lysosomes, <strong>an</strong>d reduced maturation of lysosomal enzyme ca<strong>the</strong>psin D. Consistent<br />
with a defect in lysosomal degradation, <strong>an</strong> abnormal accumulation of autolysosome-like<br />
structures was observed in snapin-deficient hippocampal slices, cultured cortical neurons, <strong>an</strong>d<br />
MEFs. Fur<strong>the</strong>rmore, we fur<strong>the</strong>r demonstrated that elevated Snapin expression facilitates dynamic<br />
<strong>for</strong>mation of tubular lysosomes by enh<strong>an</strong>cing retrograde tr<strong>an</strong>sport of late endosomes. Thus, using<br />
molecular <strong>an</strong>d cellular approaches <strong>an</strong>d time-lapse imaging in live cells combined with mouse<br />
genetic <strong>an</strong>alysis <strong>an</strong>d gene rescue experiments, our study provides mech<strong>an</strong>istic insights into <strong>the</strong><br />
spatiotemporal coordination of late endocytic tr<strong>an</strong>sport, membr<strong>an</strong>e trafficking, <strong>an</strong>d autophagylysosome<br />
function in neurons. Since impaired autophagy-lysosomal system has been implicated<br />
in neurodegeneration, Snapin-mediated up-regulation of autophagy-lysosomal function may<br />
provide a new regulatory pathway <strong>an</strong>d possible <strong>the</strong>rapies to remove aggregation-prone<br />
intracytosolic proteins that are associated with a variety of neurodegenerative diseases.<br />
(Supported by <strong>the</strong> intramural research program of NINDS, NIH)<br />
Disclosures: Q. Cai, None; L. Lu, None; J. Ti<strong>an</strong>, None; Y. Zhu, None; Z. Sheng, None.
Poster<br />
535. Aging of <strong>the</strong> Nervous System I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 535.28/N12<br />
Topic: C.11.g. Neuroprotective mech<strong>an</strong>isms: Oxidative stress<br />
Support: Health <strong>an</strong>d Labour Sciences Research Gr<strong>an</strong>ts<br />
Title: NMNAT expression in mitochondrial matrix delays Walleri<strong>an</strong> degeneration<br />
Authors: *N. YAHATA, S. YUASA, T. ARAKI;<br />
Natl. Inst. of Neuroscience, NCNP, Kodaira, Tokyo, Jap<strong>an</strong><br />
Abstract: Axonal degeneration is observed during pathogenesis of m<strong>an</strong>y neurodegenerative<br />
diseases <strong>an</strong>d after traumatic nerve injury. The active nature of axonal degeneration process was<br />
originally demonstrated by <strong>the</strong> discovery of naturally occurring mut<strong>an</strong>t mice, wld s , in which<br />
Walleri<strong>an</strong> degeneration in both central <strong>an</strong>d peripheral nervous systems shows a signific<strong>an</strong>t delay.<br />
The wld s mutation comprises <strong>an</strong> 85 kb t<strong>an</strong>dem triplication that results in overexpression of a<br />
chimeric molecule called Wld s protein. This protein consists of <strong>the</strong> N-terminal 70 amino acids of<br />
a ubiquitin ligase, Ufd2a, fused to <strong>the</strong> complete sequence of nicotinamide mononucleotide<br />
adenylyltr<strong>an</strong>sferase 1 (NMNAT1), <strong>an</strong> enzyme in <strong>the</strong> NAD syn<strong>the</strong>sis pathway. We previously<br />
showed that NMNAT overexpression in cultured neurons provides robust protection to neurite in<br />
<strong>the</strong> in vitro Walleri<strong>an</strong> degeneration model. To examine <strong>the</strong> effect of NMNAT overexpression in<br />
vivo <strong>an</strong>d to <strong>an</strong>alyze <strong>the</strong> mech<strong>an</strong>ism of axonal protection mediated by NMNAT, we generated<br />
tr<strong>an</strong>sgenic mice (Tg) overexpressing NMNAT1 (nuclear iso<strong>for</strong>m), NMNAT3 (mitochondrial<br />
iso<strong>for</strong>m), or <strong>the</strong> Wld s protein bearing a W258A mutation, which disrupts NMNAT enzymatic<br />
activity of <strong>the</strong> Wld s protein. The degree of Walleri<strong>an</strong>-degeneration delay in NMNAT3-Tg was<br />
similar to that in wld s mice, while axonal protection in NMNAT1-Tg or Wld s (W258A)-Tg was<br />
not detectable. Detailed <strong>an</strong>alysis of subcellular localization of <strong>the</strong> overexpressed proteins<br />
revealed that <strong>the</strong> axonal protection phenotype was correlated with <strong>the</strong> localization of NMNAT<br />
enzymatic activity to mitochondrial matrix. Fur<strong>the</strong>rmore, we found that isolated mitochondria<br />
<strong>from</strong> mice showing axonal protection expressed unch<strong>an</strong>ged levels of respiratory chain<br />
components, but were capable of increased ATP production. These results suggest that axonal<br />
protection by NMNAT expression in neurons is provided by modification of mitochondrial<br />
function. Alteration of mitochondrial function may constitute a novel treatment of diseases<br />
involving axonopathy.<br />
Disclosures: N. Yahata, None; S. Yuasa, None; T. Araki, None.
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.1/N13<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: The Texas A&M Institute <strong>for</strong> Genomic Medicine<br />
Title: Characterization of <strong>the</strong> Lphn3 null mut<strong>an</strong>t mouse: A model <strong>for</strong> ADHD <strong>an</strong>d addiction<br />
vulnerability?<br />
Authors: *B. SETLOW 1 , D. S. HILL 3 , L. C. ABBOTT 2 , R. H. FINNELL 4,3 , P. J. WELLMAN 1 ,<br />
D. WALLIS 4 ;<br />
1 Dept. of Psychology, 2 Vet. Integrative Biosci., Texas A&M Univ., College Station, TX; 3 Ctr.<br />
<strong>for</strong> Envrn. <strong>an</strong>d Genet. Med., Inst. of Biosci. <strong>an</strong>d Technology, Texas A&M Hlth. Sci. Ctr.,<br />
Houston, TX; 4 Texas A&M Inst. <strong>for</strong> Genomic Med., Texas A&M Hlth. Sci. Ctr., College<br />
Station, TX<br />
Abstract: Vulnerability to addiction <strong>an</strong>d ADHD are frequently comorbid, suggesting a common<br />
etiology. The LPHN3 gene has already been correlated in linkage <strong>an</strong>d association studies to both<br />
addictive phenotypes <strong>an</strong>d ADHD independently. Lphn3 is a member of <strong>the</strong> LPHN subfamily of<br />
G-protein coupled receptors. LPHN3 has been implicated in modulating neurotr<strong>an</strong>smitter release,<br />
although <strong>the</strong> endogenous lig<strong>an</strong>d is unknown. We have utilized a gene-trap embryonic stem cell<br />
line to generate mice mut<strong>an</strong>t <strong>for</strong> <strong>the</strong> Lphn3 gene. We report initial phenotypic characterization<br />
<strong>an</strong>d <strong>an</strong>alysis of this mut<strong>an</strong>t. We have begun our studies by evaluating <strong>the</strong> Lphn3 expression<br />
pattern. We find Lphn3 in <strong>the</strong> adult amygdala, hypothalamus, hippocampus, subcommissural<br />
org<strong>an</strong>, <strong>an</strong>d olfactory bulb. We have also evaluated differential gene expression between mut<strong>an</strong>t<br />
<strong>an</strong>d wildtype male littermates at postnatal day 0 in whole brain RNA. We have evaluated<br />
expression of genes commonly considered ADHD c<strong>an</strong>didate genes through TaqM<strong>an</strong> gene<br />
expression assays as well as evaluated genes identified as “neurogenesis <strong>an</strong>d neural stem cell<br />
marker” genes through <strong>the</strong> use of SABiosciences RT-PCR-array. Most notably, we find ch<strong>an</strong>ges<br />
in dopamine <strong>an</strong>d serotonin receptors <strong>an</strong>d tr<strong>an</strong>sporters (Dat1, Drd4, 5Htt, 5Ht2a), ch<strong>an</strong>ges in<br />
neurotr<strong>an</strong>smitter metabolism genes (Th, Gad1), as well as ch<strong>an</strong>ges in neural developmental<br />
genes (Nurr, Ncam,). Additional preliminary data indicate that <strong>the</strong> null mice have a hyperactive<br />
phenotype in <strong>the</strong> open field. We are following up with additional behavioral characterization, as<br />
well as brain region-specific <strong>an</strong>alyses of dopamine <strong>an</strong>d serotonin levels, which appear to be<br />
increased in <strong>the</strong> dorsal striatum of <strong>the</strong> mut<strong>an</strong>ts. Alterations in energy metabolism of monoamines<br />
have been posited as a core deficit in ADHD. Such alterations in <strong>the</strong>se mut<strong>an</strong>ts likely affect<br />
cognition, motor inhibition, <strong>an</strong>d vulnerability to addiction.
Disclosures: B. Setlow, None; D.S. Hill, None; L.C. Abbott, None; R.H. Finnell, None; P.J.<br />
Wellm<strong>an</strong>, None; D. Wallis, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.2/N14<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Title: The protective effect of fluvastatin on hydroxyl radical generation by inhibiting lowdensity<br />
lipoprotein (LDL) oxidation in <strong>the</strong> rat myocardium<br />
Authors: *T. OBATA;<br />
Analytical Chem., Ohu Univ., Koriyama, Jap<strong>an</strong><br />
Abstract: The present study examined <strong>the</strong> effect of fluvastatin on Cu 2+ -induced hydroxyl radical<br />
generation (•OH) in <strong>the</strong> extracellular fluid of rat myocardium using microdialysis technique (O<br />
system). Fluvastatin, a inhibitor of low-density lipoprotein (LDL) oxidation, was administered at<br />
dose of 5.0 mg/kg/day i.p. <strong>for</strong> 4 weeks. Rats were <strong>an</strong>es<strong>the</strong>tized <strong>an</strong>d sodium salicylate in Ringer's<br />
solution (0.5 nmol/µl/min) was infused through a microdialysis probe to detect <strong>the</strong> generation of<br />
•OH as reflected by <strong>the</strong> nonenzymatic <strong>for</strong>mation of 2,3-dihydroxybenzoic acid (DHBA) in <strong>the</strong><br />
myocardium. <strong>When</strong> CuSO4 was infused to through <strong>the</strong> microdialysis probe, CuSO4 clearly<br />
produced increase in •OH <strong>for</strong>mation trapped as 2,3-DHBA (R 2 = 0.983). However, when<br />
corresponding experiments were per<strong>for</strong>med with fluvastatin (5.0 mg/kg/day i.p. <strong>for</strong> 4 weeks)<br />
pretreated <strong>an</strong>imals, small increases in <strong>the</strong> level of 2,3-DHBA products were observed. <strong>When</strong><br />
LDL are oxidized by Cu 2+ , Cu 2+ c<strong>an</strong> be reduced to Cu 1+ by LDL. Fenton-type reactions in <strong>the</strong><br />
presence of Cu 1+ <strong>an</strong>d yields highly cytotoxic •OH. These results suggest that Cu 2+ -induced •OH<br />
generation may reduce by inhibiting LDL oxidation with fluvastatin.<br />
Disclosures: T. Obata , None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.3/N15<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH MH067938<br />
NIH HD02528<br />
NIH RR016475<br />
Title: Dietary omega-3 fatty acid content <strong>an</strong>d age affect activity <strong>an</strong>d response to novelty in rats<br />
Authors: *B. LEVANT 1 , T. J. ZARCONE 2 , S. C. FOWLER 3 ;<br />
1 Univ. K<strong>an</strong>sas Med. Ctr., K<strong>an</strong>sas City, KS; 2 Univ. of Rochester Sch. of Med. <strong>an</strong>d Dent.,<br />
Rochester, NY; 3 Univ. of K<strong>an</strong>sas, Lawrence, KS<br />
Abstract: Insufficient availability of n-3 polyunsaturated fatty acids (PUFAs) during <strong>the</strong> pre-<br />
<strong>an</strong>d neonatal periods decreases accretion of omega-3 docosahexaenoic acid (DHA, 22:6n-3) in<br />
<strong>the</strong> developing brain; <strong>an</strong>d is associated with sub-optimal sensory <strong>an</strong>d cognitive function in<br />
hum<strong>an</strong>s, altered behavior in <strong>an</strong>imals, <strong>an</strong>d may contribute to neurodevelopmental disorders<br />
including attention deficit hyperactivity disorder. In this study, male Long-Ev<strong>an</strong>s rats were raised<br />
<strong>from</strong> conception on ei<strong>the</strong>r a control diet (AIN-93G) containing α-linolenic acid (18:3n-3), <strong>the</strong><br />
dietarily essential fatty acid precursor of DHA, or a deficient diet lacking α-linolenic acid. The<br />
deficient diet resulted in a decrease in brain phospholipid DHA content of ~45% compared to<br />
controls, measured by TLC/GC. Rats were assessed in a response to novelty task at various ages<br />
(28, 35, 42, 49, 56, 70 days of age). The response to novelty task involved introduction of a<br />
novel spatial stimulus into a familiar environment, <strong>an</strong>d consisted of three time periods: (1) a<br />
novel environment - 30 min, (2) a familiar environment - 20 min, <strong>an</strong>d (3) a ch<strong>an</strong>ge in <strong>the</strong> familiar<br />
environment - 20 min. Behavior was assessed using <strong>for</strong>ce-plate actometers. There was a<br />
signific<strong>an</strong>t effect of diet on dist<strong>an</strong>ce traveled in Period 1, but this effect was not present during<br />
Period 2 <strong>an</strong>d Period 3. Diet did not interact with age in <strong>an</strong>y of <strong>the</strong> periods. In Period 1, diet<br />
signific<strong>an</strong>tly interacted with time block within <strong>the</strong> session. Within Period 1, <strong>the</strong> largest<br />
differences between <strong>the</strong> control <strong>an</strong>d deficient diets occurred in <strong>the</strong> first two time blocks of Period<br />
1, with <strong>the</strong> control diet producing less dist<strong>an</strong>ce th<strong>an</strong> <strong>the</strong> deficient diet. One interpretation of this<br />
pattern of effects <strong>for</strong> diet is that <strong>the</strong> deficient-diet <strong>an</strong>imals were more reactive to <strong>the</strong> novel<br />
environment when <strong>the</strong> novelty was at its highest intensity (early in <strong>the</strong> first exposure to <strong>the</strong><br />
recording chamber). However, in Period 3, <strong>the</strong> introduction of a ch<strong>an</strong>ge in <strong>the</strong> by-this-timethoroughly-explored<br />
(<strong>an</strong>d <strong>the</strong>re<strong>for</strong>e familiar) environment resulted in strong stimulation of<br />
movement about equally <strong>for</strong> rats on both diets. Age had a signific<strong>an</strong>t main effect on dist<strong>an</strong>ce<br />
traveled, which declined monotonically <strong>an</strong>d approximately linearly as a function of age <strong>for</strong> each<br />
of <strong>the</strong> three periods. These data suggest that this method is a useful approach <strong>for</strong> assessing<br />
activity <strong>an</strong>d response to novelty, <strong>an</strong>d that dietary n-3 PUFA content affects <strong>the</strong>se processes.
Disclosures: B. Lev<strong>an</strong>t, Friesl<strong>an</strong>d Foods, D. Speakers Bureau/Honoraria (speakers bureau,<br />
symposia, <strong>an</strong>d expert witness); Medical DecisionPoint, F. Consult<strong>an</strong>t/Advisory Board; T.J.<br />
Zarcone, I receive royalties <strong>from</strong> BASi based on <strong>the</strong>, E. Ownership Interest (stock, stock<br />
options, patent or o<strong>the</strong>r intellectual property); S.C. Fowler, I receive royalties <strong>from</strong> BASi based<br />
on <strong>the</strong>, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.4/N16<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: March of Dimes 12-FY05-1198<br />
Title: Strain-dependent ch<strong>an</strong>ges in acoustic startle response <strong>an</strong>d its plasticity across adolescence<br />
in inbred mice<br />
Authors: *S. PIETROPAOLO, W. E. CRUSIO;<br />
Bordeaux Univ., Talence, Fr<strong>an</strong>ce<br />
Abstract: Converging lines of evidence have demonstrated <strong>the</strong> critical role of adolescence in <strong>the</strong><br />
development <strong>an</strong>d expression of several hum<strong>an</strong> neuropsychiatric disorders. Fur<strong>the</strong>rmore, <strong>the</strong><br />
adolescent high vulnerability to mental illness is reportedly influenced by genetic factors,<br />
although little is known about <strong>the</strong> genetic modulation of neurobehavioural development during<br />
this ontogenetic phase. Here we investigated -<strong>for</strong> <strong>the</strong> first time in mice- <strong>the</strong> adolescent expression<br />
of acoustic startle response <strong>an</strong>d its plasticity, i.e., a behavioural domain which is altered in<br />
several neuropsychiatric pathologies, including those with a typical adolescent onset, such as<br />
schizophrenia <strong>an</strong>d ADHD. Startle reactivity, its habituation, <strong>an</strong>d prepulse inhibition (PPI) were<br />
assessed in C57BL/6J <strong>an</strong>d DBA/2J mice at three age points, i.e., at 4 (pre-pubertal age), 6 (periadolescence)<br />
<strong>an</strong>d 8 (early adulthood) weeks of age. Pre-pubertal mice displayed reduced startle<br />
reactivity <strong>an</strong>d altered PPI compared to adult <strong>an</strong>imals, but <strong>the</strong>se effects were observed only in <strong>the</strong><br />
C57BL/6J strain. Strain differences were also clearly detected <strong>for</strong> <strong>the</strong> startle response, its<br />
habituation, <strong>an</strong>d PPI. All <strong>the</strong> effects were critically modulated by <strong>the</strong> intensity of <strong>the</strong> pulse<br />
stimulus <strong>an</strong>d were not confounded by differences in <strong>an</strong>xiety-like behaviour as assessed in <strong>the</strong><br />
elevated plus maze. Our results highlight <strong>the</strong> import<strong>an</strong>ce of genetic factors <strong>an</strong>d of <strong>the</strong> early<br />
adolescent phase <strong>for</strong> <strong>the</strong> design of mouse models of sensory-motor disturb<strong>an</strong>ces.<br />
Disclosures: S. Pietropaolo, None; W.E. Crusio, None.
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.5/N17<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: Kavli Institute of Neuroscience at Yale<br />
Title: Cross-species expression profiles in alcohol intoxication models of hum<strong>an</strong> <strong>an</strong>d mouse<br />
cortical development<br />
Authors: *K. HASHIMOTO-TORII, Y. IMAMURA-KAWASAWA, P. RAKIC;<br />
Dept. Neurobiol, Sch. Med, Yale Univ., New Haven, CT<br />
Abstract: Alcohol consumption during pregn<strong>an</strong>cy is among critical environmental stressors that<br />
c<strong>an</strong> lead to devastating teratogenetic consequences during fetal development, particularly <strong>for</strong> <strong>the</strong><br />
<strong>for</strong>mation of complex neuronal org<strong>an</strong>ization <strong>an</strong>d synaptic circuitry in <strong>the</strong> brain. The perm<strong>an</strong>ent<br />
birth defects caused by alcohol misuse known as Fetal Alcohol Spectrum Disorder (FASD) are<br />
particularly prominent in <strong>the</strong> cerebral cortex. A broad spectrum of cortical dysplasia, such as<br />
microcephaly, hydrocephaly, heterotopias <strong>an</strong>d agenesis of <strong>the</strong> corpus callosum, has been<br />
observed in <strong>the</strong> FASD. In order to get insights into molecular <strong>an</strong>d cellular mech<strong>an</strong>isms causing<br />
different FASD phenotypes in <strong>the</strong> cerebral cortex, we have initiated genome-wide <strong>an</strong>alyses of<br />
fetal alcohol exposure in <strong>the</strong> developing rodent <strong>an</strong>d hum<strong>an</strong> <strong>for</strong>ebrain. In order to distinguish<br />
model-specific pseudo gene response of alcohol exposure, we screened genes conserved in both<br />
hum<strong>an</strong> in vitro chronic exposure <strong>an</strong>d mouse in vivo acute exposure models. By using fresh<br />
hum<strong>an</strong> fetal tissues, we were able to per<strong>for</strong>m genome-wide expression <strong>an</strong>alysis under chronic<br />
alcohol exposure <strong>an</strong>d compare it to <strong>the</strong> data obtained in mouse. By a combination of Weighed<br />
Average Difference method (WAD) <strong>an</strong>d Annotation Tools, we identified over thous<strong>an</strong>d gene sets<br />
which expression signific<strong>an</strong>tly ch<strong>an</strong>ged in both hum<strong>an</strong> <strong>an</strong>d mouse after alcohol exposure. Timeseries<br />
observation of gene expression levels after alcohol exposure revealed, <strong>an</strong> early peak of<br />
stress signaling genes followed by a late onset of <strong>the</strong> expression ch<strong>an</strong>ges of genes that are<br />
required <strong>for</strong> corticogenesis <strong>an</strong>d also observed some difference in gene expression between <strong>the</strong><br />
mouse <strong>an</strong>d hum<strong>an</strong>. Network <strong>an</strong>alysis of gene clustering is underway to identify functionally<br />
signific<strong>an</strong>t genes involved in each species. Our results indicate that cellular stress signaling<br />
might influence <strong>the</strong> tr<strong>an</strong>scription of genes involved in particular aspects of cortical development,<br />
leading to specific pathological pictures, <strong>an</strong>d that <strong>the</strong> intersection of listed stress-responsible<br />
genes <strong>an</strong>d cortical development genes might be a critical target in spectrum of FASD.
Disclosures: K. Hashimoto-Torii, None; Y. Imamura-Kawasawa, None; P. Rakic, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.6/N18<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: AP-HP<br />
INSERM<br />
FRM EQUIPEs 2007<br />
ANR Neuro 2005 project A05183KS<br />
ANR-06-NEURO-008-01 RPV06055ASA<br />
EMBO short-term fellowship<br />
Université Paris Descartes<br />
Title: Mutations in <strong>the</strong> β-tubulin gene TUBB2B result in asymmetrical polymicrogyria<br />
Authors: *X. H. JAGLIN 1 , K. POIRIER 1 , Y. SAILLOUR 1 , N. BAHI-BUISSON 1,2 , E.<br />
BUHLER 3,4 , G. TIAN 5 , C. FALLET-BIANCO 6 , F. PHAN-DINH-TUY 1,7 , C. CARDOSO 3 , A.<br />
REPRESA 3 , D. A. KEAYS 8 , N. J. COWAN 5 , J. CHELLY 1 ;<br />
1 INSERM U567 / CNRS UMR8104 / Univ. Paris Descartes, Paris, Fr<strong>an</strong>ce; 2 Service de<br />
Neurologie Pédiatrique, Hôpital Necker Enf<strong>an</strong>ts Malades, Paris, Fr<strong>an</strong>ce; 3 INSERM U901,<br />
Marseille, Fr<strong>an</strong>ce; 4 Plate-<strong>for</strong>me Post Génomique de l’INMED, Marseille, Fr<strong>an</strong>ce; 5 Dept. of<br />
Biochemistry, New York Univ. Med. Ctr., New York, NY; 6 Service d’Anatomie Pathologique,<br />
Hôpital Sainte Anne, Paris, Fr<strong>an</strong>ce; 7 INSERM UMR-S 839, Inst. du Fer à Moulin, Paris, Fr<strong>an</strong>ce;<br />
8 Inst. of Mol. Pathology, Vienna, Austria<br />
Abstract: The crucial role of tubulins in diverse cellular processes <strong>an</strong>d <strong>the</strong> recent association of<br />
TUBA1A mutations with a broad lissencephaly spectrum led us to hypo<strong>the</strong>size that mutations in<br />
o<strong>the</strong>r tubulin genes that are highly expressed during central nervous system development might
also result in mal<strong>for</strong>mations of cortical development.<br />
We report de novo mutations in a β-tubulin gene, TUBB2B, in four individuals <strong>an</strong>d a 27gestational-week<br />
fetus with bilateral asymmetrical polymicrogyria (PMG). PMG is a relatively<br />
common defect of cortical development usually classified among neuronal migration disorders. It<br />
is characterized by numerous small gyri <strong>an</strong>d a thick disorg<strong>an</strong>ized cortical plate lacking normal<br />
lamination. However, its etiologies <strong>an</strong>d pathogeneses remain poorly understood. We show that<br />
<strong>the</strong> five newly discovered disease-associated TUBB2B mutations involve a spectrum of tubulin<br />
heterodimer assembly defects, leading to loss of function in <strong>the</strong> cases of two mut<strong>an</strong>ts. Second,<br />
neuropathological examination of <strong>the</strong> fetus revealed <strong>an</strong> absence of cortical lamination associated<br />
with <strong>the</strong> presence of ectopic neuronal cells in <strong>the</strong> white matter. There<strong>for</strong>e, we tested <strong>the</strong><br />
implication of TUBB2B in <strong>the</strong> migration of neurons during corticogenesis using <strong>an</strong> in utero<br />
RNAi-based inactivation <strong>an</strong>d demonstrated that <strong>the</strong> expression of TUBB2B is indeed required <strong>for</strong><br />
proper migration of neurons. We also <strong>an</strong>alyzed <strong>the</strong> consequences of TUBB2B inactivation on<br />
radial glial cells morphology to relate to <strong>the</strong> neuropathological <strong>an</strong>alyses. We indeed observed<br />
ectopic neurons in <strong>the</strong> leptomeningeal spaces due to breaches in <strong>the</strong> pial basement membr<strong>an</strong>e,<br />
associated with a severe disruption of radial glial cells org<strong>an</strong>ization.<br />
These observations, toge<strong>the</strong>r with previous data, show that disruption of microtubule-based<br />
processes underlies a large spectrum of neuronal migration disorders that includes not only<br />
lissencephaly <strong>an</strong>d pachygyria, but also polymicrogyria mal<strong>for</strong>mations.<br />
Disclosures: X.H. Jaglin, None; K. Poirier, None; Y. Saillour, None; N. Bahi-Buisson,<br />
None; E. Buhler, None; G. Ti<strong>an</strong>, None; C. Fallet-Bi<strong>an</strong>co, None; F. Ph<strong>an</strong>-Dinh-Tuy, None; C.<br />
Cardoso, None; A. Represa, None; D.A. Keays, None; N.J. Cow<strong>an</strong>, None; J. Chelly, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.7/N19<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NCTR Protocol E7272<br />
Title: Cocaine preference in adult male <strong>an</strong>d female rats is not altered by adolescent<br />
methylphenidate treatment<br />
Authors: *S. A. FERGUSON 1,2 , J. BOYD-COOK 3 , S. Y. BOCTOR 1,2 ;<br />
1 Natl. Ctr. Toxicological Res., Jefferson, AR; 2 Interdisciplinary Biomed. Sci., Univ. of Ark<strong>an</strong>sas<br />
<strong>for</strong> Med. Sci., Little Rock, AR; 3 Univ. of Ark<strong>an</strong>sas at Little Rock, Little Rock, AR
Abstract: Methylphenidate (MPH) treatment in boys with Attention Deficit Hyperactivity<br />
Disorder reduces <strong>the</strong> risk of drug abuse in adulthood <strong>an</strong>d, in male rats, appears to decrease<br />
cocaine preference in conditioned place preference (CPP) tests. However, <strong>the</strong> effects of<br />
developmental MPH treatment on later subst<strong>an</strong>ce use disorders in girls/women or in female<br />
rodents using CPP assessments have not been fully examined. Here, male <strong>an</strong>d female Sprague-<br />
Dawley rats (n=34/sex/treatment) were treated orally on postnatal days (PNDs) 29-50 with water<br />
or 3 mg MPH/kg body weight 3X/day, a dose shown to produce serum levels within <strong>the</strong> hum<strong>an</strong><br />
clinical r<strong>an</strong>ge. On PND 62, all rats were assessed in <strong>an</strong> initial preconditioning session (15 min).<br />
For each rat, <strong>the</strong> least preferred chamber in this session was subsequently paired with 10 mg/kg<br />
cocaine (ip) while <strong>the</strong> preferred chamber was paired with saline (ip). On <strong>the</strong> subsequent 8 days<br />
(PNDs 63-70), each rat alternated between cocaine <strong>an</strong>d saline sessions (n=4 each; 30 min each).<br />
On PND 71, no injections occurred <strong>an</strong>d rats had access to both chambers during this<br />
postconditioning session (15 min). Pre- <strong>an</strong>d postconditioning sessions were evaluated by testers<br />
blind to treatment groups. Gonadal hormones c<strong>an</strong> alter drug effects in rodents; thus, each daily<br />
CPP assessment was followed by vaginal cytology in females while males were h<strong>an</strong>dled<br />
similarly <strong>for</strong> <strong>the</strong> same duration. MPH treatment did not alter body weight nor did it interact with<br />
CPP conditioning. All rats exhibited more time in <strong>the</strong> cocaine-paired chamber postconditioning<br />
th<strong>an</strong> preconditioning (p
NIH gr<strong>an</strong>t GM068596<br />
Title: The lissencephaly gene product, Lis1 cooperates with cytoplasmic dynein <strong>an</strong>d spectrin in<br />
<strong>the</strong> mainten<strong>an</strong>ce of <strong>the</strong> endoplasmic reticulum (ER) <strong>an</strong>d <strong>the</strong> ER-Golgi intermediate compartment<br />
Authors: *V. MURESAN, Z. MURESAN;<br />
Pharmacol. & Physiol., UMDNJ-New Jersey Med. Sch., Newark, NJ<br />
Abstract: The morphogenesis of complex membr<strong>an</strong>e tube networks, such as those of <strong>the</strong><br />
endoplasmic reticulum (ER) implicates <strong>the</strong> members of <strong>the</strong> reticulon/Yop1p families. However,<br />
<strong>the</strong>se models do not account <strong>for</strong> <strong>the</strong> participation of <strong>the</strong> microtubule cytoskeleton in <strong>the</strong><br />
mainten<strong>an</strong>ce of ER in vivo. Here, with <strong>the</strong> neuronal cell line CAD, we report that cytoplasmic<br />
dynein, spectrin, Lis1, <strong>an</strong>d <strong>the</strong> microtubules are essential <strong>for</strong> <strong>the</strong> structural integrity of <strong>the</strong> ER<br />
<strong>an</strong>d of <strong>the</strong> ER-Golgi intermediate compartment (ERGIC). Mech<strong>an</strong>istically, this result is<br />
explained by <strong>the</strong> ability of dynein-dynactin to bind at <strong>the</strong> same time to microtubules directly <strong>an</strong>d<br />
to acidic phospholipids within membr<strong>an</strong>es, via <strong>an</strong> org<strong>an</strong>elle-associated spectrin (Mures<strong>an</strong> V., et<br />
al., Molecular Cell, 2001), <strong>an</strong>d by <strong>the</strong> ability of this set of proteins to generate dynamic, complex<br />
tubular membr<strong>an</strong>e networks in vitro (Z. Mures<strong>an</strong>, B. J. Schnapp, <strong>an</strong>d V. Mures<strong>an</strong>, unpublished).<br />
Here we report that βIII spectrin <strong>an</strong>d reticulon 4 (ret4) localize in part to <strong>the</strong><br />
filamentous/vesicular structures of ER, which overlap <strong>an</strong>d intersect extensively with <strong>the</strong><br />
microtubule network. The depolymerization of microtubules induced by cold treatment<br />
redistributes ret4- <strong>an</strong>d βIII spectrin-labeled structures <strong>from</strong> <strong>the</strong> perinuclear area, into a<br />
fragmented <strong>an</strong>d diffuse pattern within <strong>the</strong> cytoplasm, a response typical <strong>for</strong> <strong>the</strong> loss of activity of<br />
cytoplasmic dynein. The cold treatment selectively disrupts acetylated microtubules, suggesting<br />
that <strong>the</strong>se dynamically stable, yet cold sensitive microtubules are essential <strong>for</strong> <strong>the</strong> mainten<strong>an</strong>ce of<br />
<strong>the</strong> ER network in vivo. Downregulation of βIII spectrin leads to redistribution <strong>an</strong>d aggregation<br />
of <strong>the</strong> ret4-associated structures to <strong>the</strong> cell periphery; <strong>the</strong> silencing of Lis1, a regulator of<br />
cytoplasmic dynein that is mutated in some <strong>for</strong>ms of lissencephaly, also disperses <strong>the</strong> ret4<br />
network. These treatments also cause delocalization of <strong>the</strong> tubulo-vesicular structures of <strong>the</strong><br />
ERGIC. Import<strong>an</strong>tly, at only slightly reduced levels of Lis1, insufficient to disrupt <strong>the</strong><br />
microtubule network, ERGIC is severely dispersed, indicating that Lis1 could regulate <strong>the</strong><br />
association of <strong>the</strong> ERGIC with <strong>the</strong> microtubules. We conclude that <strong>the</strong> reticulon/Yop1p <strong>an</strong>d <strong>the</strong><br />
dynein/spectrin/Lis1 complex cooperate in <strong>the</strong> mainten<strong>an</strong>ce of <strong>the</strong> dynamic membr<strong>an</strong>e tube<br />
network of ER, whose structural integrity requires <strong>the</strong> presence of acetylated microtubules. In<br />
addition, along with dynein <strong>an</strong>d spectrin, Lis1 appears to function to maintain <strong>the</strong> structure of<br />
ERGIC. We propose that <strong>the</strong> integrity of <strong>the</strong> ER <strong>an</strong>d of <strong>the</strong> ER-to-Golgi tr<strong>an</strong>sport may be<br />
disrupted in lissencephaly. Supported by March of Dimes (1-FY04-240) <strong>an</strong>d NIH<br />
(R01GM068596).<br />
Disclosures: V. Mures<strong>an</strong> , None; Z. Mures<strong>an</strong>, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.9/N21<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t DK49782<br />
Title: Distribution of neuropathology in beta-m<strong>an</strong>nosidase-deficient mice<br />
Authors: *K. L. LOVELL 1 , M. ZHU 3 , M. C. DRUMMOND 2 , R. C. SWITZER, III 4 , K. H.<br />
FRIDERICI 2 ;<br />
1 Neurology/Ophthalmology, 2 Microbiology & Mol. Genet., Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing,<br />
MI; 3 Genet., Harvard Med. Sch., Boston, MA; 4 NeuroScience Associates, Knoxville, TN<br />
Abstract: Beta-M<strong>an</strong>nosidosis is <strong>an</strong> autosomal recessive inherited disorder of glycoprotein<br />
catabolism, caused by a deficiency of <strong>the</strong> lysosomal enzyme beta-m<strong>an</strong>nosidase. This lysosomal<br />
storage disease is characterized by <strong>the</strong> intracellular accumulation of small oligosaccharides in<br />
selected cell types. In hum<strong>an</strong> cases of beta-m<strong>an</strong>nosidosis <strong>the</strong> clinical symptoms, including mental<br />
retardation, are variable <strong>an</strong>d relatively mild, <strong>an</strong>d little is known about <strong>the</strong> pathology. In contrast,<br />
two rumin<strong>an</strong>t <strong>an</strong>imal models (goats <strong>an</strong>d cows) have a severe clinical presentation at birth <strong>an</strong>d<br />
pathology includes severe myelin deficits as well as widespread cytoplasmic vacuolation. A<br />
mouse model was created by targeted disruption of <strong>the</strong> beta-m<strong>an</strong>nosidase gene by homologous<br />
recombination in 129X1/SvJ ES cells. Homozygous mut<strong>an</strong>t <strong>an</strong>imals had <strong>the</strong> expected enzyme<br />
deficiency, but showed normal growth, appear<strong>an</strong>ce <strong>an</strong>d lifesp<strong>an</strong>. Previous examination of mut<strong>an</strong>t<br />
<strong>an</strong>imals between 1 <strong>an</strong>d 9 months of age showed selective, variable neuronal vacuolation with no<br />
hypomyelination, closer to <strong>the</strong> hum<strong>an</strong> beta-m<strong>an</strong>nosidosis phenotype. The current study<br />
characterized distribution of brain pathology in mut<strong>an</strong>t mice between <strong>the</strong> ages of 15 <strong>an</strong>d 20<br />
months, investigating <strong>the</strong> effects of two different ES clones <strong>an</strong>d two strain backgrounds<br />
(129X1/SvJ congenic <strong>an</strong>d mixed C57BL/6J / 129X1/SvJ). After perfusion, <strong>the</strong> brains of<br />
homozygous mut<strong>an</strong>t (n=21) <strong>an</strong>d wild-type (n=4) mice were embedded in a 5 x 5 array using<br />
MultiBrain Technology (NeuroScience Associates, Knoxville, TN). Sections were stained with<br />
H&E, with Weil myelin stain, <strong>an</strong>d with amino cupric silver stain to reveal disintegrative<br />
degeneration. Analysis indicated a severe consistent pattern of neuronal vacuolation <strong>an</strong>d<br />
disintegrative degeneration in all five 129X1/SvJ mice (clone 1A2). However, <strong>the</strong> mixed<br />
background mice (1A2 <strong>an</strong>d 1B2 clones) showed subst<strong>an</strong>tial variability in <strong>the</strong> severity of<br />
pathology. In <strong>the</strong> severely affected <strong>an</strong>imals (all 129X1/SvJ mut<strong>an</strong>t mice, some mixed<br />
background mice), neuronal vacuolation was prominent in specific layers of piri<strong>for</strong>m area,<br />
retrosplenial area, <strong>an</strong>terior cingulate area, primary motor <strong>an</strong>d somatosensory cortex, selected<br />
association areas, <strong>an</strong>d in hippocampus CA3. Degeneration staining did not occur in areas of<br />
vacuolation. Silver degeneration reaction product was prominent in regions including specific<br />
cortical layers <strong>an</strong>d cerebellar molecular layer. The factors responsible <strong>for</strong> <strong>the</strong> specific pattern of<br />
neuropathology, <strong>an</strong>d <strong>the</strong> factors underlying variation in severity of pathology in different mouse
strains, have not yet been determined. Fur<strong>the</strong>r research will help to underst<strong>an</strong>d <strong>the</strong> pathogenesis<br />
of phenotypic variations.<br />
Disclosures: K.L. Lovell, None; M. Zhu, None; M.C. Drummond, None; R.C. Switzer,<br />
NeuroScience Associates, Knoxville, TN, A. Employment (full or part-time); NeuroScience<br />
Associates, Knoxville, TN, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); K.H. Friderici, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.10/N22<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: NATIONAL RESEARCH SERVICE AWARDS IN DEVELOPMENT, GENETICS<br />
AND COMPUTATIONAL BIOLOGY<br />
Title: STRADalpha regulates mTOR signaling <strong>an</strong>d lamination during corticogenesis<br />
Authors: *K. ORLOVA 1 , W. E. PARKER 1 , G. G. HEUER 2 , J. YOON 3 , M. BAYBIS 1 , R. S.<br />
FENNING 4 , K. A. STRAUSS 5 , P. B. CRINO 1 ;<br />
1 Neurol., 2 Neurosurg., Univ. of Pennsylv<strong>an</strong>ia Sch. of Med., Philadelphia, PA; 3 Col. of Arts <strong>an</strong>d<br />
Sci., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA; 4 Med., Hosp. of <strong>the</strong> Univ. of Pennsylv<strong>an</strong>ia,<br />
Philadelphia, PA; 5 The Clin. <strong>for</strong> Special Children, L<strong>an</strong>caster County, PA<br />
Abstract: Pretzel syndrome (PS), a recently described autosomal recessive disorder in Old Order<br />
Mennonite children, is characterized by abnormal brain development, severe cognitive disability,<br />
<strong>an</strong>d intractable epilepsy. All affected patients harbor a large homozygous deletion in <strong>the</strong><br />
LYK5/STRADa gene (17q23.3), which encodes <strong>the</strong> pseudokinase STRADα (STE20 Related<br />
Adaptor alpha). The underlying pathogenic mech<strong>an</strong>isms of PS <strong>an</strong>d <strong>the</strong> role of STRADα in<br />
cortical development remain unknown. The only characterized function of STRADα is to bind<br />
LKB1, effecting <strong>the</strong> subcellular localization of LKB1 <strong>an</strong>d enh<strong>an</strong>cing its catalytic activity. LKB1<br />
is a serine/threonine kinase that serves as a master upstream kinase to m<strong>an</strong>y AMPK-related<br />
kinases, including AMPK <strong>an</strong>d SAD-A/B Kinases. Phosphorylated AMPK inhibits mTOR<br />
signaling while phosphorylated SAD-A/B controls neuronal polarization <strong>an</strong>d axonogenesis. In<br />
addition, recent reports suggest that LKB1 regulates neuronal migration during corticogenesis.<br />
To define <strong>the</strong> role of STRADα during corticogenesis, we <strong>an</strong>alyzed postmortem PS brain tissue
<strong>an</strong>d examined <strong>the</strong> function of STRADα using in vitro <strong>an</strong>d in vivo strategies. Histopathological<br />
<strong>an</strong>alysis of postmortem PS brain reveals exclusively nuclear localization of LKB1, hyperactive<br />
mTOR signaling, neuronal cytomegaly, neuronal heterotopia in <strong>the</strong> subcortical white matter, <strong>an</strong>d<br />
aberr<strong>an</strong>t expression of select stem cell protein markers. Overexpression or shRNA-mediated<br />
knockdown studies in mouse neural progenitor cells (mNPCs) demonstrate that STRADα<br />
regulates mTOR signaling in <strong>an</strong> AMPK- <strong>an</strong>d rapamycin-dependent m<strong>an</strong>ner. STRADα-depleted<br />
mNPCs exhibit nuclear LKB1 staining in contrast to control cells, which express LKB1 in <strong>the</strong><br />
cytoplasmic compartment. Knockdown of STRADα in mNPCs results in cytomegaly that is<br />
prevented by rapamycin treatment. We <strong>the</strong>n utilized in utero electroporation to demonstrate that<br />
STRADα depletion at embryonic day 14 (E14) disrupts neuronal lamination during<br />
corticogenesis. Specifically, STRADα-depleted cells remain in <strong>the</strong> ventricular/subventricular<br />
zones (VZ/SVZ) at E17 <strong>an</strong>d E19 instead of assuming <strong>the</strong>ir appropriate laminar position within<br />
<strong>the</strong> cortical plate (CP). This is of particular interest, since PS cortex contains abund<strong>an</strong>t neuronal<br />
heterotopia in <strong>the</strong> subcortical white matter, fur<strong>the</strong>r supporting a critical role of STRADα in<br />
neuronal migration. These studies, <strong>for</strong> <strong>the</strong> first time, delineate a functional role of STRADα<br />
during corticogenesis <strong>an</strong>d suggest a potential <strong>the</strong>rapeutic role of rapamycin in PS.<br />
Disclosures: K. Orlova, None; W.E. Parker, None; G.G. Heuer, None; J. Yoon, None; M.<br />
Baybis, None; R.S. Fenning, None; K.A. Strauss, None; P.B. Crino, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.11/N23<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> Young Scientists (B) 21700452<br />
Title: Comprehensive phenotype <strong>an</strong>alyses of ENU induced mut<strong>an</strong>t mouse that carries a missense<br />
mutation in Grin1 gene<br />
Authors: *T. FURUSE 1 , Y. WADA 2 , K. HATTORI 3 , I. YAMADA 1 , T. KUSHIDA 1 , Y.<br />
SHIBUKAWA 1 , H. KANEDA 1 , I. MIURA 1 , K. KOBAYASHI 1 , H. MASUYA 1 , S. YUASA 3 , S.<br />
WAKANA 1 ;<br />
1 RIKEN BRC, Tsukuba/Ibaraki, Jap<strong>an</strong>; 2 Hlth. Sci. Univ., Kawaguchiko-cho, Jap<strong>an</strong>; 3 Dept. of<br />
Ultrastructural Res., Natl. Inst. of Neuroscience, Natl. Ctr. of Neurol. <strong>an</strong>d Psychiatry, Kodaira,<br />
Jap<strong>an</strong>
Abstract: Attention-deficit/hyperactivity disorder (AD/HD) is a common behavioral disorder.<br />
The main symptoms of AD/HD are persistent inattention, hyperactivity, <strong>an</strong>d impulsive behavior.<br />
There is strong evidence that genetic factors play a role in <strong>the</strong> pathogenesis of AD/HD. However,<br />
<strong>the</strong> molecular mech<strong>an</strong>isms underlying <strong>the</strong> pathogenesis <strong>an</strong>d <strong>the</strong>rapeutic efficacy of drugs <strong>for</strong><br />
AD/HD are poorly understood.<br />
In RIKEN large scale ENU mutagenesis project, we had been carrying out behavioral screening<br />
to develop novel model <strong>for</strong> psychiatric disorder. In domin<strong>an</strong>t screening, a mut<strong>an</strong>t mouse M-174<br />
that showed hyper-locomotor activity in open-field test was isolated <strong>from</strong> G1 population. In<br />
linkage <strong>an</strong>alysis, causative locus was mapped to proximal region of chromosome2. As a result of<br />
genome sequence <strong>an</strong>alyses of c<strong>an</strong>didate genes, a missense mutation with amino acid substitution<br />
in <strong>the</strong> C0 domain of Grin1 gene that encodes NMDA receptor subunit 1 (NMDAR1) was<br />
identified. At this meeting, we will report about following phenotype <strong>an</strong>alyses of M-174 mut<strong>an</strong>t<br />
mouse, general histology of brain tissue, home-cage activity test, social interaction, motor<br />
coordination, object exploration test, <strong>an</strong>d pharmacological <strong>an</strong>alyses with effects of<br />
methylphenidate (MPH) treatment in <strong>the</strong> open-field activity. MPH is one of <strong>the</strong> major <strong>the</strong>rapeutic<br />
agents <strong>for</strong> AD/HD; it is effective in 70% of AD/HD patients. Results of <strong>the</strong>se tests indicate M-<br />
174 mut<strong>an</strong>t line is a c<strong>an</strong>didate <strong>for</strong> <strong>an</strong>imal model of AD/HD. In fur<strong>the</strong>r <strong>an</strong>alyses, c-Fos expression<br />
pattern <strong>an</strong>d ERK phosphorylation level in brain tissue were examined using<br />
immunohistochemical staining <strong>an</strong>d western blot.<br />
Disclosures: T. Furuse, None; Y. Wada, None; K. Hattori, None; I. Yamada, None; T.<br />
Kushida, None; Y. Shibukawa, None; H. K<strong>an</strong>eda, None; I. Miura, None; K. Kobayashi,<br />
None; H. Masuya, None; S. Yuasa, None; S. Wak<strong>an</strong>a, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.12/N24<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: EC contract number LSH-CT-2006-037315 (EPICURE) FP6 - Thematic priority<br />
LIFESCIHEALTH<br />
Agence nationale de la recherche Neuroscience gr<strong>an</strong>t RPV06055ASA<br />
INSERM
Fédération des Maladies Orphelines<br />
Title: Etiology <strong>an</strong>d physiopathology of temporal periventricular heterotopia<br />
Authors: *C. CARDOSO 1 , A. CARABALONA 1 , A.-M. BISGAARD PEDERSEN 2 , E.<br />
PALLESI-POCACHARD 1 , E. PARRINI 3 , E. BUHLER 1 , M. KIRCHHOFF 2 , P. H. KAAD 4 , R.<br />
S. MØLLER 5 , R. GUERRINI 3 , A. REPRESA 1 ;<br />
1 INMED INSERM U901, Marseille, Fr<strong>an</strong>ce; 2 Dept. of Clin. Genet., Rigshospitalet, Copenhagen<br />
Univ. Hosp., Copenhagen, Denmark; 3 Pediatric Neurol. <strong>an</strong>d Neurogenetics Unit <strong>an</strong>d Labs.,<br />
Children's Hosp. A. Meyer-University of Florence, Florence, Italy; 4 Dept. of Paediatrics,<br />
Hjoerring Hosp., Hjørring, Denmark; 5 The Wilhelm Joh<strong>an</strong>nsen Centre, Univ. of Copenhagen,<br />
Copenhagen, Denmark<br />
Abstract: Cortical mal<strong>for</strong>mations are import<strong>an</strong>t causes of mental retardation <strong>an</strong>d account <strong>for</strong> 20-<br />
40% of drug-resist<strong>an</strong>t epilepsy in childhood. The adv<strong>an</strong>cement of high-resolution imaging has<br />
facilitated <strong>the</strong> in vivo identification of a large group of brain mal<strong>for</strong>mation phenotypes.<br />
Periventricular Heterotopia (PH) is one of <strong>the</strong>se cortical mal<strong>for</strong>mations caused by defective<br />
neuronal migration resulting in abnormal positioning of post-mitotic neurons. These ectopic<br />
neurons remain in <strong>the</strong> periventricular zone, close to <strong>the</strong>ir place of genesis, <strong>an</strong>d <strong>for</strong>m nodules of<br />
heterotopic grey matter. Most affected patients have seizures <strong>an</strong>d <strong>the</strong>ir cognitive level varies<br />
<strong>from</strong> normal to severely impaired. To date, two genes have been identified to cause PH.<br />
Mutations in FLNA (Xq28) <strong>an</strong>d ARFGEF2 (20q13) are responsible <strong>for</strong> X-linked bilateral PH<br />
<strong>an</strong>d a rare autosomal recessive <strong>for</strong>m of PH with microcephaly, respectively. Eleven additional<br />
distinct <strong>an</strong>atomoclinical PH syndromes have been described, but no genetic insights into <strong>the</strong>ir<br />
causes have been gle<strong>an</strong>ed. Recently, Cardoso et al. (<strong>2009</strong>) reported a new locus on 5q15<br />
associated with epilepsy, mental retardation <strong>an</strong>d bilateral PH in <strong>the</strong> walls of <strong>the</strong> temporal horns<br />
of <strong>the</strong> lateral ventricles in 3 unrelated patients. Investigation of additional PH patients with 5q15<br />
deletion allowed us to identify <strong>the</strong> position of <strong>the</strong> mutated loci within <strong>an</strong> interval of 4.5 Mb,<br />
which includes 10 possible c<strong>an</strong>didate genes, most of <strong>the</strong>m with unknown function.<br />
To identify <strong>the</strong> gene responsible <strong>for</strong> this domin<strong>an</strong>t <strong>for</strong>m of temporal PH, we first looked at <strong>the</strong><br />
expression of <strong>the</strong>se 10 c<strong>an</strong>didate genes in specific regions of hum<strong>an</strong> <strong>an</strong>d rodent brains. We found<br />
a basal level of expression to be ubiquitously revealed <strong>for</strong> all, with 5 being highly expressed in<br />
<strong>the</strong> posterior brain. Next, we per<strong>for</strong>med a functional screening by in utero RNAi to determine if<br />
one of <strong>the</strong>se 5 c<strong>an</strong>didate genes contributes to neuronal migration. For this purpose, we developed<br />
shRNA constructions directed against each of <strong>the</strong>se 5 c<strong>an</strong>didate genes <strong>an</strong>d injected <strong>the</strong>m in utero<br />
in <strong>the</strong> brain of rat embryos at E15 in order to induce, after electroporation, interference of <strong>the</strong><br />
target gene. This strategy allowed us to identify one good c<strong>an</strong>didate gene (that we named TPH1)<br />
with unknown function, leading to PH in rodent brain. Sequencing <strong>an</strong>alysis of <strong>the</strong> TPH1 gene in<br />
sporadic PH patients, with no mutations within FLNA <strong>an</strong>d ARFGEF2 genes, should confirm its<br />
role in temporal PH. In addition, fur<strong>the</strong>r investigations of TPH1 mediated-PH <strong>an</strong>imal model will<br />
shed light on <strong>the</strong> physiopathological mech<strong>an</strong>isms responsible <strong>for</strong> epilepsy, which have been<br />
poorly investigated by <strong>the</strong> lack of relev<strong>an</strong>t experimental models <strong>an</strong>d <strong>the</strong> limited access to patient<br />
brain tissue.
Disclosures: C. Cardoso, None; A. Carabalona, None; A. Bisgaard Pedersen, None; E.<br />
Pallesi-Pocachard, None; E. Parrini, None; E. Buhler, None; M. Kirchhoff, None; P.H.<br />
Kaad, None; R.S. Møller, None; R. Guerrini, None; A. Represa, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.13/N25<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: NIH 5P20RR017698<br />
postdoctoral fellowship, Dr. Hebbar, C<strong>an</strong>cer Research <strong>an</strong>d Prevention Foundation<br />
Title: PPARγ regulates cytoplasmic dynein in <strong>an</strong> APC-dependent m<strong>an</strong>ner<br />
Authors: S. HEBBAR, F. M. SIMMONS, *D. S. SMITH;<br />
Univ. South Carolina, Columbia, SC<br />
Abstract: Several studies indicate that <strong>the</strong> PPARγ agonist <strong>an</strong>d common diabetes drug,<br />
roziglitazone, has neuroprotective properties. Although this is typically linked to its <strong>an</strong>tiinflammatory<br />
effects, we have found that roziglitazone alters <strong>the</strong> distribution of a microtubule<br />
motor protein, cytoplasmic dynein. In addition to being a key enzyme responsible <strong>for</strong> retrograde<br />
axonal tr<strong>an</strong>sport, dynein <strong>an</strong>d its binding partners, Lis1 <strong>an</strong>d Ndel1, regulate interkinetic nuclear<br />
oscillation, nuclear envelope breakdown, <strong>an</strong>d spindle dynamics in neural stem cells, as well as<br />
<strong>the</strong> migratory behavior of newly-born neurons. Roziglitazone treatment of cultured cells affects<br />
org<strong>an</strong>elle distribution, as would be expected if it modulates dynein activity. Roziglitazone also<br />
alters mitotic spindle orientation <strong>an</strong>d migratory behavior in cultured cells. Our studies raise <strong>the</strong><br />
intriguing possibility that PPAR signaling may influence brain development, <strong>an</strong>d also that<br />
regulation of dynein could contribute to <strong>the</strong> neuroprotective effects of roziglitazone in mature<br />
brains. The roziglitazone-induced redistribution of dynein is dependent on ongoing tr<strong>an</strong>scription<br />
<strong>an</strong>d is blocked by PI3 kinase inhibition. Moreover, <strong>the</strong> impact of roziglitazone on both dynein<br />
distribution <strong>an</strong>d cell behavior is dependent on <strong>the</strong> adenomatous polyposis coli protein (APC).<br />
APC is best known <strong>for</strong> its link to colorectal c<strong>an</strong>cer, because truncating mutations in hum<strong>an</strong>s<br />
cause <strong>the</strong> disease. However, APC is import<strong>an</strong>t in maintaining polarity in a r<strong>an</strong>ge of cell types,<br />
including neural embryonic stem cells in <strong>the</strong> brain. Moreover, APC, like dynein <strong>an</strong>d Lis1,<br />
interacts with <strong>the</strong> growing plus ends of microtubules. The effects of roziglitazone described
above are ei<strong>the</strong>r absent or altered in cells expressing truncated APC. The presence of truncated<br />
APC enh<strong>an</strong>ces <strong>the</strong> brain phenotype in a Lis1-deficient mouse. Moreover, purified Lis1 <strong>an</strong>d<br />
dynein interact with a C-terminal APC fragment in vitro. Our current model is that roziglitazone<br />
stimulates dynein/Lis1 activity, <strong>an</strong>d that this is dependent on a direct or indirect interaction with<br />
APC. It remains to be determined if <strong>the</strong> effect on dynein is required <strong>for</strong> <strong>the</strong> neuroprotective<br />
characteristics of roziglitazone.<br />
Disclosures: S. Hebbar, None; F.M. Simmons, None; D.S. Smith, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.14/N26<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Title: Stem cell protein expression <strong>an</strong>d mTOR pathway activation distinguishes type I <strong>from</strong> type<br />
II cortical dysplasias<br />
Authors: *V. TSAI 1 , K. ORLOVA 2 , M. BAYBIS 2 , G. G. HEUER 3 , S. SISODIYA 4 , M.<br />
THOM 4 , K. A. STRAUSS 5 , E. ARONICA 6 , P. B. STORM 7 , P. B. CRINO 2 ;<br />
1 Univ. of Pennsylv<strong>an</strong>ia|910006410|0, Philadelphia, PA; 2 Neurol., 3 Neurosurg., Univ. of<br />
Pennsylv<strong>an</strong>ia Sch. of Med., Philadelphia, PA; 4 Dept. of Clin. <strong>an</strong>d Exptl. Epilepsy, UCL Inst. of<br />
Neurol., London, United Kingdom; 5 Clin. <strong>for</strong> Special Children, Strasburg, PA; 6 Dept. of<br />
Neuropathology, Academic Med. Ctr., Univ. of Amsterdam, Amsterdam, Ne<strong>the</strong>rl<strong>an</strong>ds;<br />
7 Neurosurg., Children's Hosp. of Philadelphia, Philadelphia, PA<br />
Abstract: Focal Cortical Dysplasias (FCDs) are classified histopathologically as ei<strong>the</strong>r type I,<br />
characterized by subtle laminar disorg<strong>an</strong>ization <strong>an</strong>d cytomegalic neurons (CNs), or type II,<br />
characterized by loss of cortical lamination <strong>an</strong>d <strong>the</strong> presence of dysmorphic neurons (DNs) <strong>an</strong>d<br />
balloon cells (BCs). 1 Cells in FCD type II have previously been shown to co-express neuronal<br />
<strong>an</strong>d glial markers, suggesting abnormalities in differentiation. 2 We hypo<strong>the</strong>sized that <strong>the</strong><br />
morphological disparities of cells in FCD type I <strong>an</strong>d type II reflect <strong>the</strong>ir differential<br />
developmental phenotypes <strong>an</strong>d that stem cell marker proteins Foxg1, N<strong>an</strong>og, Oct-4, SOX2,<br />
SOX3, <strong>an</strong>d ZNF206 would be expressed in type II but not type I FCD. We fur<strong>the</strong>r hypo<strong>the</strong>sized<br />
that stem cell protein expression in type II FCD is linked to activation of <strong>the</strong> mammali<strong>an</strong> target<br />
of rapamycin complex 1 (mTORC1) cascade. First, dissociated cell cultures were generated in<br />
vitro <strong>from</strong> a single FCD IIB specimen resected during epilepsy surgery. Cells cultured <strong>from</strong>
esected FCD IIB tissue co-expressed phosphorylated iso<strong>for</strong>m of S6 (phospho-S6), a marker of<br />
mTORC1 activation, SOX2, Oct-4, <strong>an</strong>d Nestin. Next, sporadic type I <strong>an</strong>d type II FCD, tubers<br />
<strong>from</strong> tuberous sclerosis complex (TSC), two syndromic FCD subtypes, cortical dysplasia focal<br />
epilepsy syndrome (CDFE) <strong>an</strong>d Pretzel syndrome (PS) specimens, <strong>an</strong>d tissue <strong>from</strong> resected FCD<br />
IIB case were probed with <strong>an</strong>tibodies recognizing stem cell marker proteins <strong>an</strong>d phospho-S6.<br />
Foxg1, N<strong>an</strong>og, Oct-4, SOX2, SOX3, <strong>an</strong>d ZNF206 were expressed in sporadic type II FCD,<br />
tubers, PS, but not in <strong>an</strong>y type I dysplasias. Aberr<strong>an</strong>t phospho-S6 immunoreactivity was detected<br />
in type II but not in type I FCD, control cortex, or cortex adjacent to tubers. Activated c-myc <strong>an</strong>d<br />
STAT3 (phospho-STAT3), which regulate SOX2 <strong>an</strong>d Oct-4 expression via mTORC1, were<br />
detected in type II but not type I dysplasias. These results suggest that type II but not type I<br />
FCDs express stem cell protein markers <strong>an</strong>d exhibit aberr<strong>an</strong>t activation of <strong>the</strong> mTORC1 cascade.<br />
These findings distinguish type II <strong>from</strong> type I dysplasias <strong>an</strong>d may signify a disparate<br />
pathophysiology of <strong>the</strong> two types of FCD. We demonstrate <strong>for</strong> <strong>the</strong> first time that cells <strong>from</strong> FCD<br />
c<strong>an</strong> be cultured in vitro. These results suggest that cells in type II dysplasias may retain <strong>the</strong><br />
capacity <strong>for</strong> cell division <strong>an</strong>d thus type II FCD may represent a dynamic cortical lesion.<br />
References:<br />
1.<br />
Palmini A, Najm I, Av<strong>an</strong>zini G et al. Terminology <strong>an</strong>d classification of <strong>the</strong> cortical dysplasias.<br />
Neurology. 2004;62:S2-8<br />
2.<br />
Englund C, Folkerth RD, Born D et al. Aberr<strong>an</strong>t neuronal-glial differentiation in Taylor-type<br />
focal cortical dysplasia (type IIA/B). Acta Neuropathol. 2005;109:519-533.<br />
Disclosures: V. Tsai, None; K. Orlova, None; M. Baybis, None; G.G. Heuer, None; S.<br />
Sisodiya, None; M. Thom, None; K.A. Strauss, None; E. Aronica, None; P.B. Storm,<br />
None; P.B. Crino, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.15/N27<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t MH151699<br />
NIH Gr<strong>an</strong>t NS048478<br />
STSI Gr<strong>an</strong>t U54 RR025774
NSF predoctoral fellowship<br />
Title: Lysophosphatidic acid signaling through LPA1 receptor in fetal hydrocephalus<br />
Authors: *Y. C. YUNG 1,2 , T. MUTO 1 , M.-E. LIN 1,2 , M. A. KINGSBURY 1 , J. CHUN 1 ;<br />
1 2<br />
Mol. Biol., The Scripps Res. Inst., La Jolla, CA; Biomed. Sci., UC S<strong>an</strong> Diego Sch. of Med., La<br />
Jolla, CA<br />
Abstract: Fetal hydrocephalus / ventriculomegaly is a serious medical condition that affects<br />
approximately one inf<strong>an</strong>t per 1000 live births, <strong>an</strong>d is associated with fetal intracr<strong>an</strong>ial<br />
hemorrhage, myelomeningocele (spinal bifida), <strong>an</strong>d meningitis. The cause is thought to be <strong>an</strong><br />
imbal<strong>an</strong>ce in cerebrospinal fluid (CSF) secretion <strong>an</strong>d absorption <strong>an</strong>d/or by blockage, most<br />
commonly of <strong>the</strong> cerebral aqueduct. Bleeding events prenatally have been epidemiologically<br />
linked to hydrocephalus. Intracerebral exposure to serum during fetal life, as may occur in fetal<br />
intracr<strong>an</strong>ial hemorrhage, produced hydrocephalus in some postnatal <strong>an</strong>imals. This suggested <strong>the</strong><br />
possibility that bioactive lipids like LPA, found <strong>an</strong>d produced in relatively high concentrations in<br />
serum <strong>an</strong>d during clotting, might contribute to this phenotype. Here, we report that in vivo<br />
intracerebral embryonic cortical exposure to lysophosphatidic acid (LPA) produces completely<br />
penetr<strong>an</strong>t hydrocephalus in postnatal <strong>an</strong>imals. LPA altered <strong>the</strong> cellular distribution of adhesion<br />
junction proteins, such as N-cadherin, <strong>an</strong>d induced mitotic displacement <strong>an</strong>d <strong>for</strong>mation of<br />
cellular polyps <strong>an</strong>d neurorosettes at <strong>the</strong> apical ventricular surface, which are also observed in<br />
hum<strong>an</strong> cases of hydrocephalus. Both LPA- <strong>an</strong>d blood serum-induced hydrocephalus were<br />
dramatically attenuated in LPA1 receptor null <strong>an</strong>imals. In addition, delivery of <strong>an</strong> LPA receptor<br />
<strong>an</strong>tagonist prior to LPA or serum exposure reduced <strong>the</strong> severity of cellular <strong>an</strong>d tissue defects<br />
seen in this model. These data support <strong>the</strong> hypo<strong>the</strong>sis that LPA <strong>from</strong> blood serum contributes to<br />
fetal hydrocephalus mainly through LPA1 receptor-mediated effects. LPA1 <strong>an</strong>tagonism c<strong>an</strong><br />
diminish disease severity, suggesting a possible pharmacologically based <strong>the</strong>rapeutic approach to<br />
treating fetal hydrocephalus. Supported by NIH Gr<strong>an</strong>ts MH151699 (JC), NS048478 (JC), <strong>an</strong>d<br />
STSI Gr<strong>an</strong>t U54 RR025774 (JC), <strong>an</strong>d NSF Predoctoral Fellowship (YCY).<br />
Disclosures: Y.C. Yung, None; T. Muto, None; M. Lin, None; M.A. Kingsbury, None; J.<br />
Chun, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.16/N28<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms
Support: WCMC-THMRI initiative to MER<br />
NIH NRSA NS059562 to JDG<br />
Title: Folic acid supplementation alters Wnt signaling <strong>an</strong>d c<strong>an</strong> exacerbate developmental defects<br />
Authors: *J. D. GRAY, B. SLOWINSKA-CASTALDO, M. E. ROSS;<br />
Dept. of Neurol. & Neurosci., Weill Cornell Med. Col., New York, NY<br />
Abstract: Clinical studies indicate that maternal supplementation with folic acid (FA) c<strong>an</strong><br />
reduce <strong>the</strong> prevalence of neural tube defects (NTDs) in hum<strong>an</strong> populations by as much as 70%<br />
(L<strong>an</strong>cet 338: 131, 1991). A complex genetic disorder, NTDs have been associated with more<br />
th<strong>an</strong> 190 genes in mut<strong>an</strong>t mouse lines, <strong>an</strong>d NTDs in some of <strong>the</strong>se lines c<strong>an</strong> be rescued by FA<br />
supplementation (Birth Defects Research A 85: 314, 2008). Crooked Tail (Cd), one such FArescued<br />
NTD mouse model, bears a gain of function (GOF) mutation in <strong>the</strong> Wnt co-receptor<br />
lipoprotein receptor-related protein 6 (Lrp6) (PNAS 102: 12843, 2005). To test whe<strong>the</strong>r NTD<br />
prevention in Lrp6Cd was idiosyncratic <strong>for</strong> <strong>the</strong> point mutation, background strain, or reflected a<br />
direct impact of FA on Lrp6 gene function, we examined <strong>the</strong> effect of dietary FA on Lrp6 loss of<br />
function (LOF) embryos. Elevated dietary FA (10 ppm) reduced <strong>the</strong> occurrence of NTDs <strong>an</strong>d<br />
o<strong>the</strong>r defects in Lrp6-/- embryos compared to control (2 ppm FA) by increasing early lethality in<br />
Lrp6 deficient mice (90% of Lrp6-/- embryos on 2 ppm FA were viable at E13 vs 38% on 10<br />
ppm FA, p
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: 2RO1NS046513<br />
5RO1NS049514<br />
Title: CNS gene profiling of rat prenatally exposed to methylazoxymeth<strong>an</strong>oic acid <strong>an</strong>d<br />
thalidomide<br />
Authors: *Q. FAN 1 , N. MARCHI 1 , V. FAZIO 1 , M. HOSSAIN 1 , P. FABER 2 , D. JANIGRO 1 ;<br />
1 Cell Biol., 2 Genomic Med., CCLCM, Clevel<strong>an</strong>d, OH<br />
Abstract: Pre-natal exposure to methylazoxymeth<strong>an</strong>oic acid (MAM) <strong>an</strong>d thalidomide (THAL)<br />
has been demonstrated to impede brain vasculogenesis <strong>an</strong>d neurogenesis, resulting in ectopic<br />
distribution of neurons <strong>an</strong>d vessels. We studied <strong>the</strong> molecular mech<strong>an</strong>isms of MAM <strong>an</strong>d THAL<br />
induced neuro-vascular dysplasia. We evaluated <strong>the</strong> pattern of gene expression by cDNA<br />
microarray (Illumina Ratref-12 beadchips) in rat brains harvest <strong>from</strong> early postnatal (PN 1) <strong>an</strong>d<br />
adult (PN 28) stages. A total of about 21900 genes were detected <strong>an</strong>d included in <strong>the</strong> statistical<br />
<strong>an</strong>alysis. Western blot was used to confirm <strong>the</strong> brain expression of selected genes.<br />
At PN 1, <strong>the</strong> level of 536 genes was signific<strong>an</strong>tly ch<strong>an</strong>ged (P
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.18/N30<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH<br />
JHU-BSI<br />
St<strong>an</strong>ley foundation<br />
CHDI/HighQ foundation<br />
S-R /RUSK foundation<br />
NARSAD<br />
Title: Phosphorylation of DISC1 activates a molecular switch <strong>from</strong> cell proliferation to neuronal<br />
migration in <strong>the</strong> developing cortex<br />
Authors: *K. ISHIZUKA 1 , A. KAMIYA 1 , J. ROBINSON 2 , E. OH 2 , N. MITSUMA 2 , K.<br />
FURUKORI 1 , S. SESHADRI 1 , B. HUANG 1 , K. KUBO 4 , K. NAKAJIMA 4 , N. KATSANIS 2 , A.<br />
SAWA 1,3 ;<br />
1 Dept Psychiatry, 2 McKusick-Nath<strong>an</strong>s Inst. of Genet. Med., 3 Dept Neurosci., Johns Hopkins<br />
Univ., Baltimore, MD; 4 Dept Anat., Keio Univ., Shinjuku-ku, Jap<strong>an</strong><br />
Abstract: The regulatory mech<strong>an</strong>isms governing <strong>the</strong> sequence <strong>from</strong> cell proliferation to<br />
neuronal migration during corticogenesis are poorly understood. Disrupted-In-Schizophrenia-1<br />
(DISC1), a signific<strong>an</strong>t risk factor <strong>for</strong> schizophrenia <strong>an</strong>d related mental disorders, is highly<br />
expressed in both neural progenitor cells <strong>an</strong>d post mitotic neurons in <strong>the</strong> developing cerebral<br />
cortex. Here we report that phosphorylation of DISC1 at one particular site acts as a molecular<br />
switch <strong>from</strong> maintaining proliferation of mitotic cells to activating migration of postmitotic<br />
neurons. Non-phosphorylated DISC1 at this site regulates c<strong>an</strong>onical Wnt signaling via <strong>an</strong><br />
interaction with GSK3β, whereas phosphorylation of this residue triggers interaction of DISC1 to<br />
BBS proteins <strong>an</strong>d facilitates recruitment of <strong>the</strong>se to <strong>the</strong> dynein motor complex associated with<br />
<strong>the</strong> centrosome. Suppression of DISC1 causes deficits in both cell proliferation <strong>an</strong>d neuronal<br />
migration, however phospho-dead mut<strong>an</strong>t DISC1 c<strong>an</strong> only rescue <strong>the</strong> <strong>for</strong>mer, while phosphomimic<br />
mut<strong>an</strong>t DISC1 rescues specifically <strong>the</strong> latter. These data highlight a dual role <strong>for</strong> DISC1<br />
in corticogenesis <strong>an</strong>d suggest that phosphorylation at this site represents a key developmental<br />
switch.<br />
Disclosures: K. ishizuka, None; A. Kamiya, S-R foudndation, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);
NARSAD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Robinson, None; E. Oh, None; N. Mitsuma,<br />
None; K. Furukori, None; S. Seshadri, None; B. Hu<strong>an</strong>g, None; K. Kubo, None; K. Nakajima,<br />
None; N. Kats<strong>an</strong>is, NICHD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); NIDDK, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); NIH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Macular Vision Research Foundation, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received); Foundation <strong>for</strong> Fighting Blindness, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); A. Sawa, NIH,<br />
B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well<br />
as gr<strong>an</strong>ts already received); JHU-BSI, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); HighQ/CHDI, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received); NARSAD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); St<strong>an</strong>ley, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); S-R/RUSK, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.19/N31<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t NS046521<br />
Title: Loss of CCM3 function in neuroglia leads to cerebral cavernous mal<strong>for</strong>mations<br />
Authors: *A. LOUVI, A. M. TWO, L. CHEN, W. MIN, M. GUNEL;<br />
Yale Sch. of Med., New Haven, CT<br />
Abstract: CCM3/PDCD10 is one of three genes known to cause familial cerebral cavernous<br />
mal<strong>for</strong>mations (CCM), common vascular lesions that affect <strong>the</strong> central nervous system <strong>an</strong>d may<br />
lead to neurological problems including hemorrhage <strong>an</strong>d seizures. Ccm3 is a cytoplasmic protein<br />
expressed in neural cells (including cortical pyramidal neurons <strong>an</strong>d astrocytes) in <strong>the</strong> central
nervous system, as well as in <strong>the</strong> arterial endo<strong>the</strong>lium. Targeted inactivation of Ccm3 in neuronal<br />
<strong>an</strong>d glial progenitors results in abnormal cortical org<strong>an</strong>ization, indicating defects in neuronal<br />
migration. In primary astrocytes Ccm3 loss results in increased proliferation <strong>an</strong>d resist<strong>an</strong>ce to<br />
apoptosis through a cell autonomous mech<strong>an</strong>ism. Surprisingly, loss of Ccm3 function in neural<br />
cells also leads to generalized vascular pathology <strong>an</strong>d <strong>for</strong>mation of multiple vascular lesions that<br />
closely resemble hum<strong>an</strong> CCMs through a cell non-autonomous mech<strong>an</strong>ism. Both cell<br />
autonomous <strong>an</strong>d non-autonomous effects are a consequence of Akt pathway activation. These<br />
observations implicate neural cells in CCM pathogenesis <strong>for</strong> <strong>the</strong> first time <strong>an</strong>d point to a role of<br />
Ccm3 <strong>an</strong>d its interactions with <strong>the</strong> Akt pathway in neural/vascular coupling within <strong>the</strong><br />
neurovascular unit.<br />
Disclosures: A. Louvi, None; A.M. Two, None; L. Chen, None; W. Min, None; M. Gunel,<br />
None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.20/N32<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: HD 35471<br />
MH-58846<br />
Yerkes Base Gr<strong>an</strong>t NIH 00165<br />
Center <strong>for</strong> Behavioral Neuroscience gr<strong>an</strong>t NSF IBN-9876754<br />
Title: Neonatal hippocampal <strong>an</strong>d amygdala lesions alter <strong>the</strong> development of <strong>the</strong> corpus callosum:<br />
An MRI study in adult monkeys<br />
Authors: L. CIRILLI, C. PAYNE, *J. BACHEVALIER;<br />
Yerkes Natl. Primate Rese Cr, Atl<strong>an</strong>ta, GA<br />
Abstract: Dysfunction of <strong>the</strong> amygdala <strong>an</strong>d hippocampus as well as o<strong>the</strong>r brain areas have been<br />
consistently associated with hum<strong>an</strong> development disorders associated with impairment in stress<br />
<strong>an</strong>d <strong>an</strong>xiety, social communications <strong>an</strong>d cognitive functions, such as autism, schizophrenia <strong>an</strong>d<br />
Williams syndrome (Bachevalier et al., Neurosci Biobehav Rev 2006:). Because <strong>the</strong>
neuropathology in <strong>the</strong>se hum<strong>an</strong> developmental disorders is widely distributed ra<strong>the</strong>r th<strong>an</strong><br />
localized, <strong>an</strong>imal models may provide <strong>the</strong> unique opportunity to directly observe how <strong>the</strong> brain<br />
re-org<strong>an</strong>izes after damage to a specific structure in inf<strong>an</strong>cy. Here, we report how selective<br />
neonatal amygdala <strong>an</strong>d hippocampal lesions have impacted <strong>the</strong> normal development of <strong>the</strong><br />
corpus callosum (CC) using non-invasive magnetic reson<strong>an</strong>ce imaging (MRI) techniques. Six<br />
<strong>an</strong>imals had sham operations (SHAM: 3 male, 3 female) <strong>an</strong>d twelve received neurotoxic lesions<br />
of ei<strong>the</strong>r <strong>the</strong> hippocampus (HIPP; 4 males, 2 females) or <strong>the</strong> amygdala (AMYG; 3 males, 3<br />
females) when <strong>the</strong>y were 8 to 10 days of age. At 1 year of age, <strong>the</strong>ir brain was sc<strong>an</strong>ned (1.5 Tesla<br />
Magnet) at 1 mm intervals in <strong>the</strong> coronal <strong>an</strong>d sagittal pl<strong>an</strong>es using a 3D High Resolution T1weighted<br />
imaging sequence. The midsagittal image was used to measure <strong>the</strong> total midsagittal<br />
surface area (SA) <strong>an</strong>d to segment <strong>the</strong> CC into seven subregions (rostrum, genu, rostral body,<br />
<strong>an</strong>terior midbody, posterior midbody, isthmus, <strong>an</strong>d splenium; Witelson et al., Brain, 1989,<br />
112:799). All measurements were done using Image J software <strong>an</strong>d data were <strong>an</strong>alyzed with <strong>an</strong>d<br />
without correction <strong>for</strong> brain size (SA). Neonatal amygdala lesions resulted in a reduction in <strong>the</strong><br />
<strong>an</strong>terior midbody SA (AMYG < SHAM, p = .078). In contrast, neonatal hippocampal lesions<br />
resulted in ch<strong>an</strong>ges more posteriorly in <strong>the</strong> CC. First, <strong>the</strong> posterior midbody SA was smaller in<br />
<strong>an</strong>imals with neonatal hippocampal lesions as compared to controls (p =.05). Second, <strong>the</strong><br />
combined SA of <strong>the</strong> posterior midbody <strong>an</strong>d isthmus was correlated with <strong>the</strong> extent of<br />
hippocampal lesions (p =.01). Overall, <strong>the</strong>se data demonstrate that <strong>the</strong> postnatal development of<br />
<strong>the</strong> corpus callosum is differently affected by neonatal amygdala <strong>an</strong>d hippocampal lesions.<br />
Although <strong>the</strong> amygdala lesions impacted <strong>the</strong> motor component of <strong>the</strong> CC, <strong>the</strong> hippocampal<br />
lesions impacted <strong>the</strong> superior temporal <strong>an</strong>d parietal components of <strong>the</strong> CC. These findings<br />
suggest that <strong>the</strong> effects of neonatal damage to <strong>the</strong> amygdala <strong>an</strong>d hippocampus on <strong>the</strong> CC are not<br />
widespread but ra<strong>the</strong>r specific to motor <strong>an</strong>d parietal areas.<br />
Disclosures: L. Cirilli, None; C. Payne, None; J. Bachevalier, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.21/N33<br />
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Title: Characterization of <strong>the</strong> overlapping expression of LIS1 orthologs in <strong>the</strong> zebrafish nervous<br />
system
Authors: H. M. WIORA, C. M. DRERUP, *J. A. MORRIS;<br />
Pediatrics, Northwestern Univ. <strong>an</strong>d Children's Mem. Res. Ctr., Chicago, IL<br />
Abstract: Lissencephaly is a neurodevelopmental birth defect caused by abnormal neuronal<br />
migration resulting in severe mental retardation, epilepsy, <strong>an</strong>d brain mal<strong>for</strong>mation. One in 30,000<br />
children are born with this devastating disorder <strong>an</strong>d depending on <strong>the</strong> degree of <strong>the</strong> brain<br />
mal<strong>for</strong>mation, m<strong>an</strong>y of <strong>the</strong>se children die be<strong>for</strong>e two years of age. Classic lissencephaly is due to<br />
mutations in <strong>the</strong> LIS1 gene. The LIS1 protein is highly conserved across multiple species,<br />
including, hum<strong>an</strong>s, mice, flies <strong>an</strong>d fungus. In <strong>the</strong> CNS, it has been shown to have a role in<br />
neuroblast proliferation, neuronal morphology <strong>an</strong>d migration. We are using <strong>the</strong> zebrafish model<br />
system to examine <strong>the</strong> developmental function of LIS1 in <strong>the</strong> whole embryo. We have cloned<br />
<strong>an</strong>d characterized <strong>the</strong> zebrafish orthologs of LIS1. Zebrafish have been recognized as <strong>an</strong><br />
import<strong>an</strong>t vertebrate model <strong>for</strong> hum<strong>an</strong> development <strong>an</strong>d disease. There are m<strong>an</strong>y adv<strong>an</strong>tages of<br />
using zebrafish as a model including low cost to maintain, rapid life cycle, ease of genetic<br />
m<strong>an</strong>ipulation <strong>an</strong>d external tr<strong>an</strong>sparent embryonic development. We have determined that <strong>the</strong><br />
LIS1 orthologs are expressed in overlapping temporal <strong>an</strong>d partially overlapping spatial patterns<br />
in <strong>the</strong> zebrafish nervous system. While lis1a is primarily expressed in <strong>the</strong> developing central<br />
nervous system <strong>an</strong>d <strong>the</strong> eye, lis1b is highly expressed in <strong>the</strong> peripheral nervous system as well as<br />
<strong>the</strong> Rohon-beard neurons. Rohon-beard neurons are <strong>the</strong> early sensory system of <strong>the</strong> embryo. We<br />
postulate that underst<strong>an</strong>ding <strong>the</strong> functions of Lis1 in <strong>the</strong> whole embryo will provide better insight<br />
into <strong>the</strong> genetic <strong>an</strong>d neurodevelopmental basis of lissencephaly. This will not only aid in <strong>the</strong><br />
development of <strong>the</strong>rapeutic interventions but will also contribute to <strong>the</strong> general underst<strong>an</strong>ding of<br />
brain development.<br />
Disclosures: H.M. Wiora, None; C.M. Drerup, None; J.A. Morris, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.22/N34<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t NS054210<br />
Title: Age of increased propensity <strong>for</strong> epilepti<strong>for</strong>m activity in mal<strong>for</strong>med cortex<br />
Authors: *A. BELL, K. M. JACOBS;<br />
Anat. <strong>an</strong>d Neurobio., Virginia Commonwealth Univ., Richmond, VA
Abstract: Determining <strong>the</strong> timing of increased propensity <strong>for</strong> epilepti<strong>for</strong>m activity in<br />
developmentally mal<strong>for</strong>med cortex is critical <strong>for</strong> underst<strong>an</strong>ding which cellular abnormalities<br />
contribute to epileptogenesis <strong>an</strong>d which ones might be caused by hyperexcitable circuits. Using<br />
<strong>the</strong> rat freeze lesion model of cortical microgyria, we have previously shown that epilepti<strong>for</strong>m<br />
field potentials recorded in normal aCSF c<strong>an</strong>not be evoked until postnatal day (P)12. Recordings<br />
of mEPSCs have suggested that <strong>the</strong>re is <strong>an</strong> increase in excitatory afferents to <strong>the</strong> paramicrogyral<br />
region (PMG) on P10 that is not apparent at P7-9. The purpose of this study was to determine <strong>the</strong><br />
age at which <strong>the</strong> PMG shows <strong>an</strong> increased propensity <strong>for</strong> ei<strong>the</strong>r interictal- or ictal-like<br />
epilepti<strong>for</strong>m activity when challenged with a second ‘hit’ in excitability. A low Mg+2 aCSF was<br />
applied to control (unlesioned) <strong>an</strong>d mal<strong>for</strong>med cortical slices <strong>from</strong> four age groups (P7-9, P10-<br />
11, P12-15, <strong>an</strong>d P30-60, adult) in order to compare time of onset <strong>an</strong>d incidence of field potential<br />
epilepti<strong>for</strong>m activity (100 min of exposure, reported as Me<strong>an</strong> ±SEM, N slices, t-tests). In <strong>the</strong> P7-<br />
9 age group, <strong>the</strong>re was no difference between control <strong>an</strong>d PMG on <strong>an</strong>y measure (N= 3 PMG <strong>an</strong>d<br />
3 control, NS). In <strong>the</strong> P10-11 age group, PMG slices showed signific<strong>an</strong>tly more ictal-like activity<br />
in response to a low (threshold) intensity extracellular stimulus to deep layers (0.03 Hz, 47 ±10%<br />
vs 11 ±4% of sweeps in 5 PMG <strong>an</strong>d 5 control, respectively, p
Title: The role of filamin <strong>an</strong>d <strong>for</strong>mins in cortical development<br />
Authors: *G. LIAN 1 , J. LU 1 , M. DETTENHOFER 2 , J. NEAL 1 , R. FERLAND 3 , V. SHEEN 1 ;<br />
1 Dept. of Neurology, Beth Israel Deaconess Med. Center, Harvard Med. S, Boston, MA; 2 Dept.<br />
of Genetics, Harvard Med. Sch., Boston, MA; 3 Dept. of Biology, Ctr. <strong>for</strong> Biotech. <strong>an</strong>d<br />
Interdisciplinary Studies, Rensselaer Polytechnic Inst., Troy, NY<br />
Abstract: Periventricular heterotopia (PH) is a mal<strong>for</strong>mation of cortical development<br />
characterized by nodules of neurons aberr<strong>an</strong>tly situated along <strong>the</strong> lateral ventricles of <strong>the</strong> brain.<br />
Hum<strong>an</strong> mutations in <strong>the</strong> actin-binding protein filamin A (FLNA) cause PH. The localization of<br />
<strong>the</strong> heterotopic nodules along <strong>the</strong> ventricular zone during development suggests that FLNA plays<br />
some role in neural progenitor proliferation <strong>an</strong>d initial migration. Although FLNA interacts with<br />
a variety of tr<strong>an</strong>smembr<strong>an</strong>e receptors (including integrins) <strong>an</strong>d tr<strong>an</strong>smits extracellular signal to<br />
cytoskeleton <strong>an</strong>d nucleus, <strong>the</strong> mech<strong>an</strong>ism by which FLNA regulates <strong>the</strong>se signal tr<strong>an</strong>duction<br />
pathways <strong>an</strong>d how disruption of <strong>the</strong>se processes gives rise to PH are not fully understood. Here,<br />
we report that FLNA specifically interacts <strong>an</strong>d binds to <strong>the</strong> Rho GTPase, Rho A, but not Rac1<br />
<strong>an</strong>d Cdc42 by co-IP <strong>an</strong>d pull-down assays. Endogenous RhoA activation of <strong>the</strong> integrin receptors<br />
through laminin is dependent on FLNA in neural progenitor cells. Moreover, we also find that<br />
both activated RhoA <strong>an</strong>d FLNA bind to <strong>for</strong>min 2 (FMN2), a member of a group of proteins<br />
involved in actin polymerization. Activated RhoA competes with <strong>the</strong> binding of FMN2 Cterminus<br />
to <strong>the</strong> FMN2 N-terminus <strong>the</strong>reby activating FMN2 dependent actin nucleation. FLNA<br />
appears to direct localization of both FMN2 <strong>an</strong>d RhoA toward <strong>the</strong> cell membr<strong>an</strong>e <strong>an</strong>d vesicles.<br />
At <strong>the</strong> tissue level, FLNA <strong>an</strong>d FMN2 co-localize along <strong>the</strong> ventricular lining <strong>an</strong>d pial surface of<br />
<strong>the</strong> developing mouse cortex. Consistent with <strong>the</strong> co-localization results, immunostaining data<br />
show that both null FLNA <strong>an</strong>d FLNA/FMN2 double knockout mice exhibited defects in <strong>the</strong><br />
lining of <strong>the</strong> lateral ventricles, implying that both proteins play a vital role in <strong>the</strong> localization of<br />
membr<strong>an</strong>e proteins along <strong>the</strong> neuroepi<strong>the</strong>lium. Overall, <strong>the</strong>se preliminary results suggest that a<br />
FLNA, FMN2 <strong>an</strong>d RhoA complex might regulate <strong>the</strong> actin cytoskeleton <strong>an</strong>d assist in <strong>the</strong><br />
mainten<strong>an</strong>ce of apical surface proteins within neural progenitors along <strong>the</strong> neuroepi<strong>the</strong>ium.<br />
Disruption of <strong>the</strong>se functions may contribute to impaired proliferation <strong>an</strong>d migration seen in PH.<br />
Disclosures: G. Li<strong>an</strong>, None; J. Lu, None; M. Dettenhofer, None; J. Neal, None; R. Ferl<strong>an</strong>d,<br />
None; V. Sheen, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.24/N36
Topic: C.06.d. Developmental disorders affecting brain size : Lissencephaly, microcephaly <strong>an</strong>d<br />
macrocephaly<br />
Support: Webster Family<br />
BrainChild Foundation<br />
Title: Pulsation absorber mech<strong>an</strong>ism in patients with normal pressure hydrocephalus<br />
Authors: *E.-H. PARK 1 , P. K. EIDE 2 , J. R. MADSEN 1 ;<br />
1 2<br />
Children's Hosp. Boston, Boston, MA; Dept. of Neurosurg., Rikshospitalet Univ. Hosp., Oslo,<br />
Norway<br />
Abstract: Systems <strong>an</strong>alysis has revealed that, under normal physiological conditions, pulsatile<br />
intracr<strong>an</strong>ial cerebrospinal fluid (CSF) movement may play <strong>an</strong> import<strong>an</strong>t role in absorbing strong<br />
arterial pulsations within <strong>the</strong> brain. This protective mech<strong>an</strong>ism, or pulsation absorber, is similar<br />
to a notch filter where <strong>the</strong> notch is in <strong>the</strong> r<strong>an</strong>ge of primary cardiac frequency. Using a newly<br />
developed metric, “cardiac pulsation absorb<strong>an</strong>ce (CPA)”, of gain of <strong>the</strong> pulsation absorber<br />
evaluated at <strong>the</strong> cardiac frequency, we have shown that development of hydrocephalus c<strong>an</strong> be<br />
correlated with signific<strong>an</strong>tly deteriorated per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong> pulsation absorber.<br />
In a series of clinical studies per<strong>for</strong>med with NPH patients, Eide has shown that high intracr<strong>an</strong>ial<br />
pressure (ICP) pulsatile amplitudes (> 4mmHg) predicted better response to shunting th<strong>an</strong> seen<br />
in patients with lower ICP amplitudes. We hypo<strong>the</strong>size that <strong>an</strong> impairment of <strong>the</strong> pulsation<br />
absorber could be a mech<strong>an</strong>ism behind <strong>the</strong> elevated intracr<strong>an</strong>ial pulsatility observed in <strong>the</strong> shunt<br />
responders. To test this hypo<strong>the</strong>sis, we have applied <strong>the</strong> time-varying tr<strong>an</strong>sfer function (TVTF)<br />
method using arterial blood pressure (ABP) input <strong>an</strong>d ICP output obtained <strong>from</strong> ten patients<br />
undergoing evaluations of presumed normal pressure hydrocephalus (NPH). ICP pulse wave<br />
amplitude was also calculated in <strong>the</strong> same samples to examine its relationship with <strong>the</strong> notch<br />
filter per<strong>for</strong>m<strong>an</strong>ce. The preliminary results show that <strong>the</strong> pulsation absorber appears to be present<br />
in <strong>the</strong> hum<strong>an</strong> brain <strong>an</strong>d that <strong>the</strong> tendency towards relatively large frequency mismatch between<br />
<strong>the</strong> notch frequency <strong>an</strong>d <strong>the</strong> primary cardiac frequency shown in hydrocephalic c<strong>an</strong>ines has also<br />
been observed in all patients studied. In two patients with sufficient data <strong>for</strong> comparison,<br />
amplitude is inversely related to CPA where higher amplitude (> 4 mmHg) indicates lower<br />
per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong> cardiac pulsation absorber. The results suggest that a notch filter c<strong>an</strong> be<br />
found in <strong>the</strong> hum<strong>an</strong> intracr<strong>an</strong>ial system <strong>an</strong>d that shunt response may be directly related to <strong>the</strong><br />
level of pulsation absorb<strong>an</strong>ce which c<strong>an</strong> be indicated by <strong>the</strong> amplitude of ICP pulsations.<br />
Disclosures: E. Park, None; P.K. Eide, dPCom AS (Sensometrics software), E. Ownership<br />
Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); J.R. Madsen, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.25/N37<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship Gr<strong>an</strong>t<br />
Americ<strong>an</strong> Association of Neurological Surgeons Medical Student Summer Research<br />
Fellowship Gr<strong>an</strong>t<br />
Title: TSC2 knockdown in developing cortex replicates <strong>the</strong> cellular phenotype of cortical tubers<br />
<strong>an</strong>d interferes with normal cortical lamination: Novel in vitro <strong>an</strong>d in vivo approaches<br />
Authors: *W. E. PARKER 1 , K. ORLOVA 1 , G. HEUER 1,2 , L. ATKINSON 1 , V. TSAI 1 , J.<br />
YOON 1 , M. BAYBIS 1 , G. H. BALTUCH 2 , P. B. CRINO 3 ;<br />
2 Dept. of Neurosurg., 3 Dept. of Neurol., 1 Univ. of Pennsylv<strong>an</strong>ia Sch. of Med., Philadelphia, PA<br />
Abstract: Tuberous Sclerosis Complex (TSC) is <strong>an</strong> autosomal domin<strong>an</strong>t multisystem disorder<br />
characterized neurologically by cortical tubers <strong>an</strong>d epilepsy (Crino et al 2006). Tubers exhibit<br />
abnormal hexalaminar structure <strong>an</strong>d contain dysplastic neurons <strong>an</strong>d cytomegalic cells termed<br />
gi<strong>an</strong>t cells. TSC is caused by mutations in TSC1 or TSC2, <strong>the</strong> protein products of which function<br />
as heterodimers that inhibit mTOR signaling. Gi<strong>an</strong>t cells found in tubers are highly<br />
immunoreactive <strong>for</strong> downstream effectors of mTOR, suggesting that abnormal hyperactive<br />
mTOR signaling may be central to <strong>the</strong> pathogenesis of TSC. Previous studies in cell lines <strong>an</strong>d<br />
mature neurons show that depletion of TSC1 or TSC2 proteins results in hyperactivation of<br />
mTOR (Onda et al 2002, Tavazoie et al 2005). However, since patients with TSC often show<br />
neurological m<strong>an</strong>ifestations at a very early age (<strong>an</strong>d tubers have even been detected in utero,<br />
Hengstschlager 2006), it is imperative to study <strong>the</strong> function of <strong>the</strong> disease-causing genes in<br />
immature neuronal precursors. We defined <strong>the</strong> effects of TSC2 depletion in mouse neural<br />
progenitor cells (mNPCs) in vitro by tr<strong>an</strong>sfection <strong>an</strong>d in vivo by in utero electroporation of E14<br />
mouse embryos with a GFP-tagged shRNA. Knockdown of TSC2 in mNPCs in vitro resulted in<br />
hyperphosphorylation of S6 <strong>an</strong>d 4E-BP1 <strong>an</strong>d increased expression of VEGF, suggesting<br />
hyperactive mTOR signaling. Knockdown of TSC2 in vivo not only resulted in mTOR<br />
hyperactivity in tr<strong>an</strong>sfected subventricular zone (SVZ) cells in cortex, but interestingly, also<br />
interfered with normal cortical lamination, causing tr<strong>an</strong>sfected cells to remain in <strong>the</strong> SVZ ra<strong>the</strong>r<br />
th<strong>an</strong> migrate out to <strong>the</strong> cortical plate. Thus, we have been able to create <strong>an</strong>d characterize a ra<strong>the</strong>r<br />
effective model <strong>for</strong> TSC cortical tuber pathogenesis in <strong>the</strong> developing brain, replicating both
mTOR hyperactivity at <strong>the</strong> cellular level <strong>an</strong>d a focal defect in cortical lamination <strong>an</strong>d cellular<br />
migration.<br />
Disclosures: W.E. Parker, None; K. Orlova, None; G. Heuer, None; L. Atkinson, None; V.<br />
Tsai, None; J. Yoon, None; M. Baybis, None; G.H. Baltuch, None; P.B. Crino, None.<br />
Poster<br />
536. Neurodevelopmental Disorders Affecting Brain Size <strong>an</strong>d Patterning<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 536.26/N38<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t 1 F31 NS061619-0 1A1<br />
Doris Duke Clinical Investigator Award<br />
NIH 1R21HD054347<br />
Title: The molecular roles of Filamin A <strong>an</strong>d Arfgef2 in periventricular heterotopia <strong>for</strong>mation<br />
Authors: *J. NEAL 1,2 , G. LIAN 2 , R. FERLAND 3 , C. A. WALSH 2,4 , V. L. SHEEN 2 ;<br />
1 Cambridge, MA; 2 Neurol., Beth Israel Deaconess Med. Center, Harvard Med. Sch., Boston,<br />
MA; 3 Biol., Rensselaer Polytechnic Inst., Troy, NY; 4 Howard Hughes Med. Inst. <strong>an</strong>d Div. of<br />
Genetics, Childrens Hospital, Harvard Med. Sch., Boston, MA<br />
Abstract: Periventricular heterotopia (PH) is a mal<strong>for</strong>mation of cortical development<br />
characterized by <strong>the</strong> <strong>for</strong>mation of ectopic neuronal nodules, in part due to <strong>the</strong> disruption of <strong>the</strong><br />
lateral ventricular neuroepi<strong>the</strong>lium. Mutations in <strong>the</strong> Filamin A (FLNA) gene are <strong>the</strong> most<br />
common cause of <strong>the</strong> congenital disorder. FLNA encodes a 280kDa actin-binding protein which
inds various cytosolic <strong>an</strong>d membr<strong>an</strong>e proteins at its C-terminus. A rarer <strong>for</strong>m of PH is caused<br />
by mutations in <strong>the</strong> Arfgef2 gene which encodes Brefeldin A-inhibited gu<strong>an</strong>ine nucleotide<br />
exch<strong>an</strong>ge factor (BIG2), a 180 kDa Protein Kinase A (PKA) <strong>an</strong>choring protein that regulates<br />
vesicle budding <strong>from</strong> cellular membr<strong>an</strong>es through catalyzing <strong>the</strong> exch<strong>an</strong>ge of GDP-to-GTP on<br />
ADP-ribosylation factors 1-3 (ARFs). The extent to which FLNA <strong>an</strong>d BIG2 participate in a<br />
common signaling pathway to regulate <strong>the</strong> integrity of <strong>the</strong> neuroepi<strong>the</strong>lium has not been<br />
established. Here we show that FLNA <strong>an</strong>d BIG2 physically interact <strong>an</strong>d colocalize along <strong>the</strong><br />
neuroepi<strong>the</strong>lium <strong>an</strong>d within <strong>the</strong> perinuclear <strong>an</strong>d peripheral regions of neural progenitor cells.<br />
PKA-dependent phosphorylation of FLNA (Serine 2152) induces membr<strong>an</strong>e ruffling in neural<br />
progenitors <strong>an</strong>d BIG2 possesses multiple PKA binding domains, suggesting that BIG2 regulates<br />
FLNA localization. Conversely, BIG2 redistribution towards <strong>the</strong> cell periphery also requires<br />
FLNA expression <strong>an</strong>d BIG2-FLNA colocalization at membr<strong>an</strong>e ruffles after <strong>for</strong>skolin treatment<br />
(cAMP activator) requires <strong>the</strong> FLNA-binding domain of BIG2. Prior studies have shown that<br />
engagement of ephrin-B lig<strong>an</strong>ds to EphB receptors regulates neural cell adhesion <strong>an</strong>d ARF1<br />
activation. We now find that FLNA binds Ephrin-B1 <strong>an</strong>d that activation of ARF1 through<br />
Ephrin-B1 is dependent on FLNA. Loss of BIG2 inhibits ARF1 activation. Finally, we show that<br />
domin<strong>an</strong>t negative inhibition of ARF1 <strong>an</strong>d BIG2 mislocalizes membr<strong>an</strong>e adherens junction<br />
proteins (beta-catenin, E-cadherin) to <strong>the</strong> golgi in polarized MDCK cells. These results suggest<br />
that FLNA regulates BIG2 localization <strong>an</strong>d function, <strong>an</strong>d BIG2 reciprocally determines FLNA<br />
location. Toge<strong>the</strong>r, <strong>the</strong>se proteins help tr<strong>an</strong>sduce extrinsic signals (i.e. Eph) <strong>an</strong>d direct vesicle<br />
trafficking of adhesion molecules. Loss of cell adhesion disrupts neural progenitor development<br />
<strong>an</strong>d contributes to PH <strong>for</strong>mation.<br />
Disclosures: J. Neal, None; G. Li<strong>an</strong>, None; R. Ferl<strong>an</strong>d, None; C.A. Walsh, None; V.L.<br />
Sheen, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.1/O1<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: MRC UK G0400298<br />
Wellcome Trust UK<br />
Title: What's <strong>the</strong> matter with gray matter? Neurobiological risk factors <strong>for</strong> specific l<strong>an</strong>guage<br />
impairment
Authors: *K. E. WATKINS 1 , N. A. BADCOCK 1 , M. J. HARDIMAN 1 , J. G. BARRY 2 , D. V.<br />
M. BISHOP 1 ;<br />
1 2<br />
Exptl. Psychology, Univ. Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom; MRC Inst. of Hearing Res.,<br />
Nottingham, United Kingdom<br />
Abstract: Specific l<strong>an</strong>guage impairment (SLI) is a highly heritable disorder in which <strong>the</strong>re are<br />
delays in l<strong>an</strong>guage development <strong>for</strong> no obvious reason. Typically, no brain abnormalities are<br />
evident on gross inspection. We conducted computational morphometric <strong>an</strong>alyses on MRI sc<strong>an</strong>s<br />
obtained in 10 families with a child affected by SLI <strong>an</strong>d compared <strong>the</strong>m with sc<strong>an</strong>s <strong>from</strong> 10<br />
control families. We used voxel-based morphometry to compare gray matter in 10 children with<br />
SLI (aged 8-17 years), 6 unaffected siblings (aged 12-22 years), <strong>an</strong>d 16 control children (aged 6-<br />
25 years). In <strong>the</strong> adults, we compared 7 l<strong>an</strong>guage-impaired parents (aged 40-61 years), 6<br />
unaffected parents of children with SLI (aged 37-53 years) <strong>an</strong>d 13 parents of controls (aged 38-<br />
55 years), who were not l<strong>an</strong>guage impaired. Age <strong>an</strong>d sex were entered as covariates in both sets<br />
of <strong>an</strong>alysis. T1-weighted 1-mm isotropic structural images of <strong>the</strong> whole brain were acquired at<br />
1.5T. Gray-matter segments were nonlinearly tr<strong>an</strong>s<strong>for</strong>med to a template, modulated by <strong>the</strong><br />
Jacobi<strong>an</strong> determin<strong>an</strong>t <strong>an</strong>d smoo<strong>the</strong>d (10-mm FWHM). A t-test between groups was per<strong>for</strong>med<br />
voxel-wise <strong>an</strong>d thresholded at p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.2/O2<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: IZKF, Jena, Germ<strong>an</strong>y<br />
Title: Active <strong>an</strong>d passive auditory l<strong>an</strong>guage perception in dyslexia as studied with fMRI <strong>an</strong>d<br />
EEG<br />
Authors: *M. TRAUTMANN 1,2 , C. LIGGES 2 , B. BLANZ 2 ;<br />
1 2<br />
Jacobs-University, Bremen, Germ<strong>an</strong>y; Child <strong>an</strong>d Adolescent Psychiatry, Univ. Hosp., Jena,<br />
Germ<strong>an</strong>y<br />
Abstract: Aside <strong>from</strong> deficits during phonological processing in subjects with dyslexia<br />
(Shaywitz et al., 1998, Ligges & Bl<strong>an</strong>z, 2007) <strong>the</strong>re is always a subgroup that shows deficits in<br />
auditory perception (Ramus, 2003, Mody et al., 2008). Studies with functional magnetic<br />
reson<strong>an</strong>ce imaging (fMRI) showed reduced temporo-parietal activation patterns during <strong>the</strong><br />
phonological processing of visual presented lingual stimuli (Shaywitz et al., 1998), but also in<br />
auditory processing (Corina et al., 2001). In passive auditory presentation of l<strong>an</strong>guage stimuli,<br />
Schulte-Körne <strong>an</strong>d colleagues found reduced mismatch negativity over <strong>the</strong> frontal cortex<br />
measured by electro encephalogram (EEG) (Schulte-Körne et al., 2001). The aim of this study<br />
was <strong>the</strong> identification of neural activation differences <strong>an</strong>d <strong>the</strong> comparison of behavioral<br />
per<strong>for</strong>m<strong>an</strong>ce parameters between children with <strong>an</strong>d without dyslexia during <strong>the</strong> processing of<br />
auditory l<strong>an</strong>guage stimuli. The investigation of <strong>the</strong> neural processing during active <strong>an</strong>d passive<br />
auditory presented l<strong>an</strong>guage stimuli as studied with fMRI <strong>an</strong>d EEG on one sample fills a gap in<br />
<strong>the</strong> causal research of dyslexia. With a passive oddball paradigm <strong>the</strong> difficulty of attentional<br />
processing <strong>an</strong>d visual l<strong>an</strong>guage perception in children with dyslexia has been considered. Results<br />
showed a reduced negativity over P3, Pz <strong>an</strong>d P4 during <strong>the</strong> active perception of auditory stimuli<br />
as measured with EEG. Beside <strong>the</strong>se results a widespread activation pattern in <strong>the</strong> right temporal<br />
cortex was found as measured with fMRI. These results lead to <strong>the</strong> conclusion that auditory<br />
l<strong>an</strong>guage perception is disrupted in children with dyslexia. For diagnosis <strong>an</strong>d prevention of<br />
dyslexia not only phonological but also auditory speech processing has to be tested <strong>an</strong>d trained.<br />
Disclosures: M. Trautm<strong>an</strong>n , None; C. Ligges, None; B. Bl<strong>an</strong>z, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 537.3/O3<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: MHLW Gr<strong>an</strong>t 19A-8<br />
Title: A functional magnetic reson<strong>an</strong>ce imaging study on reading acquisition of <strong>the</strong> Jap<strong>an</strong>ese<br />
phonogram: Different trajectory in dyslexic children<br />
Authors: *A. SEKI 1,2 , H. UCHIYAMA 1,2 , T. KOEDA 1,2 , T. KOCHIYAMA 3 , M. INAGAKI 4 ;<br />
1 2<br />
Tottori Univ., Tottori, Jap<strong>an</strong>; Tottori Med. Center, Natl. Hosp. Org<strong>an</strong>ization, Tottori, Jap<strong>an</strong>;<br />
3 4<br />
Adv<strong>an</strong>ced Telecommunications Res. Inst. Intl., Kyoto, Jap<strong>an</strong>; Natl. Inst. of Mental Health,<br />
NCNP, Tokyo, Jap<strong>an</strong><br />
Abstract: In alphabetic l<strong>an</strong>guages, <strong>the</strong>re is a consensus regarding neural substrates associated<br />
with reading acquisition: <strong>the</strong> left temporoparietal region contributes to reading in <strong>the</strong> early stage<br />
of reading acquisition, in which children rely on decoding; <strong>the</strong> left fusi<strong>for</strong>m contributes to skilled<br />
reading with whole word identification. Dyslexic children show weaker activation of both <strong>the</strong>se<br />
regions. The purpose of this study is to identify <strong>the</strong> developmental ch<strong>an</strong>ges in reading acquisition<br />
of k<strong>an</strong>a, <strong>the</strong> Jap<strong>an</strong>ese phonogram, <strong>an</strong>d reveal <strong>the</strong> different trajectory in Jap<strong>an</strong>ese dyslexic<br />
children.<br />
Methods: The study included 11 non-impaired (NI) children (6 boys; age, 9.6 ± 1.7 years) <strong>an</strong>d 9<br />
children with reading difficulties (RD; 7 boys; age, 9.4 ± 3.3 years). During f-MRI, <strong>the</strong> subjects<br />
had to judge whe<strong>the</strong>r given pictures <strong>an</strong>d words matched. Over 4 sessions, we conducted 20 trials<br />
of 5 trial categories: familiar words, unfamiliar words, pseudowords, letter strings, <strong>an</strong>d<br />
me<strong>an</strong>ingless figures. The trials were r<strong>an</strong>domized <strong>an</strong>d presented at 8-second intervals using <strong>the</strong><br />
Presentation software. We used a Siemens 1.5T sc<strong>an</strong>ner with gradient echo EPI (TR = 4000 ms)<br />
to acquire f-MRI data, <strong>an</strong>d <strong>an</strong>alyzed using <strong>the</strong> SPM5. After <strong>an</strong> event-related single-subject<br />
<strong>an</strong>alysis, group <strong>an</strong>alysis was per<strong>for</strong>med using a r<strong>an</strong>dom effect model to identify differences<br />
between NI <strong>an</strong>d RD children. Signal increase <strong>for</strong> each particip<strong>an</strong>t was measured as <strong>the</strong> contrast<br />
estimate, <strong>an</strong>d was compared with <strong>the</strong> particip<strong>an</strong>t’s reading skills measured after <strong>the</strong> f-MRI.<br />
Results: In all <strong>the</strong> reading trials, activation was observed in <strong>the</strong> inferior to middle frontal gyri, <strong>the</strong><br />
inferior parietal lobuli (IPLs), <strong>an</strong>d fusi<strong>for</strong>m gyri, bilaterally. The activation in <strong>the</strong> bilateral<br />
fusi<strong>for</strong>m gyri <strong>an</strong>d <strong>the</strong> left pons were more evident in NI th<strong>an</strong> in RD. In NI, <strong>the</strong> activation in <strong>the</strong><br />
left fusi<strong>for</strong>m gyrus was greater with familiar words th<strong>an</strong> with letter strings, while <strong>the</strong> left IPL<br />
was more active with unfamiliar words th<strong>an</strong> familiar words. The IPL activation signific<strong>an</strong>tly<br />
correlated with <strong>the</strong> speed of decoding k<strong>an</strong>a letters in NI. Compared to NI, <strong>the</strong> activation in <strong>the</strong><br />
fusi<strong>for</strong>m gyri were poor <strong>for</strong> all trial categories in RD.<br />
Conclusion: In children with normal reading ability, <strong>the</strong> left IPL activity correlates to <strong>the</strong> skill of<br />
decoding <strong>the</strong> Jap<strong>an</strong>ese phonogram <strong>an</strong>d <strong>the</strong> fusi<strong>for</strong>m activation is related to reading familiar<br />
words. These results indicate that fusi<strong>for</strong>m activation enh<strong>an</strong>ces as <strong>the</strong> skill of phonogram reading<br />
improves. The poor activation in <strong>the</strong> bilateral fusi<strong>for</strong>m gyri <strong>an</strong>d <strong>the</strong> left IPL was revealed in<br />
Jap<strong>an</strong>ese children who have <strong>the</strong> difficulty in phonogram reading.<br />
Disclosures: A. Seki, None; H. Uchiyama, None; T. Koeda, None; T. Kochiyama, None; M.<br />
Inagaki, None.
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.4/O4<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NARSAD Independent Investigator Award (JAS)<br />
Children Research Center of Michig<strong>an</strong> (JAS)<br />
Title: Evidence of a developmental deviation in <strong>the</strong> integration of cortico-striatal-thalamic<br />
networks in ADHD: A whole-brain, multi-voxel 31 P spectroscopy study at 4 Tesla<br />
Authors: *J. A. STANLEY, D. KHATIB, R. M. DICK, O. MCGARRAGLE, F. P.<br />
MACMASTER, A. L. ROBIN, D. R. ROSENBERG;<br />
Psych & Behav Neurosci, Wayne State Univ. Sch. Med., Detroit, MI<br />
Abstract: BACKGROUND<br />
Attention-deficit/hyperactivity disorder (ADHD) is one of <strong>the</strong> most prevalent<br />
neurodevelopmental disorders in children. We recently reported subcortical <strong>an</strong>d cortical deficits<br />
in <strong>the</strong> metabolism of membr<strong>an</strong>e phospholipids (MPL) that are suggestive of <strong>an</strong> alteration of <strong>an</strong><br />
earlier developing region influencing <strong>the</strong> maturational integration of later or slower developing<br />
regions, using in vivo 31 P spectroscopy, which is sensitive in detecting biochemical ch<strong>an</strong>ges in a<br />
developing brain. In this study, <strong>the</strong> purpose is to fur<strong>the</strong>r our investigation by using a high-field<br />
system, which dramatically improves (a) spatial resolution to assess cortico-striatal-thalamic<br />
networks <strong>an</strong>d (b) <strong>the</strong> biochemical resolution [e.g., phosphoeth<strong>an</strong>olamine (PE) <strong>an</strong>d<br />
phosphocholine (PC)].<br />
SUBJECTS AND METHODS<br />
In vivo 31 P metabolite levels were qu<strong>an</strong>tified bilaterally in 5 different brain areas within <strong>the</strong><br />
prefrontal cortex <strong>an</strong>d <strong>the</strong> subcortex of 18 children with DSM-IV ADHD (12M+6F; me<strong>an</strong> age<br />
9.2±1.2 yrs) <strong>an</strong>d 15 healthy controls (HC; 4M+11F; 9.2±2 yrs of age) using a 3D multi-voxel 31 P<br />
spectroscopy acquisition method at 4 Tesla.<br />
RESULTS<br />
PE levels tended to be lower in ADHD children compared with HC in <strong>the</strong> striatum (p=0.075) <strong>an</strong>d<br />
thalamus (p=0.079), but were signific<strong>an</strong>tly lower on <strong>the</strong> right side of <strong>the</strong>se areas (p=0.046 <strong>an</strong>d<br />
p=0.0077). In <strong>the</strong> dorsal <strong>an</strong>terior cingulate (dACC), PE levels were signific<strong>an</strong>tly higher in <strong>the</strong><br />
ADHD children compared with HC (p=0.0066). Also, <strong>the</strong>re was a signific<strong>an</strong>t group-by-age<br />
interaction <strong>for</strong> PE in dACC (p=0.019) <strong>an</strong>d <strong>for</strong> PC in <strong>the</strong> ventro-medial prefrontal cortex (vmPFC;
p=0.43) with converging values in <strong>the</strong> younger subjects <strong>an</strong>d diverging values with increasing<br />
age.<br />
CONCLUSIONS<br />
In healthy development, PE decreases with age reflecting a possible reduction in <strong>the</strong> dem<strong>an</strong>d<br />
MPL syn<strong>the</strong>sis of neuronal <strong>an</strong>d synaptic processes. In contrast, PC increases followed by a<br />
decrease with age (inverted “U”) in prefrontal cortices, which appear to reflect growth spurts in<br />
<strong>the</strong>se relatively later developing brain areas. The lower PE in subcortical areas in ADHD may<br />
reflect reduced density of dendrites <strong>an</strong>d synaptic connections due to <strong>an</strong> underdevelopment of <strong>the</strong><br />
striatum <strong>an</strong>d thalamus. In <strong>the</strong> dACC, <strong>the</strong> non-signific<strong>an</strong>t difference in PE levels in <strong>the</strong> younger<br />
subjects followed by a lack of progressive decreasing PE levels in ADHD with age suggests a<br />
deviation in <strong>the</strong> developmental trajectory. The increasing PC levels in <strong>the</strong> vmPFC of HC are<br />
consistent with that of prefrontal areas experiencing a developmental growth spurt, which<br />
appears underachieved in <strong>the</strong> ADHD children. Conducting longitudinal measurements will<br />
enable us to better address temporal developmental deviations within subjects.<br />
Disclosures: J.A. St<strong>an</strong>ley, None; D. Khatib, None; R.M. Dick, None; O. McGarragle,<br />
None; F.P. MacMaster, None; A.L. Robin, None; D.R. Rosenberg, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.5/O5<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Title: Applying novel functional brain imaging <strong>an</strong>d brain network <strong>an</strong>alysis <strong>for</strong> objective<br />
profiling of ADHD<br />
Authors: N. PINCHUK 1 , A. RECHES 2,1 , G. BEN BASHAT 1 , G. SHAHAF 3 , A. KANTER 1 , *J.<br />
J. SCHULMAN 1 , T. FISHER 2 , J. AHARON 3 , A. B. GEVA 4,1 , H. PRATT 2 ;<br />
1 Elminda, Ltd., Haifa, Israel; 2 Technion -- Israel Inst. <strong>for</strong> Technol., Haifa, Israel; 3 Rambam Med.<br />
Ctr., Haifa, Israel; 4 Ben Gurion Univ. of <strong>the</strong> Negev, Beersheba, Israel<br />
Abstract: Introduction: Ongoing research has supported <strong>the</strong> st<strong>an</strong>ce that functional networks<br />
sp<strong>an</strong>ning spatial <strong>an</strong>d temporal scales are central to sensory, cognitive <strong>an</strong>d emotional experience.<br />
Underst<strong>an</strong>ding <strong>the</strong> nature of <strong>the</strong>se networks in normal <strong>an</strong>d pathological states using<br />
electrophysiology has become increasingly feasible with adv<strong>an</strong>ces in conceptual <strong>an</strong>d<br />
computational methods. The need <strong>for</strong> tools of this type is particularly acute in neurobehavioral<br />
disorders due to diagnostic ambiguity <strong>an</strong>d <strong>the</strong> need <strong>for</strong> long-term treatment strategies.
ADHD is a common neurobehavioral disorder with signific<strong>an</strong>t morbidity <strong>an</strong>d social cost. Despite<br />
convergent data <strong>from</strong> neuroimaging, neuropsychology, genetic <strong>an</strong>d neurochemical studies which<br />
support <strong>the</strong> involvement of cognitive-attention network dysfunction as <strong>the</strong> neurobiological core<br />
of this disorder, <strong>the</strong>re is currently no objective physiological measure of ADHD. Instead,<br />
diagnosis <strong>an</strong>d monitoring in clinical practice continue to rely on behavioral assessments. In <strong>an</strong><br />
ef<strong>for</strong>t to address this need, we developed <strong>an</strong>d tested EEG <strong>an</strong>alysis tools focused on network<br />
properties to objectively classify brain activity in ADHD.<br />
Methods: 13 ADHD patients <strong>an</strong>d matched controls were recorded with 21-lead EEG (Ceegraph<br />
IV, Biologic Systems Corp., IL, USA) while per<strong>for</strong>ming <strong>an</strong> auditory go/no-go task. ADHD<br />
subjects fulfilled DSM-IV <strong>an</strong>d Conners’ Adult ADHD Rating Scales (tr<strong>an</strong>slated) criteria <strong>an</strong>d<br />
were unmedicated at <strong>the</strong> time of recording.<br />
EEG signals were processed using st<strong>an</strong>dard methods <strong>an</strong>d blind source separation to derive<br />
spectral <strong>an</strong>d spatiotemporal patterns in typical frequency b<strong>an</strong>ds. Following discretization of<br />
statistically-signific<strong>an</strong>t features in <strong>the</strong> EEG signal, a novel algorithm was used to identify<br />
patterns of activity <strong>an</strong>d derive m<strong>an</strong>y-to-m<strong>an</strong>y network maps across regions.<br />
Results: Preliminary results show well-defined activation patterns <strong>an</strong>d network activity dynamics<br />
in distributed neural networks subserving go/no-go task per<strong>for</strong>m<strong>an</strong>ce. In particular, in <strong>the</strong> no-go<br />
condition, activation of a distinct network in <strong>the</strong> delta r<strong>an</strong>ge was observed in controls. In<br />
contrast, evoked activity in <strong>the</strong> no-go condition <strong>for</strong> ADHD subjects was present in <strong>the</strong> <strong>the</strong>ta<br />
r<strong>an</strong>ge in fronto-parietal areas. Both activation patterns were specific <strong>an</strong>d sensitive <strong>an</strong>d correlated<br />
with behavioral assessments.<br />
Conclusion: These results suggest that development of objective profiling of patients' brain<br />
network activity may enh<strong>an</strong>ce <strong>the</strong> ability to make in<strong>for</strong>med diagnostic <strong>an</strong>d <strong>the</strong>rapeutic decisions<br />
based on underlying brain physiology. EEG is a promising technology <strong>for</strong> this application due to<br />
its non-invasiveness, high temporal resolution, usability <strong>an</strong>d low cost.<br />
Disclosures: N. Pinchuk, Elminda, Ltd., A. Employment (full or part-time); A. Reches,<br />
Elminda, Ltd., A. Employment (full or part-time); G. Ben Bashat, Elminda, Ltd., A.<br />
Employment (full or part-time); G. Shahaf, Elminda, Ltd., E. Ownership Interest (stock, stock<br />
options, patent or o<strong>the</strong>r intellectual property); A. K<strong>an</strong>ter, Elminda, Ltd., A. Employment (full or<br />
part-time); J.J. Schulm<strong>an</strong>, Elminda, Ltd., A. Employment (full or part-time); T. Fisher,<br />
None; J. Aharon, None; A.B. Geva, Elminda, Ltd., E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); H. Pratt, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.6/O6
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: Sigrid Jusélius Foundation<br />
Academy of Finl<strong>an</strong>d<br />
Finnish Graduate School of Neuroscience<br />
Knut <strong>an</strong>d Alice Wallenberg Foundation<br />
Swedish Research Council<br />
Hjärnfonden (Sweden)<br />
Swedish Royal B<strong>an</strong>k Tercentennial Foundation<br />
Title: DCDC2 affects <strong>the</strong> length of <strong>an</strong>d signaling through primary cilia in neurons<br />
Authors: M.-E. HOKKANEN 1 , S. MASSINEN 2 , J. KERE 4 , *E. CASTREN 3 ;<br />
1 Neurosci. Ctr., 2 Dept. of Med. Genet., Univ. of Helsinki, Helsinki, Finl<strong>an</strong>d; 3 Univ. of Helsinki,<br />
00014 Helsinki, Finl<strong>an</strong>d; 4 Dept. of Biosci. <strong>an</strong>d Nutr., Karolinska Institutet, Stockholm, Sweden<br />
Abstract: DCDC2, doublecortin domain containing 2, is one of <strong>the</strong> six genes associated to<br />
developmental dyslexia, <strong>the</strong> most common learning disorder. Knock-down of Dcdc2 in <strong>the</strong><br />
developing rodent brain leads to impaired neuronal migration. DCDC2 has also been shown to<br />
bind to <strong>an</strong>d stabilize microtubules but o<strong>the</strong>r th<strong>an</strong> this its cellular function has remained largely<br />
unknown.<br />
We show that hum<strong>an</strong> DCDC2 localizes to <strong>the</strong> primary cilium when expressed in primary rat<br />
hippocampal neurons or NIH3T3 cells with some protein also found in <strong>the</strong> cytoplasm. The<br />
primary cilium is a cell org<strong>an</strong>elle consisting of a microtubule scaffold, <strong>the</strong> axoneme, encased by<br />
<strong>the</strong> cell membr<strong>an</strong>e. Primary cilia are found on most cells in different types of org<strong>an</strong>s in mammals<br />
including neurons in <strong>the</strong> central nervous system. Its function in m<strong>an</strong>y tissues is not clear but it’s<br />
characterized by specific markers in different cell types e.g. adenylyl cyclase 3 <strong>an</strong>d somatostatin<br />
receptor 3 in neurons. The primary cilium has been shown to be essential <strong>for</strong> Sonic Hedgehog<br />
(Shh) signaling while it seems to have a bal<strong>an</strong>cing role between c<strong>an</strong>onical <strong>an</strong>d non-c<strong>an</strong>onical<br />
Wnt signaling.<br />
We found that overexpression of DCDC2 signific<strong>an</strong>tly increases <strong>the</strong> length of primary cilia both<br />
in neurons <strong>an</strong>d NIH3T3 cells while knock-down by shRNA had no effect on ciliary length. We<br />
also studied if overexpression or knock-down of Dcdc2 in primary cortical neurons affects Shh<br />
or Wnt signalling, two import<strong>an</strong>t developmental signaling pathways linked to ciliary function.<br />
Overexpression of DCDC2 increases Shh signaling, as measured by a luciferase expression<br />
vector system responsive to Gli activator function, but did not influence c<strong>an</strong>onical Wnt signaling,<br />
as measured by a TCF/Lef dependent luciferase expression vector assay. Conversely, knockdown<br />
of DCDC2 by shRNA had no effect on ciliary length or Shh signaling, but it increased<br />
c<strong>an</strong>onical Wnt signaling. This indicates that knock-down has <strong>an</strong> affect on <strong>the</strong> functionality of <strong>the</strong><br />
primary cilium.
Our results show that DCDC2 has a ciliary localization <strong>an</strong>d influences both structure <strong>an</strong>d<br />
function of <strong>the</strong> primary cilium. These findings are <strong>the</strong> first indication that <strong>the</strong> primary cilium<br />
could have a role in <strong>the</strong> etiology of dyslexia.<br />
Disclosures: M. Hokk<strong>an</strong>en, None; S. Massinen, None; J. Kere, None; E. Castren, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.7/O7<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: HD20806 to RHF<br />
UConn Research Foundation to DT<br />
Title: Behavioral implications of knockout <strong>for</strong> <strong>the</strong> dyslexia-risk gene Dcdc2 in mice<br />
Authors: D. T. TRUONG 1 , C. E. CLEARY 1 , C. A. HILL 1 , S. W. THRELKELD 3 , Y. WANG 2 ,<br />
J. J. LOTURCO 2 , *R. FITCH 1 ;<br />
1 2 3<br />
Psyc/Behav Neurosci, Physiol. <strong>an</strong>d Neurobio., Univ. of Connecticut, Storrs, CT; Brown Univ.,<br />
Providence, RI<br />
Abstract: Several genetic linkage <strong>an</strong>d epidemiological studies have provided strong evidence<br />
that DCDC2 is a c<strong>an</strong>didate gene <strong>for</strong> developmental dyslexia--a disorder that impairs a person’s<br />
reading ability despite adequate intelligence, education, <strong>an</strong>d socio-economic status. Studies<br />
investigating embryonic intra-ventricular RNA interference (RNAi) of Dcdc2 (a rat homolog of<br />
<strong>the</strong> DCDC2 gene in hum<strong>an</strong>s) indicate disruptions in neuronal migration in <strong>the</strong> rat cortex during<br />
development. Interestingly, <strong>the</strong>se <strong>an</strong>atomical disruptions are consistent with post mortem<br />
histological <strong>an</strong>omalies seen in hum<strong>an</strong> dyslexic patients. O<strong>the</strong>r rodent studies of cortical<br />
developmental disruption using RNAi <strong>for</strong> related genes have shown impairments in rapid<br />
auditory processing <strong>an</strong>d spatial learning affected subjects.<br />
The current study investigates <strong>the</strong> rapid auditory processing abilities of mice heterozygous <strong>for</strong><br />
Dcdc2 (i.e., one functioning Dcdc2 allele), as well as mice with a homozygous knockout of<br />
Dcdc2 (i.e., no functioning Dcdc2 allele). It is import<strong>an</strong>t to note that behavioral assessments of<br />
this genetic model are still in pilot stages. However, preliminary results suggest that mice with a<br />
genetic mutation of Dcdc2 exhibit impaired rapid auditory processing, as well as non-spatial<br />
maze learning <strong>an</strong>d memory ability, as compared to wildtypes. By genetically knocking out
Dcdc2 in mice, behavioral features associated with Dcdc2 c<strong>an</strong> be characterized, along with o<strong>the</strong>r<br />
neurological abnormalities that may arise due to <strong>the</strong> loss of this functioning gene.<br />
Disclosures: D.T. Truong, None; C.E. Cleary, None; C.A. Hill, None; S.W. Threlkeld,<br />
None; Y. W<strong>an</strong>g, None; J.J. LoTurco, None; R. Fitch, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.8/O8<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NIH Gr<strong>an</strong>t HD20806<br />
Title: Effects of test experience <strong>an</strong>d neocortical microgyria on spatial <strong>an</strong>d non-spatial learning in<br />
rats<br />
Authors: *S. W. THRELKELD 1 , D. T. TRUONG 2 , C. A. HILL 2 , C. E. CLEARY 2 , G. D.<br />
ROSEN 3 , R. H. FITCH 2 ;<br />
1 Women <strong>an</strong>d Inf<strong>an</strong>ts' Hosp. of Rhode Isl<strong>an</strong>d, Warren Alpert Med. Sch. Brown Uni, Providence,<br />
RI; 2 Psychology, Behavioral Neurosci., Univ. of Connecticut, Storrs, CT; 3 Neurol., Beth Israel<br />
Deaconess Med. Center, Harvard Med. Sch., Boston, MA<br />
Abstract: Cortical neuronal migration <strong>an</strong>omalies such as microgyria <strong>an</strong>d heterotopia have been<br />
associated with developmental l<strong>an</strong>guage learning impairments in hum<strong>an</strong>s, <strong>an</strong>d rapid auditory<br />
processing deficits in rodent models. Similar processing impairments have been suggested to<br />
play a causal role in hum<strong>an</strong> l<strong>an</strong>guage impairment. Recent data <strong>from</strong> our group has shown<br />
persistent spatial working memory deficits associated with neocortical microgyria in rats, which<br />
are also identified in some l<strong>an</strong>guage learning impaired hum<strong>an</strong>s. To fur<strong>the</strong>r explore <strong>the</strong> extent of<br />
learning deficits associated with cortical neuronal migration <strong>an</strong>omalies, we evaluated <strong>the</strong> effects<br />
of neocortical microgyria <strong>an</strong>d test order experience using spatial (Morris water maze, MWM)<br />
<strong>an</strong>d non-spatial water maze learning paradigms. Two independent groups were employed (G1 or<br />
G2) incorporating both microgyria <strong>an</strong>d sham conditions. G1 received spatial testing <strong>for</strong> five days<br />
followed by non-spatial testing, while <strong>the</strong> reverse order was followed <strong>for</strong> G2. Initial <strong>an</strong>alysis,<br />
including both test groups <strong>an</strong>d both maze conditions, revealed a main effect of Treatment, with<br />
microgyric rats per<strong>for</strong>ming signific<strong>an</strong>tly worse th<strong>an</strong> shams. The overall <strong>an</strong>alysis also revealed a<br />
task by order interaction, suggesting that each group per<strong>for</strong>med better on <strong>the</strong> second task as<br />
compared to <strong>the</strong> first, regardless of which task was presented first. Interestingly, when both
groups were combined <strong>for</strong> <strong>an</strong>alysis on each task independently, a signific<strong>an</strong>t effect of Treatment<br />
(microgyria worse th<strong>an</strong> sham) was observed only <strong>for</strong> <strong>the</strong> spatial water maze condition. Results<br />
indicate that prior maze experience (regardless of task type) leads to better subsequent<br />
per<strong>for</strong>m<strong>an</strong>ce. Fur<strong>the</strong>r, results suggest that learning impairments associated with microgyria<br />
extend beyond auditory <strong>an</strong>d working memory deficits seen in previous studies, to include spatial<br />
but not non-spatial learning impairments.<br />
Disclosures: S.W. Threlkeld, None; D.T. Truong, None; C.A. Hill, None; C.E. Cleary,<br />
None; G.D. Rosen, None; R.H. Fitch, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.9/O9<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NIH Gr<strong>an</strong>t HD20806<br />
Title: The behavioral effects of knockdown of Kiaa0319, a c<strong>an</strong>didate dyslexia susceptibility<br />
gene<br />
Authors: *C. E. CLEARY 1 , C. G. FIONDELLA 1 , D. T. TRUONG 1 , J. J. LOTURCO 1 , G. D.<br />
ROSEN 2 , R. H. FITCH 1 ;<br />
1 Univ. Connecticut, Storrs, CT; 2 BIDMC/Harvard Med. Sch., Cambridge, MA<br />
Abstract: Subtle alterations in <strong>the</strong> development of <strong>the</strong> neocortex are associated with cognitive<br />
deficits in hum<strong>an</strong>s <strong>an</strong>d o<strong>the</strong>r mammals. A number of genes contribute to <strong>the</strong> development of <strong>the</strong><br />
neocortex, <strong>an</strong>d research into <strong>the</strong> behavioral phenotype associated with specific gene<br />
m<strong>an</strong>ipulations is adv<strong>an</strong>cing rapidly. Recent findings include evidence that <strong>an</strong>omalies in four<br />
hum<strong>an</strong> genes - DYX1C1, ROBO1, DCDC2, <strong>an</strong>d KIAA0319 - may be associated with <strong>an</strong><br />
increased incidence of dyslexia. Concurrent research has shown that <strong>the</strong> rat homologs <strong>for</strong> <strong>the</strong>se<br />
genes modulate critical parameters of early cortical development. Moreover, recent studies have<br />
shown deficits in auditory processing, spatial learning, <strong>an</strong>d working memory in rats following in<br />
utero tr<strong>an</strong>sfection of <strong>an</strong> RNA interference vector (RNAi) of <strong>the</strong> rat homolog Dyx1c1 gene.<br />
However, <strong>the</strong> role of DYX1C1 in <strong>the</strong> etiology of dyslexia in hum<strong>an</strong> populations remains<br />
controversial due to difficulty replicating <strong>the</strong> original genetic linkage findings. More recent<br />
research has focused on <strong>the</strong> potential role of one of <strong>the</strong> o<strong>the</strong>r c<strong>an</strong>didate dyslexia susceptibility<br />
genes, KIAA0319, in <strong>the</strong> development of this reading disorder. The clinical evidence supporting
<strong>the</strong> role <strong>for</strong> this gene in <strong>the</strong> etiology of dyslexia is more compelling th<strong>an</strong> what has been found <strong>for</strong><br />
DYX1C1. It is located on <strong>the</strong> 6p22.2 chromosome, <strong>the</strong> most replicated region of linkage to<br />
developmental dyslexia. Vari<strong>an</strong>ts in this gene have been associated with dyslexia in broader<br />
populations as compared to DYX1C1. Additionally, this gene has recently been associated with<br />
variation in reading ability within <strong>the</strong> general population. Animal models using in utero RNAi<br />
against Kiaa0319 (<strong>the</strong> rodent homolog of <strong>the</strong> hum<strong>an</strong> gene) have shown that interference with this<br />
gene leads to aberr<strong>an</strong>t neuronal migration in <strong>the</strong> neocortex, resulting in <strong>for</strong>mation of heterotopia<br />
in white matter. In order to fur<strong>the</strong>r pursue possible neurobehavioral relationships between<br />
genetic <strong>an</strong>omalies <strong>an</strong>d subsequent behavioral disruptions that may be relev<strong>an</strong>t to hum<strong>an</strong> clinical<br />
conditions such as dyslexia, <strong>the</strong> current study examined <strong>the</strong> effects of in utero RNAi <strong>for</strong><br />
Kiaa0319 on rapid auditory processing as well as spatial <strong>an</strong>d nonspatial learning abilities, in 28<br />
male Wistar-Kyoto rats. Behavioral results <strong>for</strong> Kiaa0319 RNAi versus sham subjects will be<br />
presented, <strong>an</strong>d <strong>the</strong> findings discussed in <strong>the</strong> context of improving our underst<strong>an</strong>ding of <strong>the</strong><br />
genetic contributions to <strong>the</strong> behavioral expression of dyslexia in hum<strong>an</strong>s.<br />
Disclosures: C.E. Cleary, None; C.G. Fiondella, None; D.T. Truong, None; J.J. LoTurco,<br />
None; G.D. Rosen, None; R.H. Fitch, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.10/O10<br />
Topic: C.06.i. Non- syndromic <strong>for</strong>ms of general cognitive dysfunction<br />
Support: NIDA R21 DA15878<br />
NIDA R01 DA017831<br />
March of Dimes 6FY2008-50<br />
NCRR P41 RR13642<br />
NINDS NS3753<br />
NIH &54 RR021813<br />
Title: Decreased activation associated with verbal working memory mainten<strong>an</strong>ce in children<br />
prenatally exposed to methamphetamine <strong>an</strong>d/or alcohol
Authors: *C. NUNEZ 1 , A. STARR 1 , E. D. O'HARE 2 , S. Y. BOOKHEIMER 3,4 , M. J.<br />
O'CONNOR 4 , L. M. SMITH 5 , E. R. SOWELL 1,3 ;<br />
1 2<br />
Lab. Neuro Imaging, UCLA Dept Neurol, Los Angeles, CA; Helen Willis Neurosci. Inst.,<br />
Univ. of Cali<strong>for</strong>nia, Berkeley, Berkeley, CA; 3 Interdepartmental Ph.D. Program <strong>for</strong> Neurosci.,<br />
4 5<br />
Dept. of Psychiatry <strong>an</strong>d Biobehavioral Sci., UCLA, Los Angeles, CA; Dept. of Pediatrics,<br />
Harbor-UCLA Med. Ctr., Torr<strong>an</strong>ce, CA<br />
Abstract: Cortical activation during a verbal working memory task was evaluated in children<br />
prenatally exposed to methamphetamine (MA) <strong>an</strong>d non-exposed children (CON). M<strong>an</strong>y MAexposed<br />
children are also exposed to alcohol prenatally, so a contrast group of alcohol-exposed<br />
children (ALC) was also evaluated. It was hypo<strong>the</strong>sized that <strong>the</strong> MA group would demonstrate<br />
different activation patterns th<strong>an</strong> <strong>the</strong> CON <strong>an</strong>d ALC groups. Forty-nine children (ages 7-16,<br />
me<strong>an</strong> age=10.7, SD=2.5; 25 females) participated. The MA group had 18 children, with 15 of<br />
<strong>the</strong>m also exposed to ALC in utero. Eight children with signific<strong>an</strong>t prenatal ALC exposure<br />
comprised <strong>the</strong> ALC group, <strong>an</strong>d 23 children were in <strong>the</strong> CON group. There were no differences in<br />
age or gender between groups, but me<strong>an</strong> IQ in <strong>the</strong> ALC group was signific<strong>an</strong>tly lower th<strong>an</strong> <strong>the</strong><br />
CON group. With fMRI at 3T (EPI gradient echo, slice thickness=3mm/1 skip, 36 total slices,<br />
TR=3.0s), particip<strong>an</strong>ts per<strong>for</strong>med a Sternberg verbal working memory task presented in a<br />
blocked design. fMRI data were <strong>an</strong>alyzed using FSL 4.1, <strong>an</strong>d group effects <strong>for</strong> <strong>the</strong> verbal<br />
working memory vs. rest contrast were evaluated <strong>for</strong> MA vs. CON <strong>an</strong>d MA vs. ALC. Z-statistic<br />
images were thresholded using clusters determined by Z > 2.3 <strong>an</strong>d corrected cluster signific<strong>an</strong>ce<br />
of p=0.05. There were no group differences in reaction time or accuracy. The CON group<br />
activated in regions typically observed in working memory studies, including <strong>the</strong> frontal-parietal<br />
attention system, visual <strong>an</strong>d motor cortex. Activation in <strong>the</strong> ALC group was more diffuse th<strong>an</strong><br />
<strong>the</strong> CON group with greater activation in dorsal frontal, parietal, <strong>an</strong>d temporal regions. MA<br />
group activation was signific<strong>an</strong>tly lower th<strong>an</strong> <strong>the</strong> CON group in temporal, parietal <strong>an</strong>d occipital<br />
regions, <strong>an</strong>d activation was decreased more globally when compared to <strong>the</strong> ALC group. Children<br />
with prenatal MA exposure had different patterns of cortical activation on a verbal working<br />
memory mainten<strong>an</strong>ce task when compared to unexposed children <strong>an</strong>d children exposed to ALC<br />
prenatally. MA-exposed children had decreased activation in frontal-parietal attention networks,<br />
visual <strong>an</strong>d motor cortex when compared to unexposed children. The MA group also had a<br />
distinct activation pattern <strong>from</strong> <strong>the</strong> ALC group with less global activation, suggesting that<br />
differences are not a result of concomit<strong>an</strong>t ALC exposure. While all groups per<strong>for</strong>med similarly<br />
on <strong>the</strong> task, MA exposed children relied less on regions that are typically recruited <strong>for</strong><br />
attention/working memory tasks, <strong>an</strong>d this may reflect differences in org<strong>an</strong>ization of <strong>the</strong> attention<br />
system.<br />
Disclosures: C. Nunez, None; A. Starr, None; E.D. O'Hare, None; S.Y. Bookheimer,<br />
None; M.J. O'Connor, None; L.M. Smith, None; E.R. Sowell, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.11/O11<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: CIHR<br />
NSERC<br />
Title: Neonatal medial frontal cortex lesions disrupt circadi<strong>an</strong> activity patterns<br />
Authors: *V. M. SMITH 1 , C. I. PHILLIPS 1 , M. C. ANTLE 1,2,3 , R. H. DYCK 1,2,4 ;<br />
1 2 3 4<br />
Psychology, Hotchkiss Brain Inst., Physiol. & Pharmacol., Cell Biol. & Anat., Univ. of<br />
Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: The medial frontal cortex (MFC) is involved in <strong>the</strong> temporal org<strong>an</strong>ization of<br />
behaviour, such as sequential motor acts that are directed towards pups or potential mates<br />
(Afonso et al., 2007; Hern<strong>an</strong>dez-Gonzalesz et al., 2005; Stamm, 1955; Sylvester et al., 2002). It<br />
receives timing in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> master circadi<strong>an</strong> clock, located in <strong>the</strong> suprachiasmatic<br />
nucleus (SCN), <strong>an</strong>d exhibits daily oscillations in gene expression (Antle & Silver, 2005;<br />
Sylvester et al., 2002). The present study examined various properties of <strong>the</strong> circadi<strong>an</strong> rhythms<br />
of locomotor activity following neonatal or adult MFC aspiration lesions. <strong>When</strong> entrained to a 12<br />
hr light/12 hr dark lighting cycle, mice with neonatal lesions were more active during <strong>the</strong> day<br />
(lights on) th<strong>an</strong> mice with adult lesions (p < .05), <strong>an</strong>d less active during <strong>the</strong> early night (first 4<br />
hours following lights off) th<strong>an</strong> ei<strong>the</strong>r mice with adult lesions or control mice (p < .05). Mice<br />
with adult lesions had a shorter duration to <strong>the</strong>ir active phase (alpha) th<strong>an</strong> <strong>an</strong>imals with neonatal<br />
lesions (p < .05) <strong>an</strong>d showed a trend towards a shorter duration of alpha th<strong>an</strong> control <strong>an</strong>imals (p<br />
= .086). Relative to controls, mice with neonatal lesions exhibited signific<strong>an</strong>tly smaller phase<br />
delays to <strong>an</strong> early-night light pulse (4 hours after activity onset; p < .05) <strong>an</strong>d marginally larger<br />
phase adv<strong>an</strong>ces to a late-night light pulse (10 hours after activity onset; p = .070). There was no<br />
difference between mice with adult lesions <strong>an</strong>d control mice in <strong>the</strong> magnitude of phase shifts<br />
elicited by a light pulse at ei<strong>the</strong>r time. These results suggest that <strong>the</strong> timing of behaviour is<br />
determined by <strong>an</strong> interaction between <strong>the</strong> MFC <strong>an</strong>d <strong>the</strong> SCN, <strong>an</strong>d that injury early in life has a<br />
signific<strong>an</strong>t effect on <strong>the</strong> ability of <strong>an</strong>imals to temporally org<strong>an</strong>ize <strong>the</strong>ir behaviour.<br />
Disclosures: V.M. Smith, None; C.I. Phillips, None; M.C. Antle, None; R.H. Dyck, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.12/O12<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NIH Gr<strong>an</strong>t MH082123<br />
NIAAA Gr<strong>an</strong>t AA0011873<br />
Title: Immature error-regulatory function in young men with childhood histories of ADHD<br />
Authors: *K. VELANOVA 1 , B. LUNA 1 , T. WILSON 1 , K. KINGSLEY 1 , E. GNAGY 2 , N.<br />
NAWARAWONG 1 , W. PELHAM 2 , B. MOLINA 1 ;<br />
1 2<br />
Dept. of Psychiatry, Univ. of Pittsburgh, Pittsburgh, PA; Dept. of Psychology, SUNY Buffalo,<br />
Buffalo, NY<br />
Abstract: Recent research indicates that children with Attention Deficit Hyperactivity Disorder<br />
(ADHD) show atypical signaling in <strong>an</strong>terior cingulate cortex (ACC) during functional magnetic<br />
reson<strong>an</strong>ce imaging (fMRI; e.g., Pliszka et al., 2006; Smith et al., 2008), <strong>an</strong>d that adults with <strong>the</strong><br />
disorder show both atypical ACC connectivity during resting-state fMRI (Castell<strong>an</strong>os et al.,<br />
2008) <strong>an</strong>d striking reductions in ACC volume (~14%; Siedem<strong>an</strong> et al., 2006) relative to controls.<br />
These findings contribute to a growing body of evidence implicating ACC in <strong>the</strong><br />
pathophysiology of ADHD. Here we examine functional activity in ACC associated with error<br />
commission in a cohort of young men who have been followed since childhood as part of <strong>the</strong><br />
Pittsburgh ADHD Longitudinal Study (AA11873; DA12414). Particip<strong>an</strong>ts were 24 young men<br />
aged 18 to 25 years, 16 of whom met DSM-III-R or DSM-IV diagnostic criteria <strong>for</strong> ADHD in<br />
childhood. Event-related fMRI was conducted as particip<strong>an</strong>ts per<strong>for</strong>med <strong>the</strong> <strong>an</strong>tisaccade task--<strong>an</strong><br />
oculomotor test of inhibitory control. On each of 54 (jittered) <strong>an</strong>tisaccade trials presented over<br />
three sc<strong>an</strong>ned runs, particip<strong>an</strong>ts’ task was to inhibit <strong>the</strong> prepotent tendency to look toward a<br />
briefly presented peripheral stimulus, <strong>an</strong>d to instead look toward <strong>the</strong> mirror location. Regions of<br />
interest associated with oculomotor task per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d with error detection <strong>an</strong>d regulation<br />
were derived <strong>from</strong> <strong>an</strong> independent fMRI study of <strong>an</strong>tisaccade per<strong>for</strong>m<strong>an</strong>ce in typical young<br />
adults. Timecourses of activity <strong>for</strong> correctly per<strong>for</strong>med <strong>an</strong>tisaccades, <strong>an</strong>d <strong>for</strong> (corrected) errors<br />
were estimated within regions, <strong>an</strong>d effects confirmed in whole-brain voxelwise <strong>an</strong>alyses. Young<br />
men with ADHD showed only modestly increased error rates relative to age- <strong>an</strong>d IQ-matched<br />
controls. However, <strong>the</strong>y were less likely to correct <strong>the</strong>ir errors. As a corollary, <strong>the</strong>se young men<br />
showed atypical signaling in ACC when errors were committed; Whereas typical young adults<br />
show extended activity in dorsal ACC following error commission--a signal thought to indicate<br />
need <strong>for</strong> per<strong>for</strong>m<strong>an</strong>ce adjustment--activity of this sort was attenuated in young men with ADHD,<br />
<strong>an</strong>d resembled more <strong>the</strong> pattern of activity reported in children (Vel<strong>an</strong>ova et al., 2008). The<br />
present findings suggest a possible delay in maturation, or dysmaturation, of error-regulatory<br />
functions in young men with ADHD. Fur<strong>the</strong>r, our findings highlight <strong>the</strong> need <strong>for</strong> future work
examining functional-<strong>an</strong>atomic development in ADHD <strong>an</strong>d support emerging hypo<strong>the</strong>ses<br />
implicating ACC <strong>an</strong>d ACC dysfunction in <strong>the</strong> pathophysiology of ADHD.<br />
Disclosures: K. Vel<strong>an</strong>ova, None; B. Luna, None; T. Wilson, None; K. Kingsley, None; E.<br />
Gnagy, None; N. Nawarawong, None; W. Pelham, None; B. Molina, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.13/O13<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: NSERC<br />
CIHR<br />
Title: Behavioural phenotyping of adult mice following medial frontal cortex lesions induced in<br />
<strong>the</strong> neonatal or adult period<br />
Authors: C. I. PHILLIPS 1 , *R. H. DYCK 4,2,3 ;<br />
1 Psychology, 2 Cell Biol. & Anat., 3 Hotchkiss Brain Inst., Univ. of Calgary, Calgary, AB,<br />
C<strong>an</strong>ada; 4 Univ. Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: Damage to <strong>the</strong> medial frontal cortex (MFC) results in a variety of behavioural deficits,<br />
including ch<strong>an</strong>ges in learning, <strong>an</strong>xiety <strong>an</strong>d activity levels (e.g. Brito & Brito, 1990; Deacon et al.,<br />
2003; Jinks & McGregor, 1997, Kolb et al., 1996). Kolb <strong>an</strong>d colleagues (1996) have found that,<br />
in rats, neonatal MFC aspiration lesions result in remarkable functional recovery. The present<br />
study examined <strong>the</strong> effects of neonatal (postnatal day 7) compared to adult MFC aspiration<br />
lesions, in a mouse model, on battery of tasks: open field, elevated plus maze, wireh<strong>an</strong>g, rotarod,<br />
horizontal ladder, Morris water maze, prepulse inhibition, step-through active avoid<strong>an</strong>ce task,<br />
<strong>an</strong>d step-through passive avoid<strong>an</strong>ce task. In <strong>the</strong> open field, mice with adult lesions were<br />
hyperactive relative to mice with neonatal lesions <strong>an</strong>d controls. Mice with adult lesions travelled<br />
far<strong>the</strong>r th<strong>an</strong> ei<strong>the</strong>r mice with neonatal lesions or control mice. They also travelled faster th<strong>an</strong><br />
both mice with neonatal lesions <strong>an</strong>d control mice. MFC lesions also decreased <strong>an</strong>xiety on <strong>the</strong><br />
elevated plus maze task. Control mice spent less time on <strong>the</strong> open arms th<strong>an</strong> mice with adult<br />
lesions <strong>an</strong>d showed a trend towards a similar relationship when compared to mice with neonatal<br />
lesions. Also on <strong>the</strong> elevated plus maze, mice with adult lesions entered <strong>the</strong> open arms more<br />
frequently (corrected <strong>for</strong> increased activity) th<strong>an</strong> control mice. On <strong>the</strong> step-through active
avoid<strong>an</strong>ce task <strong>the</strong>re was a signific<strong>an</strong>t interaction between surgical type <strong>an</strong>d trial, mice with<br />
neonatal lesions spent less time in error th<strong>an</strong> adults. Finally, <strong>the</strong>re was a signific<strong>an</strong>t interaction<br />
between surgical type <strong>an</strong>d trial on <strong>the</strong> step-through passive avoid<strong>an</strong>ce task. On <strong>the</strong> acquisition<br />
trial, mice with neonatal lesions had longer latencies to cross to <strong>the</strong> opposite chamber th<strong>an</strong> both<br />
mice with adult lesions <strong>an</strong>d control mice. Forty-eight hours after acquisition, control mice had<br />
longer latencies to cross to <strong>the</strong> opposite chamber th<strong>an</strong> mice with adult lesions. Seven days after<br />
acquisition <strong>the</strong> groups did not differ. These data indicate that lesions of <strong>the</strong> mouse MFC result in<br />
behavioural deficits regardless of <strong>the</strong> age of <strong>the</strong> <strong>an</strong>imal at <strong>the</strong> time of <strong>the</strong> lesion. However, <strong>the</strong><br />
deficits exhibited by neonatal injured <strong>an</strong>imals differ <strong>from</strong> those resulting <strong>from</strong> injury in<br />
adulthood. The differences in <strong>the</strong> behavioural profiles could reflect compensatory mech<strong>an</strong>isms<br />
<strong>an</strong>d/or functional recovery.<br />
Disclosures: C.I. Phillips, None; R.H. Dyck, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.14/O14<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NIMH P50 MH077248<br />
Title: Structural correlates of response inhibition <strong>an</strong>d symptom severity in children with ADHD<br />
Authors: E. M. MILLER 1 , A. GALVAN 1 , S. Y. BOOKHEIMER 2 , R. M. BILDER 2 , J. T.<br />
MCCRACKEN 2 , *R. A. POLDRACK 1 ;<br />
1 Psychology, 2 Psychiatry <strong>an</strong>d Biobehavioral Sci., UCLA, Los Angeles, CA<br />
Abstract: Attention-deficit hyperactivity disorder (ADHD) is characterized by varying deficits<br />
in response inhibition, attention, <strong>an</strong>d behavior regulation. In <strong>the</strong> current study, we examine<br />
whe<strong>the</strong>r successful response inhibition <strong>an</strong>d symptom severity in children with ADHD is related<br />
to differences in brain structure. 42 subjects aged 7 to 14 years participated in <strong>the</strong> experiment.<br />
All met DSM-IV criteria <strong>for</strong> ADHD, <strong>an</strong>y subtype. Severity of ADHD symptoms off-medication<br />
was assessed using <strong>the</strong> inattentive <strong>an</strong>d hyperactive-impulsive symptom rating scales <strong>from</strong> <strong>the</strong><br />
ADHD-RS. Subjects’ ability to inhibit pre-potent responses was assessed via a stop-signal<br />
paradigm that used a simple choice response as <strong>the</strong> primary task <strong>an</strong>d <strong>an</strong> auditory stop-signal<br />
(occurring on 25% of trials). For each subject, a staircase procedure was used to determine <strong>the</strong><br />
delay value between <strong>the</strong> primary stimulus <strong>an</strong>d <strong>the</strong> stop-signal at which <strong>the</strong> individual failed to
inhibit on 50% of stop-trials. Stop-signal reaction time (SSRT), <strong>the</strong> time necessary <strong>for</strong> a<br />
particip<strong>an</strong>t to inhibit his or her response, was determined <strong>from</strong> this delay value according to <strong>the</strong><br />
race model of Log<strong>an</strong> <strong>an</strong>d Cow<strong>an</strong> (1984). Gray matter volume was assessed <strong>from</strong> T1-weighted<br />
MRI images, which were spatially normalized using high-dimensional nonlinear registration<br />
with <strong>the</strong> FSL FNIRT tool. The relationship between gray matter volume <strong>an</strong>d symptom ratings<br />
was assessed via voxel-wise correlation with permutation testing to control <strong>for</strong> multiple<br />
comparisons. Inattentive symptom severity was negatively correlated bilaterally with gray matter<br />
volume in <strong>the</strong> superior parietal cortex, precuneus, <strong>an</strong>d left lateral parietal cortex, such that<br />
children with more severe inattentive symptoms showed decreased gray matter volume in <strong>the</strong>se<br />
areas. No signific<strong>an</strong>t relationship was found with hyperactive-impulsive symptoms at a corrected<br />
threshold. Region of interest <strong>an</strong>alysis of <strong>the</strong> right inferior frontal gyrus, a region previously<br />
implicated in successful response inhibition, indicated a positive correlation between gray matter<br />
<strong>an</strong>d SSRT. These results suggest that discrete structural differences may account <strong>for</strong> symptom<br />
severity <strong>an</strong>d cognitive deficits associated with ADHD. These data also highlight <strong>the</strong> regional <strong>an</strong>d<br />
network specificity underlying dimensions of <strong>the</strong> ADHD phenotype.<br />
Disclosures: E.M. Miller, None; A. Galv<strong>an</strong>, None; S.Y. Bookheimer, None; R.M. Bilder,<br />
None; J.T. McCracken, None; R.A. Poldrack, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.15/O15<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: Hong Kong Research Gr<strong>an</strong>ts Council Gr<strong>an</strong>t 750107<br />
The University of Hong Kong internal gr<strong>an</strong>t<br />
National Strategic Basic Research Program (‘‘973’’ Program) of <strong>the</strong> Ministry of<br />
Science <strong>an</strong>d Technology of China Gr<strong>an</strong>t 2005CB522802<br />
Title: The co-occurrence of visuospatial <strong>an</strong>d phonological deficits in Chinese dyslexic children<br />
Authors: *W. SIOK 1 , Z. JIN 2 , L. TAN 1 ;<br />
1 Univ. of Hong Kong, Hong Kong, Hong Kong; 2 Beijing 306 Hosp., Beijing, China
Abstract: Developmental dyslexia is a neurobiologically based disorder that is characterized by<br />
severe problems with reading, writing <strong>an</strong>d spelling. In alphabetic l<strong>an</strong>guages, <strong>the</strong> central problem<br />
of dyslexics lies in <strong>the</strong>ir phonological skills. Import<strong>an</strong>tly, this phonological deficit does not coexist<br />
with visual processing dysfunctions in a majority of dyslexics (Ramus et al., 2003).<br />
Cognitive-neuro<strong>an</strong>atomical profiles of developmental dyslexia vary across l<strong>an</strong>guages (Paulesu et<br />
al., 2001; Siok et al., 2004). All writing systems map graphic units to speech sounds, implying<br />
that <strong>the</strong> phonological deficit may be universal. This mapping, however, occurs at different levels.<br />
While English <strong>an</strong>d o<strong>the</strong>r alphabetic writing systems map letters onto phonemes (i.e., minimal<br />
speech sounds), logographic Chinese maps characters onto syllables which usually correspond to<br />
morphemes (i.e. me<strong>an</strong>ings). Visually, Chinese characters possess a number of intricate strokes<br />
that are packed into a square shape, often having <strong>the</strong>ir me<strong>an</strong>ings suggested by visual<br />
configurations. Learning to read Chinese thus may call <strong>for</strong> both phonological as well as<br />
visuospatial <strong>an</strong>alysis of characters <strong>an</strong>d words.<br />
We have previously shown that, similar to dyslexia in <strong>an</strong> alphabetic l<strong>an</strong>guage, dyslexia in<br />
Chinese is m<strong>an</strong>ifested by a phonological deficit, though cortical regions mediating this deficit in<br />
Chinese <strong>an</strong>d alphabetic l<strong>an</strong>guages are spatially separated (Siok et al., 2004, 2008). Here we set<br />
out to test <strong>the</strong> hypo<strong>the</strong>sis that disordered phonological processes co-exist with abnormal<br />
perceptual processing of visuospatial in<strong>for</strong>mation in Chinese dyslexia. Twelve dyslexic children<br />
<strong>an</strong>d 12 age-matched control subjects (average age 11) participated in this fMRI study. In a<br />
previous study (Siok et al., 2008), <strong>the</strong>se dyslexic subjects were found to have phonological<br />
problems as demonstrated by weaker activation of <strong>the</strong> left middle frontal gyrus in a rhyme<br />
judgment task. In <strong>the</strong> current study, <strong>the</strong>se subjects were asked to decide whe<strong>the</strong>r two characters<br />
viewed simult<strong>an</strong>eously had <strong>the</strong> same physical size (“physical size decision”). Results showed<br />
that normal controls exhibited stronger activations th<strong>an</strong> dyslexic readers in left intraparietal<br />
sulcus, bilateral extrastriate cortex, <strong>an</strong>d cuneus. The dyslexic group, however, exhibited<br />
signific<strong>an</strong>tly greater activation th<strong>an</strong> normal subjects in right intraparietal sulcus.<br />
The current findings, combined with our previous study with <strong>the</strong> same groups of subjects,<br />
suggested that developmental dyslexia in Chinese may be associated with <strong>the</strong> comorbidity of a<br />
nonlinguistic visuospatial deficit <strong>an</strong>d a linguistics-related phonological disorder.<br />
Disclosures: W. Siok, None; Z. Jin, None; L. T<strong>an</strong>, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.16/O16<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior
Support: Finnish Cultural Foundation<br />
Academy of Finl<strong>an</strong>d<br />
Title: Plastic neural ch<strong>an</strong>ges <strong>an</strong>d improvement in reading related skills caused by a short training<br />
period in Finnish six-year-old children<br />
Authors: *R. K. LOVIO 1 , A. HALTTUNEN 2 , H. LYYTINEN 3 , R. NÄÄTÄNEN 4 , T.<br />
KUJALA 4 ;<br />
1 2<br />
Univ. Helsinki, Helsinki, Finl<strong>an</strong>d; Policlinic of Child Psychiatry, HUCH, V<strong>an</strong>taa, Finl<strong>an</strong>d;<br />
3 4<br />
Dept. of Psychology, Univ. of Jyväskylä, Jyväskylä, Finl<strong>an</strong>d; Cognitive Brain Res. Unit, Univ.<br />
of Helsinki, Helsinki, Finl<strong>an</strong>d<br />
Abstract: Objective: This study aimed at determining whe<strong>the</strong>r <strong>an</strong> intervention game Literate that<br />
streng<strong>the</strong>ns sound-letter connections has a remedial effect on reading-related skills <strong>an</strong>d central<br />
auditory processing in children with <strong>an</strong> elevated risk <strong>for</strong> dyslexia. Children who played <strong>the</strong><br />
intervention game were hypo<strong>the</strong>sized to per<strong>for</strong>m better th<strong>an</strong> <strong>the</strong> control group children in<br />
reading-related tests <strong>an</strong>d also have increased mismatch negativity responses (MMNs) to speech<br />
sound ch<strong>an</strong>ges in Finnish syllables after <strong>the</strong> three hour training period.<br />
Methods: A group of 59 six-year-old children were assessed in <strong>the</strong>ir pre-reading skills with tests<br />
of phonological processing, letter knowledge <strong>an</strong>d recognition, reading <strong>an</strong>d writing syllables, nonwords<br />
<strong>an</strong>d words, rapid naming, <strong>an</strong>d working memory. In addition, <strong>the</strong> parents of <strong>the</strong> children<br />
were carefully interviewed on <strong>the</strong>ir family history of dyslexia in order to evaluate children’s<br />
familial risk <strong>for</strong> dyslexia. Of <strong>the</strong>se children, 35 children who could not read <strong>an</strong>d had problems in<br />
<strong>the</strong>ir pre-reading skills were included in <strong>the</strong> study. The children were divided into two groups not<br />
differing in IQ, age, sex or in reading-related skills. The intervention group played <strong>the</strong> game<br />
Literate <strong>an</strong>d <strong>the</strong> control group a game with ma<strong>the</strong>matical exercises. A total of 16 children in <strong>the</strong><br />
intervention group <strong>an</strong>d 13 in <strong>the</strong> control group finished <strong>the</strong> training period of altoge<strong>the</strong>r three<br />
hours during three weeks. In addition to behavioral tests, children’s mismatch negativity<br />
responses (MMNs) to five ch<strong>an</strong>ges (vowel, vowel-duration, conson<strong>an</strong>t, frequency (F0) <strong>an</strong>d<br />
intensity) were recorded both be<strong>for</strong>e <strong>an</strong>d after <strong>the</strong> training period. The stimuli were presented<br />
both in <strong>an</strong> oddball- <strong>an</strong>d in a multi-feature paradigm.<br />
Results: The intervention game group improved <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce statistically signific<strong>an</strong>tly in<br />
tests of letter knowledge, letter recognition, writing words <strong>an</strong>d writing non words where as <strong>the</strong><br />
control group improved <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> test of letter knowledge. Preliminary results also<br />
suggest that after <strong>the</strong> training period, <strong>the</strong> children who had played <strong>the</strong> intervention game had<br />
enh<strong>an</strong>ced MMN responses <strong>for</strong> <strong>the</strong> vowel <strong>an</strong>d vowel-duration ch<strong>an</strong>ges. Similar effects were not<br />
seen in <strong>the</strong> control group.<br />
Conclusions: The present results are encouraging by suggesting that already be<strong>for</strong>e <strong>the</strong> school<br />
start a short training period may improve per<strong>for</strong>m<strong>an</strong>ce in reading-related skills <strong>an</strong>d also cause<br />
improved cortical auditory discrimination. In future, it is import<strong>an</strong>t to study how <strong>the</strong> positive<br />
effects of <strong>the</strong> intervention game c<strong>an</strong> be optimized <strong>an</strong>d to what extent <strong>the</strong> intervention prevents<br />
reading deficits at school.<br />
Disclosures: R.K. Lovio, None; A. Halttunen, None; H. Lyytinen, None; R. Näätänen,<br />
None; T. Kujala, None.
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.17/O17<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: NS046565<br />
Title: Multiple reading-related factors differentially influence cortical <strong>an</strong>d subcortical white<br />
matter structure in normal <strong>an</strong>d dyslexic readers<br />
Authors: *R. E. FRYE 1 , K. M. HASAN 2 ;<br />
2 Dept. of Radiology, 1 Univ. of Texas, Houston, TX<br />
Abstract: Diffusion tensor imaging (DTI) studies have demonstrated inconsistent difference in<br />
white matter microstructure between dyslexic readers (DRs) <strong>an</strong>d typical readers (TRs). We<br />
hypo<strong>the</strong>sized that <strong>the</strong>se inconsistencies arise <strong>from</strong> <strong>the</strong> fact that both a history of reading disability<br />
<strong>an</strong>d actual per<strong>for</strong>m<strong>an</strong>ce on reading-related tasks are related to white matter microstructure in<br />
opposing directions, <strong>the</strong>reby making it difficult to find microstructure differences if both factors<br />
are not accounted <strong>for</strong> simult<strong>an</strong>eously. To test this hypo<strong>the</strong>sis we examined reading-related skills<br />
<strong>an</strong>d white matter structure in 16 DRs <strong>an</strong>d 20 age-equivalent TRs. Using a diffusion tensor<br />
imaging atlas-based algorithm volumetric microstructure <strong>an</strong>d macrostructure indices were<br />
calculated <strong>for</strong> major white matter pathways. The association of reading group, hemisphere <strong>an</strong>d<br />
reading-related skill on fractional <strong>an</strong>isotropy (FA), radial diffusivity (LT), <strong>an</strong>d volume of white<br />
matter tracts were <strong>an</strong>alyzed using linear regression in order to account <strong>for</strong> <strong>the</strong> influence of <strong>the</strong>se<br />
several factors simult<strong>an</strong>eously. DRs demonstrated lower FA, higher LT <strong>an</strong>d lower volume, as<br />
compared to TRs in <strong>the</strong> corona radiata. Better per<strong>for</strong>m<strong>an</strong>ce on reading-related tasks was<br />
associated with lower FA <strong>an</strong>d higher LT in <strong>the</strong> corona radiata <strong>an</strong>d <strong>the</strong> superior longitudinal<br />
fasciculus. This study suggests that multiple white matter pathways are associated with multiple<br />
reading-related factors. Most import<strong>an</strong>tly, we have identified that a history of dyslexia primary<br />
influences deep white matter tracts, specifically <strong>the</strong> corona radiata. This suggests that <strong>the</strong><br />
primary disturb<strong>an</strong>ces associated with dyslexia may selectively affect <strong>the</strong> deep white matter <strong>an</strong>d<br />
that microstructure ch<strong>an</strong>ges in o<strong>the</strong>r white matter tracts may result <strong>from</strong> secondary processes.<br />
Disclosures: R.E. Frye, None; K.M. Has<strong>an</strong>, None.
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.18/O18<br />
Topic: C.06.j. ADHD, SLI, dyslexia <strong>an</strong>d o<strong>the</strong>r specific disorders of neurobehavior<br />
Support: MH059803<br />
Title: Sensory gating <strong>an</strong>d premonitory urges in Tourette syndrome<br />
Authors: *N. R. SWERDLOW 1 , E. C. MIGUEL 2 , A. N. SUTHERLAND OWENS 1 ;<br />
1 UCSD Sch. of Medicine, Dept of Psychiatry, La Jolla, CA; 2 Rua Ouvidio Pires de Campos S/N,<br />
Dept. de Psiquiatria da Faculdade de Medicina da USP, Sao Paulo, Brazil<br />
Abstract: Overview: Sensory <strong>an</strong>d sensorimotor gating deficits characterize several<br />
neuropsychiatric disorders, including Tourette Syndrome <strong>an</strong>d schizophrenia. Premonitory urges<br />
(PU) in TS are uncom<strong>for</strong>table <strong>an</strong>d intrusive subjective feelings that c<strong>an</strong> be associated to sensory<br />
sensations that may reflect deficits in <strong>the</strong> automatic inhibit or "gating" of perceptual experiences.<br />
Two scales developed to characterize <strong>an</strong>d assess PU severity in TS are <strong>the</strong> University of Sao<br />
Paulo Sensory Phenomena Scale (USP-SPS; Rosario et al., in press, CNS Spectrums), <strong>an</strong>d <strong>the</strong><br />
Premonitory Urge <strong>for</strong> Tics Scale (PUTS: Woods, et al., 2005). We assessed <strong>the</strong> relationship<br />
between <strong>the</strong>se scales in TS patients <strong>an</strong>d measures of two scales used to assess sensory gating in<br />
schizophrenia: <strong>the</strong> Sensory Gating Inventory (SGI) or <strong>the</strong> Structured Interview <strong>for</strong> Assessing<br />
Perceptual Anomalies (SIAPA).<br />
Methods: Carefully characterized TS adults <strong>an</strong>d children <strong>an</strong>d healthy comparison subjects<br />
(HCS) (total n=40, age r<strong>an</strong>ge 10-41 y) participated in this study. Patients completed <strong>the</strong> USP-<br />
SPS, PUTS, SGI <strong>an</strong>d SIAPA, as well as adult or child versions of symptom severity scales (Yale<br />
Global Tic Severity Scale (YGTSS) <strong>an</strong>d <strong>the</strong> Yale-Brown Obsessive Compulsive Scale<br />
(YBOCS)). HCS completed <strong>the</strong> SGI <strong>an</strong>d SIAPA. Group differences were assessed by ANOVA;<br />
regression <strong>an</strong>alyses were used to investigate relationships among clinical <strong>an</strong>d subjective scale<br />
scores.<br />
Results: TS patients endorsed greater subjective difficulty in sensory gating, measured by <strong>the</strong><br />
SGI, compared to HCS (p < 0.0003). Specific SGI subscales provided greatest group separation<br />
<strong>an</strong>d subscale/item <strong>an</strong>alyses are in progress. In contrast, SIAPA total scores did not differ<br />
signific<strong>an</strong>tly across groups. Despite this, <strong>the</strong>re was a signific<strong>an</strong>t positive correlation between<br />
SIAPA total <strong>an</strong>d SGI total scores (p
signific<strong>an</strong>tly with traditional measures of TS symptoms (YGTSS, YBOCS).<br />
Conclusion: TS patients endorse difficulties in sensory gating compared to non-TS particip<strong>an</strong>ts,<br />
but <strong>the</strong>re is no clear relationship between <strong>the</strong>se subjective impressions, <strong>an</strong>d ei<strong>the</strong>r measures of<br />
premonitory urges or traditional symptom measures of TS. Conceivably, sensory gating<br />
disturb<strong>an</strong>ces, premonitory urges <strong>an</strong>d motor/vocal tics represent three separable features of TS.<br />
Disclosures: N.R. Swerdlow, S<strong>an</strong>ofi/Aventis, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); MH059803, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); Allerg<strong>an</strong>, Inc., B. Research Gr<strong>an</strong>t (principal investigator, collaborator<br />
or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Pfizer Pharmaceuticals, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); E.C. Miguel, None; A.N. Su<strong>the</strong>rl<strong>an</strong>d Owens, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.19/O19<br />
Topic: C.06.h. Angelm<strong>an</strong> <strong>an</strong>d o<strong>the</strong>r developmental disorders<br />
Support: CIHR Gr<strong>an</strong>t ELA-80227<br />
Title: Oculomotor control assessed with fMRI in children with fetal alcohol spectrum disorders<br />
Authors: *N. ALAHYANE, S. ROMAN, R. TITMAN, J. N. REYNOLDS, D. P. MUNOZ;<br />
Ctr. Neurosci. Studies, Queen's Univ., Kingston, ON, C<strong>an</strong>ada<br />
Abstract: Prenatal alcohol exposure c<strong>an</strong> result in a r<strong>an</strong>ge of cognitive <strong>an</strong>d behavioural deficits<br />
that are collectively termed fetal alcohol spectrum disorders (FASD). Most notably, children<br />
with FASD present with deficits in executive functions. However, <strong>the</strong> underlying neural<br />
substrate responsible <strong>for</strong> <strong>the</strong>se behavioural deficits is still almost completely unknown. Yet,<br />
exploring <strong>the</strong> functional integrity of <strong>the</strong> brain circuitry in FASD is crucial to guide <strong>the</strong>rapeutic<br />
treatment <strong>an</strong>d rehabilitation. To address this issue, we combined functional magnetic reson<strong>an</strong>ce<br />
imaging (fMRI) <strong>an</strong>d eye movement recording in children with FASD (10-14 years) <strong>an</strong>d age/sex<br />
matched controls. Volunteers per<strong>for</strong>med <strong>the</strong> <strong>an</strong>tisaccade task (inhibit <strong>the</strong> prepotent saccade to a<br />
peripheral visual stimulus <strong>an</strong>d instead initiate a voluntary saccade in <strong>the</strong> opposite direction) to<br />
investigate executive control of behaviour. This task was compared to <strong>the</strong> prosaccade task in<br />
which particip<strong>an</strong>ts were instructed to per<strong>for</strong>m <strong>the</strong> automatic response, i.e., to look directly at <strong>the</strong>
stimulus. In our fMRI design, prosaccade <strong>an</strong>d <strong>an</strong>tisaccade trials were pseudo-r<strong>an</strong>domly<br />
interleaved based on a central instructional visual cue. Catch trials (containing only <strong>the</strong><br />
instructional cue, i.e., no peripheral stimulus) were also included, requiring no saccades but<br />
instead mainten<strong>an</strong>ce of central fixation until <strong>the</strong> end of <strong>the</strong> trial. Children with FASD had<br />
difficulties in initiating voluntary <strong>an</strong>tisaccades <strong>an</strong>d inhibiting inappropriate saccades, compared<br />
to control children. These deficits were represented by longer <strong>an</strong>tisaccade reaction times, higher<br />
intra-individual variability in saccade reaction times, more errors (i.e., saccades to <strong>the</strong> stimulus)<br />
in <strong>the</strong> <strong>an</strong>tisaccade task, <strong>an</strong>d difficulties in maintaining fixation in catch trials. These behavioural<br />
deficits were associated with altered patterns of activation in <strong>the</strong> frontostriatal <strong>an</strong>d frontoparietal<br />
networks, both essential to per<strong>for</strong>m <strong>an</strong>tisaccades successfully. Indeed, when compared to<br />
controls, children with FASD showed lower activation in <strong>the</strong> frontal eye field (FEF),<br />
supplementary eye field (SEF), parietal eye field (PEF), dorsolateral prefrontal cortex (DLPFC)<br />
<strong>an</strong>d striatum, our regions of interest. Thus <strong>the</strong> core brain systems <strong>for</strong> oculomotor control are in<br />
place in FASD, but <strong>the</strong>y are inefficient <strong>an</strong>d easily subjected to errors. As a consequence, children<br />
with FASD would be unable to control efficiently <strong>the</strong>ir saccade behaviour. Supported by CIHR<br />
Gr<strong>an</strong>t ELA-80227.<br />
Disclosures: N. Alahy<strong>an</strong>e, None; S. Rom<strong>an</strong>, None; R. Titm<strong>an</strong>, None; J.N. Reynolds,<br />
None; D.P. Munoz, None.<br />
Poster<br />
537. Developmental Disorders of L<strong>an</strong>guage, Attention, <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 537.20/O20<br />
Topic: F.01.e. Emotion<br />
Title: Lesch-Nyh<strong>an</strong> syndrome, a metabolic <strong>an</strong>d behavioral correlation<br />
Authors: *L. JIMENEZ BOTELLO 1,2 , M. AVILA-COSTA 1 , J. CARRILLO-RUIZ 2 , J.<br />
CORREA-BASURTO 3 , O. PICAZO 3 ;<br />
1 Lab. Neuromorfologia, FES-Iztacala UNAM, Tlalnep<strong>an</strong>tla, Mexico; 2 Lab. de Neurociencias,<br />
Univ. Anahuac, Huixquiluc<strong>an</strong>, Estado de Mexico, Mexico; 3 Seccion de Estudios de Posgrado e<br />
Investigacion, ESM-IPN, Ciudad de Mexico, Mexico<br />
Abstract: Background<br />
It is well know that compulsive conducts have direct relation with alterations in limbic system<br />
<strong>an</strong>d D2 dopaminergic(D2) receptors in accumbens nuclei. Previous works have been confirmed a<br />
functional <strong>an</strong>tagonism between D2 dopaminergic receptors <strong>an</strong>d A2A adenosine(A2A) receptors. In
fact A2A <strong>an</strong>tagonists c<strong>an</strong> increase agonist D2 receptor mediated effects.<br />
Metilx<strong>an</strong>tines, like caffeine c<strong>an</strong> block unselectively A2A receptors <strong>an</strong>d high doses chronic<br />
administration c<strong>an</strong> induce self-mutilation behavior in rats.<br />
Lesch-Nyh<strong>an</strong> Syndrome(LNS), is a rare X chromosome related disease which causes absence of<br />
HGPRT enzyme, indispensable in “de novo” purine biosyn<strong>the</strong>sis. Main m<strong>an</strong>ifestations are, high<br />
uric acid levels in serum, notable mental delay <strong>an</strong>d most relev<strong>an</strong>t, a serious compulsive selfmutilation<br />
behavior. In LNS, a notable increase in hypox<strong>an</strong>thin <strong>an</strong>d x<strong>an</strong>thin serum levels also<br />
exist, which are precursors of uric acid.<br />
If hypox<strong>an</strong>thin <strong>an</strong>d x<strong>an</strong>thin are molecules similar to Metilx<strong>an</strong>thines, it is possible that could have<br />
action as A2A <strong>an</strong>tagonist receptors.<br />
Objective<br />
To predict by Docking, if hypox<strong>an</strong>thin <strong>an</strong>d x<strong>an</strong>thin have affinity <strong>for</strong> A2A receptors <strong>an</strong>d compare<br />
it with caffeine.<br />
Methodology<br />
Docking by Autodock3 was realized, molecular simulation was made between A2A receptor <strong>an</strong>d<br />
molecules, x<strong>an</strong>thin, hypox<strong>an</strong>thin <strong>an</strong>d caffeine, <strong>an</strong>d <strong>the</strong>ir atomic interaction was <strong>an</strong>alyzed.<br />
Results<br />
Results established that hipox<strong>an</strong>thine <strong>an</strong>d x<strong>an</strong>thin have affinity <strong>for</strong> A2A receptors even similar to<br />
caffeine.<br />
Conclusions<br />
It is possible that high serum levels of hypox<strong>an</strong>thin <strong>an</strong>d x<strong>an</strong>thin, have direct relation with selfmutilation<br />
behavior, observed in Lesch-Nyh<strong>an</strong> syndrome, maybe acting as A2A receptor<br />
<strong>an</strong>tagonists, never<strong>the</strong>less pharmacological <strong>an</strong>d behavioral studies are required to obtain a<br />
concrete affirmation.<br />
Disclosures: L. Jimenez Botello, None; M. Avila-Costa, None; J. Carrillo-Ruiz, None; J.<br />
Correa-Basurto, None; O. Picazo, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.1/O21<br />
Topic: C.07.i. Predisposing factors<br />
Support: AHFMR postdoctoral fellowship<br />
CIHR research gr<strong>an</strong>t
Killam scholarship<br />
NSERC scholarship<br />
Ruby doctoral scholarship of University of Calgary<br />
Title: Viral-like brain inflammation during development increases seizure susceptibility in adult<br />
rats<br />
Authors: *K. RIAZI 1 , M. A. GALIC 2 , A. K. HENDERSON 3 , S. TSUTSUI 3 , Q. J. PITTMAN 3 ;<br />
1 Physiol. <strong>an</strong>d Biophysics, 2 Neurosci., 3 Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: Viral infections of <strong>the</strong> CNS c<strong>an</strong> cause both immediate <strong>an</strong>d long-term neurological<br />
side-effects including increased risk <strong>for</strong> seizures <strong>an</strong>d epilepsy despite bench-mark <strong>the</strong>rapy. Brain<br />
inflammation subsequent to CNS infection (encephalitis) in children has been suspected to<br />
mediate chronic ch<strong>an</strong>ges in neuronal excitability; however, <strong>the</strong> mech<strong>an</strong>isms underlying such<br />
effects are not completely understood. To examine <strong>the</strong> effects of CNS inflammation on seizure<br />
susceptibility, we injected polyinosinic: polycytidylic acid (POLY I:C) intracerebroventricularly<br />
to mimic a viral CNS infection in 14 day-old male rats. This caused <strong>an</strong> elevation in body<br />
temperature <strong>an</strong>d <strong>an</strong> increase in <strong>the</strong> pro-inflammatory cytokine, interleukin (IL)-1beta in<br />
comparison to saline-treated matched controls. Five weeks later, we tested <strong>the</strong>ir susceptibility to<br />
lithium-pilocarpine <strong>an</strong>d pentylenetetrazole-induced seizures <strong>an</strong>d found a signific<strong>an</strong>t increase in<br />
seizure sensitivity. A marked increase in specific glutamate receptor mRNA was found in <strong>the</strong><br />
adult hippocampus, including subunits <strong>for</strong> NMDA (NR2A <strong>an</strong>d C) <strong>an</strong>d AMPA (GluR1). Both <strong>the</strong><br />
increase in seizure susceptibility <strong>an</strong>d <strong>the</strong> alterations in glutamate receptor subunit mRNAs could<br />
be blocked if minocycline, a tetracycline derivative capable of suppressing CNS inflammation,<br />
was given systemically at <strong>the</strong> time of <strong>the</strong> POLY I:C administration. The systemic administration<br />
of minocycline was also able to attenuate <strong>the</strong> neonatal POLY I:C-induced IL-1beta increase in<br />
<strong>the</strong> hippocampus, but not <strong>the</strong> febrile process. The long-term increase in seizure susceptibility was<br />
also associated with deficits in fear conditioning per<strong>for</strong>m<strong>an</strong>ce, but not linked to <strong>an</strong>y chronic<br />
effect on cytokine (IL-1beta, TNFalpha) levels. Thus a novel model of viral CNS inflammation<br />
using POLY I:C c<strong>an</strong> yield import<strong>an</strong>t insights into <strong>the</strong> pathophysiological relationship between<br />
encephalitis <strong>an</strong>d seizures, <strong>an</strong>d supports <strong>the</strong> possible use of adjunctive <strong>an</strong>ti-inflammatory agents<br />
like minocycline.<br />
Disclosures: K. Riazi, None; M.A. Galic, None; A.K. Henderson, None; S. Tsutsui,<br />
None; Q.J. Pittm<strong>an</strong>, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.2/O22<br />
Topic: C.07.j. Animal models<br />
Title: LPS-induced inflammation triggers epileptogenic mech<strong>an</strong>isms in pilocarpine non<br />
responsive treated rats<br />
Authors: *F. SCHIO, G. NAVARRO MORA, M. PELLITTERI, A. CHAKIR, L. ZANETTI,<br />
R. LODICO, A. SBARBATI, P. FABENE;<br />
Dept. of Morphological <strong>an</strong>d Biomed. Sci., Univ. of Verona (Italy), Verona, Italy<br />
Abstract: Systemic administration of a single dose of pilocarpine in rat c<strong>an</strong> lead to status<br />
epilepticus (SE), a condition that seems to be essential to develop chronic epilepsy. But,<br />
preliminary data in our lab suggest that spont<strong>an</strong>eous <strong>an</strong>d recurrent seizures (SRS) c<strong>an</strong> evolve<br />
even in absence of SE. Moreover, our recent studies have suggested a link between vascular<br />
inflammation <strong>an</strong>d pro-epileptogenic events. In this study, we have investigated <strong>the</strong> effect of<br />
chronic (three/week, 1 mg/kg) <strong>an</strong>d acute (twice/month, 30 mg/kg) i.p. administration of LPS <strong>for</strong><br />
40 days in control (saline injected) rats <strong>an</strong>d pilocarpine-injected <strong>an</strong>imals without SE to<br />
investigate how inflammation conditioned <strong>the</strong> epileptogenic process. In particular, our<br />
hypo<strong>the</strong>sis was that inflammatory challenges might modulate neuronal excitability after <strong>an</strong> initial<br />
priming. We have recorded EEG, body temperature <strong>an</strong>d circadi<strong>an</strong> rhythms <strong>for</strong> up to 2 months,<br />
<strong>an</strong>d after sacrifice we have per<strong>for</strong>med microarray on inflammatory cytokine, chemokine <strong>an</strong>d<br />
adhesion molecules to <strong>an</strong>alyze <strong>the</strong> inflammatory response. Our hypo<strong>the</strong>sis was confirmed: even<br />
in absence of SE, pilocarpine injected rats exhibit SRS both in acute <strong>an</strong>d chronic LPS<br />
administration. The higher number of seizures was observed with chronic subclinical injection.<br />
These results subst<strong>an</strong>tiate that LPS inflammatory insults trigger epileptogenic mech<strong>an</strong>ism in<br />
pilocarpine non responsive treated rats <strong>an</strong>d microarray <strong>an</strong>alysis confirm <strong>the</strong> involvement of<br />
vascular inflammation <strong>an</strong>d molecular adhesion in epilepsy.<br />
Disclosures: F. Schio, None; G. Navarro Mora, None; M. Pellitteri, None; A. Chakir,<br />
None; L. Z<strong>an</strong>etti, None; R. Lodico, None; A. Sbarbati, None; P. Fabene, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.3/O23
Topic: C.07.j. Animal models<br />
Support: ZNZ PhD Fellowship<br />
Title: Absence of T-cells aggravates epileptogenesis <strong>an</strong>d neurodegeneration in a mouse model of<br />
temporal lobe epilepsy<br />
Authors: M. ZATTONI 1,2 , M. MURA 1 , *J.-M. FRITSCHY 1 , K. FREI 2 ;<br />
1 Univ. Zurich, CH-8057 Zurich, Switzerl<strong>an</strong>d; 2 Univ. Hosp. Zurich, Zurich, Switzerl<strong>an</strong>d<br />
Abstract: Mesial temporal lobe epilepsy (MTLE) is a prevalent adult seizure disorders <strong>an</strong>d,<br />
when associated with hippocampal sclerosis (HS), is <strong>the</strong> most refractory to pharmaco<strong>the</strong>rapy.<br />
There is ample evidence <strong>for</strong> a contribution of inflammatory processes to <strong>the</strong> pathophysiology of<br />
TLE. Here, we used a mouse model of TLE, based on a unilateral intrahippocampal injection of<br />
kainic acid (KA), to investigate <strong>the</strong> recruitment of leukocytes to <strong>the</strong> epileptic focus <strong>an</strong>d <strong>the</strong>ir<br />
contribution to epileptogenesis. In this model, gradual neurodegeneration in CA1 <strong>an</strong>d CA3 <strong>an</strong>d<br />
strong activation of astrocytes <strong>an</strong>d microglia leads to a focal lesion <strong>an</strong>d onset of spont<strong>an</strong>eous<br />
recurrent seizures after a latent phase of 2 weeks. Immunohistochemical <strong>an</strong>alysis revealed a<br />
tr<strong>an</strong>sient recruitment of CD4 + <strong>an</strong>d CD8 + T cells, but not B cells, in <strong>the</strong> lesioned hippocampus,<br />
peaking at <strong>the</strong> time of seizure onset. To clarify <strong>the</strong> possible link between T cell invasion,<br />
structural ch<strong>an</strong>ges in <strong>the</strong> epileptic focus, <strong>an</strong>d seizure activity, we investigated immunodeficient<br />
mice (RAG1-KO) lacking both B <strong>an</strong>d T cells. KA-induced neurodegeneration was more severe<br />
in RAG1-KO th<strong>an</strong> in wild-type mice, as seen after 2 weeks. It was accomp<strong>an</strong>ied by pronounced<br />
microglia activation, <strong>an</strong>d most strikingly, by invasion of Gr-1 + neutrophils into <strong>the</strong> lesion. As a<br />
result, <strong>the</strong> affected hippocampus was gradually destroyed in <strong>the</strong>se mice. Moreover, RAG1-KO<br />
mice presented with a dramatic shortening of <strong>the</strong> latent phase prior to onset of recurrent seizures,<br />
which occurred within 2-days post-KA, well be<strong>for</strong>e synaptic reorg<strong>an</strong>ization has occurred in <strong>the</strong><br />
lesioned hippocampus.<br />
These results indicate that <strong>the</strong> absence of T cells aggravates both <strong>the</strong> initial effects of <strong>the</strong> KA<br />
lesion <strong>an</strong>d <strong>the</strong> long-term degeneration of <strong>the</strong> epileptic hippocampus. There<strong>for</strong>e, activation of<br />
innate immune responses is exacerbated in <strong>the</strong> absence of acquired immunity <strong>an</strong>d might<br />
contribute to neutrophil-mediated neurodegeneration <strong>an</strong>d to enh<strong>an</strong>ced epileptogenesis.<br />
Disclosures: M. Zattoni, None; M. Mura, None; J. Fritschy , None; K. Frei, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.4/O24
Topic: C.07.j. Animal models<br />
Title: New evidences of a possible role of vascular alterations <strong>an</strong>d cytokines in absence epilepsy<br />
Authors: *M. PELLITTERI 1 , F. SCHIO 1 , R. LODICO 1 , E. NICOLATO 1 , F. BOSCHI 1 , G.<br />
NAVARRO MORA 1 , A. CHAKIR 1 , F. MERIGO 1 , E. MOSCONI 1 , P. MARZOLA 1 , R.<br />
VASTYANOV 2 , A. SHANDRA 2 , L. GODLEVSKY 2 , A. SBARBATI 1 , G. VAN<br />
LUIJTELAAR 3 , P. F. FABENE 1 ;<br />
1 Dept. of Morphological <strong>an</strong>d Biomed. Sci., Univ. of Verona, Verona, Italy; 2 Physiol. Dept., State<br />
Med. Univ., Odessa, Ukraine; 3 Donders Inst. <strong>for</strong> Brain, Radboud Univ. Nijmegen, Nijmegen,<br />
Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: The actions of proinflammatory cytokines in <strong>the</strong> CNS are only partially discovered.<br />
Some cytokines have been recently shown to affect neurotr<strong>an</strong>smitters or are required to preserve<br />
<strong>the</strong> synaptic strength at excitatory synapses, or affect <strong>the</strong> expression of various neuropeptides <strong>an</strong>d<br />
neurotrophic factors in several brain regions. Ch<strong>an</strong>ges in <strong>the</strong> immune system may ch<strong>an</strong>ge <strong>the</strong><br />
excitability of <strong>the</strong> CNS <strong>an</strong>d alter <strong>the</strong> susceptibility of exogenous induced or genetically<br />
determined types of epilepsy. Here we investigate <strong>the</strong> role of two cytokines, IL-1β <strong>an</strong>d TNF-α in<br />
WAG/Rij rats, a genetic model of absence epilepsy, <strong>the</strong> most pure <strong>for</strong>m of generalized epilepsy<br />
characterized in <strong>the</strong> EEG by widespread bilaterally synchronous spike-wave discharges (SWDs)<br />
caused by thalamo-cortical oscillations.<br />
Our hypo<strong>the</strong>sis is that cerebral blood flow alterations <strong>an</strong>d cytokines/chemokines release c<strong>an</strong><br />
modulate <strong>the</strong> occurrence of SWDs. In this view, gene-array <strong>an</strong>alysis <strong>for</strong> cell signaling pathways<br />
involved in SWDs in different cerebral areas, <strong>an</strong>d qPCR data were correlated with EEG <strong>an</strong>d<br />
MRS data. Fur<strong>the</strong>rmore, we <strong>an</strong>alyzed <strong>the</strong> blood serum by ELISA method <strong>for</strong> TNF-α <strong>an</strong>d IL-1β in<br />
control <strong>an</strong>d epileptic <strong>an</strong>imals; in addition, we injected i.p. both controls <strong>an</strong>d WAG/Rij rats by<br />
TNF-α (2µg/kg) <strong>an</strong>d IL-1β (2µg/kg).<br />
Our results indicate that a) cerebral blood volume <strong>an</strong>d flow are altered in epileptic <strong>an</strong>imals vs<br />
controls; b) slight differences have been noticed in TNF-α basal serum levels in early<br />
developmental stages (2 months) in WAG/Rij vs controls; c) TNF-α <strong>an</strong>d IL-1β injections<br />
induced a large increase in <strong>the</strong> number of SDWs in WAG/Rij <strong>an</strong>imals following a different timepattern;<br />
d) MRS data indicate that WAG/Rij rats have higher levels of Glutamate vs controls,<br />
both in <strong>the</strong> thalamus <strong>an</strong>d in <strong>the</strong> cerebral cortex; e) since cytokines have been reported to alter <strong>the</strong><br />
neurotr<strong>an</strong>smitters levels, we would like to fur<strong>the</strong>r investigate <strong>the</strong> possible role of <strong>the</strong>se<br />
mech<strong>an</strong>isms in this <strong>an</strong>imal model.Summarizing, we hereby reported some evidences on <strong>the</strong><br />
possible neuromodulator functions of inflammatory cytokines <strong>an</strong>d absence epilepsy. Fur<strong>the</strong>r<br />
study will allow to better elucidate <strong>the</strong>se “liaisons d<strong>an</strong>gereuses”.<br />
Disclosures: M. Pellitteri, None; F. Schio, None; R. Lodico, None; E. Nicolato, None; F.<br />
Boschi, None; G. Navarro Mora, None; A. Chakir, None; F. Merigo, None; E. Mosconi,<br />
None; P. Marzola, None; R. Vasty<strong>an</strong>ov, None; A. Sh<strong>an</strong>dra, None; L. Godlevsky, None; A.<br />
Sbarbati, None; G. v<strong>an</strong> Luijtelaar, None; P.F. Fabene, None.<br />
Poster
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.5/O25<br />
Topic: C.07.j. Animal models<br />
Support: NIH Gr<strong>an</strong>t NS36981<br />
National Health <strong>an</strong>d Medical Research Council ID 465423<br />
Title: The cortical innate immune response increases local neuronal excitability leading to<br />
seizures<br />
Authors: *K. M. RODGERS, M. R. HUTCHINSON, A. NORTHCUTT, S. F. MAIER, L. R.<br />
WATKINS, D. S. BARTH;<br />
Behav Neurosci, Univ. Colorado, Boulder, CO<br />
Abstract: Brain glial cells, which are five times more prevalent th<strong>an</strong> neurons, have recently<br />
received attention <strong>for</strong> <strong>the</strong>ir potential involvement in epileptic seizures. Microglia <strong>an</strong>d astrocytes,<br />
associated with inflammatory innate immune responses, are responsible <strong>for</strong> surveill<strong>an</strong>ce of brain<br />
damage that frequently results in seizures. Thus, <strong>an</strong> intriguing suggestion has been put <strong>for</strong>ward<br />
that seizures may be facilitated <strong>an</strong>d perhaps triggered by brain immune responses. Indeed, recent<br />
evidence strongly implicates innate immune responses in lowering seizure threshold in<br />
experimental models of epilepsy, yet, <strong>the</strong>re is no proof that <strong>the</strong>y c<strong>an</strong> play <strong>an</strong> independent role in<br />
initiating seizures in vivo. Here we show that cortical innate immune responses alone produce<br />
profound increases of brain excitability resulting in focal seizures. We found that cortical<br />
application of lipopolysaccharide (LPS), binding to toll-like receptor 4 (TLR4), triples evoked<br />
field potential amplitudes <strong>an</strong>d produces focal epilepti<strong>for</strong>m discharges. These effects are<br />
prevented by pre-application of interleukin-1 (IL-1) receptor <strong>an</strong>tagonist (IL-1ra). Our results<br />
demonstrate how <strong>the</strong> innate immune response may participate in acute seizures, increasing<br />
neuronal excitability through IL-1 release in response to TLR4 detection of <strong>the</strong> d<strong>an</strong>ger signals<br />
associated with infections of <strong>the</strong> central nervous system <strong>an</strong>d with brain injury. These results<br />
suggest <strong>an</strong> import<strong>an</strong>t role of innate immunity in epileptogenesis <strong>an</strong>d focus on glial inhibition,<br />
through pharmacological blockade of TLR4 <strong>an</strong>d <strong>the</strong> proinflammatory mediators released by<br />
activated glia, in <strong>the</strong> study <strong>an</strong>d treatment of seizure disorders in hum<strong>an</strong>s.<br />
Disclosures: K.M. Rodgers, None; M.R. Hutchinson, None; A. Northcutt, None; S.F. Maier,<br />
None; L.R. Watkins, None; D.S. Barth, None.<br />
Poster
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.6/O26<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: Lundbeck Foundation<br />
Title: Neuroprotective properties of a novel, non-hematopoietic agonist of <strong>the</strong> erythropoietin<br />
(EPO) receptor<br />
Authors: S. PANKRATOVA 1 , K. SONN 2 , V. SOROKA 1 , D. KIRYUSHKO 1 , L. B. KOHLER 1 ,<br />
M. RATHJE 1 , B. GU 1 , O. CLAUSEN 1 , A. ZHARKOVSKY 2 , E. BOCK 1 , *V. BEREZIN 1 ;<br />
1 2<br />
Protein Lab, INF, Copenhagen Univ., Copenhagen N, Denmark; Dept. of Pharmacology, Univ.<br />
of Tartu, Tartu, Estonia<br />
Abstract: Erythropoietin (EPO), a member of <strong>the</strong> type 1 cytokine superfamily, controls<br />
proliferation <strong>an</strong>d differentiation of erythroid progenitor cells through binding to <strong>an</strong>d dimerization<br />
of <strong>the</strong> EPO receptor (EPOR). EPO <strong>an</strong>d EPOR are also expressed in <strong>the</strong> central nervous system,<br />
where <strong>the</strong>y are involved in tissue protection. However, <strong>the</strong> use of EPO as a neuroprotective agent<br />
may be hampered by its erythropoietic activity. There<strong>for</strong>e, developing EPO mimetics with no<br />
hematopoietic activity is import<strong>an</strong>t. Based on <strong>the</strong> known crystal structure of <strong>the</strong> EPO-EPOR<br />
complex, we designed a peptide, termed Epotris, corresponding to <strong>the</strong> C α-helix region (amino<br />
acid residues 92-111) of hum<strong>an</strong> EPO. This peptide, which mimics binding site 2 of EPO,<br />
specifically binds to EPOR <strong>an</strong>d induces neurite outgrowth <strong>from</strong> cerebellar <strong>an</strong>d hippocampal<br />
primary neurons. This induction depends on <strong>the</strong> integrity of EPOR downstream signaling<br />
mech<strong>an</strong>isms. We showed that Epotris promotes neuronal survival in in vitro <strong>an</strong>d in vivo models<br />
of kainic acid-induced neurotoxicity. Treatment with Epotris attenuated seizures <strong>an</strong>d decreased<br />
<strong>the</strong> kainic acid-induced mortality. Systemically injected Epotris was found in cerebral spinal<br />
fluid <strong>an</strong>d, in contrast to EPO, did not stimulate erythropoiesis upon chronic administration. Thus,<br />
Epotris was found to mimic <strong>the</strong> neuroprotective, but not erythropoietic, activity of EPO.<br />
Disclosures: S. P<strong>an</strong>kratova, None; K. Sonn, None; V. Soroka, None; D. Kiryushko,<br />
None; L.B. Kohler, None; M. Rathje, None; B. Gu, None; O. Clausen, None; A. Zharkovsky,<br />
None; E. Bock, Neoloch Pharmaceuticals ApS, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); V. Berezin , Neoloch Pharmaceuticals ApS, E. Ownership<br />
Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.7/O27<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: NINDS Gr<strong>an</strong>t NS35633 to TJM<br />
Title: Adeno-associated virus-mediated expression <strong>an</strong>d constitutive secretion of interleukin-10<br />
(IL-10) suppresses kainic-acid seizure-induced inflammatory glial activation in <strong>the</strong> brain<br />
Authors: *M. S. WEINBERG, S. B. FOTI, B. L. BLAKE, R. J. SAMULSKI, T. J. MCCOWN;<br />
Gene Therapy Ctr., Univ. of North Carolina At Chapel Hill, Chapel Hill, NC<br />
Abstract: Growing evidence supports <strong>the</strong> notion that seizures may result <strong>from</strong> <strong>an</strong>d contribute to<br />
a pathological imbal<strong>an</strong>ce of glial-based cytokine release in <strong>the</strong> brain. This cytokine imbal<strong>an</strong>ce<br />
results in impaired neurochemical tr<strong>an</strong>smission, feed-<strong>for</strong>ward inflammatory cascades, leakage of<br />
<strong>the</strong> blood-brain barrier, <strong>an</strong>d / or neuronal damage, which, in part or whole, may contribute to<br />
epileptogenesis. The limbic system, including <strong>the</strong> hippocampus, piri<strong>for</strong>m cortex, <strong>an</strong>d amygdala,<br />
is particularly susceptible to damage in patients suffering <strong>from</strong> temporal lobe epilepsy. An<br />
<strong>an</strong>imal model of temporal lobe epilepsy, in which rats are injected peripherally with kainic acid,<br />
results in a well-characterized progression of glial activation, followed by cell damage <strong>an</strong>d death<br />
in <strong>the</strong> limbic system. Thus, <strong>the</strong> glial-cytokine inflammatory pathway involvement in <strong>the</strong> kainic<br />
acid-based model represents <strong>an</strong> import<strong>an</strong>t <strong>the</strong>rapeutic target <strong>for</strong> exploration of novel <strong>an</strong>d more<br />
effective treatments <strong>for</strong> temporal lobe epilepsy. In <strong>the</strong> present studies, we demonstrate <strong>the</strong><br />
efficacy of adeno-associated viral vectors (AAV) expressing <strong>an</strong>d constitutively secreting full<br />
length or a truncated <strong>for</strong>m of interleukin 10 (IL-10) in limbic areas of kainic acid-treated rats. IL-<br />
10 is known to exert potent ‘<strong>an</strong>ti-inflammatory’ effects via suppression of proinflammatory<br />
cytokine <strong>an</strong>d chemokine production, <strong>an</strong>d upregulation of immunosuppressive mediators such as<br />
interleukin-1 receptor <strong>an</strong>tagonist. Unilateral infusion of <strong>the</strong> viral vector, which consists of<br />
promoter-driven full or truncated IL-10 along with <strong>the</strong> fibronectin secretory amino acid sequence<br />
(Haberm<strong>an</strong> et al., 2003), leads to clear localized <strong>an</strong>d lateralized suppression of microglial<br />
activation (based on OX-42 / CD11b immunoreactivity) in kainic acid-treated rats. Thus,<br />
expression <strong>an</strong>d constitutive secretion of IL-10 signific<strong>an</strong>tly modifies <strong>the</strong> neuroimmune response<br />
to seizure activity.<br />
Disclosures: M.S. Weinberg, None; S.B. Foti, None; B.L. Blake, None; R.J. Samulski, None;<br />
T.J. McCown, The University of North Carolina holds a patent on <strong>the</strong> secretory signal vector in<br />
<strong>the</strong> names of TJ McCown <strong>an</strong>d Rebecca Haberm<strong>an</strong>, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property).
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.8/O28<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: NIH Gr<strong>an</strong>t NS047385<br />
Americ<strong>an</strong> Epilepsy <strong>Society</strong> (JGH)<br />
Title: Hippocampal <strong>an</strong>d neocortical cytokine expression after neonatal hypoxia-induced seizures<br />
Authors: J. G. HEIDA 1,2,3 , K. KATKI 1,2,3 , K. ZHANG 1,2,3 , *R. M. SANCHEZ 1,2,3 ;<br />
1 Dept Surgery, Texas A&M Hlth. Sci. Ctr., Temple, TX; 2 Dept of Neurosurg., Scott & White<br />
Hosp., Temple, TX; 3 Central Texas Veter<strong>an</strong>s Hlth. Care Syst., Temple, TX<br />
Abstract: The incidence of seizure is highest in <strong>the</strong> neonate, <strong>an</strong>d hypoxia is <strong>the</strong> most common<br />
cause of neonatal seizures. Neonatal seizures associated with hypoxia increase <strong>the</strong> risk of<br />
developing epilepsy, but <strong>the</strong> mech<strong>an</strong>isms by which such epileptogenesis occurs are not well<br />
understood. There<strong>for</strong>e, <strong>the</strong>re are no <strong>the</strong>rapeutic strategies to prevent epilepsy following neonatal<br />
seizure-inducing hypoxia. Recent studies indicate that neuroinflammatory processes may play a<br />
critical role in epileptogenesis consequent to experimental seizures through alterations of<br />
neuro<strong>an</strong>atomical circuits <strong>an</strong>d possibly direct modulation of synaptic tr<strong>an</strong>smission. To examine<br />
<strong>the</strong> role of neuroinflammation in neonatal hypoxic seizures (HS) we aimed to characterize <strong>the</strong><br />
cytokine response in <strong>the</strong> hippocampus <strong>an</strong>d neocortex of rats after HS. In P10 Long-Ev<strong>an</strong>s rat<br />
pups, HS were induced by exposure to 5-7% oxygen <strong>for</strong> 14-16min. Control littermate <strong>an</strong>imals<br />
were h<strong>an</strong>dled identically but exposed to room air <strong>for</strong> <strong>the</strong> same period. Animals were sacrificed at<br />
2hrs, 6hrs, 24hrs, 3 days, <strong>an</strong>d 7 days after hypoxia treatment, <strong>the</strong> brains removed, <strong>an</strong>d <strong>the</strong><br />
hippocampii <strong>an</strong>d a section of neocortex rapidly dissected out <strong>an</strong>d frozen. Tissue was later<br />
homogenized, centrifuged, <strong>an</strong>d <strong>the</strong> supernat<strong>an</strong>t collected <strong>for</strong> multi-plexing immunoassays using<br />
Milliplex(TM) map cytokine p<strong>an</strong>els <strong>an</strong>d a BioRad Bioplex-200 system. This allowed us<br />
simult<strong>an</strong>eously to measure concentrations of <strong>the</strong> cytokines IL-1beta, IL-2, IL-9, IL-10, <strong>an</strong>d TNFalpha.<br />
In <strong>the</strong> hippocampus we found that IL-1beta was signific<strong>an</strong>tly elevated at 24hrs, 3 <strong>an</strong>d 7<br />
days after HS (p < 0.05, 0.001, <strong>an</strong>d 0.01 compared to control, respectively). Additionally <strong>the</strong>re<br />
were also signific<strong>an</strong>t increases in IL-2 <strong>an</strong>d IL-9 at 24hrs <strong>an</strong>d 3 days after HS (both p < 0.01),<br />
while IL-10 <strong>an</strong>d TNF-alpha were only increased at 3 days after seizure (both p < 0.001). In <strong>the</strong><br />
neocortex we found a signific<strong>an</strong>t increase of IL-1beta at 7 days after HS (p < 0.05) without <strong>an</strong>y<br />
ch<strong>an</strong>ges in <strong>the</strong> levels of <strong>the</strong> o<strong>the</strong>r cytokines at <strong>an</strong>y of <strong>the</strong> time points examined. These results<br />
show that <strong>the</strong>re is a dynamic neuroinflammatory response within <strong>the</strong> brain after neonatal HS.<br />
This is evident by alterations in <strong>the</strong> pattern <strong>an</strong>d amount of cytokines present at specific times in
different brain regions. This study provides a basis to examine fur<strong>the</strong>r <strong>the</strong> specific roles of each<br />
of <strong>the</strong>se cytokines in neuro<strong>an</strong>atomical <strong>an</strong>d functional perturbations to hippocampal <strong>an</strong>d<br />
neocortical development <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>se may offer accessible <strong>the</strong>rapeutic targets to prevent<br />
epileptogenesis after neonatal HS.<br />
Disclosures: J.G. Heida, None; K. Katki, None; K. Zh<strong>an</strong>g, None; R.M. S<strong>an</strong>chez, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.9/O29<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: NINDS R01 NS036604 to RD<br />
Title: 3′UTR-specific inhibition of GluR2 mRNA tr<strong>an</strong>slation by <strong>an</strong>tibiotics<br />
Authors: *H. A. IRIER, J. J. YOO, R. DINGLEDINE;<br />
Mol. Systems & Pharmacol, Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Two major GluR2 RNA species of 5.4 kb <strong>an</strong>d 4.8 kb have been detected in Nor<strong>the</strong>rn<br />
Blots of <strong>the</strong> rat hippocampus <strong>an</strong>d cortex. GluR2 tr<strong>an</strong>scripts exist as mRNA subpopulations<br />
bearing alternative 5’- <strong>an</strong>d 3’-untr<strong>an</strong>slated regions (UTRs), which are uni<strong>for</strong>mly distributed in<br />
<strong>the</strong> rat hippocampus. GluR2 mRNAs with at least two different 3'UTRs, differing only in length<br />
(long ~2750 bases <strong>an</strong>d short ~750 bases) exist in <strong>the</strong> brain. GluR2 expression is regulated by <strong>the</strong><br />
presence of GU repeats residing in <strong>the</strong> longer 5' UTRs of GluR2 mRNAs, however, in <strong>the</strong><br />
absence of <strong>the</strong> GU repeats, <strong>the</strong> long but not short 3’UTRs c<strong>an</strong> repress tr<strong>an</strong>slation in <strong>an</strong> oocyte<br />
reporter system. Moreover, endogenous GluR2 tr<strong>an</strong>scripts bearing <strong>the</strong> long 3’UTRs undergo<br />
seizure-induced de-repression in <strong>the</strong> rat hippocampus. These findings, overall, suggest that<br />
regulatory mech<strong>an</strong>isms targeting different populations of GluR2 tr<strong>an</strong>scripts exist, <strong>an</strong>d that <strong>the</strong><br />
alternative 5’-<strong>an</strong>d 3’UTRs in <strong>the</strong>se tr<strong>an</strong>scripts confer distinct tr<strong>an</strong>slation profiles. To fur<strong>the</strong>r<br />
investigate <strong>the</strong> role of alternative UTRs on tr<strong>an</strong>slation regulation, we used rabbit reticulocyte<br />
lysates to examine <strong>the</strong> expression profiles of firefly reporters bearing naturally existing<br />
alternative combinations of GluR2 5’<strong>an</strong>d 3’ UTRs in <strong>the</strong> presence of <strong>an</strong>tibiotics targeting<br />
initiation, elongation or termination steps of <strong>the</strong> mRNA tr<strong>an</strong>slation. At relatively high<br />
concentrations (1-10ng/ul), cycloheximide, a tr<strong>an</strong>slation elongation inhibitor, indiscriminately<br />
inhibited all <strong>the</strong> reporter mRNAs. However, at lower concentrations of cycloheximide (0.1-<br />
0.01ng/ul), only <strong>the</strong> reporters bearing short GluR2 3’UTRs were signific<strong>an</strong>tly repressed. We did
not observe a detectible difference in <strong>the</strong> expression profiles among <strong>the</strong> reporters when treated<br />
with Tetracycline or Hygromycine B. These findings suggest that elongation is <strong>the</strong> rate limiting<br />
step in <strong>the</strong> tr<strong>an</strong>slation of reporter mRNAs bearing <strong>the</strong> short, but not long, GluR2 3’UTRs, <strong>an</strong>d<br />
that tr<strong>an</strong>slation initiation is a target <strong>for</strong> potential regulatory mech<strong>an</strong>isms ultimately causing a<br />
sizeable tr<strong>an</strong>slation repression of <strong>the</strong> GluR2 mRNAs with <strong>the</strong> long 3’UTRs.<br />
Disclosures: H.A. Irier, None; J.J. Yoo, None; R. Dingledine, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.10/O30<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: T32 NS007480<br />
NIH U01 NS058158-02<br />
Title: EP2 receptor activation reduces neuropathologies associated with status epilepticus<br />
Authors: *R. N. SHAW, A. ROJAS, N. LELUTIU, R. DINGLEDINE;<br />
Emory Univ. Sch. of Med., Atl<strong>an</strong>ta, GA<br />
Abstract: An import<strong>an</strong>t characteristic of status epilepticus is <strong>the</strong> up-regulation of<br />
cyclooxygenase-2 (COX-2), <strong>the</strong> rate limiting enzyme in <strong>the</strong> production of Prostagl<strong>an</strong>din E2<br />
(PGE2). PGE2 is <strong>an</strong> import<strong>an</strong>t mediator of m<strong>an</strong>y physiological functions. PGE2 elucidates its<br />
functions via four different G-protein coupled receptors (EP1, EP2, EP3, <strong>an</strong>d EP4). Recent<br />
studies have suggested that <strong>the</strong> EP2 receptor opposes ischemia-induced damage in vivo <strong>an</strong>d in<br />
vitro. In our lab we found that EP2 receptor activation by butaprost (a specific EP2 agonist)<br />
reduces neuronal injury observed following LPS-induced cell damage in vitro (unpublished data)<br />
also suggesting that EP2 activation may be neuroprotective. Here, we test <strong>the</strong> hypo<strong>the</strong>sis that<br />
EP2 receptor activation opposes neuropathologies associated with pilocarpine induced status<br />
epilepticus (SE) in vivo. Intralateral ventricular c<strong>an</strong>nulated (ICV) rats were injected<br />
subcut<strong>an</strong>eously with pilocarpine (380mg/kg) to induce SE <strong>for</strong> 90 minutes. After stopping SE<br />
with pentobarbital <strong>the</strong> rats were injected with 2µl of 1µM butaprost or saline ICV <strong>an</strong>d were<br />
allowed to recover. The next day <strong>the</strong> brains were harvested <strong>an</strong>d bisected. Half of <strong>the</strong> brain was<br />
subjected to histology with fluorojade to detect neurodegeneration in <strong>the</strong> hippocampus. RNA<br />
was isolated <strong>from</strong> <strong>the</strong> hippocampus of <strong>the</strong> o<strong>the</strong>r half of <strong>the</strong> brain. The results confirmed that
pilocarpine-induced SE leads to hippocampal neurodegeneration that c<strong>an</strong> be detected with<br />
fluorojade. The results also demonstrated that activation of <strong>the</strong> EP2 receptor with butaprost<br />
administered just after SE reduces <strong>the</strong> amount of hippocampal neurodegeneration in <strong>the</strong> CA1 <strong>an</strong>d<br />
CA3 primary cell layers 24 hours post-SE. Thus, activation of <strong>the</strong> EP2 receptor results in<br />
neuroprotection in <strong>the</strong> hippocampus following SE. EP2 activation ch<strong>an</strong>ged <strong>the</strong> mRNA<br />
expression level of three inflammatory mediators: complement component 3 (C3) <strong>an</strong>d<br />
macrophage migration inhibitory factor (MIF) were up-regulated, <strong>an</strong>d interleukin-15 (IL-15) was<br />
down-regulated .These results support fur<strong>the</strong>r investigation into <strong>the</strong>rapeutic modalities <strong>for</strong> <strong>the</strong><br />
activation of EP2 in neuronal hyperexcitation <strong>an</strong>d epilepsy.<br />
Disclosures: R.N. Shaw, None; A. Rojas, None; N. Lelutiu, None; R. Dingledine, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.11/O31<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: NIH Gr<strong>an</strong>t 5U01NS058158 (RD)<br />
NIH Gr<strong>an</strong>t 3U54HG003918 (RD)<br />
Title: Development of novel selective allosteric potentiator <strong>for</strong> prostagl<strong>an</strong>din receptor EP2<br />
Authors: *J. JIANG 1 , T. GANESH 2 , Y. DU 3 , P. THEPCHATRI 3 , A. ROJAS 1 , I. LEWIS 3 , S.<br />
KURTKAYA 3 , L. LI 3 , M. QUI 3 , R. N. SHAW 1 , A. SUN 2 , R. J. DINGLEDINE 1 ;<br />
1 2 3<br />
Pharmacol., Emory Univ. Sch. of Med., Atl<strong>an</strong>ta, GA; Chem., Chem. Biol. Discovery Ctr.,<br />
Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Prostagl<strong>an</strong>din E2 (PGE2), one of <strong>the</strong> major enzymatic products of COX2, c<strong>an</strong> activate<br />
four G protein-coupled receptors (GPCRs): EP1-4. Activation of EP2 receptor by PGE2 has been<br />
demonstrated to be neuroprotective effects following ischemia. Additionally, EP2 activation has<br />
been shown to spatial learning, improve survival of epi<strong>the</strong>lia cells after radiation injury, <strong>an</strong>d<br />
improve renal function in <strong>an</strong> HgCl2 model of chronic renal failure. EP2 is a Gαs-coupled<br />
receptor <strong>an</strong>d increases cellular cAMP when activated. Only one selective agonist <strong>for</strong> EP2 has<br />
been identified - butaprost, which has a prostagl<strong>an</strong>din-like structure. Developing allosteric<br />
modulators has been successful <strong>for</strong> lig<strong>an</strong>d-gated ion ch<strong>an</strong>nels <strong>an</strong>d o<strong>the</strong>r GPCRs. The allosteric<br />
modulators spatiotemporally affect receptor activation in <strong>the</strong> presence of agonist. We
hypo<strong>the</strong>size that a small molecule allosteric potentiator of EP2 could beneficially exert broad<br />
cell-protective effects. We developed a C6 glioma cell line (C6G) stably expressing <strong>the</strong> hum<strong>an</strong><br />
EP2 receptor <strong>an</strong>d a TR-FRET assay measuring <strong>the</strong> cAMP induced by EP2 activation <strong>for</strong> high<br />
throughput screening in a 1536-well <strong>for</strong>mat. Screening of 292,000 compounds at 20 µM revealed<br />
112 confirmed hits that: 1) potentiated <strong>the</strong> response to a low concentration of PGE2 by >50%; 2)<br />
had no effect on o<strong>the</strong>r GPCRs like EP4 or β2 adrenergic receptors; 3) had no effect on <strong>the</strong> cAMP<br />
TR-FRET assay itself or <strong>the</strong> untr<strong>an</strong>sfected cell line; <strong>an</strong>d 4) increased <strong>the</strong> potency of PGE2 on<br />
EP2 receptors at least 3-fold. A thiophene acetamide scaffold was selected <strong>from</strong> <strong>the</strong> confirmed<br />
hits <strong>for</strong> chemical modification, <strong>an</strong>d 94 compounds were designed <strong>an</strong>d syn<strong>the</strong>sized to explore <strong>the</strong><br />
SAR of target scaffold. The most active compounds increased <strong>the</strong> potency of PGE2 on EP2<br />
receptors 4 to 6-fold at 10-20 µM. Fur<strong>the</strong>r tests in cell lines which stably express EP4 <strong>an</strong>d β2adrenergic<br />
receptors revealed that none of <strong>the</strong> compounds had <strong>an</strong>y effect on <strong>the</strong> potency of PGE2<br />
or isoproterenol <strong>for</strong> <strong>the</strong>ir cognate receptors at 20 µM. These EP2 allosteric potentiators also show<br />
no cellular toxicity in vivo up to at least 100 µM. Interestingly, <strong>the</strong>se EP2 potentiators could<br />
partially prevent <strong>the</strong> neuronal injury induced by NMDA (30 µM) treatment in cultured<br />
hippocampal neurons. These novel compounds represent <strong>the</strong> first allosteric modulators of a PGE2<br />
receptor.<br />
Disclosures: J. Ji<strong>an</strong>g, None; T. G<strong>an</strong>esh, None; Y. Du, None; P. Thepchatri, None; A. Rojas,<br />
None; I. Lewis, None; S. Kurtkaya, None; L. Li, None; M. Qui, None; R.N. Shaw, None; A.<br />
Sun, None; R.J. Dingledine, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.12/O32<br />
Topic: C.07.g. Post-seizure modifications<br />
Support: NIH-NINDS<br />
Title: Cyclooxygenase-2 in principal neurons modulates brain inflammation after status<br />
epilepticus<br />
Authors: *G. E. SERRANO, S. COCHI, R. SHAW, R. DINGLEDINE;<br />
Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Temporal lobe epilepsy (TLE) is a type of epilepsy often associated with brain<br />
inflammation. In <strong>the</strong> murine model of status epilepticus (SE), systemically administered
pilocarpine induces prolonged SE resulting in robust astrogliosis, microglial activation,<br />
leukocyte infiltration, <strong>an</strong>d rapid induction of numerous mediators of inflammation as observed in<br />
hum<strong>an</strong> TLE. One of <strong>the</strong> inflammatory mediators that is rapidly induced after SE is <strong>the</strong><br />
multifunctional neuronal modulator, cyclooxygenase-2 (COX2). Its induction occurs mainly in<br />
principal neurons of <strong>the</strong> hippocampus, cortex, <strong>an</strong>d amygdaloid complex. In this study we explore<br />
<strong>the</strong> role of neuron-derived COX2 in seizure-induced pathology using a conditional knockout<br />
(cKO) mouse created in <strong>the</strong> laboratory that lacks COX2 in <strong>the</strong> principal <strong>for</strong>ebrain neurons after<br />
P15. In this study, we used <strong>the</strong> conditional knockout to investigate <strong>the</strong> involvement of neuronderived<br />
COX2 in <strong>the</strong> inflammatory reactions observed after SE. We explored <strong>the</strong> ch<strong>an</strong>ges of <strong>the</strong><br />
principal brain cells involved in inflammation using immunohistochemical techniques, <strong>an</strong>d <strong>the</strong><br />
regulation of inflammatory cytokines <strong>an</strong>d <strong>the</strong>ir receptor expression using RT-PCR. Mice<br />
experienced SE <strong>for</strong> one hour <strong>an</strong>d were sacrificed one day later. After brain dissection, half of <strong>the</strong><br />
<strong>for</strong>ebrain was fixed overnight with 4% para<strong>for</strong>maldehyde <strong>an</strong>d processed with rabbit <strong>an</strong>ti-GFAP<br />
or rabbit <strong>an</strong>ti-Iba1. The o<strong>the</strong>r half was used <strong>for</strong> RNA extraction <strong>an</strong>d first str<strong>an</strong>d DNA syn<strong>the</strong>sis<br />
<strong>for</strong> RT-PCR. An array of 84 inflammatory genes <strong>from</strong> SA-Bioscience was used <strong>for</strong> each <strong>an</strong>imal.<br />
The latency to reach SE, <strong>the</strong> behavior be<strong>for</strong>e <strong>an</strong>d during SE, <strong>an</strong>d <strong>the</strong> percentage mortality were<br />
similar in wt <strong>an</strong>d cKO <strong>an</strong>imals. Never<strong>the</strong>less, RNA expression <strong>from</strong> several inflammatory<br />
factors, as well as <strong>the</strong> number of reactive astrocytes, were up-regulated after SE to a higher<br />
extent in wt mice th<strong>an</strong> in cKO. These results underline <strong>an</strong> import<strong>an</strong>t neuronal component during<br />
seizure-induced brain inflammation. In addition, we previously demonstrated that COX2<br />
induction after SE results in degeneration of somatostatin-labeled GABAergic interneurons in<br />
<strong>the</strong> dentate hilus <strong>an</strong>d CA1 stratum oriens. This toge<strong>the</strong>r with <strong>the</strong> present findings demonstrates a<br />
detrimental outcome of neuron-derived COX2 induction after SE. We conclude that neuronderived<br />
COX2 induction after SE strongly contributes to <strong>the</strong> pro-inflammatory events observed<br />
after SE that might lead to neuronal degeneration <strong>an</strong>d/or synaptic remodeling.<br />
Disclosures: G.E. Serr<strong>an</strong>o, None; S. Cochi, None; R. Shaw, None; R. Dingledine, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.13/O33<br />
Topic: C.07.i. Predisposing factors<br />
Support: EU GRANT 7th FP-HEALTH-F2-2007-202167<br />
Telethon Gr<strong>an</strong>t GGP07278
Title: Role of interleukin 1β in <strong>the</strong> initiation of ictal discharges<br />
Authors: A. CHIAVEGATO 1 , G. LOSI 2 , M. GOMEZ-GONZALO 1 , *G. CARMIGNOTO 1 ;<br />
1 Univ. Padova, 35121 Padova, Italy; 2 CNR-IN-Univ Padova, 35121 Padova, Italy<br />
Abstract: Interleukin 1Β (IL-1Β ) is a proinflammatory cytokine that is involved in both acute<br />
<strong>an</strong>d chronic epilepsies. IL-1Β is overexpressed in brain epileptic areas <strong>an</strong>d has pro-convulsive<br />
properties. Its natural <strong>an</strong>tagonist IL-1ra has also powerful <strong>an</strong>ti-convulsive effects. Recently, it<br />
has been shown that IL-1Β c<strong>an</strong> directly increase neuronal excitability by acting on both NMDA<br />
<strong>an</strong>d GABAA receptors. To investigate <strong>the</strong> effect of IL-1Β on <strong>the</strong> generation of <strong>the</strong> ictal, seizurelike<br />
discharge, we used a new model of ictogenesis in entorhinal cortex slices obtained <strong>from</strong><br />
P14-P18 rats. This model used pressure pulses applied to <strong>an</strong> NMDA-containing patch pipette in<br />
<strong>the</strong> presence of 100 µM 4-aminopyridine <strong>an</strong>d 0.5 mM Mg2+, to stimulate <strong>an</strong> episode of high<br />
neuronal activity in layer V-VI EC neurons. As revealed by imaging <strong>the</strong> Ca2+ signal <strong>from</strong><br />
neurons, two successive NMDA pulses applied with a 3 s interval, but not a single pulse,<br />
stimulated <strong>an</strong> intense neuronal response that soon evolved into a propagating ictal discharge.<br />
However, after IL-1Β was applied <strong>for</strong> 5-10 min ei<strong>the</strong>r locally or through bath perfusion, a single<br />
NMDA pulse, that was regularly ineffective be<strong>for</strong>e IL-1Β application, could trigger <strong>an</strong> ictal<br />
discharge (in 32 out of 49 single NMDA pulse, 12 experiments). We also observed that <strong>the</strong> me<strong>an</strong><br />
duration of ictal discharges triggered after IL-1Β by single NMDA pulse stimulations was<br />
signific<strong>an</strong>tly longer with respect to that of ictal discharges evoked be<strong>for</strong>e IL-1Β application by<br />
double NMDA pulse stimulations (65 ± 10.1 <strong>an</strong>d 79,4 ± 17, 5 be<strong>for</strong>e <strong>an</strong>d after IL-1Β<br />
respectively; p < 0.05). To gain fur<strong>the</strong>r insights into <strong>the</strong> effects of IL-1Β on neuronal excitability,<br />
we are currently investigating by patch clamp recordings <strong>an</strong>d calcium imaging <strong>from</strong> entorhinal<br />
cortex pyramidal neurons <strong>the</strong> possible IL-1Β effect on NMDA receptor function. These results<br />
hint at <strong>the</strong> possibility that IL-1Β favors focal seizure generation by lowering <strong>the</strong> ictal discharge<br />
threshold.<br />
Disclosures: A. Chiavegato, None; G. Losi, None; M. Gomez-Gonzalo, None; G.<br />
Carmignoto, None.<br />
Poster<br />
538. Epilepsy: Inflammation <strong>an</strong>d Immune System<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 538.14/O34<br />
Topic: C.07.h. In vivo <strong>an</strong>d behavior<br />
Support: NS056304-02
NS051445-04<br />
Title: Increased incidence of status epilepticus in interleukin-1β mut<strong>an</strong>t mice<br />
Authors: *R. J. CLAYCOMB, S. J. HEWETT, J. A. HEWETT;<br />
Univ. Connecticut Hl, Farmington, CT<br />
Abstract: Mice deficient in interleukin-1β (IL-1β) signaling exhibit <strong>an</strong> increase in <strong>the</strong> incidence<br />
of acute convulsive seizures following treatment with <strong>the</strong> GABAA <strong>an</strong>tagonist, pentylenetetrazole<br />
(Claycomb et al., J. Neurochem., 108 (Suppl. 1) p 130-131, <strong>2009</strong>), suggesting that IL-1β<br />
possesses <strong>an</strong>ticonvuls<strong>an</strong>t properties in <strong>the</strong> central nervous system (CNS). The purpose of <strong>the</strong><br />
present study was to explore this possibility fur<strong>the</strong>r in a model of status epilepticus (SE) induced<br />
by <strong>the</strong> glutamate receptor agonist, kainic acid (KA). The role of IL-1β was assessed using<br />
homozygous null (-/-) male mice (n = 13-14) derived <strong>from</strong> heterozygous IL-1β gene-targeted<br />
mut<strong>an</strong>t mice. Comparisons were made to wild-type (+/+) littermate controls (n = 14-18). To<br />
elicit reproducible SE, a titrating dosing paradigm was developed wherein <strong>an</strong> initial dose of 20<br />
mg/kg KA (i.p.) was followed 30, 60, 90 <strong>an</strong>d 150 min later by booster doses of KA (5 mg/kg,<br />
i.p.). Motor seizure activity was terminated 90min after <strong>the</strong> last KA dose by administration of<br />
diazepam (5 mg/kg, i.p.). Animals were monitored continuously throughout <strong>the</strong> protocol by <strong>an</strong><br />
investigator blinded to genotype <strong>an</strong>d <strong>the</strong> maximal seizure score attained during each 10 min bin<br />
of <strong>the</strong> 240 min observation period was recorded. Seizure behavior was scored as follows: 0 = no<br />
behavioral ch<strong>an</strong>ge, 1 = hypomobility, 2 = myoclonus, 3 = rearing with no clonus, 4 = rearing<br />
with clonus/convulsions, 5 = convulsions with loss of righting reflex, 6 = continuous convulsive<br />
seizures (SE). The medi<strong>an</strong> seizure scores steadily rose to 5 over <strong>the</strong> 150 min dosing period<br />
irrespective of genotype. During <strong>the</strong> subsequent 90 minutes, however, <strong>the</strong> medi<strong>an</strong> seizure score<br />
<strong>for</strong> IL-1β +/+ mice decreased to 3, whereas <strong>the</strong> medi<strong>an</strong> scores in <strong>the</strong> IL-1β -/- null group<br />
increased to 5.5 (different at p < 0.05, comparison of fit test). Strikingly, SE occurred in 72%<br />
(13/18) of IL-1β -/- mice, compared to only 36% [5/14] of <strong>the</strong>ir +/+ littermate controls (different<br />
at p < 0.05, Chi-squared test). This increase in SE incidence was associated with <strong>an</strong> increase in<br />
mortality. One-third of all IL-1β -/- mice (33% [6/18]), but only one IL-1β +/+ mouse (7%<br />
[1/14]) died during or within <strong>the</strong> 72 hr period following <strong>the</strong> SE paradigm (different at p < 0.05<br />
Chi-squared test). Results <strong>from</strong> this study provide additional compelling evidence to support <strong>the</strong><br />
conclusion that endogenous IL-1β possesses <strong>an</strong>ticonvuls<strong>an</strong>t properties in <strong>the</strong> CNS.<br />
Disclosures: R.J. Claycomb, None; S.J. Hewett, None; J.A. Hewett, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 539.1/O35<br />
Topic: C.07.i. Predisposing factors<br />
Support: NIH R15<br />
Title: Neuronal cell survival induced by VEGFR2 inhibition attenuates oxidative stress in a rat<br />
model of status epilepticus<br />
Authors: T. HAO 1 , J. EDELSTEIN 1 , E. SALERNI 2 , J. LENZER 2 , Q. CAO 1 , S. CROLL 2 , *P.<br />
ROCKWELL 1 ;<br />
1 Hunter Col. CUNY, New York, NY; 2 Queens Col., New York, NY<br />
Abstract: Neuronal death in adult org<strong>an</strong>isms is observed after ischemia, oxidative insults <strong>an</strong>d in<br />
neurodegenerative disorders. Identifying <strong>the</strong> cell death mech<strong>an</strong>isms that are triggered by harmful<br />
stimuli is import<strong>an</strong>t <strong>for</strong> developing strategies to halt <strong>the</strong> neurodegenerative process. One method<br />
<strong>for</strong> attenuating neuronal death is to administer neuroprotective growth factors. One such factor is<br />
vascular endo<strong>the</strong>lial growth factor (VEGF), a well established <strong>an</strong>giogenic factor in endo<strong>the</strong>lial<br />
cells, which has been shown to reduce neuronal death in in vitro <strong>an</strong>d in vivo models of<br />
neurodegeneration. In several paradigms of neurodegeneration, VEGF signals survival through<br />
<strong>the</strong> activation of its cognate receptor VEGFR2. To this end, we investigated <strong>the</strong> signaling<br />
mech<strong>an</strong>ism associated with VEGF-mediated neuroprotection in a rat model of status epilepticus.<br />
In this model, VEGF provides protection against hippocampal neuronal cell loss resulting <strong>from</strong><br />
pilocarpine-induced seizures (Nicoletti, et al. Neuroscience 151:232-41, 2008). More recent<br />
studies revealed that seizures were accomp<strong>an</strong>ied by increased levels of <strong>the</strong> proinflammatory<br />
enzyme cycloxygenase-2 (COX-2) in <strong>the</strong> hippocampus while inducing <strong>an</strong> upregulation in <strong>the</strong><br />
<strong>an</strong>tioxid<strong>an</strong>t enzyme Heme oygenase-1 (HO-1) <strong>an</strong>d <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> nitric oxide-derived<br />
oxidative product 3-nitrotyrosine (3-NT). Consistent with <strong>the</strong> survival data, Western blot<br />
<strong>an</strong>alyses revealed that VEGF treatments were associated with decreased levels of HO-1 <strong>an</strong>d<br />
COX-2. There<strong>for</strong>e, we hypo<strong>the</strong>sized that VEGF suppressed inflammation <strong>an</strong>d oxidative stress to<br />
mediate neuroprotection through VEGFR2 activation. However, treatments with a small<br />
molecular weight inhibitor of VEGFR2, SU1498, failed to counteract <strong>the</strong> protection mediated by<br />
VEGF in rats undergoing seizures. Instead, Western blot <strong>an</strong>d immunofluorescent assays showed<br />
that SU1498 augmented <strong>the</strong> neuronal cell survival mediated by VEGF <strong>an</strong>d abrogated <strong>the</strong> high<br />
levels of COX-2, HO-1 <strong>an</strong>d 3-NT induced by pilocarpine. These findings suggest that VEGFR2<br />
signals inflammation <strong>an</strong>d oxidative injury in hippocampal neurons in response to pilocarpine<br />
while treatments with VEGF mediate neuroprotection through <strong>an</strong> alternate receptor(s) such as<br />
VEGFR1. Thus, VEGF mediates a complex pattern of signaling events during status epilepticus<br />
where its capacity to elicit beneficial or damaging effects appears to depend upon <strong>the</strong> cognate<br />
receptor activated. There<strong>for</strong>e, delineating VEGF-mediated signaling pathways that relay<br />
protection <strong>an</strong>d oxidative stress during status epilepticus would provide valuable insight on <strong>the</strong><br />
role of VEGF in <strong>the</strong> neurodegenerative process associated with epilepsy.<br />
Disclosures: T. Hao, None; J. Edelstein, None; E. Salerni, None; J. Lenzer, None; Q. Cao,<br />
None; S. Croll, Regeneron, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or<br />
o<strong>the</strong>r in-kind support); P. Rockwell, None.
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.2/O36<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: NIH NINDS Gr<strong>an</strong>t R15NS059016<br />
Title: Inhibition of VEGFR2 protects against status epilepticus-induced hippocampal neuronal<br />
loss<br />
Authors: *E. A. SALERNI 1,3 , T. HAO 7,4 , J. LENZER 3,9 , J. EDELSTEIN 8,4 , G. OLIVARES 2 , E.<br />
FELDMAN 2 , P. ROCKWELL 10,5 , S. D. CROLL 9,6 ;<br />
2 Psychology, 1 Queens Col., Flushing, NY; 3 Neuropsychology, 4 Biochem., 5 Biol. & Biochem.,<br />
6 Neuropsychology & Neurosci., Grad. Ctr. of CUNY, New York, NY; 8 Biol., 7 Hunter Col., New<br />
York, NY; 9 Psychology, Queens Col. of CUNY, New York, NY; 10 Biol., Hunter Col. of CUNY,<br />
New York, NY<br />
Abstract: Vascular endo<strong>the</strong>lial growth factor (VEGF) is <strong>an</strong> <strong>an</strong>giogenic factor with known<br />
neuroprotective effects. Previous research in our lab has shown that intrahippocampal infusions<br />
of VEGF protects hippocampal pyramidal neurons <strong>from</strong> death 24 hours after pilocarpine-induced<br />
status epilepticus (SE). VEGF’s known signal-tr<strong>an</strong>sducing receptors are VEGFR1 <strong>an</strong>d VEGFR2.<br />
In vitro studies suggest that VEGF’s neuroprotective effects are mediated by VEGFR2. The<br />
current experiments were conducted to determine whe<strong>the</strong>r VEGFR2 activation also mediates its<br />
in vivo protective effects. Bic<strong>an</strong>nulae were impl<strong>an</strong>ted into dorsal hippocampus <strong>an</strong>d <strong>an</strong>imals<br />
received continuous infusion of VEGF or inactivated VEGF control through one c<strong>an</strong>nula, <strong>an</strong>d<br />
ei<strong>the</strong>r <strong>the</strong> VEGFR2-selective tyrosine kinase inhibitor SU1498 or 75% DMSO vehicle through<br />
<strong>the</strong> o<strong>the</strong>r c<strong>an</strong>nula during pilocarpine-induced SE. Hippocampal tissue was taken 24 hours after<br />
SE <strong>an</strong>d prepared <strong>for</strong> histological <strong>an</strong>alysis. Brain sections were stained with cresyl violet <strong>an</strong>d<br />
assessed <strong>for</strong> neuronal loss using previously published methods. Results showed that VEGFR2<br />
inhibition not only failed to prevent VEGF’s neuroprotective effects, but also was<br />
neuroprotective on its own. These data suggest not only that VEGFR2 does not mediate VEGF’s<br />
protective effects in this in vivo model, but that it might actually produce a harmful effect in this<br />
context. Future studies will explore <strong>the</strong> possibility that it is VEGFR1, not VEGFR2, which<br />
mediates VEGF’s protective effects against SE-induced neuronal death.<br />
Disclosures: E.A. Salerni, None; T. Hao, None; J. Lenzer, None; J. Edelstein, None; G.<br />
Olivares, None; E. Feldm<strong>an</strong>, None; P. Rockwell, None; S.D. Croll, VEGF was provided by
Regeneron, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind<br />
support); Regeneron shareholder, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); Regeneron consult<strong>an</strong>t, F. Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.3/O37<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: NIH R01 NS063360<br />
NIH R01 EB007082-02<br />
Wilder Center of Excellence <strong>for</strong> Epilepsy Research<br />
Children's Miracle Network<br />
Title: Convection-enh<strong>an</strong>ced delivery as a local <strong>an</strong>ti-convuls<strong>an</strong>t delivery treatment <strong>for</strong> epilepsy<br />
Authors: *S. KANTOROVICH 1 , G. W. ASTARY 2 , T. H. MARECI 3 , M.<br />
SARNTINORANONT 4 , P. R. CARNEY 5 ;<br />
1 Neurosci., 2 Biomed. Engin., 3 Biochem. <strong>an</strong>d Mol. Biology, Biomed. Engin., 4 Mech<strong>an</strong>ical <strong>an</strong>d<br />
Aerospace Engin., 5 Pediatric Neurology, Biomed. Engineering, Neurosci., Univ. of Florida,<br />
Gainesville, FL<br />
Abstract: Rationale: Epilepsy is a chronic neurological disorder that is characterized by<br />
recurrent unprovoked seizures. It c<strong>an</strong> be controlled with <strong>an</strong>tiepileptic drugs, but <strong>the</strong>se<br />
medications may cause side effects <strong>an</strong>d systemic toxicity. A local drug delivery method, such as<br />
convection-enh<strong>an</strong>ced delivery (CED), c<strong>an</strong> be used to overcome <strong>the</strong>se barriers. CED is <strong>the</strong><br />
infusion of <strong>the</strong>rapeutic agents directly into <strong>the</strong> extracellular space of tissues under a positive<br />
pressure gradient. This method c<strong>an</strong> be employed to bypass <strong>the</strong> blood-brain-barrier, distribute<br />
<strong>the</strong>rapeutic agents within <strong>the</strong> CNS, overcome slow diffusivity of macromolecules, <strong>an</strong>d provide<br />
greater control over <strong>the</strong> distribution of <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t with minimal structural damage. In this<br />
study, we investigated distribution patterns of Gd-DTPA labeled albumin that was targeted to <strong>the</strong><br />
rat hippocampus using CED tr<strong>an</strong>sport.<br />
Methods: Male Sprague-Dawley rats were infused with 5 µl of Gd-DTPA labeled, Ev<strong>an</strong>s Blue<br />
tagged, albumin at a rate of 0.3 µl/min into <strong>the</strong> left ventral (CA3 subfield) <strong>an</strong>d right dorsal
(dentate gyrus subfield) hippocampus. Imaging studies were <strong>the</strong>n per<strong>for</strong>med in vivo to determine<br />
volume distributions using <strong>an</strong> 11.1T magnet system to acquire high-resolution T1-weighted<br />
images with diffusion tensor imaging (DTI). These images were validated with fluorescence<br />
microscopy, <strong>an</strong>d Cresyl violet <strong>an</strong>d Black-Gold staining to visualize <strong>the</strong> tracer’s distribution with<br />
regard to cell bodies <strong>an</strong>d fiber tracks, respectively.<br />
Results: Our results demonstrate that <strong>the</strong> contrast agent infuses primarily along fiber directions,<br />
which correspond to predomin<strong>an</strong>t diffusion directions measured using DTI. The orientation of<br />
<strong>the</strong> fibers contribute to <strong>the</strong> distribution pattern of <strong>the</strong> contrast agent, <strong>an</strong>d <strong>the</strong> infusate is limited to<br />
regions of densely packed cell bodies such as <strong>the</strong> CA1, CA3 pyramidal cell layers, <strong>an</strong>d <strong>the</strong><br />
dentate gr<strong>an</strong>ule cell layer. A preferential distribution was also observed into hippocampal<br />
fissures.<br />
Conclusion: These preliminary results suggest <strong>the</strong> distribution profiles of infused agents are<br />
highly dependent on <strong>the</strong> location of <strong>the</strong> infusion site <strong>an</strong>d <strong>the</strong> underlying structural <strong>an</strong>atomy of <strong>the</strong><br />
hippocampus. These infusion studies, taken toge<strong>the</strong>r with computational modeling, may guide<br />
clinical decisions regarding <strong>the</strong> local targeting of specific regions with <strong>the</strong>rapeutic agents <strong>for</strong> <strong>the</strong><br />
treatment of epilepsy.<br />
Disclosures: S. K<strong>an</strong>torovich, None; G.W. Astary, None; T.H. Mareci, None; M.<br />
Sarntinor<strong>an</strong>ont, None; P.R. Carney, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.4/O38<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: National Basic Research Program of China (973 Program), No. 2006CB504506<br />
National Natural Science Foundation of China, No. 30472133<br />
Title: A new concept of tr<strong>an</strong>scut<strong>an</strong>eous vagus nerve stimulation <strong>for</strong> epileptic seizure<br />
Authors: *W. HE, B. ZHU, P. RONG;<br />
Inst. of Acup-Moxi, CACMS, Beijing, China<br />
Abstract: Vagus nerve stimulation (VNS) using <strong>the</strong> impl<strong>an</strong>ted Neurocybernetic Pros<strong>the</strong>sis<br />
(NCP),has been approved by <strong>the</strong> U.S. Food <strong>an</strong>d Drug Administration as <strong>an</strong> adjunctive treatment<br />
<strong>for</strong> refractory partial-onset of epileptic seizures. Besides, its <strong>the</strong>rapeutic effect, adverse events
<strong>an</strong>d stimulation-induced effects of VNS, have recently been reported. To address <strong>the</strong><br />
disadv<strong>an</strong>tage of <strong>the</strong> state-of-<strong>the</strong>-art VNS device, a new concept of tr<strong>an</strong>scut<strong>an</strong>eous vagus nerve<br />
stimulation (TVNS) <strong>for</strong> epileptic seizure is proposed.<br />
The present study is designed to observe <strong>the</strong> effect of tr<strong>an</strong>scut<strong>an</strong>eous stimulation of <strong>the</strong> auricular<br />
br<strong>an</strong>ch of <strong>the</strong> vagus nerve (ABVN) at <strong>the</strong> left tragus on <strong>the</strong> epidural electroencephalogram (EEG)<br />
of pentylenetrazole (PTZ) induced epilepsy rats. Male SD rats weighing 250-320 g were<br />
<strong>an</strong>aes<strong>the</strong>tized by 10% ureth<strong>an</strong>e (1.2 g/kg, i.p.). Epidural EEG was recorded with two silver globe<br />
electrodes placed over <strong>the</strong> dura through two small holes (AP: ±1.0 mm, ML: 2.0 mm)<br />
respectively. Epileptic seizure was induced by intraperitoneal injection of PTZ (40mg/kg). The<br />
left vagus nerve was dissected <strong>an</strong>d placed over a cuff electrode <strong>for</strong> VNS. Two silver slice<br />
electrodes were sticked on <strong>the</strong> <strong>the</strong> left tragus <strong>for</strong> TVNS. Stimulation parameters of frequency 20<br />
Hz; pulse width 0.5 ms, strength 1.0 mA <strong>an</strong>d duration of stimulation 30s were used. The results<br />
showed that 1) when PTZ was administered without stimulation delivered, highly synchronous,<br />
large-amplitude activity in epidural EEG traces occurred. 2) VNS <strong>an</strong>d TVNS both subst<strong>an</strong>tially<br />
reduced PTZ-induced seizure activity in epidural EEG traces, compared with that of control<br />
periods. And <strong>the</strong> <strong>an</strong>tiseizure effects of VNS <strong>an</strong>d TVNS outlasted <strong>the</strong> period during stimulation is<br />
provided. 3) The average duration of <strong>an</strong>tiseizure effect of VNS <strong>an</strong>d TVNS were 15.29±2.24 min<br />
<strong>an</strong>d 14.87±2.83 min, respectively. There was no signific<strong>an</strong>t difference between VNS <strong>an</strong>d TVNS<br />
in <strong>the</strong> average duration of acute <strong>an</strong>tiseizure effect (p>0.05) .<br />
The technique <strong>for</strong> vagal nerve stmulation, despite of being effective, has several disadv<strong>an</strong>tages<br />
such as high cost of <strong>the</strong> equipment, surgical risks, or unknown long term effects of <strong>the</strong> chronic<br />
electrical stimulation, etc. Perhaps it is a new concept of noninvasive vagus nerve stimulation at<br />
<strong>the</strong> left tragus to suppress epileptic seizures. In addition, chronic <strong>an</strong>tiseizure effect of TVNS<br />
versus VNS needs to be observed in <strong>the</strong> future.<br />
Disclosures: W. He, None; B. Zhu, None; P. Rong, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.5/P1<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Title: Poisson distributed high frequency hippocampal stimulation suppresses epileptic seizures<br />
in <strong>the</strong> kainate treated rat
Authors: *T. M. WYCKHUYS 1 , P. BOON 1 , R. RAEDT 1 , B. VAN NIEUWENHUYSE 1 , K.<br />
VONCK 1 , W. WADMAN 2 ;<br />
1 Ghent Univ., Ghent, Belgium; 2 Univ. of Amsterdam, Amsterdam, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Temporal lobe epilepsy is one of <strong>the</strong> most difficult to treat <strong>for</strong>ms of epilepsy. One<br />
third of <strong>the</strong> patients is or becomes refractory to <strong>an</strong>ti-epileptic drugs, emphasizing <strong>the</strong> need <strong>for</strong><br />
new <strong>the</strong>rapeutic strategies, among which hippocampal deep brain stimulation (DBS). In this<br />
study we demonstrate <strong>the</strong> efficacy of a new stimulation paradigm: Poisson distributed<br />
stimulation (PDS) in <strong>the</strong> kainic acid model, a validated model <strong>for</strong> hum<strong>an</strong> temporal lobe epilepsy.<br />
Status epilepticus (SE) was induced by intraperitoneal injection of kainic acid, leading to<br />
epileptogenesis. After fifty days, rats with spont<strong>an</strong>eous seizures were impl<strong>an</strong>ted with depth<br />
stimulation <strong>an</strong>d recording electrodes in <strong>the</strong> hippocampus. After 15 days of continuous baseline<br />
EEG monitoring, 13 rats received continuous PDS (me<strong>an</strong> frequency 130 Hz) <strong>an</strong>d 11 received<br />
regular High Frequency Stimulation (HFS at 130 Hz) during <strong>the</strong> following 10 days. The<br />
maximum stimulus intensity at which rats could be stimulated without experiencing EEG <strong>an</strong>d/or<br />
behavioral side effects was signific<strong>an</strong>tly lower <strong>for</strong> PDS th<strong>an</strong> <strong>for</strong> HFS (p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.6/P2<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: NIH Gr<strong>an</strong>t F33NS062617<br />
INSERM U751<br />
Title: NADH imaging of zero Mg2+ induced epilepti<strong>for</strong>m events in intact, immature mouse<br />
hippocampus in vitro <strong>an</strong>d response to beta-hydroxybutyrate, a ketone body<br />
Authors: A. IVANOV 1 , C. BERNARD 1 , *D. A. TURNER 2 ;<br />
1 Epilepsie et Cognition, INSERM U751, Marseille, Fr<strong>an</strong>ce; 2 Duke Univ. Med. Ctr., Durham, NC<br />
Abstract: The intact, in vitro immature hippocampus facilitates <strong>the</strong> development of spont<strong>an</strong>eous,<br />
ictal epilepti<strong>for</strong>m events (ictal-like events, ILEs) under zero Mg 2+ conditions, resembling a<br />
generalized epilepsy model (Quilichini et al., 2002). We hypo<strong>the</strong>sized that metabolic imaging<br />
may facilitate identifying <strong>the</strong> origin of <strong>the</strong>se events within <strong>the</strong> hippocampus, <strong>an</strong>d that this model<br />
may respond to elements of <strong>the</strong> ketogenic diet, specifically <strong>the</strong> ketone body betahydroxybutryate.<br />
Our methods focused on using <strong>the</strong> immature (ie, P5-P9) mouse hippocampus maintained in vitro<br />
at 33 O C, with extracellular field recordings. ILEs were induced by zero Mg 2+ conditions. The<br />
initiation <strong>an</strong>d spread of <strong>the</strong>se events in <strong>the</strong> intact hippocampus was evaluated with NADH<br />
tr<strong>an</strong>smission imaging (ex = 290-270 nm, em = > 400 nm) using a macro lens attached to a cooled<br />
12-bit CCD camera (Sensicam).<br />
ILE events typically occurred every 10-12 minutes. The NADH imaging of each ILE showed a<br />
short oxidation (decrease) followed by a prolonged reduction (increase); <strong>the</strong> duration of <strong>the</strong><br />
response was proportional to <strong>an</strong>d coincident with <strong>the</strong> degree <strong>an</strong>d timing of epilepti<strong>for</strong>m spiking.<br />
There was often <strong>an</strong> initiation site within <strong>the</strong> hippocampus <strong>for</strong> <strong>the</strong> ILE events, <strong>the</strong>n rapid spread of<br />
<strong>the</strong> metabolic signal along <strong>the</strong> axis of <strong>the</strong> hippocampus. These events were <strong>the</strong>n <strong>an</strong>alyzed in<br />
response to a ketone body, beta-hydroxybutyrate (10 mM racemic mixture). If <strong>the</strong> betahydroxybutyrate<br />
was started after <strong>the</strong> ILE events had begun, <strong>the</strong>re was a signific<strong>an</strong>t decrease in<br />
<strong>the</strong> rate of subsequent ILE events (52%, p < 0.001, n=12 experiments) compared to control<br />
hippocampi, though events were not abolished. The ketone body required at least 30 minutes <strong>for</strong><br />
<strong>an</strong> effect to occur, suggesting a metabolic modification underlying <strong>the</strong> activity. Addition of ei<strong>the</strong>r<br />
pyruvate or lactate also modified <strong>the</strong> ILE profile <strong>an</strong>d frequency.<br />
This generalized, in vitro epilepsy model in immature hippocampus may be useful to examine<br />
mech<strong>an</strong>isms underlying <strong>the</strong> ketogenic diet. The preliminary results suggest a modest reduction in<br />
seizure frequency by ~ 50%, similar to in vivo responses in children with generalized epilepsy.<br />
Additionally, NADH imaging parallels <strong>the</strong> field potential response associated with <strong>the</strong> ictal like<br />
events, providing a measure of intensity <strong>an</strong>d spread of <strong>the</strong> ictal like events within <strong>the</strong> axis of <strong>the</strong><br />
intact hippocampus.<br />
Disclosures: A. Iv<strong>an</strong>ov, None; C. Bernard, None; D.A. Turner, None.
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.7/P3<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Title: Evaluating <strong>the</strong> efficacy of <strong>the</strong> ketogenic diet in mouse models of epilepsy<br />
Authors: *S. B. DUTTON 1 , A. ESCAYG 1 , K. BORGES 2 ;<br />
1 Emory Univ., Decatur, GA; 2 Sch. of Biomed. Sci., Univ. of Queensl<strong>an</strong>d, St Lucia, Australia<br />
Abstract: Generalized epilepsy with febrile seizures plus (GEFS+) is <strong>an</strong> inherited idiopathic<br />
epilepsy syndrome caused, in part, by mutations in <strong>the</strong> voltage-gated sodium ch<strong>an</strong>nel SCN1A.<br />
Symptoms of GEFS+ include febrile seizures that persist beyond <strong>the</strong> age of 6, as well as <strong>the</strong><br />
development of afebrile seizures. Affected members of GEFS+ families exhibit a broad r<strong>an</strong>ge of<br />
disease severities <strong>an</strong>d c<strong>an</strong> differ in <strong>the</strong>ir response to <strong>an</strong>ti-epilepsy medications. The ketogenic<br />
diet (KD) has been demonstrated to be effective in <strong>the</strong> treatment of some types of epilepsy;<br />
however, little is known about <strong>the</strong> efficacy of <strong>the</strong> KD in <strong>the</strong> treatment of epilepsy subtypes that<br />
are due to SCN1A dysfunction. We evaluated <strong>the</strong> ability of <strong>the</strong> KD to raise seizure thresholds in<br />
mice with Scn1a mutations that serve as a model of GEFS+. Thresholds to flurothyl-induced<br />
seizures were compared between Scn1a mut<strong>an</strong>ts <strong>an</strong>d wildtype (WT) littermates that were fed<br />
ei<strong>the</strong>r a 6:1 KD or st<strong>an</strong>dard rodent chow <strong>for</strong> 2 weeks. Mut<strong>an</strong>ts on st<strong>an</strong>dard rodent chow exhibited<br />
a 26% reduction (p= 0.01) in latency to flurothyl-induced generalized tonic clonic seizures<br />
(GTCS) when compared to WT littermates. However <strong>the</strong> latency to <strong>the</strong> GTCS in mut<strong>an</strong>ts on <strong>the</strong><br />
KD was not statistically different to that of WT mice on st<strong>an</strong>dard rodent chow, indicating that <strong>the</strong><br />
diet was able to restore more normal seizure thresholds.<br />
Since <strong>the</strong> KD is not equally effective in all patients, we also examined whe<strong>the</strong>r genetic factors<br />
might influence efficacy. To explore this possibility, seizure thresholds were compared between<br />
5 inbred mouse strains (C57BL/6J, NIH Swiss, DBA/2J, A/J, <strong>an</strong>d 12956/SVEVTAC) that were<br />
fed ei<strong>the</strong>r <strong>the</strong> 6:1 KD or st<strong>an</strong>dard rodent chow <strong>for</strong> 2 weeks. C57Bl/6J mice exhibited increased<br />
seizure thresholds. In contrast, decreased seizure thresholds were observed in DBA/2J, NIH<br />
Swiss, <strong>an</strong>d A/J mice.<br />
Disclosures: S.B. Dutton, None; A. Escayg, None; K. Borges, None.<br />
Poster
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.8/P4<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: CURE 2008 Quest Award<br />
Title: The effect of <strong>an</strong>aplerotic diet in mouse epilepsy models<br />
Authors: S. WILLIS 1 , L. SWEETMAN 2 , *J. STOLL 1 , K. BORGES 3 ;<br />
1 2 3<br />
Texas Tech. Sch. Pharm., Amarillo, TX; Baylor Res. Inst., Dallas, TX; Univ. of Queensl<strong>an</strong>d,<br />
Brisb<strong>an</strong>e, Australia<br />
Abstract: The Citric Acid Cycle (CAC) is critical <strong>for</strong> oxidative metabolism in neurons. Also,<br />
CAC intermediates are <strong>the</strong> precursor of neurotr<strong>an</strong>smitters. Impaired CAC activity leads to<br />
decreases in GABA <strong>an</strong>d ATP that could lead to generation/progression of epileptic disorders.<br />
Anaplerosis is <strong>the</strong> metabolic replenishment of CAC catalytic intermediates. We w<strong>an</strong>ted to<br />
determine if <strong>an</strong> <strong>an</strong>aplerotic diet is <strong>an</strong>ticonvuls<strong>an</strong>t <strong>an</strong>d/or <strong>an</strong>tiepileptogenic. Mice were fed ad<br />
libitum ei<strong>the</strong>r control diet or diet containing 35% calories <strong>from</strong> trihept<strong>an</strong>oin (C7), <strong>an</strong> <strong>an</strong>aplerotic<br />
molecule. Metabolic studies showed <strong>the</strong>re was no statistically signific<strong>an</strong>t difference in body<br />
weights or kilocalories consumed/kg/day on ei<strong>the</strong>r diet, which indicates <strong>the</strong> diets are calorically<br />
equivalent. Hept<strong>an</strong>oylcarnitine was elevated ten-fold in mice on <strong>the</strong> C7 diet, demonstrating<br />
metabolic availability. The C7 diet was not <strong>an</strong>ticonvuls<strong>an</strong>t in three acute seizure models; <strong>the</strong><br />
pentylenetetrazole, 6 Hertz, or <strong>the</strong> fluorothyl models, as demonstrated by <strong>the</strong> lack of effect on<br />
seizure thresholds. The effect of <strong>the</strong> C7 diet was also tested in two chronic seizure models,<br />
pilocarpine-induced status epilepticus (SE) or corneal kindling. In <strong>the</strong> first model, mice that<br />
developed SE in response to pilocarpine were subsequently fed <strong>the</strong> C7 or control diet <strong>for</strong> three<br />
weeks. Mice on <strong>the</strong> C7 diet showed statistically signific<strong>an</strong>t increases in β-hydroxybutyrate <strong>an</strong>d<br />
<strong>the</strong> <strong>an</strong>aplerotic CAC precursors, propionyl-CoA <strong>an</strong>d methylmalonyl-CoA. SE mice were <strong>the</strong>n<br />
tested <strong>for</strong> seizure susceptibility upon administration of pentylenetetrazole. SE mice were more<br />
sensitive th<strong>an</strong> non-SE mice to pentylenetetrazole-induced seizures. SE mice fed C7 diet had <strong>an</strong><br />
increase in seizure threshold compared to SE mice fed <strong>the</strong> control diet, but it did not quite reach<br />
statistical signific<strong>an</strong>ce. Ongoing experiments will assess if hippocampal neurodegeneration is<br />
reduced by <strong>the</strong> diet. In <strong>the</strong> second model, mice on ei<strong>the</strong>r diet were kindled by corneal<br />
electroshock. The <strong>an</strong>aplerotic diet produced a statistically signific<strong>an</strong>t delay in kindling <strong>an</strong>d<br />
decrease in medi<strong>an</strong> seizure score. This data shows <strong>an</strong> <strong>an</strong>aplerotic diet may be <strong>an</strong>tiepileptic <strong>an</strong>d<br />
beneficial <strong>for</strong> <strong>the</strong> treatment of hum<strong>an</strong> seizure disorders.<br />
Disclosures: S. Willis, None; L. Sweetm<strong>an</strong>, None; J. Stoll, None; K. Borges, None.
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.9/P5<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: NIH U01 NS058204<br />
R01 NS040337<br />
Title: Treatment of status epilepticus by somatostatin type 2 receptor agonists<br />
Authors: *J. KAPUR 1 , C. BALINT 2 ;<br />
1 2<br />
Dept Neurol, Univ. Virginai Hlth. Sci. Ctr., Charlottesville, VA; Neurol., Univ. of Virginia,<br />
Charlottesville, VA<br />
Abstract: Status Epilepticus (SE) is a neurological emergency characterized by prolonged<br />
seizure that c<strong>an</strong> cause death or disability. Approximately 30-40% of patients are refractory to<br />
current <strong>the</strong>rapies of SE <strong>an</strong>d new treatments are needed. We have previously demonstrated that<br />
somatostatin <strong>an</strong>d SST2 receptor agonists diminish glutamate release <strong>from</strong> presynaptic terminals.<br />
We tested <strong>an</strong>ticonvuls<strong>an</strong>t action of somatostatin <strong>an</strong>d Octreotide. Animals were impl<strong>an</strong>ted with <strong>an</strong><br />
intracerebroventricular infusion c<strong>an</strong>nula <strong>an</strong>d a pair of hippocampal EEG electrodes. Status<br />
epilepticus was induced by a combination of lithium <strong>an</strong>d pilocarpine. Animals were ei<strong>the</strong>r treated<br />
with intracerebro-ventricular infusion of sterile saline or somatostatin suspended in saline.<br />
Animals were treated with intraperitoneal injection of lithium (1.5 mEq/ Kg) 4-6 hours prior to<br />
pilocarpine infusion. Drug or saline infusion into <strong>the</strong> ventricle was initiated 90 minutes be<strong>for</strong>e<br />
pilocarpine injection (50 mg/Kg). Somatostatin (0.4mg/m l) was infused at a rate of 0.06ml/hr<br />
over <strong>the</strong> course of three hours. Animals <strong>the</strong>re<strong>for</strong>e received 0.18ml of somatostatin, which<br />
corresponds to doses of 72 µg dose. EEG recordings were obtained <strong>from</strong> <strong>the</strong> hippocampal<br />
electrodes <strong>an</strong>d infusion c<strong>an</strong>nula continuously <strong>from</strong> <strong>the</strong> beginning of infusion <strong>for</strong> 24 hours.<br />
Pattern of continuous electrographic seizures lasting more th<strong>an</strong> 30 minutes were considered<br />
status epilepticus. Additionally, behavioral seizures were observed <strong>an</strong>d time to first grade 5<br />
seizure (Racine scale) recorded.<br />
In 9 control <strong>an</strong>imals treated with lithium <strong>an</strong>d pilocarpine, infusion of saline into <strong>the</strong> ventricle did<br />
not protect against status epilepticus in <strong>an</strong>y <strong>an</strong>imal <strong>an</strong>d 4 <strong>an</strong>imals died during seizures. In 11<br />
<strong>an</strong>imals infused with 72 µg of somatostatin, no seizures occurred <strong>an</strong>d status epilepticus did not<br />
develop in 5<strong>an</strong>imals, all of which survived. Remaining 6 <strong>an</strong>imals developed status epilepticus, 4<br />
of which died during seizures. Overall somatostatin treated <strong>an</strong>imals were less likely to develop<br />
status epilepticus more likely to survive (χ 2 test). Adding 10 mg/Kg of diazepam (i.p.)<br />
administered after first grade 5 seizure, to <strong>the</strong> regimen increased survival to 80%.
We tested whe<strong>the</strong>r selective SST2 Receptor agonist octreotide would be more efficacious th<strong>an</strong><br />
somatostatin. Initial experiments with 16 µg of octreotide infused in 10 <strong>an</strong>imals revealed that<br />
seizures were prevented in 3 <strong>an</strong>imals (30%). We <strong>the</strong>n increased <strong>the</strong> dose to 50 µg <strong>an</strong>d 4/6 (66%)<br />
of <strong>the</strong> <strong>an</strong>imals were treated. Higher doses were not more efficacious.<br />
These studies suggest that somatostatin <strong>an</strong>d SST2 receptor agonists could effectively treat status<br />
epilepticus.<br />
Disclosures: J. Kapur, None; C. Balint, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.10/P6<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Support: NIH gr<strong>an</strong>t AA16852A<br />
NIH gr<strong>an</strong>t P20AA011997-07<br />
Citizens United <strong>for</strong> Research on Epilepsy (CURE)<br />
Title: Activation of Group 2 metabotropic glutamate receptors inhibits status epilepticus<br />
Authors: *E. H. CAULDER 1 , D. GODWIN 2 ;<br />
1 2<br />
Wake Forest Univ., winston salem, NC; Neurobio. <strong>an</strong>d Anat., Wake Forest Univ., Winston<br />
salem, NC<br />
Abstract: Pilocarpine-induced status epilepticus (SE) in mice is <strong>an</strong> often-used model of hum<strong>an</strong><br />
temporal lobe epilepsy <strong>an</strong>d epileptogenesis. Acute SE in <strong>an</strong>imal models is characterized by<br />
clonic/tonic (C/T) seizure activity <strong>an</strong>d electroencephalographic (EEG) activity with signific<strong>an</strong>tly<br />
increased power in <strong>the</strong> <strong>the</strong>ta <strong>an</strong>d gamma b<strong>an</strong>dwidths that lasts <strong>for</strong> upwards of 5 to 6 hours.<br />
Previously, we have demonstrated that agonists to Group 2 metabotropic glutamate receptors<br />
(mGluRs) reduce epileptic activity in a model of absence epilepsy <strong>an</strong>d that mGluR2 specifically<br />
acts a presynaptic autoreceptor to modulate excessive amounts of corticothalamic input<br />
(Neuroscience. 141:501-13,2006). Here, we tested <strong>the</strong> effects of <strong>the</strong> Group 2 mGluR agonist<br />
LY379268, <strong>an</strong>d <strong>the</strong> mGluR2-specific positive allosteric modulator BINA in vivo in C57Bl/6<br />
mice that underwent pilocarpine-induced SE. C57Bl/6 mice (8 weeks) were injected with <strong>the</strong><br />
muscarinic <strong>an</strong>tagonist scopolamine (1 mg/kg, i.p.) followed by pretreatment with ei<strong>the</strong>r vehicle
(saline, 0.2 mls, i.p.), LY379268 (10 mg/kg, i.p.), or BINA (10 mg/kg, i.p.). Pilocarpine (330<br />
mg/kg, i.p.) was <strong>the</strong>n administered to induce SE <strong>an</strong>d <strong>the</strong>se <strong>an</strong>imals were observed in order to<br />
record <strong>the</strong> latency to <strong>the</strong> first C/T seizure, <strong>the</strong> number of C/T seizures <strong>an</strong>d <strong>the</strong> average Racine<br />
score. Mice treated with LY379268 (n= 9) prior to pilocarpine exhibited a longer latency to <strong>the</strong><br />
first C/T seizure (me<strong>an</strong>= 68.75 minutes) as compared to saline injected mice (n= 10, me<strong>an</strong>=<br />
39.13 minutes, t-test, p
59 Sprague-Dawley rats were divided into 4 experimental groups. From E12 to birth pregn<strong>an</strong>t<br />
mo<strong>the</strong>rs received ei<strong>the</strong>r 54mg/kg s.c. LEV q8h or 54mg/kg s.c. LO60 q8h (inactive LEV<br />
ent<strong>an</strong>tiomer). Half of <strong>the</strong> mo<strong>the</strong>rs were irradiated with 145cGy in utero on E17. On PND 60 all<br />
offsprings were given a subconvulsive dose (50 mg/kg s.c.) of pentylenetetrazol (PTZ; “second<br />
hit”) <strong>an</strong>d <strong>the</strong>ir behavior <strong>an</strong>d EEG were monitored <strong>for</strong> a subsequent 8 hours, be<strong>for</strong>e being<br />
sacrificed. Immunocytochemistry <strong>for</strong> SV2A (<strong>the</strong> LEV binding site in <strong>the</strong> presynaptic vesicle),<br />
Cresyl-violet <strong>an</strong>d Timm staining were per<strong>for</strong>med.<br />
Results<br />
Rats who received LEV in utero displayed in greater numbers signific<strong>an</strong>tly less severe seizures<br />
th<strong>an</strong> those who did not. CV staining confirmed CD in irradiated rats. The hippocampal hilus <strong>an</strong>d<br />
CA3 regions of LEV-pretreated irradiated rats were enriched with SV2A in contrast to those that<br />
did not receive LEV during gr<strong>an</strong>ule cell development (E14-E17). Likewise, on Timm staining<br />
those LEV-pretreated irradiated rats did not show increased mossy fiber staining in contrast to<br />
irradiated rats who had not received LEV.<br />
Conclusions<br />
Pre-treatment with LEV during hippocampal gr<strong>an</strong>ule cell development impedes <strong>the</strong> expression of<br />
acute seizures induced by a “second hit” in CD rats. Mossy fiber sprouting appears to be<br />
inhibited in those <strong>an</strong>imals that received LEV during gr<strong>an</strong>ule cell development, despite irradiation<br />
<strong>an</strong>d seizure provocation. These findings suggest a disease-modifying effect of LEV in this<br />
<strong>an</strong>imal model <strong>an</strong>d provide tentative evidence <strong>for</strong> a possible mech<strong>an</strong>ism of action. Fur<strong>the</strong>r<br />
confirmatory cellular <strong>an</strong>d <strong>an</strong>imal studies using LEV & o<strong>the</strong>r <strong>an</strong>tiseizure agents are ongoing to<br />
elucidate fur<strong>the</strong>r <strong>the</strong> effects of in utero LEV, <strong>an</strong>d to determine whe<strong>the</strong>r <strong>the</strong> observed effect is<br />
LEV-specific.<br />
Disclosures: R. O'Dwyer, None; S. Yates, None; I. Najm, None; A. Alexopoulos, UCB<br />
Pharma Inc., B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.12/P8<br />
Topic: C.07.k. Anticonvuls<strong>an</strong>t <strong>an</strong>d <strong>an</strong>tiepileptic <strong>the</strong>rapies<br />
Title: Effect of serotonergic system in <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t effect of adenosine against<br />
pentylenetetrazol-induced seizure threshold in mice
Authors: *K. K. AKULA 1 , S. K. KULKARNI 2 ;<br />
2 Univerity Inst. of Pharmaceut. Sci., 1 P<strong>an</strong>jab Univ., Ch<strong>an</strong>digarh, India<br />
Abstract: Purpose: Recent studies indicate <strong>the</strong> role of serotonergic system in subsiding<br />
convulsions in several seizure types. Adenosine is one of <strong>the</strong> endogenous inhibitory<br />
neuromodulators with potent <strong>an</strong>ticonvuls<strong>an</strong>t activity. The present study was per<strong>for</strong>med to<br />
examine <strong>the</strong> possible interaction of serotonergic system with adenosine against pentylenetetrazol<br />
(PTZ) seizure threshold in mice.<br />
Method: In <strong>the</strong> present study, minimal dose of PTZ (iv, mg/kg) needed to induce different<br />
phases (myoclonic jerks, generalized clonus <strong>an</strong>d tonic extension) of convulsions were recorded<br />
as <strong>an</strong> index of seizure threshold. The experiments were approved by Institutional Animal Ethics<br />
Committee.<br />
Results: Adenosine (100 or 200 mg/kg ip) produced a signific<strong>an</strong>t increase in seizure threshold<br />
<strong>for</strong> convulsions induced by PTZ iv infusion. Quipazine (5 mg/kg ip) [5-HT1B, 5-HT2 <strong>an</strong>d 5-HT3<br />
agonist] produced synergistic <strong>an</strong>ticonvuls<strong>an</strong>t effect with sub-effective dose of adenosine (50<br />
mg/kg ip). 8-OH-DPAT (0.5 mg/kg ip) [5-HT1A agonist] or pindolol (20 mg/kg ip) [5-HT1A/1B/βadrenoceptor<br />
<strong>an</strong>tagonist] or DOI (1 mg/kg ip) [preferential 5-HT2A agonist] produced a<br />
synergistic <strong>an</strong>ticonvuls<strong>an</strong>t effect with a sub-effective dose of adenosine (50 mg/kg ip) against<br />
PTZ seizure threshold paradigm in mice. However, concomit<strong>an</strong>t administration of ket<strong>an</strong>serin (5<br />
mg/kg ip) or seg<strong>an</strong>serin (1 mg/kg ip) [both 5-HT2A/2C <strong>an</strong>tagonists] had no effect on <strong>the</strong><br />
<strong>an</strong>ticonvuls<strong>an</strong>t effect of adenosine (100 mg/kg ip) in <strong>the</strong> PTZ seizure threshold paradigm. PCPA<br />
(100mg/kg ip) [<strong>an</strong> inhibitor of serotonin syn<strong>the</strong>sis <strong>for</strong> four consecutive days] pretreatment also<br />
prevented <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t effect of adenosine (100 mg/kg ip) in <strong>the</strong> PTZ seizure threshold<br />
paradigm.<br />
Conclusion: The results indicate that <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t effect of adenosine in <strong>the</strong> PTZ seizure<br />
threshold paradigm appears to be mediated, at least in part, by <strong>an</strong> interaction with 5-HT receptors<br />
<strong>an</strong>d this combined approach may prove to be effective in targeting convulsions refractory to<br />
existing <strong>the</strong>rapies.<br />
Disclosures: K.K. Akula, None; S.K. Kulkarni, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.13/P9<br />
Topic: C.07.j. Animal models<br />
Title: Anticonvulsive effect of acute stress in rats
Authors: *N. BUKIA 1 , M. BUTSKHRIKIDZE 2 , I. BILANISHVILI 2 , N. KHIZANISHVILI 2 , L.<br />
MACHAVARIANI 2 , Z. NANOBASHVILI 2 ;<br />
1 Neurophysiol., 2 I.Beritashvili Inst. of Physiol., Tbilisi, Georgia<br />
Abstract: Determining a link between stress <strong>an</strong>d epileptic activity is <strong>an</strong> import<strong>an</strong>t problem of<br />
current neurology. The <strong>an</strong>atomical structures, which are implicated in stress - septum,<br />
hippocamp, amygdala, medial prefrontal <strong>an</strong>d orbital cortex, raphe nuclei, spino-thalamo-cortical<br />
tract, are also involved in <strong>the</strong> propagation of epileptic seizures.<br />
Investigation of this problem is complicated by <strong>the</strong> following factors: 1) Reliable measurement<br />
of a stress levels is practically impossible, 2) Susceptibility to <strong>the</strong> stressogenic factors is highly<br />
individual, 3) Ethical restrictions, as well as <strong>an</strong>tiepileptic drug <strong>the</strong>rapy. In order to solve this<br />
problem, it is expedient to use <strong>the</strong> <strong>an</strong>imal models of epilepsy.<br />
In <strong>the</strong> kindling model we endeavoured to determine alterations in <strong>the</strong> electroencephalographic<br />
(EEG) <strong>an</strong>d behavioral parameters induced by <strong>the</strong> stressogenic factors, in different time intervals.<br />
Experiments were carried out in male albino inbred rats; weighing 200-250 g (n=10). Stainless<br />
steel electrodes were impl<strong>an</strong>ted into <strong>the</strong> hippocampus <strong>an</strong>d neocortex, in conditions of general<br />
<strong>an</strong>es<strong>the</strong>sia. Following 7-10 days after <strong>the</strong> surgery, <strong>the</strong> ventral hippocampus was electrically<br />
stimulated 40 times with intervals of 5 min, within one-day session. On <strong>the</strong> next day, 2 <strong>an</strong>d 3<br />
weeks <strong>the</strong>reafter <strong>the</strong> test-stimulations were applied. As a result, were obtained <strong>the</strong> <strong>an</strong>imals with<br />
epileptogenic focus.<br />
Following establishing <strong>an</strong> epileptogenic focus, a noxious electrical stimulation was applied on<br />
<strong>the</strong> paws (45 V, with 15 s interval, during 1 min). In different experimental days, following 6<br />
min, 3 hr <strong>an</strong>d 6 hr after noxious stimulation, <strong>the</strong> EEG <strong>an</strong>d behavioral repertory of epileptic<br />
activity was observed.<br />
Experiments have shown that 6 min after application of a noxious stimulus, epileptic reactions<br />
decreased as compared to those reactions, which were induced by <strong>the</strong> test-stimulation in kindling<br />
procedure; this trend remained after 3 hours too. That is, statistically signific<strong>an</strong>t decrease of both<br />
EEG seizures <strong>an</strong>d behavioral reactions was recorded (P
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.14/P10<br />
Topic: C.07.j. Animal models<br />
Support: Gr<strong>an</strong>t-GNSF/ST 07/6-238<br />
Title: Anticonvulsive effects of pregn<strong>an</strong>cy on <strong>an</strong>imal model of kindling in <strong>the</strong> rats<br />
Authors: *M. BUTSKHRIKIDZE 1 , N. BUKIA 2 , Z. MACHAVARIANI 2 , Z.<br />
NANOBASHVILI 2 ;<br />
1 Neurophysiol., 2 I.Beritashvili Inst. of Physiol., Tbilisi, Georgia<br />
Abstract: Interrelation between pregn<strong>an</strong>cy <strong>an</strong>d epilepsy is one of <strong>the</strong> pressing problems of<br />
current neurology. Those mech<strong>an</strong>isms, which suppress or amplify <strong>the</strong> seizure reactions in<br />
pregn<strong>an</strong>cy, have not been yet determined experimentally. Investigation of <strong>the</strong> interrelation<br />
between <strong>the</strong> epileptic fits <strong>an</strong>d pregn<strong>an</strong>cy in women is complicated by <strong>the</strong> bioethical<br />
considerations, as well as by <strong>the</strong> <strong>an</strong>tiepileptic <strong>the</strong>rapy. There<strong>for</strong>e, in order to solve this problem,<br />
<strong>an</strong> implementing <strong>the</strong> <strong>an</strong>imal models seems expedient; moreover that this problem is scarcely<br />
investigated so far.<br />
The goal of present work was investigation of impact of gestation <strong>an</strong>d <strong>the</strong> postpartum period on<br />
initiation <strong>an</strong>d development of convulsive reactions in <strong>the</strong> experimental <strong>an</strong>imal model. Because of<br />
specificity of experiments, <strong>the</strong> part of experiments will be carried out on female rats grown up<br />
isolated <strong>from</strong> males (n=12). Female rats were subjected to kindling : 40 times electrical<br />
stimulations of ventral hippocampus with 5 min intervals during a day; <strong>an</strong>imals were received 5<br />
test-stimulations in ventral hippocampus with 5 min intervals at 24 hr, 2 <strong>an</strong>d 3 weeks <strong>the</strong>reafter.<br />
Parameters of Electroencephalographic (EEG) <strong>an</strong>d behavioral seizures were assessed. Behavioral<br />
m<strong>an</strong>ifestation of seizures was scored according to a modification of <strong>the</strong> scale of Racine.<br />
Following establishing <strong>an</strong> epileptogenic focus, <strong>for</strong> fertilization female rats was copulated with<br />
male rats. To determine <strong>the</strong> state of pregn<strong>an</strong>cy we were inspected smear <strong>from</strong> uterus <strong>an</strong>d by<br />
palpation.<br />
The EEG <strong>an</strong>d behavioral repertory of epileptic activity was observed in various time intervals: at<br />
<strong>the</strong> end of 2 weeks, 3 weeks <strong>an</strong>d after postpartum period.<br />
Differences in parameters were evaluated using <strong>the</strong> ANOVA computer program. Epileptic<br />
reactions were signific<strong>an</strong>tly suppressed during gestation (2 <strong>an</strong>d 3 weeks). The data on <strong>the</strong><br />
ch<strong>an</strong>ges are concerned behavioral reactions <strong>an</strong>d EEG seizure activity. At <strong>the</strong> postpartum period<br />
seizure expression were facilitated in kindled <strong>an</strong>imals.<br />
It has been proposed that increase of progesterone level during pregn<strong>an</strong>cy modulates activity of<br />
<strong>the</strong> brain neurons, as <strong>the</strong> main metabolite of progesterone - allopregn<strong>an</strong>olone has a high affinity<br />
to <strong>the</strong> GABA type receptors. Activation of <strong>the</strong> GABA-ergic synapses suppresses generalized<br />
convulsions inducing by <strong>the</strong> activation of limbic structures.<br />
During parturition <strong>an</strong>d in post-delivery period content of progesterone was decreased. Response<br />
this EEG seizure <strong>an</strong>d behavioral activity were enh<strong>an</strong>ced.<br />
There<strong>for</strong>e, generalization of <strong>the</strong> seizure activity is blocked during pregn<strong>an</strong>cy.
Disclosures: M. Butskhrikidze, None; N. Bukia, None; Z. Machavari<strong>an</strong>i, None; Z.<br />
N<strong>an</strong>obashvili, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.15/P11<br />
Topic: C.07.d. Glutametergic neurotr<strong>an</strong>smission<br />
Support: Epilepsy Research UK PhD studentship<br />
Title: The role of purinergic P2 receptors in <strong>the</strong> modulation of seizure activity<br />
Authors: *J. LOPATAR, N. DALE, B. G. FRENGUELLI;<br />
Dept. of Biol. Sci., Univ. of Warwick, Coventry, United Kingdom<br />
Abstract: The role of <strong>the</strong> purine nucleoside adenosine as <strong>an</strong> import<strong>an</strong>t endogenous<br />
<strong>an</strong>ticonvuls<strong>an</strong>t is well established. It is not clear however whe<strong>the</strong>r ATP, as a potential source of<br />
adenosine, c<strong>an</strong> participate in <strong>the</strong> regulation of neuronal excitability <strong>an</strong>d seizure activity. The aim<br />
of this work is to investigate <strong>the</strong> possible role of P2 receptors in modulating epilepti<strong>for</strong>m activity.<br />
As <strong>an</strong> in vitro model of seizure activity, hippocampal slices (600 µm) were ba<strong>the</strong>d in Mg 2+ -free<br />
aCSF <strong>an</strong>d seizures were evoked electrically (high frequency stimulation, HFS, 60Hz, 2s). The<br />
model consisted of two sets of three seizures, <strong>the</strong> first in <strong>the</strong> absence <strong>an</strong>d <strong>the</strong> second in <strong>the</strong><br />
presence of a drug. Two parameters were measured: number of events (spikes or bursts) per 1<br />
second interval, <strong>an</strong>d seizure duration. Broad-spectrum P2 receptor <strong>an</strong>tagonists (RB-2, PPADS),<br />
as well as a specific <strong>an</strong>tagonist against P2X1, P2X2/3 <strong>an</strong>d P2X3 receptors (NF023), <strong>an</strong>d a<br />
specific P2Y1 receptor <strong>an</strong>tagonist (MRS 2179) have been used to establish <strong>the</strong> possible action of<br />
ATP on its receptors during seizures. RB-2 <strong>an</strong>d PPADS (both at 10 µM) signific<strong>an</strong>tly decreased<br />
<strong>the</strong> duration of seizures (by 33 <strong>an</strong>d 25 %, respectively) with seizures being less intense. In<br />
addition, similar effects on seizures were made with <strong>the</strong> more selective <strong>an</strong>tagonist NF023 (a 24%<br />
ch<strong>an</strong>ge in duration). A specific P2Y1 receptor <strong>an</strong>tagonist MRS 2179 (30 µM) did not ch<strong>an</strong>ge<br />
measured parameters. In contrast, <strong>the</strong> P2X receptor-specific agonist α,β-methylene ATP (30 µM)<br />
caused little or no ch<strong>an</strong>ge in seizure characteristics.<br />
Enzyme-based microelectrode biosensors <strong>for</strong> real-time detection of purines have been used to<br />
detect purine release during seizures. This method revealed considerable adenosine release<br />
following HFS-induced seizures in magnesium-free conditions. However, <strong>the</strong> sensors failed to<br />
reveal <strong>an</strong>y release of ATP during seizures.<br />
To establish if <strong>the</strong> rapid breakdown of ATP limited <strong>the</strong> action <strong>an</strong>d detection of ATP, we used <strong>an</strong>
ecto-ATPase inhibitor ARL 67156. In <strong>the</strong> presence of ARL 67156 (100 µM) seizures were<br />
shorter <strong>an</strong>d less intense. This observation may be explained by <strong>the</strong> strong depression of synaptic<br />
tr<strong>an</strong>smission caused by ARL 67156, possibly involving non-specific actions on <strong>the</strong> fibre volley<br />
(a 15 % decrease, n=6, p
Disclosures: L. Khuchua, None; E. Saralidze, None; T. Ioseli<strong>an</strong>i, None.<br />
Poster<br />
539. Epilepsy: Anticonvuls<strong>an</strong>t Strategies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 539.17/P13<br />
Topic: C.07.j. Animal models<br />
Support: NIH R21-NS059669<br />
Interdepartmental Program in Neuroscience<br />
Title: Differential response to <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t effect of NAX-5055, a rationally designed<br />
gal<strong>an</strong>in <strong>an</strong>alog, following chronic treatment in mice<br />
Authors: *S. P. FLYNN 1 , E. A. SCHOLL 4,2 , G. BULAJ 3 , H. S. WHITE 2,1 ;<br />
1 Interdepartmental Program in Neurosci., 2 Dept. of Pharmacol. <strong>an</strong>d Toxicology, 3 Dept. of<br />
Medicinal Chem., Univ. of Utah, Salt Lake City, UT; 4 NeuroAdjuv<strong>an</strong>ts, Inc., Salt Lake City, UT<br />
Abstract: The neuropeptide gal<strong>an</strong>in <strong>an</strong>d its receptors are novel targets <strong>for</strong> <strong>the</strong> treatment of<br />
epilepsy given <strong>the</strong>ir potent modulatory activity on neurotr<strong>an</strong>smission <strong>an</strong>d expression in seizure<br />
prone areas. NAX-5055, a modified gal<strong>an</strong>in <strong>an</strong>alog, is a GalR1 preferring agonist that is<br />
systemically bioavailable <strong>an</strong>d blood-brain-barrier (BBB) penetr<strong>an</strong>t with <strong>an</strong>ticonvuls<strong>an</strong>t activity<br />
in <strong>an</strong>imal models after acute treatment. However, <strong>the</strong> efficacy of NAX-5055 is reduced after<br />
chronic treatment. We hypo<strong>the</strong>sized that <strong>the</strong> loss of efficacy in chronic studies results <strong>from</strong> <strong>the</strong><br />
development of toler<strong>an</strong>ce to NAX-5055.<br />
To test this hypo<strong>the</strong>sis, male CF-1 mice were used in two groups (n=8, per group) matched with<br />
controls (n=8, per group). Experimental mice were given 4 mg/kg NAX-5055 <strong>an</strong>d controls 0.9%<br />
saline by i.p. injection. In group 1, mice received 6 doses (2 times daily <strong>for</strong> 3 days), <strong>an</strong>d in group<br />
2, mice received 3 doses (once daily every o<strong>the</strong>r day). After completion of <strong>the</strong> repeated dosing,<br />
treated mice along with a naive group (n=8, per group) received a challenge dose of 4 mg/kg<br />
NAX-5055 (test-day). Sixty minutes post-injection all mice were given a corneal stimulation<br />
sufficient to produce a limbic seizure (6 Hz, 32 mA, 3 sec). Mice not displaying a seizure were<br />
considered protected. After test-day, <strong>an</strong>imals in all groups were permitted a drug- <strong>an</strong>d<br />
stimulation-free washout period. After 3 days of washout, mice were challenged with 4 mg/kg<br />
NAX-5055 <strong>an</strong>d stimulated, followed by <strong>an</strong> additional 3 day washout, challenge <strong>an</strong>d stimulation<br />
(7 day washout).<br />
On test-day, 12.5% (2/16) of control mice were protected, <strong>an</strong>d 93.8% (15/16) of naive mice were
protected confirming <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t activity of NAX-5055. Protection was reduced to 12.5%<br />
in group 1 <strong>an</strong>d 25% in group 2. After <strong>the</strong> 3 day washout, <strong>the</strong> percent protection increased in both<br />
groups to 50% (3/8, group 1; 5/8, group 2) <strong>an</strong>d remained at similar levels at 7 days (4/8, group 1;<br />
5/8, group 2). Individually, mice segregated into one of three groups; 1) toler<strong>an</strong>t on test-day with<br />
no recovery, 2) protected after all stimulations, <strong>an</strong>d 3) toler<strong>an</strong>t on test day but protected after<br />
both washouts.<br />
These results suggest that some mice develop toler<strong>an</strong>ce to <strong>the</strong> <strong>an</strong>ticonvuls<strong>an</strong>t effect of NAX-<br />
5055 following chronic administration, while o<strong>the</strong>rs never develop toler<strong>an</strong>ce or recover <strong>from</strong><br />
toler<strong>an</strong>ce <strong>an</strong>d remain non-toler<strong>an</strong>t. We hypo<strong>the</strong>size that this segregation may result <strong>from</strong> ch<strong>an</strong>ges<br />
in drug tr<strong>an</strong>sporters <strong>an</strong>d/or decreased gal<strong>an</strong>in receptor sensitivity. To test this hypo<strong>the</strong>sis, we are<br />
evaluating <strong>the</strong> effect of chronic, intracerebroventricular infusion of NAX-5055 to bypass <strong>the</strong><br />
BBB <strong>an</strong>d microarrays to evaluate <strong>the</strong> role of tr<strong>an</strong>sporters <strong>an</strong>d receptors in <strong>the</strong> differential<br />
response to NAX-5055.<br />
Disclosures: S.P. Flynn, None; E.A. Scholl, NeuroAdjuv<strong>an</strong>ts, Inc., SLC, UT, A. Employment<br />
(full or part-time); G. Bulaj, NeuroAdjuv<strong>an</strong>ts, Inc., SLC, UT, Scientific Co-founder, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); H.S. White,<br />
Ortho-McNeil Neurologics, Titusville, NJ, Speakers Bureau, D. Speakers Bureau/Honoraria<br />
(speakers bureau, symposia, <strong>an</strong>d expert witness); NeuroAdjuv<strong>an</strong>ts, Inc., SLC, UT, Scientific Cofounder,<br />
E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
Johnson & Johnson Pharmaceutical Res & Devel. (PND), Springhouse, PA, Consult<strong>an</strong>t, F.<br />
Consult<strong>an</strong>t/Advisory Board; Vale<strong>an</strong>t Pharmaceuticals, Costa Mesa, CA, Consult<strong>an</strong>t, F.<br />
Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.1/P14<br />
Topic: C.08.f. Inflammation<br />
Support: NIH Gr<strong>an</strong>ts R01NS38118<br />
Gr<strong>an</strong>t<br />
NIH Gr<strong>an</strong>ts R01NS048216<br />
AHA Established-Investigator Award Gr<strong>an</strong>t N0540154<br />
University of Wisconsin Institute <strong>for</strong> Clinical <strong>an</strong>d Tr<strong>an</strong>slational Research (ICTR) Pilot
Title: Inhibition of sodium-hydrogen exch<strong>an</strong>ger activity reduces microglia activation after focal<br />
cerebral ischemia<br />
Authors: *Y. SHI 1,2 , P. A. FERRAZZANO 3 , D. SUN 1,2 ;<br />
1 2 3<br />
Neurolog. Surgery, Neurosci. Training Program, Pediatrics, Univ. of Wisconsin Med. Sch. <strong>an</strong>d<br />
Waism<strong>an</strong> Ctr., Madison, WI<br />
Abstract: Our previous studies have shown that inhibition of Na + /H + exch<strong>an</strong>ger iso<strong>for</strong>m 1<br />
(NHE-1) is neuroprotective in focal cerebral ischemia. Regulation of H + homeostasis is<br />
import<strong>an</strong>t <strong>for</strong> macrophage/microglia activation. In <strong>the</strong> present study, we investigated whe<strong>the</strong>r<br />
NHE-1 plays a role in activation of microglia <strong>an</strong>d promotes proinflammatory responses<br />
following cerebral ischemia. In this study, tr<strong>an</strong>sient focal cerebral ischemia was induced in mice<br />
by 60 min occlusion of left middle cerebral artery (MCAO) followed by 1-24 h reperfusion. In<br />
<strong>the</strong> drug treatment groups, a potent NHE-1 inhibitor HOE 642 (0.5 mg/kg, i.p.) was<br />
administrated 30 min prior to onset of reperfusion (Rp). Animals were sacrificed at 1, 6, or 24 h<br />
Rp <strong>an</strong>d brain sections (35 µm) prepared. Microglia proliferation <strong>an</strong>d morphological ch<strong>an</strong>ges<br />
were characterized by immunostaining assay with <strong>an</strong>ti-CD11b <strong>an</strong>tibody (against <strong>the</strong> microglial<br />
specific integrin Mac-1). Proliferation of microglia was detected in <strong>the</strong> ipsilateral cortex <strong>an</strong>d<br />
striatum at as early as 1 h Rp. Activated microglia, as evidenced by a morphological ch<strong>an</strong>ge <strong>from</strong><br />
“ramified” to “amoeboid”, were detected in ischemic cortex <strong>an</strong>d striatum <strong>an</strong>d tended to occur in<br />
clusters during 1-24 h Rp. In <strong>the</strong> HOE-642-treated brains, <strong>the</strong>re was tr<strong>an</strong>sient microglial<br />
activation at 1 h Rp, similar to <strong>the</strong> vehicle controls. However, at 6 h Rp, activated microglia were<br />
only detected in <strong>the</strong> ipsilateral striatum but not in <strong>the</strong> cortex. By 24 h Rp, <strong>the</strong> number of activated<br />
microglia was sharply decreased in <strong>the</strong> ipsilateral hemisphere, becoming indistinguishable <strong>from</strong><br />
<strong>the</strong> uninjured control hemisphere. Taken toge<strong>the</strong>r, our preliminary results suggest that NHE-1<br />
activity plays a role in activation <strong>an</strong>d proliferation of microglia during early reperfusion, <strong>an</strong>d that<br />
<strong>the</strong> neuroprotective effects of NHE-1 inhibition may in part result <strong>from</strong> blocking <strong>the</strong> activation of<br />
microglia following cerebral ischemia.<br />
Disclosures: Y. Shi, None; P.A. Ferrazz<strong>an</strong>o, None; D. Sun, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.2/P15<br />
Topic: C.08.f. Inflammation<br />
Support: NIH Gr<strong>an</strong>ts R01NS38118
NIH Gr<strong>an</strong>ts R01NS048216<br />
AHA Established-Investigator Award Gr<strong>an</strong>t N0540154<br />
Title: Activation of microglia following in vitro ischemia depends on Na + /H + exch<strong>an</strong>gemediated<br />
H + homeostasis<br />
Authors: Y. LIU 1 , D. B. KINTNER 1 , X. CHEN 2 , J. ALGHARABLI 1 , J. OLSON 1 , *D. SUN 1,2 ;<br />
1 Dept Neurosurg., 2 Neurosci. Training Program, Univ. Wisconsin Med. Sch., Madison, WI<br />
Abstract: Regulation of H + homeostasis is import<strong>an</strong>t <strong>for</strong> macrophage/microglia activation. In<br />
this study, we investigated <strong>the</strong> role of Na + /H + exch<strong>an</strong>ger iso<strong>for</strong>m 1 (NHE-1) in activation of<br />
microglia following in vitro ischemia. NHE-1 expression was detected in CD11b-positive<br />
immortalized M4T.4 microglia <strong>an</strong>d mouse primary microglia. NHE-1 was import<strong>an</strong>t in<br />
mainten<strong>an</strong>ce of basal pHi in microglia. Pharmacological inhibition of NHE-1 activity with <strong>the</strong><br />
potent inhibitor cariporide (HOE 642) not only decreased <strong>the</strong> basal pHi (<strong>from</strong> 7.1-7.2 to ~ 6.8)<br />
but also abolished pHi regulatory function following NH4 + /NH3-induced acidification.<br />
Lipopolysaccharide (LPS) activated <strong>the</strong> microglia <strong>an</strong>d ch<strong>an</strong>ged <strong>the</strong> morphology of <strong>the</strong> microglia<br />
<strong>from</strong> “ramified” to “amoeboid”, which was accomp<strong>an</strong>ied with <strong>an</strong> increase in pHi <strong>an</strong>d H +<br />
extrusion following acidification. Blocking NHE-1 activity abolished <strong>the</strong>se ch<strong>an</strong>ges.<br />
Interestingly, oxygen/glucose deprivation <strong>an</strong>d reoxygenation (OGD/REOX) signific<strong>an</strong>tly<br />
stimulated NHE-1 activity in microglia, resulting in 2-3 fold increases in H + extrusion rates.<br />
Exposure of microglia to LPS or OGD/REOX also triggered a signific<strong>an</strong>t elevation in [Ca 2+ ]i <strong>an</strong>d<br />
[Na + ]i <strong>an</strong>d production of superoxide <strong>an</strong>ion, which was attenuated by inhibition of NHE-1. Taken<br />
toge<strong>the</strong>r, <strong>the</strong>se results show that NHE-1 activity is increased in activated microglia. Function of<br />
NHE-1 may be import<strong>an</strong>t in mainten<strong>an</strong>ce of pHi homeostasis <strong>an</strong>d triggering Ca 2+ i signaling<br />
necessary <strong>for</strong> <strong>the</strong> microglial “respiratory” burst.<br />
Disclosures: Y. Liu, None; D.B. Kintner, None; X. Chen, None; J. Algharabli, None; J.<br />
Olson, None; D. Sun, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.3/P16<br />
Topic: C.08.b. Molecular mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t NS47309
NIH Gr<strong>an</strong>t NS061108<br />
Title: Microglial toll-like receptor-4 <strong>an</strong>d ischemic preconditioning<br />
Authors: *J. R. WEINSTEIN 1 , A. SAVOS 1 , R. LEE 1 , T. MÖLLER 1 , S. P. MURPHY 2 ;<br />
1 Dept Neurol, 2 Dept Neurolog. Surgery, Univ. Washington, Seattle, WA<br />
Abstract: Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a<br />
brief period of cerebral ischemia confers tr<strong>an</strong>sient toler<strong>an</strong>ce to subsequent ischemic challenge.<br />
Inflammatory responses in brain are critical in stroke pathophysiology. Microglia, <strong>the</strong> brain’s<br />
resident tissue macrophages, are central in <strong>the</strong>se responses. Toll-like receptors (TLRs) mediate<br />
powerful immune responses to a variety of exogenous <strong>an</strong>d endogenous lig<strong>an</strong>ds. TLR4 is<br />
expressed by microglia <strong>an</strong>d has been implicated in stroke pathophysiology. However, <strong>the</strong> role of<br />
microglial TLR4 in IPC is unknown. We hypo<strong>the</strong>size that microglial TLR4 signaling is required<br />
<strong>for</strong> both optimal IPC-mediated neuroprotection <strong>an</strong>d IPC-mediated attenuation of inflammatory<br />
responses seen following stroke. We fur<strong>the</strong>r postulate that hypoxia-inducible factor-1 (HIF-1), a<br />
central regulator of gene expression under low oxygen tension, is a key downstream mediator of<br />
TLR4 signaling in microglia <strong>an</strong>d contributes to <strong>the</strong> TLR4-dependent component of IPC-induced<br />
neuroprotection. To test <strong>the</strong>se hypo<strong>the</strong>ses, we per<strong>for</strong>med middle cerebral artery occlusion<br />
(MCAO) on TLR4 -/- <strong>an</strong>d wild-type (WT) mice using established paradigms <strong>for</strong> stroke <strong>an</strong>d IPC.<br />
We first assessed infarct volume <strong>an</strong>d neurobehavioral outcome. We <strong>the</strong>n used ex vivo flow<br />
cytometry to characterize <strong>the</strong> inflammatory infiltrate in ischemic cortex following stroke alone or<br />
IPC followed by stroke. IPC induced 35% <strong>an</strong>d 4% reductions in infarct volume in WT <strong>an</strong>d TLR4 -<br />
/- mice, respectively. Myeloid cellular content <strong>an</strong>d phenotype in ipsilateral cortex was markedly<br />
influenced by both IPC <strong>an</strong>d strain. We also carried out in vitro experiments on cultured primary<br />
microglia <strong>from</strong> TLR4 -/- <strong>an</strong>d WT mice to characterize <strong>the</strong> role of TLR4 in modulating <strong>the</strong><br />
microglial response to hypoxia/hypoglycemia (ischemia). Using microarray oligonucleotide<br />
hybridization methods <strong>an</strong>d gene set <strong>an</strong>alysis, we found that ischemia induced characteristic<br />
profiles of genomic ch<strong>an</strong>ges that were markedly disparate in microglia derived <strong>from</strong> TLR4 -/- <strong>an</strong>d<br />
WT mice. In WT microglia, we also found that activation of TLR4 with exogenous agonists<br />
synergistically enh<strong>an</strong>ced ischemia-induced expression of both HIF-1 itself <strong>an</strong>d HIF-1 gene<br />
targets. Results of <strong>the</strong>se studies will provide insights into <strong>the</strong> pathophysiologic state of microglia<br />
in <strong>the</strong> ischemic penumbra <strong>an</strong>d may help identify molecular targets <strong>for</strong> <strong>the</strong>rapeutic intervention in<br />
stroke.<br />
Disclosures: J.R. Weinstein, None; A. Savos, None; R. Lee, None; T. Möller, None; S.P.<br />
Murphy, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.4/P17<br />
Topic: C.08.b. Molecular mech<strong>an</strong>isms<br />
Support: NIH<br />
Title: Activation <strong>an</strong>d neurotoxicity of microglial NF-κB <strong>an</strong>d purinergic receptors in oxygen<br />
glucose deprivation: Differential roles of extracellular nucleotides<br />
Authors: *C. M. WEBSTER, M. A. YENARI;<br />
Neurol., UCSF-VAMC, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: The inflammatory response following brain ischemia includes activation of microglia,<br />
<strong>the</strong> brain’s resident immune cell, followed by activation of a key inflammatory nuclear<br />
tr<strong>an</strong>scription factor, NF-κB, <strong>an</strong>d upregulation of inflammatory cytokines, release of reactive<br />
oxygen species, <strong>an</strong>d o<strong>the</strong>r immune mediators. Triggering of this inflammatory response is<br />
thought to be through oxidative stress or necrotic cell lysis, leading to <strong>the</strong> release of extracellular<br />
nucleotides. Previous work has shown that extracellular purines activate microglia by inducing<br />
chemotaxis via <strong>the</strong> purinergic, P2Y12 receptor, towards regions of cell damage, or phagocytosis,<br />
via <strong>the</strong> P2Y6 receptor, of dead cells or debris. Here, we show that <strong>the</strong> addition of primary<br />
microglia or BV-2 cells to mixed cultures of neurons <strong>an</strong>d astrocytes increases <strong>the</strong> amount of<br />
neuron damage following in vitro ischemia (OGD, oxygen glucose deprivation) compared to<br />
neuron-glia cells alone. By loading neurons with <strong>the</strong> fluorescent label CM-DiI, we found that<br />
microglia (identified by CD11b) phagocytosed neurons following OGD. Knocking down P2Y12<br />
in BV-2 cells, using siRNA, <strong>the</strong>n co-culturing with primary astrocytes <strong>an</strong>d primary neurons, <strong>an</strong>d<br />
exposing cultures to OGD, protected neurons <strong>from</strong> microglia mediated damage at a level similar<br />
to <strong>the</strong> uninjured controls (n=6, p
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.5/P18<br />
Topic: C.08.f. Inflammation<br />
Support: CIHR<br />
Title: Role of galectin-3 in microglial activation after cerebral ischemia<br />
Authors: *M. LALANCETTE-HEBERT, Y. WENG, S. SATO, J. KRIZ;<br />
Laval University, CHUL, Quebec, QC, C<strong>an</strong>ada<br />
Abstract: Microglial cells are <strong>the</strong> main effectors of <strong>the</strong> innate response after CNS injuries,<br />
including ischemia. After ischemic brain injury, microglial activation is characterized by ch<strong>an</strong>ges<br />
of morphology, massive exp<strong>an</strong>sion <strong>an</strong>d migration of <strong>the</strong> resident microglial cell population. All<br />
<strong>the</strong>se processes lead to <strong>an</strong> inflammatory cascade promoting <strong>the</strong> syn<strong>the</strong>sis <strong>an</strong>d <strong>the</strong> secretion of<br />
inflammatory cytokines but also of trophics factors. Recent studies by o<strong>the</strong>rs <strong>an</strong>d us put <strong>for</strong>ward<br />
a neuroprotective role <strong>for</strong> a particular microglial population, <strong>the</strong> galectin-3 (Mac2) positive cells.<br />
After cerebral ischemia, this galactosidase-binding lectin is only expressed on activated <strong>an</strong>d<br />
resident microglial cell restricted to <strong>the</strong> stroke area region. The galectin-3 + cell population is also<br />
associated with microglial proliferative response which c<strong>an</strong> provide neuroprotection to adjacent<br />
injured neurons by <strong>the</strong> secretion of trophic molecules, such as IGF-1. However, despite <strong>the</strong> fact<br />
that galectin-3 may be implicated in numerous cellular function, such as <strong>the</strong> regulation of<br />
apoptosis <strong>an</strong>d control of <strong>the</strong> cell cycle in <strong>the</strong> periphery, <strong>the</strong> function of galectin-3 in <strong>the</strong> central<br />
nervous system <strong>an</strong>d in <strong>the</strong> pathology of cerebral ischemia is still ambiguous. Here, we show that<br />
<strong>the</strong> genetic depletion of galectin-3 causes <strong>an</strong> increase in <strong>the</strong> size of ischemic lesion when<br />
compared to <strong>the</strong> WT. This exacerbation in <strong>the</strong> stroke area is accomp<strong>an</strong>ied by <strong>an</strong> increase in <strong>the</strong><br />
number of apoptotic cells, preferentially neurons. In addition, we observed a lower rate of<br />
microglial proliferation after BrdU injection. Interestingly, a protein <strong>an</strong>alysis of <strong>the</strong> stroke area<br />
region at different time point after cerebral ischemia showed marked alterations in IGF-<br />
1signaling pathway in galectin-3 deficient mice. These results suggest <strong>an</strong> import<strong>an</strong>t role of<br />
galectin-3 in brain response to ischemic injury. Galectin-3 positive microglia appears to plays a<br />
protective role in <strong>the</strong> context of brain injuries suggesting that <strong>the</strong> m<strong>an</strong>ipulation of this particular<br />
cell subpopulation may help in finding neuroprotective strategies after cerebral ischemia.<br />
Disclosures: M. Lal<strong>an</strong>cette-Hebert, None; Y. Weng, None; S. Sato, None; J. Kriz, None.
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.6/Q1<br />
Topic: C.08.g. Neuroprotection<br />
Title: Repeated treatment with Yamabushitake ameliorates neurologic impairment through<br />
activated microglia expressing a high-mobility group box1-inhibiting mech<strong>an</strong>ism<br />
Authors: *A. KATAOKA 1,2 , K. HAYAKAWA 3 , M. HAZEKAWA 2 , K. IRIE 3 , S. HIGUCHI 2 ,<br />
M. ENOKI 2 , T. NAKANO 2 , R. FURUTA 2 , K. MISHIMA 2,3 , K. IWASAKI 2,3 , M. FUJIWARA 2 ;<br />
1 2 3<br />
Fukuoka, Jap<strong>an</strong>; Fukuoka Univ., Fukuoka, Jap<strong>an</strong>; Adv<strong>an</strong>ced Materials Institute, Fukuoka<br />
Univ., Fukuoka, Jap<strong>an</strong><br />
Abstract: Hericium erinaceum ( Yamabushitake ) is <strong>the</strong> mushroom used as food in Jap<strong>an</strong> <strong>an</strong>d<br />
China. Hericenones <strong>an</strong>d erinacines with nerve growth factor (NGF) syn<strong>the</strong>tic promotion are<br />
included in yamabushitake. These results were expected <strong>the</strong> usefulness of yamabushitake <strong>for</strong> <strong>the</strong><br />
prevention <strong>an</strong>d treatment of dementia. In <strong>the</strong> present study, we investigated cerebroprotective<br />
effect of yamabushitake on post-ischemic injury induced by 4h middle cerebral artery (MCA)<br />
occlusion mice. Recently, high-mobility group box1(HMGB1) has been massively released into<br />
<strong>the</strong> extracellular space immediately after ischemic insult <strong>an</strong>d to induce neuroinflammation <strong>an</strong>d<br />
microglial activation in <strong>the</strong> postischemic brain. There<strong>for</strong>e, it is import<strong>an</strong>t to investigate <strong>the</strong> effect<br />
of Yamabushitake on <strong>the</strong> released HMGB1 during postischemic injury.<br />
One day after 4h MCA occlusion, yamabushitake (100, 300mg/kg) was administrated orally <strong>for</strong><br />
14 days. Neurological score, survival rate, body weight were measured <strong>for</strong> 14 days after cerebral<br />
ischemia. Motor coordination was evaluated at 1, 3, 7, 14th day after cerebral ischemia by rotarod<br />
test at 10rpm. Activated microglia was evaluated in immunofluorescent stain with Iba1, at<br />
14th day. HMGB1 were also evaluated by western blot <strong>an</strong>d immunostaining at 14th day after<br />
cerebral ischemia.<br />
The neurological score <strong>an</strong>d rota-rod test were impaired in MCA-occluded mice during <strong>the</strong><br />
experimental periods. Yamabushitake (100, 300mg/kg, p.o. <strong>for</strong> 2weeks) improved neurological<br />
score, motor coordination on rota-rod test, <strong>an</strong>d survival rates <strong>an</strong>d inhibited decrease of <strong>the</strong> weight<br />
in a dose-dependent m<strong>an</strong>ner. Yamabushitake was signific<strong>an</strong>tly reduced brain atrophy induced by<br />
MCA-occlusion in a dose-dependent m<strong>an</strong>ner. Moreover, yamabushitake decreased activated<br />
microglia expressing HMGB1 within <strong>the</strong> brain, <strong>an</strong>d also decreased both brain <strong>an</strong>d plasma<br />
HMGB1.<br />
Our results suggest that repeated treatment with yamabushitake prevented <strong>the</strong> brain damage by<br />
inhibiting activated microglia expressing HMGB1. These findings suggest that yamabushitake<br />
may be clinically useful <strong>for</strong> curative to cerebrovascular accident.
Disclosures: A. Kataoka, None; K. Hayakawa, None; M. Hazekawa, None; K. Irie, None; S.<br />
Higuchi, None; M. Enoki, None; T. Nak<strong>an</strong>o, None; R. Furuta, None; K. Mishima, None; K.<br />
Iwasaki, None; M. Fujiwara, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.7/Q2<br />
Topic: C.08.g. Neuroprotection<br />
Support: NIH/NINDS NS44025<br />
AHA GIA 0855235F<br />
Title: Are activated microglial cells a part of endogenous neuroprotection after neonatal focal<br />
stroke?<br />
Authors: J. FAUSTINO 1 , C. JOHNSON 1 , A. KLIBANOV 4 , N. DERUGIN 2 , M.<br />
WENDLAND 3 , *Z. S. VEXLER 1 ;<br />
1 2 3 4<br />
Dept Of Neurol., Neurosurg., Radiology, UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; Univ. of Virginia,<br />
Virginia, VA<br />
Abstract: Accumulation of activated microglial cells <strong>an</strong>d invading macrophages in injured brain<br />
regions are thought to contribute to <strong>the</strong> pathophysiology of stroke. The relative role of local brain<br />
macrophages_activated microglial cells_<strong>an</strong>d invading cells of monocyte lineage after injury<br />
remains poorly understood. In <strong>the</strong> neonatal brain after focal tr<strong>an</strong>sient cerebral ischemia,<br />
macrophage accumulation occurs rapidly but macrophages are comprised predomin<strong>an</strong>tly of<br />
activated microglia ra<strong>the</strong>r th<strong>an</strong> invaded macrophages (Denker et al, J. Neurochem. 2007). Aim:<br />
We set-out to determine if depletion of microglial cells affects injury after acute <strong>an</strong>d subacute<br />
neonatal focal stroke. Methods: Postnatal day 5 (P5) rats were injected with liposomeencapsulated<br />
clodronate or empty liposomes into <strong>the</strong> brain. At P7, rats were subjected to a 3 hr<br />
middle cerebral artery occlusion (MCAO). Injury volume was determined 24 or 72 hr postreperfusion<br />
in <strong>an</strong>imals with confirmed injury, as determined by diffusion-weighted MRI (DWI)<br />
during MCAO. Depletion of microglial cells by clodronate liposomes was confirmed in adjacent<br />
brain sections. Cytokine concentrations (8-plex) <strong>an</strong>d caspase-3 <strong>an</strong>d calpain activation were<br />
determined in injured <strong>an</strong>d contralateral cortex at 24 hrs. Results: At 24 hr post-reperfusion, injury<br />
volumes were not signific<strong>an</strong>tly different between clodronate- <strong>an</strong>d vehicle-treated groups<br />
(51.1±7.4% <strong>an</strong>d 54.8±8.3%, respectively, n=5-6 per group). A 9.9±5.8 fold increase in calpain-
dependent degradation of spectrin was observed in ischemia-reperfused tissue in PBS-treated<br />
pups. Clodronate treatment signific<strong>an</strong>tly increased calpain-dependent spectrin degradation<br />
(p
of cytotoxic brain edema in ischemia <strong>an</strong>d, along with vasogenic edema, may contribute to<br />
increased intracr<strong>an</strong>ial pressure, brain herniation, <strong>an</strong>d additional ischemic injuries. C6 cultures<br />
were exposed to oxygen-glucose deprivation (OGD) <strong>for</strong> 5hr <strong>an</strong>d cell swelling was determined 90<br />
min after <strong>the</strong> end of OGD using <strong>the</strong> 3-O-methyl-glucose method. OGD-induced increase in glial<br />
swelling was signific<strong>an</strong>tly blocked by both quercetin <strong>an</strong>d myricetin. Increased free radical<br />
production, a contributing factor in cell swelling following ischemic injury, was also<br />
signific<strong>an</strong>tly reduced by myricetin <strong>an</strong>d quercetin. Mitochondrial dysfunction, <strong>an</strong>o<strong>the</strong>r key feature<br />
of ischemic injury, is hypo<strong>the</strong>sized to contribute to glial swelling. Depolarization of <strong>the</strong> inner<br />
mitochondrial membr<strong>an</strong>e potential (∆Ψm) was assessed immediately at <strong>the</strong> end of OGD using<br />
TMRE, a fluorescent dye. However, OGD-induced decline in ∆Ψm was signific<strong>an</strong>tly attenuated<br />
by myricetin but not quercetin. An import<strong>an</strong>t feature of glial cells is mainten<strong>an</strong>ce of extracellular<br />
glutamate levels. Reduced glutamate uptake after ischemia is <strong>an</strong> import<strong>an</strong>t mech<strong>an</strong>ism that c<strong>an</strong><br />
lead to excitotoxic damage. Myricetin, but not quercetin, attenuated <strong>the</strong> OGD-induced decrease<br />
in glutamate uptake. Additionally, cyclosporin A, a blocker of <strong>the</strong> mitochondrial permeability<br />
tr<strong>an</strong>sition pore, but not FK506, attenuated declines in glutamate uptake after OGD. Our results<br />
indicate that while both flavonoids signific<strong>an</strong>tly attenuate cell swelling following ischemic<br />
injury, <strong>the</strong>y likely employ different intracellular mech<strong>an</strong>isms to exert <strong>the</strong>ir effects. In addition,<br />
<strong>the</strong> differential effects of myricetin <strong>an</strong>d quercetin on OGD-induced reduction in glutamate uptake<br />
may be due to <strong>the</strong>ir differential effects on mitochondria.<br />
Disclosures: K.S. P<strong>an</strong>ickar, None; M.M. Pol<strong>an</strong>sky, None; R.A. Anderson, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.9/Q4<br />
Topic: C.08.b. Molecular mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t RO1 HL082511 (SC)<br />
Burke Foundation<br />
Title: CD36 is involved in glial scar <strong>for</strong>mation after injury<br />
Authors: *Y. BAO 1 , L. QIN 1 , E. KIM 1 , M. FEBBRAIO 2 , S. CHO 1,3 ;<br />
1 Burke Med. Res. Inst., White Plains, NY; 2 Cell Biol., Lerner Res. Institute, Clevel<strong>an</strong>d Clin.,<br />
Clevel<strong>an</strong>d, OH; 3 Neurosci., Weill Cornell Med. Col., New York, NY
Abstract: CD36, a multi-functional scavenger receptor, contributes to brain injury following<br />
ischemic stroke. In addition to its expression in microvascular endo<strong>the</strong>lial cells <strong>an</strong>d CD11b+<br />
microglia/macrophages, it is also expressed on astrocytes in <strong>the</strong> peri-infarct area where glial scar<br />
<strong>for</strong>ms (Cho et al, 2005), suggesting <strong>the</strong> potential function of CD36 in astrocytic activity after<br />
brain injury. Glial scar <strong>for</strong>mation is a reactive cellular process upon injury, which is<br />
characterized by proliferation of astrocytes in <strong>the</strong> vicinity of damaged tissue. The purpose of this<br />
study is to investigate whe<strong>the</strong>r CD36 is involved in glial scar <strong>for</strong>mation using <strong>the</strong> astrocyte cell<br />
line C8-D1A in <strong>an</strong> in vitro scratch wound model. After scratch injury, <strong>the</strong> level of GFAP mRNA<br />
was increased (2.0 fold at 24 h, p
Abstract: Neurotrauma <strong>an</strong>d stroke both result in oxygen <strong>an</strong>d glucose deprivation to <strong>the</strong> brain.<br />
We are interested in underst<strong>an</strong>ding how neurons <strong>an</strong>d glia respond to reductions in oxygen <strong>an</strong>d<br />
glucose, <strong>an</strong>d which pathways may be modulated to provide <strong>the</strong>rapeutic benefit. To better<br />
underst<strong>an</strong>d <strong>the</strong> molecular mech<strong>an</strong>isms that lead to cell death after stroke, we have developed cell<br />
type specific dissociated hippocampal cell cultures <strong>from</strong> postnatal mouse to parallel our studies<br />
in <strong>the</strong> org<strong>an</strong>otypical slice culture.<br />
Experiments were per<strong>for</strong>med with mixed cultures (35% neurons, 49% astrocytes), neuronal<br />
enriched cultures (with only 10% astrocytes), <strong>an</strong>d pure astrocyte cultures (< 1% neurons), all<br />
prepared <strong>from</strong> 7 day old mouse hippocampi.<br />
In mixed cultures, signific<strong>an</strong>t cell death (measured with Live/dead assay) was not seen with 30<br />
minutes oxygen-glucose deprivation (OGD). However, death did signific<strong>an</strong>tly increase at 60 or<br />
90 minutes OGD, at <strong>the</strong> same time that total cell number declined <strong>an</strong>d <strong>the</strong> number of apoptotic<br />
cells increased.<br />
Double label immunostaining <strong>for</strong> neurons (MAP2, βIII-tubulin) <strong>an</strong>d astrocytes (GFAP) was used<br />
to compare <strong>the</strong> vulnerability of neurons <strong>an</strong>d astrocytes to OGD. In mixed cultures, signific<strong>an</strong>t<br />
loss or apoptosis of neurons was not observed following 30 minutes of OGD, although durations<br />
of 60 or 90 minutes OGD resulted in signific<strong>an</strong>t apoptosis of neurons. In <strong>the</strong> same cultures,<br />
astrocytes were unaffected at <strong>an</strong>y duration. Relatively pure cultures of astrocytes also did not<br />
show <strong>an</strong>y ch<strong>an</strong>ges in cell number or apoptosis after OGD durations of 30 to 150 minutes.<br />
However, in neuronal enriched cultures, OGD durations as short as 20 <strong>an</strong>d 30 minutes led to<br />
increased numbers of apoptotic neurons compared to mixed cultures. Thus, neurons are more<br />
vulnerable to OGD in <strong>the</strong> absence of astrocytes, <strong>an</strong>d astrocytes appear resist<strong>an</strong>t to OGD.<br />
In mixed cultures, ischemic toler<strong>an</strong>ce (IT) c<strong>an</strong> be produced by 30 min of nonlethal OGD<br />
administered 24 hr prior to a 60 min OGD insult. However, IT is not evident in neuronal<br />
enriched cultures. Taken toge<strong>the</strong>r, <strong>the</strong>se data suggest that astrocytes may be involved in <strong>the</strong><br />
phenomenon of ischemic toler<strong>an</strong>ce, as well as neuroprotection following OGD. Ongoing studies<br />
are examining <strong>the</strong> effects of astrocytes <strong>an</strong>d astrocyte conditioned medium on OGD <strong>an</strong>d ischemic<br />
toler<strong>an</strong>ce.<br />
Disclosures: S.M. Jones, None; A.E. Novak, None; J.P. Elliott, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.11/Q6<br />
Topic: C.08.f. Inflammation
Title: Different mononuclear cell populations constitute <strong>the</strong> brain inflammatory cell status at<br />
early <strong>an</strong>d late time points after cerebral ischemia<br />
Authors: *A. ARAC, M. P. PEREIRA, T. M. BLISS, G. K. STEINBERG;<br />
Neurosurg., St<strong>an</strong><strong>for</strong>d Univ., St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Introduction: After cerebral ischemia, inflammation plays <strong>an</strong> import<strong>an</strong>t role. Here we<br />
investigated <strong>the</strong> cellular inflammatory response in brain <strong>an</strong>d blood at different time points after<br />
stroke.<br />
Methods: 12-week-old, male C57BL6J mice were subjected to 30 minutes middle cerebral artery<br />
occlusion (MCAo). Mononuclear cells (MNCs) were isolated <strong>from</strong> brain <strong>an</strong>d blood <strong>from</strong> naïve<br />
mice <strong>an</strong>d <strong>from</strong> mice 8hr, 24hr, 1week <strong>an</strong>d 2week after stroke. The cells were stained <strong>an</strong>d<br />
<strong>an</strong>alyzed by flow cytometry.<br />
Results: We investigated <strong>the</strong> temporal profile of CNS mononuclear cells in <strong>the</strong> brain after<br />
cerebral ischemia. We found four populations of MNCs in <strong>the</strong> brain after stroke that differed in<br />
<strong>the</strong>ir expression of CD45, CD11b, Gr1 <strong>an</strong>d F4/80. Three of <strong>the</strong>se populations have been<br />
previously reported but we also detected a novel population that becomes more prominent at<br />
later times after stroke. The different MNC populations exhibit different temporal responses to<br />
ischemia. The largest population, <strong>the</strong> resident microglia, exhibited a peak increase in number by<br />
8h post-stroke <strong>an</strong>d returned to baseline by one week. In contrast, <strong>the</strong> infiltrating MNC<br />
populations had begun to increase at 8h, <strong>an</strong>d continued to increase over <strong>the</strong> next 1 - 14 days. The<br />
three infiltrating MNC populations showed distinct temporal ch<strong>an</strong>ge patterns over time after<br />
stroke, but all remained signific<strong>an</strong>tly elevated at 2 week post-stroke. The response of MNC<br />
populations in <strong>the</strong> blood also exhibited distinct ch<strong>an</strong>ges over time.<br />
Conclusion: The mononuclear cell profile in <strong>the</strong> brain ch<strong>an</strong>ges with time after ischemic stroke<br />
<strong>an</strong>d involves 4 different sub-populations of MNCs. Ch<strong>an</strong>ges in <strong>the</strong> inflammatory profile of <strong>the</strong><br />
brain <strong>an</strong>d blood occur in <strong>the</strong> acute stage of stroke <strong>an</strong>d do not return to baseline by 2 weeks after<br />
stroke.<br />
Disclosures: A. Arac, None; M.P. Pereira, None; T.M. Bliss, None; G.K. Steinberg, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.12/Q7<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Support: The Ministry of Knowledge Economy, Republic of Korea 10030064
Title: Micro <strong>an</strong>d macroglial attitudes in <strong>the</strong> rat retinas injured by increased ocular pressure <strong>an</strong>d<br />
by <strong>the</strong> middle cerebral artery occlusion<br />
Authors: J.-M. SHIN, J.-H. LEE, F.-S. QUAN, Y.-J. SHIN, *M.-Y. LEE, M.-H. CHUN, S.-J.<br />
OH;<br />
Dept Anat, Catholic Univ. Med. Col., Seoul 137-701, Republic of Korea<br />
Abstract: Introduction<br />
Glaucomatous degeneration of <strong>the</strong> retina is known to be progressed by ischemia. It was<br />
generated mostly by <strong>the</strong> elevation of <strong>the</strong> intraocular pressure (EIOP) with or without blockage of<br />
aqueous humor drainage ch<strong>an</strong>nels <strong>an</strong>d on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d glaucomatous individuals with normal<br />
IOP were recently increased. Ischemic injury models of <strong>the</strong> rodent retina by different routes may<br />
provide signific<strong>an</strong>t clues <strong>for</strong> studying <strong>the</strong> pathophysiology of diverse glaucomatous<br />
degenerations. Regarding <strong>the</strong> point, we are purposed to examine whe<strong>the</strong>r <strong>the</strong> microglia <strong>an</strong>d<br />
macroglial cells in ischemic retinas evoked by EIOP or arterial occlusion are differentially<br />
activated.<br />
Materials <strong>an</strong>d Methods<br />
EIOP was induced by ischemia-reperfusion injury using Sprague-Dawley rats aged of 8weeks.<br />
Arterial occlusion was done by a tr<strong>an</strong>sient middle cerebral artery occlusion (MCAO), produced<br />
by 3-0 rounded tip nylon suture into <strong>the</strong> right common carotid artery. After each injury, <strong>the</strong><br />
<strong>an</strong>imals were cared <strong>for</strong> 1 week. All retina tissues were taken <strong>from</strong> parts of superior-nasal<br />
quadr<strong>an</strong>t, 5 mm apart <strong>from</strong> <strong>the</strong> optic disc to reduce <strong>the</strong> regional differences error. Glial reaction<br />
was evaluated with lectin histochemistry <strong>an</strong>d immunohistochemistry using by <strong>an</strong>tibodies against<br />
some glial specific marker molecules.<br />
Results <strong>an</strong>d Conclusion<br />
Nestin was expressed in <strong>the</strong> end feet of <strong>the</strong> Mueller glial cells of <strong>the</strong> normal, whereas in <strong>the</strong><br />
proximal processes in <strong>the</strong> MCAO retina <strong>an</strong>d even in <strong>the</strong> distal radial processes in <strong>the</strong> EIOP<br />
retina. GFAP immunoreactivity was limited into <strong>the</strong> nerve fiber layer <strong>an</strong>d in some end feet of<br />
Mueller glial cells, whereas appeared stoutly in <strong>the</strong> radial processes in <strong>the</strong> EIOP <strong>an</strong>d MCAO<br />
retinas. Isolectin B4 <strong>from</strong> Griffonia simplicifolia was expressed in <strong>the</strong> endo<strong>the</strong>lial cells of <strong>the</strong><br />
blood vessels of <strong>the</strong> retinas of normal <strong>an</strong>d EIOP. Interestingly, GSIB4 was also expressed faintly<br />
in some profiles of microglia in <strong>the</strong> inner plexi<strong>for</strong>m layer of MCAO retina. Expression of GS <strong>an</strong>d<br />
GLAST associated with glutamate metabolism was up-regulated in <strong>the</strong> Mueller cells of EIOP<br />
retina, whereas down-regulated in those of MCAO retina.<br />
Our findings demonstrate that EIOP ischemia evokes mainly macroglia reaction <strong>an</strong>d MCAO<br />
ischemia additionally a mild microglial reaction. These results may suggest that EIOP ischemia<br />
is accomp<strong>an</strong>ied with <strong>an</strong> excitotoxic injury to neural components.<br />
Disclosures: J. Shin, None; J. Lee, None; F. qu<strong>an</strong>, None; Y. Shin, None; M. Lee, None; M.<br />
Chun, None; S. Oh, None.<br />
Poster<br />
540. Ischemia: Glia
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.13/Q8<br />
Topic: C.08.g. Neuroprotection<br />
Support: VR 521-2006-2783<br />
Title: Dual role of reactive astrocytes in hypoxic-ischemic brain injury in neonatal mice<br />
Authors: *K. PUGLISI 1 , C. I. ROUSSET 2 , M. FAIZ 3 , U. WILHELMSSON 3 , M. PEKNA 4 , C.<br />
MALLARD 1 , H. HAGBERG 2,5 , M. PEKNY 3 ;<br />
1 Inst. of Neurosci. <strong>an</strong>d Physiol., Go<strong>the</strong>nburg, Sweden; 2 Reproductive Biol., Imperial Col.,<br />
London, United Kingdom; 3 Ctr. <strong>for</strong> Brain Repair <strong>an</strong>d Rehabilitation, Dept. of Clin. Neurosci. <strong>an</strong>d<br />
Rehabil., Neurosci. <strong>an</strong>d Physiology, Sahlgrenska Academy, Univ. of Go<strong>the</strong>nburg, Go<strong>the</strong>nburg,<br />
Sweden; 4 Med. Chem. <strong>an</strong>d Cell Biol., Biomedicine, Sahlgrenska Academy, Univ. of<br />
Go<strong>the</strong>nburg, Go<strong>the</strong>nburg, Sweden; 5 Dept. of Obstetrics <strong>an</strong>d Gynecology, Sahlgrenska Academy,<br />
Univ. of Go<strong>the</strong>nburg, Go<strong>the</strong>nburg, Sweden<br />
Abstract: Astroglial cells are activated following injury <strong>an</strong>d up-regulate <strong>the</strong> expression of <strong>the</strong><br />
intermediate filament proteins glial fibrillary acidic protein (GFAP) <strong>an</strong>d vimentin. Adult mice<br />
lacking <strong>the</strong> intermediate filament proteins GFAP <strong>an</strong>d vimentin show attenuated reactive gliosis<br />
<strong>an</strong>d reduced glial scar development after neurotrauma in <strong>the</strong> brain or spinal cord. They also show<br />
improved regeneration of neuronal synapses after partial denervation of <strong>the</strong> hippocampus. We<br />
have recently shown that reactive astrocytes have a protective role in adult brain ischemia.<br />
However, <strong>the</strong> role of astrocyte intermediate filaments in <strong>the</strong> injured developing brain is<br />
unknown.<br />
Here we examined <strong>the</strong> role of astrocyte intermediate filaments on <strong>the</strong> outcome in a neonatal<br />
model of hypoxia-ischemia (HI).<br />
On postnatal day (PND) 9, GFAP -/- Vim -/- <strong>an</strong>d wild type (WT) mice were exposed to unilateral<br />
HI. Bromodeoxyuridine (BrdU) was injected intraperitoneally twice daily <strong>from</strong> PND9 to PND12<br />
(25mg/kg). On PND12 <strong>an</strong>d 31, <strong>the</strong> <strong>an</strong>imals were perfused intracardially. Immunohistochemistry<br />
with MAP-2, BrdU, NeuN, S100 <strong>an</strong>d Iba-1 <strong>an</strong>tibodies was per<strong>for</strong>med on coronal sections.<br />
We found that at <strong>the</strong> level of <strong>the</strong> hippocampus, GFAP -/- Vim -/- mice had a more pronounced lack<br />
of growth of <strong>the</strong> HI compared to <strong>the</strong> contralateral hemisphere (35.2±4.6%; n=21) versus <strong>the</strong> WT<br />
mice (21.5±4.2%; n=17, p
after HI, but increases neurogenesis <strong>an</strong>d benefits cell survival during <strong>the</strong> later phase of recovery.<br />
These findings suggest that reactive gliosis is <strong>an</strong> import<strong>an</strong>t component in neonatal HI.<br />
Disclosures: K. Puglisi, None; C.I. Rousset, None; M. Faiz, None; U. Wilhelmsson, None; M.<br />
Pekna, None; C. Mallard, None; H. Hagberg, None; M. Pekny, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.14/Q9<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Title: Hyper<strong>the</strong>rmia worsens ischemic brain injury by aggravating glial reaction <strong>an</strong>d increasing<br />
mcirovascular damage in rats<br />
Authors: *C. C. PEGG 1,2 , M. A. COLEMAN 3,2 , C. HE 1,2 , K. A. KATTNER 2 , A. R.<br />
STROINK 2 , C. WANG 1,2 ;<br />
1 2 3<br />
Illinois State Univ., Normal, IL; Central Illinois Neurosci. Fndn., Bloomington, IL; Illinois<br />
Wesly<strong>an</strong> Univ., Bloomington, IL<br />
Abstract: Both clinical <strong>an</strong>d <strong>an</strong>imal studies have shown that hyper<strong>the</strong>rmia is detrimental to<br />
outcome in ischemic brain injury. In <strong>the</strong> present study, we have been examining whe<strong>the</strong>r<br />
hyper<strong>the</strong>rmia worsens <strong>the</strong> injury <strong>an</strong>d underlying mech<strong>an</strong>isms. Focal ischemia is induced by<br />
inserting a silicon-coated nylon suture into <strong>the</strong> internal carotid artery (ICA) through <strong>the</strong> external<br />
carotid artery (ECA) until <strong>the</strong> opening of middle cerebral artery (MCA) is blocked. At <strong>the</strong> end of<br />
each experiment, <strong>the</strong> brain is collected <strong>an</strong>d sectioned with a cryostat. The infarct <strong>an</strong>d brain<br />
swelling are measured in H & E stained brain sections. Glial reactions <strong>an</strong>d damage of<br />
microvessels are determined with immunohistochemistry. Activated astrocytes <strong>an</strong>d microglia are<br />
detected with <strong>an</strong>ti-glial fibrillary acidic protein (GFAP) <strong>an</strong>d ED1, respectively. Damage of<br />
microvascular basal membr<strong>an</strong>e is detected with <strong>an</strong>ti-fibronectin. Preliminary results show that<br />
blocking blood flow resulted in infarction in <strong>the</strong> brain, mainly in <strong>the</strong> territory irrigated by <strong>the</strong><br />
MCA. Hyper<strong>the</strong>rmia, 39ºC <strong>for</strong> a period of 3 hours, signific<strong>an</strong>tly increased infarct size.<br />
Experiments <strong>for</strong> detecting glial reactions <strong>an</strong>d microvascular damages are still on going. The<br />
present study thus shows that hyper<strong>the</strong>rmia is harmful <strong>for</strong> <strong>the</strong> injured brain under ischemic<br />
conditions. Fur<strong>the</strong>r research is still required to underst<strong>an</strong>d relationship of vasculature damage,<br />
glial reactions <strong>an</strong>d hyper<strong>the</strong>rmic-aggravated injury processes during ischemic brain injury.
Disclosures: C.C. Pegg, None; M.A. Colem<strong>an</strong>, None; C. He, None; K.A. Kattner, None; A.R.<br />
Stroink, None; C. W<strong>an</strong>g, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.15/Q10<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Title: Effects of ischemic factors <strong>an</strong>d estradiol on cell volume of cortical astrocytes<br />
Authors: *J. RUTKOWSKY, M. O'DONNELL;<br />
Physiol. <strong>an</strong>d Membr<strong>an</strong>e Biol., UC Davis, Davis, CA<br />
Abstract: During <strong>the</strong> initial hours of <strong>an</strong> ischemic stroke, edema <strong>for</strong>mation occurs in <strong>the</strong> presence<br />
of <strong>an</strong> intact blood brain barrier (BBB) by a process involving secretion of Na, Cl, <strong>an</strong>d<br />
osmotically obliged water across <strong>the</strong> barrier <strong>from</strong> blood into brain <strong>an</strong>d swelling of astrocytes.<br />
Our previous studies have demonstrated that BBB Na-K-Cl cotr<strong>an</strong>sporter <strong>an</strong>d Na/H exch<strong>an</strong>ger<br />
activities are stimulated by ischemic factors including hypoxia, aglycemia <strong>an</strong>d arginine<br />
vasopressin (AVP). Also, we have shown that estradiol, known to be neuroprotective, attenuates<br />
ischemia-induced cerebral edema <strong>an</strong>d infarct, <strong>an</strong>d abolishes ischemia stimulation of BBB<br />
cotr<strong>an</strong>sporter <strong>an</strong>d exch<strong>an</strong>ger activities. While ischemic factors induce a rapid increase in BBB<br />
secretion of Na, Cl <strong>an</strong>d water we have also found that <strong>the</strong>y cause a gradual swelling of BBB<br />
endo<strong>the</strong>lial cells with signific<strong>an</strong>t increases observed after 3 or more hours of hypoxia. Ischemiainduced<br />
swelling of astrocytes is also <strong>an</strong> integral part of cerebral edema. However, <strong>the</strong> effects of<br />
ischemic factors on astrocyte cell volume have not been well characterized <strong>an</strong>d little is known<br />
about estradiol effects on astrocyte volume. In <strong>the</strong> present study we evaluated <strong>the</strong> effects of<br />
hypoxia, aglycemia, AVP <strong>an</strong>d estradiol on astrocyte intracellular volume. Cortical astrocytes<br />
were exposed to hypoxia (7.5% O2) <strong>for</strong> 1 or 5 hr, or to aglycemia, AVP (100 nM) or OGD (a<br />
combination of 2% O2 <strong>an</strong>d aglycemia) <strong>for</strong> 30 min in <strong>an</strong> O2-controlled glove box <strong>an</strong>d cell volume<br />
was assessed using 3 H-3-0-methyl-D-glucose as a marker of water space. We found that hypoxia,<br />
aglycemia, AVP <strong>an</strong>d OGD all caused signific<strong>an</strong>t increases in intracellular volume (28%, 32%,<br />
15%, <strong>an</strong>d 28%, respectively). Acute exposure to 17β-estradiol (1 or 10nM during <strong>the</strong> cell volume<br />
assay) attenuated hypoxia-induced swelling in a time- <strong>an</strong>d dose-dependent m<strong>an</strong>ner. Estradiol (1-<br />
100 nM) also abolished AVP-induced but not aglycemia-induced astrocyte swelling. The<br />
aquaporin 4 (AQP4) water ch<strong>an</strong>nel has been shown to participate in ischemia-induced astrocyte<br />
swelling during edema <strong>for</strong>mation. Thus, we also evaluated <strong>the</strong> effect of exposing astroctyes to<br />
estradiol (1-100 nM, 7 d) on AQP4 abund<strong>an</strong>ce <strong>an</strong>d found that 10 <strong>an</strong>d 100 nM estradiol caused <strong>an</strong>
~30% reduction in AQP4 abund<strong>an</strong>ce. These findings suggest that hypoxia, aglycemia <strong>an</strong>d AVP<br />
all contribute to ischemia-induced astrocyte swelling <strong>an</strong>d fur<strong>the</strong>r, that <strong>the</strong> edema-reducing effects<br />
of estradiol include reduction of cortical astrocyte AQP4 abund<strong>an</strong>ce.<br />
Disclosures: J. Rutkowsky, None; M. O'Donnell, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.16/Q11<br />
Topic: C.08.g. Neuroprotection<br />
Support: Swedish Research Council<br />
The Nordic Centre of Excellence Program in Molecular Medicine<br />
The Persson Family Foundation<br />
HKH Kronprinsess<strong>an</strong> Lovisas förening för barnsjukvård<br />
Stiftelsen Axel Tielm<strong>an</strong>s Minnesfond<br />
Title: Erythropoietin modulates astrocyte metabotropic glutamate receptor signaling<br />
Authors: Y. SONG 1 , E. GUNNARSON 1 , A. APERIA 1 , *M. ZELENINA 1,2 ;<br />
1 2<br />
Karolinska Institutet, Stockholm, Sweden; Dept. of Applied Physics, Royal Inst. of Technol.,<br />
Stockholm, Sweden<br />
Abstract: Erythropoietin (EPO) is a promising neuroprotective agent in stroke, trauma <strong>an</strong>d<br />
hypoxia, conditions characterized by a sustained increase in extracellular glutamate level in <strong>the</strong><br />
brain. Our recent data suggest that EPO might exert its neuroprotective effect partly by<br />
modulating astrocyte function. We have shown that glutamate <strong>an</strong>d metabotropic glutamate<br />
receptor (mGluR) group I agonist DHPG trigger intracellular Ca 2+ oscillations in primary<br />
astrocytes. EPO interferes with this signaling by altering <strong>the</strong> frequency of <strong>the</strong> oscillations. We<br />
have shown that one of <strong>the</strong> immediate downstream effects of mGluR activation is <strong>an</strong> increase of<br />
astrocyte water permeability <strong>an</strong>d that pretreatment with EPO is able to counteract this effect both<br />
in primary astrocytes <strong>an</strong>d in acute hippocampal slices. Here we report that, along with <strong>the</strong> shortterm<br />
effects, EPO might also modulate long-term effects of astrocyte mGluR activation. We
show that DHPG activates extracellular signal-regulated kinase (ERK) phosphorylation in<br />
primary astrocytes. Pretreatment with EPO signific<strong>an</strong>tly attenuates <strong>the</strong> DHPG effect.<br />
Interestingly, EPO alone also activates ERK1/2 phosphorylation in <strong>the</strong> astrocytes. Activation of<br />
ERK is known to have multiple long-term effects mediated by phosphorylation of nuclear<br />
tr<strong>an</strong>scription factors. By interfering with MEK/ERK pathway, EPO might modulate astrocyte<br />
gene expression patterns evoked by excessive glutamate exposure <strong>an</strong>d <strong>the</strong>reby counteract longterm<br />
effects of glutamate on astrocyte function. Our results indicate that astrocytes might be a<br />
component of <strong>the</strong> long-term neuroprotective effect of EPO.<br />
Disclosures: Y. Song, None; E. Gunnarson, None; A. Aperia, None; M. Zelenina, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.17/Q12<br />
Topic: C.08.b. Molecular mech<strong>an</strong>isms<br />
Support: Heart <strong>an</strong>d Stroke Foundation<br />
Title: Regulation of MMP9 expression <strong>an</strong>d activity in astrocytes<br />
Authors: *E. M. MALCOMSON 1 , J. LEI 2 , H. XIONG 2 , D. CALLAGHAN 2 , Q. LIU 2 , W.<br />
ZHANG 3,2 ;<br />
1 Neurosci, Univ. Ottawa, Natl. Res. Council, Ottowa, ON, C<strong>an</strong>ada; 2 Neurobio. Program, Inst. <strong>for</strong><br />
Biol. Sciences, Natl. Res. Council of C<strong>an</strong>ada, Ottawa, ON, C<strong>an</strong>ada; 3 NSC Program, Dept. of<br />
Cell. & Mol. Medicine, Univ. of Ottawa, Ottawa, ON, C<strong>an</strong>ada<br />
Abstract: Ischemic stroke is characterized by thrombotic or embolic disruption of blood supply<br />
to a part of <strong>the</strong> brain tissue, which leads to a focal ischemic infarct. During ischemic stroke, high<br />
levels of MMP9 are produced by neurons, glial <strong>an</strong>d endo<strong>the</strong>lial cells. Increased MMP9 activity<br />
degrades neurovascular extracellular matrix, vascular lamina membr<strong>an</strong>e, <strong>an</strong>d tight junction of <strong>the</strong><br />
BBB <strong>an</strong>d is responsible <strong>for</strong> damages to <strong>the</strong> blood-brain barrier (BBB). The loss of BBB integrity<br />
consequently leads to brain edema, infiltration of inflammatory cells <strong>an</strong>d secondary brain<br />
damage. The role of MMP9 in BBB disruption has been well elucidated; however, little is known<br />
about <strong>the</strong> molecular mech<strong>an</strong>isms that regulate MMP9 expression in brain cells/tissues during<br />
ischemic stroke. Since MAPK-AP1 signaling pathway is activated in ischemic brain tissue, we<br />
believe that MAPK-AP1 signaling pathway is involved in <strong>the</strong> regulation of MMP9 expression in<br />
brain cells during ischemic stroke. It is known that <strong>the</strong> only effective <strong>the</strong>rapy <strong>for</strong> ischemic stroke
is <strong>the</strong> administration of <strong>the</strong> tissue plasminogen activator (tPA). However, tPA stimulates <strong>the</strong><br />
expression <strong>an</strong>d activity of MMP9 which fur<strong>the</strong>r compromises <strong>the</strong> BBB <strong>an</strong>d results in vascular<br />
rupture <strong>an</strong>d hemorrhagic tr<strong>an</strong>s<strong>for</strong>mations. Inhibition of MMP9 activity during early phase of<br />
ischemic stroke may reduce BBB permeability <strong>an</strong>d inhibit tPA-mediated adverse effects. In this<br />
study, we investigate <strong>the</strong> role of MAPK signaling in <strong>the</strong> regulation of MMP9 expression in<br />
cultured astrocytes <strong>an</strong>d <strong>the</strong> inhibition of MMP9 activity by a fragment <strong>from</strong> <strong>the</strong> tissue inhibitor<br />
of metalloproteinase-1 (TIMP-1). Activation of MAPK signaling by PMA strongly up-regulated<br />
MMP9 expression in astrocytes. In vitro assay shows that MMP9 activity was increased in<br />
culture media <strong>from</strong> PMA-treated cells compared to controls. Analysis of hum<strong>an</strong> MMP9 gene<br />
allowed us to identify two AP-1 binding sites in <strong>the</strong> promoter region of <strong>the</strong> gene. The AP-1<br />
binding sites were cloned into a reporter gene vector. The construct <strong>an</strong>d <strong>an</strong> empty vector were<br />
tr<strong>an</strong>siently tr<strong>an</strong>sfected into HEK293 cells, respectively. Luciferease reporter gene assay shows<br />
that treatment of <strong>the</strong> cells with PMA strongly activated <strong>the</strong> promoter of MMP9 gene. To inhibit<br />
MMP9 activity, hum<strong>an</strong> TIMP-1 gene was cloned <strong>an</strong>d overexpressed in HEK293-6EWCB cells<br />
<strong>an</strong>d a 15kDa recombin<strong>an</strong>t fragment of TIMP-1 protein was purified <strong>from</strong> <strong>the</strong> tr<strong>an</strong>sfected cells.<br />
MMP9 activity assay shows that <strong>the</strong> fragment was capable of inhibiting MMP9 activity. The<br />
results suggest that MAPK-AP1 signaling pathway is involved in <strong>the</strong> regulation of MMP9<br />
expression in astrocytes <strong>an</strong>d <strong>the</strong> TIMP-1 fragment may have <strong>the</strong>rapeutic applications in<br />
inhibiting MMP9 activity.<br />
Disclosures: E.M. Malcomson, None; J. Lei, None; H. Xiong, None; D. Callagh<strong>an</strong>, None; Q.<br />
Liu, None; W. Zh<strong>an</strong>g, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.18/Q13<br />
Topic: B.11.b. Cell biology <strong>an</strong>d signaling<br />
Support: Center <strong>for</strong> Adv<strong>an</strong>ced Medical Education (BK21 project)<br />
Title: Ethyl pyruvate confers protection against oxidative stress-induced astrocyte cell death via<br />
hemeoxygenase 1 induction<br />
Authors: *J.-H. SHIN 1 , S.-W. KIM 2 , Y. JIN 2 , J.-Y. JUNG 2 , C.-K. SUH 3 , J.-K. LEE 2 ;<br />
2 Anat., 3 Physiol. <strong>an</strong>d Biophysics, 1 Inha medical school, Inchon, Republic of Korea
Abstract: Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to act as<br />
<strong>an</strong> <strong>an</strong>ti-inflammatory molecule in various pathological conditions, which include sepsis or<br />
hemorrhagic shock. We have shown that ethyl pyruvate has a neuroprotective effect in <strong>the</strong><br />
postischemic brain <strong>an</strong>d also in KA-induced pathogenesis in <strong>the</strong> brain. In <strong>the</strong> present study, we<br />
examined protective effect of ethyl pyruvate in astrocytes under oxidative stress. Ethyl pyruvate<br />
treatment reduced hydrogen peroxide-induced C6 astroglioma cell death in a dose-dependent<br />
m<strong>an</strong>ner, wherein 5 mM ethyl pyruvate suppressed it by 60%. Protective effect of ethyl pyruvate<br />
was evident when it was pre-treated, <strong>the</strong> longer <strong>the</strong> pretreating time <strong>the</strong> higher <strong>the</strong> protection<br />
level. To investigate <strong>the</strong> molecular mech<strong>an</strong>ism underlying <strong>the</strong> protective effects of ethyl<br />
pyruvate, we examined <strong>the</strong> level of hemeoxygenase 1 (HO-1), <strong>an</strong> <strong>an</strong>tioxid<strong>an</strong>t enzyme which<br />
catalyzes <strong>the</strong> degradation of heme, in H2O2-treated C6 astroglioma cell. Hemeoxygenase 1<br />
expression was notably increased after hydrogen peroxide treatment, which was fur<strong>the</strong>r increased<br />
by ethyl pyruvate treatment. Moreover, hemeoxygenase 1 expression was continuously <strong>an</strong>d<br />
signific<strong>an</strong>tly increased after ethyl pyruvate treatment in normal C6 astroglioma cell. The level of<br />
cell death in ethyl pyruvate-pretreated C6 astroglioma cell was notably low compared to that of<br />
untreated control. Fur<strong>the</strong>rmore, <strong>the</strong> repression of hemeoxygenase 1 expression by<br />
hemeoxygenase 1 siRNA tr<strong>an</strong>sfection suppressed <strong>the</strong> protective effect of ethyl pyruvate<br />
pretreatment, indicating that hemeoxygenase 1 is responsible <strong>for</strong> <strong>the</strong> protection against H2O2induced<br />
cell death. Fur<strong>the</strong>r investigation regarding molecular mech<strong>an</strong>isms underlying <strong>the</strong> ethyl<br />
pyruvate-mediated hemeoxygenase 1 induction in C6 astroglioma cells is underway.<br />
Disclosures: J. Shin, None; S. Kim, None; Y. Jin, None; J. Jung, None; C. Suh, None; J. Lee,<br />
None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.19/Q14<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Support: NINDS RO1 NS056839<br />
Title: Reorg<strong>an</strong>ization of perisynaptic astrocytes in layer V of perilesion motor cortex after<br />
ischemic lesions <strong>an</strong>d motor rehabilitation<br />
Authors: *S. KIM 1 , J. E. HSU 3 , T. A. JONES 1,2 ;<br />
1 Inst. <strong>for</strong> Neurosci., 2 Dept. of Psycholgy, Univ. Texas Austin, Austin, TX; 3 Dept. of Gen.<br />
Surgery, Univ. of Texas Med. Sch., Houston, TX
Abstract: Motor rehabilitation after ischemic sensorimotor cortex lesions enh<strong>an</strong>ces functional<br />
recovery <strong>an</strong>d induces synaptic restructuring in layer V of perilesion cortex of adult rats (Hsu et<br />
al., 2007, <strong>Society</strong> <strong>for</strong> Neuroscience Abstract #899.15). This includes increases in <strong>the</strong> densities of<br />
axodendritic synapses, including per<strong>for</strong>ated synapses, presumed to be a particularly efficacious<br />
synapse subtype. The objective of this study was to determine how this synaptic structural<br />
reorg<strong>an</strong>ization is coordinated with that of perisynaptic astrocytes. Using <strong>the</strong> same electron<br />
micrographs as used in Hsu et al. (2007) <strong>an</strong>d stereological methods, we estimated <strong>the</strong> volume<br />
fraction <strong>an</strong>d surface density of astrocytic processes as well as <strong>the</strong> fraction of per<strong>for</strong>ated <strong>an</strong>d nonper<strong>for</strong>ated<br />
synapses with direct astrocytic apposition. Despite overall increases in synaptic<br />
density in <strong>the</strong> perilesion cortex, <strong>the</strong> surface area of astrocytic coverage per synapse was very<br />
similar to that found in intact (sham-operated) <strong>an</strong>imals, indicating that <strong>the</strong> perisynaptic astrocytes<br />
must adapt to maintain normal coverage of synapses in <strong>the</strong> presence of synapse addition.<br />
Reaching per<strong>for</strong>m<strong>an</strong>ce was signific<strong>an</strong>tly correlated with <strong>the</strong> fraction of synapses with direct<br />
astrocytic apposition (r=0.474, p
Title: The relative roles of neuronal HIF-1α <strong>an</strong>d HIF-2α in hypoxic-preconditioning<br />
Authors: P. G. W. KEDDY, *G. S. ROBERTSON;<br />
Dalhousie Univ., Halifax, NS, C<strong>an</strong>ada<br />
Abstract: Hypoxia inducible factor (HIF) comprises a family of tr<strong>an</strong>scriptional regulating<br />
factors that mediate adaptive cellular responses to hypoxia resulting <strong>from</strong> insufficient oxygen.<br />
We are exploring <strong>the</strong> cell type-specific roles of individual subunits of this tr<strong>an</strong>scriptional<br />
complex known as HIF-1α <strong>an</strong>d HIF-2α in neurons <strong>an</strong>d astrocytes. Primary cerebellar gr<strong>an</strong>ule<br />
neuron (CGN) cultures lacking HIF-1α or HIF-2α were used to assess <strong>the</strong> relative roles of <strong>the</strong>se<br />
factors in hypoxia-induced cell death <strong>an</strong>d <strong>the</strong> neuroprotective effects of exposure to a sub-lethal<br />
(short) period of hypoxia, a phenomenon known as hypoxic-preconditioning. Hypoxia was<br />
induced by exposure to <strong>an</strong> atmosphere containing 0.5% oxygen. In <strong>the</strong> case of wild-type (WT)<br />
CGNs, loss of cell viability occurred after 12-24h, but not after 6h of hypoxia. Interestingly, WT<br />
<strong>an</strong>d HIF-1α null neurons displayed <strong>an</strong> equal loss of cell viability (40%) after 12h of hypoxia,<br />
despite <strong>the</strong> fact that levels of <strong>the</strong> potent neuroprotective protein known as vascular endo<strong>the</strong>lial<br />
growth factor were reduced in <strong>the</strong> absence of HIF-1α. This finding suggests that compensatory<br />
mech<strong>an</strong>isms developed in <strong>the</strong> absence of HIF-1α. Similarly, WT <strong>an</strong>d HIF-2α null CGNs showed<br />
<strong>an</strong> equivalent loss of viability after 12h of hypoxia. To produce hypoxic-preconditioning CGNs<br />
were exposed to sub-lethal hypoxia (3h). The next day (24h), CGNs were exposed to lethal<br />
hypoxia (12h). Unexpectedly, WT CGNs failed to display hypoxic-preconditioning, whereas<br />
CGNs lacking ei<strong>the</strong>r HIF-1α or HIF-2α were protected. We hypo<strong>the</strong>size that HIF-2α activation<br />
in astrocytes compensates <strong>for</strong> <strong>the</strong> loss of neuronal HIF-1α or HIF-2α by facilitating <strong>the</strong><br />
expression of <strong>an</strong>o<strong>the</strong>r neuroprotective factor, erythropoietin.<br />
Disclosures: P.G.W. Keddy, None; G.S. Robertson, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.21/Q16<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Support: NS038475<br />
1DP10D003347<br />
The Alberta Heritage Foundation <strong>for</strong> Medical Research
Title: Temporal <strong>an</strong>d spatial patterns of oligodendrocyte degeneration following hypoxiaischemia<br />
in neonatal PLP-EGFP tr<strong>an</strong>sgenic mice<br />
Authors: *L. L. JANTZIE, J. J. VOLPE, F. E. JENSEN;<br />
Neurol., Children's Hosp. Boston, Boston, MA<br />
Abstract: Neonatal hypoxia-ischemia (HI) <strong>an</strong>d/or sepsis are a major cause of injury to <strong>the</strong><br />
developing white matter (WM) in preterm inf<strong>an</strong>ts, with coincident injury to cortical <strong>an</strong>d<br />
subcortical gray matter also commonly observed (Volpe, <strong>2009</strong> L<strong>an</strong>cet). While injury to WM<br />
often m<strong>an</strong>ifests as periventricular leukomalacia (PVL) <strong>an</strong>d may lead to motor disabilities such as<br />
cerebral palsy, HI cell death in gray matter is associated with cognitive, intellectual <strong>an</strong>d<br />
neurodevelopmental disabilities. The age window of greatest susceptibility to PVL is between<br />
24-32 weeks gestation, when <strong>the</strong> WM is predomin<strong>an</strong>tly populated by oligodendrocyte (OL)<br />
precursors <strong>an</strong>d immature OLs. In order to precisely follow <strong>the</strong> response of <strong>the</strong> OL cell population<br />
to HI in neonatal <strong>an</strong>imals, we used tr<strong>an</strong>sgenic mice overexpressing enh<strong>an</strong>ced green fluorescent<br />
protein (EGFP) in OLs. In <strong>the</strong>se mice, proteolipid protein (PLP) promoter regulatory elements<br />
<strong>an</strong>d PLP 3’ untr<strong>an</strong>slated regions are ligated to EGFP (Mallon et al, 2002 J Neurosci). Similar to<br />
our rat model of premature WM injury (M<strong>an</strong>ning et al, 2008 J Neurosci), post-natal day 6 PLP-<br />
EGFP mice were subjected to perm<strong>an</strong>ent unilateral common carotid ligation followed by<br />
systemic hypoxia (7% O2 <strong>for</strong> 1h), <strong>an</strong>d euth<strong>an</strong>ized 6h 24h, 72h <strong>an</strong>d 7d following HI (n=8). Brains<br />
were assessed <strong>for</strong> EGFP in WM <strong>an</strong>d cortex, oligodendrocyte developmental stage (O4, O1),<br />
maturation of myelination (MBP), apoptotic cell death (TUNEL, cleaved caspase-3) <strong>an</strong>d<br />
neuronal survival. Activated microglia <strong>an</strong>d astrocytes were also examined. Semi-qu<strong>an</strong>titative<br />
histopathologic <strong>an</strong>alyses reveal EGFP-positive OLs are signific<strong>an</strong>tly depleted in <strong>the</strong> corpus<br />
callosum <strong>an</strong>d overlying cortex ipsilateral to carotid ligation 24h, 72h <strong>an</strong>d 7d post-HI (p
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.22/Q17<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t R01NS061983<br />
NIH Gr<strong>an</strong>t R01ES015988<br />
NMSS<br />
Shriners Gr<strong>an</strong>ts<br />
Title: Axon-glia synapses are signific<strong>an</strong>t targets in injury to immature white matter<br />
Authors: *Y. SHEN, X.-B. LIU, W. DENG;<br />
Univ. Cali<strong>for</strong>nia, Davis, Sacramento, CA<br />
Abstract: Recent studies have surprisingly shown that oligodendrocyte precursor cells (OPCs)<br />
<strong>for</strong>m synaptic contacts with axons in <strong>the</strong> corpus callosum in neonatal rodents. At <strong>the</strong> axon-glia<br />
synapses, callosal axons derived <strong>from</strong> cortical pyramidal cells release excitatory neurotr<strong>an</strong>smitter<br />
glutamate that activates AMPA subtype glutamate receptors on OPCs. The function of <strong>the</strong>se<br />
newly discovered synapses is largely unknown. Here, we determined whe<strong>the</strong>r <strong>the</strong> axo-OPC<br />
synapses are sensitive targets in white matter injury using a mouse model of hypoxic/ischemic<br />
neonatal brain damage. Axo-OPC synapses were localized by immune-electronic microscopy in<br />
external capsule of <strong>the</strong> neonatal mouse. VgluT1 <strong>an</strong>d VgluT2 were used as markers <strong>for</strong><br />
glutamatergic synapses. Decreased number of synapses <strong>an</strong>d down-regulated expression of<br />
Vglut1 <strong>an</strong>d Vglut2 were found in ipsilateral white matter in <strong>an</strong>imals subjected to<br />
hypoxia/ischemia compared to contralteral white matter in <strong>the</strong> same <strong>an</strong>imals. Few VGluT2<br />
immunopostive terminals were found in <strong>the</strong> injured side, <strong>an</strong>d <strong>the</strong>y showed only few vesicles in<br />
<strong>the</strong> terminals <strong>an</strong>d less prominent postsynaptic densities compared to those in <strong>the</strong> contralateral<br />
side. In addition, much less myelinated axons were found in <strong>the</strong> injured side compared <strong>the</strong><br />
contralateral side. These preliminary results show that hypoxia/ischemia cause damage to axo-<br />
OPC synapses <strong>an</strong>d suggest that <strong>the</strong> reducing number of synaptic contacts between axons <strong>an</strong>d<br />
OPCs may be involved in <strong>the</strong> pathogenesis of white matter injury in neonates.<br />
Disclosures: Y. Shen, None; X. Liu, None; W. Deng, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.23/Q18<br />
Topic: C.08.a. Cellular mech<strong>an</strong>isms<br />
Support: Wellcome Trust<br />
EU<br />
Title: Testing <strong>the</strong> mech<strong>an</strong>isms of glutamate release <strong>an</strong>d inward current activation in<br />
oligodendrocytes during white matter ischaemia<br />
Authors: K. KOLODZIEJCZYK, *D. I. ATTWELL;<br />
Univ. Col. London, London WC1E 6BT, United Kingdom<br />
Abstract: Ischaemia causes irreversible brain <strong>an</strong>d spinal cord damage during stroke,<br />
periventricular leukomalacia (leading to cerebral palsy) <strong>an</strong>d spinal cord injury. In <strong>the</strong> grey<br />
matter, ischaemia results in abnormal ion gradients, pH ch<strong>an</strong>ges <strong>an</strong>d a massive release of<br />
glutamate into <strong>the</strong> extracellular space. However, <strong>the</strong>re is controversy over <strong>the</strong> mech<strong>an</strong>isms by<br />
which glutamate is released <strong>an</strong>d damage occurs in white matter ischaemia.<br />
Glutamate release in white matter ischaemia could occur by reversal of glutamate uptake (1), by<br />
NKCC1 co-tr<strong>an</strong>sporters causing astrocytes to die <strong>an</strong>d release glutamate (2), by cystine-glutamate<br />
<strong>an</strong>tiporters (3) or by exocytosis <strong>from</strong> unmyelinated axons (4,5). In addition, factors o<strong>the</strong>r th<strong>an</strong><br />
glutamate may contribute to <strong>the</strong> effects of ischaemia on <strong>the</strong> white matter, e.g. ATP acting<br />
through P2X7 receptors (6), a decrease of extracellular pH acting through ASICs (7), or GABA<br />
acting through GABAA receptors (8).<br />
We whole-cell patch-clamped white matter oligodendrocytes with myelinating morphology in rat<br />
P12 cerebellar slices. In ischaemic solution, glucose was replaced with sucrose, <strong>an</strong>d oxygen with<br />
nitrogen, <strong>an</strong>d iodoacetate <strong>an</strong>d <strong>an</strong>timycin B were added (to block ATP production by glycolysis<br />
<strong>an</strong>d oxidative phosphorylation).<br />
Ischaemia evoked a peak inward current of 229±24 pA after 7.3±0.6 min. The effect of drugs<br />
blocking various mech<strong>an</strong>isms was tested by applying <strong>the</strong> drugs <strong>from</strong> 2 mins be<strong>for</strong>e ischaemia.<br />
The ischaemia-evoked inward current was largely blocked by D-AP5 (50µM) <strong>an</strong>d NBQX<br />
(25µM), while bicuculline (100µM, a GABAA receptor blocker), BBG (10µM, a P2X7 receptor<br />
blocker) <strong>an</strong>d amiloride (100µM, <strong>an</strong> ASIC blocker) had no effect.<br />
A contribution to glutamate release <strong>an</strong>d/or inward current generation <strong>from</strong> NKCC1 cotr<strong>an</strong>sporters,<br />
cystine-glutamate <strong>an</strong>tiporters or exocytosis <strong>from</strong> unmyelinated axons was excluded<br />
because <strong>the</strong> appropriate blockers (50µM bumet<strong>an</strong>ide, 50µM CPG, <strong>an</strong>d 100µM Cd2+,<br />
respectively) had no effect on <strong>the</strong> ischaemia-evoked current. However, <strong>the</strong> current was<br />
successfully inhibited by pre-incubation <strong>for</strong> 1h with PDC (1mM), a blocker of reversed<br />
glutamate uptake1.<br />
These results show that glutamate is <strong>the</strong> main cause of <strong>the</strong> inward current occuring in<br />
oligodendrocytes during white matter ischaemia, <strong>an</strong>d that <strong>the</strong> major source of this<br />
neurotr<strong>an</strong>smitter is a reversal of glutamate tr<strong>an</strong>sporters.<br />
1 Li et al., 1999, J Neurosci, 19, RC16
2 Wilke et al., 2004, J Neuropathol Exp Neurol, 63, 872<br />
3 Domercq et al., 2007, J Immunol, 178, 6549<br />
4 Kukley et al., 2007, Nat Neurosci, 10, 311<br />
5 Ziskin et al., 2007, Nat Neurosci, 10, 321<br />
6 W<strong>an</strong>g et al., 2004, Nat Med, 10, 821<br />
7 Allen <strong>an</strong>d Attwell, 2002, J Physiol, 543, 521<br />
8 Allen et al., 2004, J Neurosci, 24, 3837<br />
Disclosures: K. Kolodziejczyk, None; D.I. Attwell, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.24/R1<br />
Topic: C.08.g. Neuroprotection<br />
Support: Intramural Research Program, NIMH, NIH<br />
Title: HDAC inhibitors protect oligodendrocytes <strong>an</strong>d stimulate oligodendrogenesis in a rat<br />
model of cerebral ischemia<br />
Authors: *H. KIM 1 , D.-M. CHUANG 2 ;<br />
1 2<br />
Dept. Neurol., Childrens Hosp. Harvard Med. Univ., Boston, MA; Natl. Inst. of Mental Health,<br />
Natl. Inst. of Hlth., Be<strong>the</strong>sda, MD<br />
Abstract: Oligodendrocyte injury occurs rapidly in response to cerebral ischemia, in part<br />
through microglia activation <strong>an</strong>d glutamate-induced excitotoxicity in <strong>the</strong> white matter.<br />
Previously, we demonstrated that post-insult treatment with inhibitors of histone deacetylases<br />
(HDACs) decreased brain infarct volume <strong>an</strong>d suppressed neuroinflammation in a rat middle<br />
cerebral artery occlusion (MCAO) model of stroke. Fur<strong>the</strong>r, HDAC inhibitors enh<strong>an</strong>ced MCAOinduced<br />
cell proliferation in multiple ischemic brain regions including <strong>the</strong> subventricular zone<br />
(SVZ), <strong>an</strong>d some of <strong>the</strong>se newborn cells exhibited phenotypes of neuronal progenitors <strong>an</strong>d<br />
astrocytes. Since <strong>the</strong> SVZ consists of multi-potent progenitors/stem cells <strong>an</strong>d is <strong>an</strong> import<strong>an</strong>t<br />
source of myelinating oligodendrocytes, we tested whe<strong>the</strong>r treatment with HDAC inhibitors<br />
would protect against ischemia-induced damage of oligodendrocytes <strong>an</strong>d promote<br />
oligodendrogenesis <strong>from</strong> newborn cells in various brain regions of <strong>the</strong> MCAO rat. Male rats<br />
were subjected to perm<strong>an</strong>ent MCAO <strong>an</strong>d immediately treated with <strong>the</strong> HDAC inhibitor, sodium<br />
butyrate (SB) (300 mg/kg, s.c.) or trichostatin A (TSA, 0.2 mg/kg, s.c.), followed by daily
injections. BrdU (50 mg/kg, i.p.) was administered daily <strong>from</strong> day 3 to 7 <strong>an</strong>d <strong>an</strong>imals were <strong>the</strong>n<br />
sacrificed. We found that MCAO resulted in a marked loss of myelin basic protein (MBP)(+)<br />
cells, determined by confocal microscopy, in <strong>the</strong> ipsilateral SVZ, <strong>an</strong>terior SVZ, striatum, corpus<br />
callosum <strong>an</strong>d frontal cortex 7 days after ischemia. Treatment with SB blocked <strong>the</strong> MCAOinduced<br />
decrement of MBP(+) cells. Fur<strong>the</strong>r, SB treatment enh<strong>an</strong>ced ischemia-induced BrdU<br />
labeling in various regions <strong>an</strong>d some of BrdU(+) <strong>an</strong>d MBP(+) cells were colocalized, notably in<br />
<strong>the</strong> SVZ, corpus callosum <strong>an</strong>d striatum. Similar effects were observed in various ipsilateral brain<br />
regions of MCAO rats treated with TSA. SB-induced increase in BrdU/MBP labeling in <strong>the</strong><br />
ischemic brain regions was blocked by pretreatment with K252a, <strong>an</strong> <strong>an</strong>tagonist of BDNF-TrkB<br />
signaling. Vascular endo<strong>the</strong>lial growth factor (VEGF), which is known to stimulate <strong>an</strong>giogenesis<br />
<strong>an</strong>d neurogenesis, was also upregulated by SB in <strong>the</strong> ischemic SVZ, <strong>an</strong>terior SVZ <strong>an</strong>d striatum in<br />
a BDNF-TrkB-dependent m<strong>an</strong>ner. Finally, SB treatment blocked ischemia-induced microglia<br />
activation (detected by Ox42-staining), macrophage/monocyte infiltration (detected by ED1staining)<br />
<strong>an</strong>d caspase-3 activation in <strong>the</strong> ischemic brain. Taken toge<strong>the</strong>r, our results demonstrate<br />
that HDAC inhibitors protect oligodendrocytes <strong>an</strong>d stimulate oligodendrogenesis by multiple<br />
mech<strong>an</strong>isms after cerebral ischemia.<br />
Disclosures: H. Kim, None; D. Chu<strong>an</strong>g, None.<br />
Poster<br />
540. Ischemia: Glia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 540.25/R2<br />
Topic: B.11.c. In vivo approaches<br />
Support: NIH gr<strong>an</strong>t HL 079555<br />
NIH gr<strong>an</strong>t HL080492<br />
Title: Oligodendrocyte unfolded protein response injury <strong>an</strong>d myelin disruption in murine model<br />
of sleep apnea<br />
Authors: *S. C. VEASEY, Y. ZHU, P. FENIK;<br />
Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Obstructive sleep apnea is present in over 2% of <strong>the</strong> population in developed<br />
countries. At <strong>the</strong> same time, this disorder is associated with a vast array of neurobehavioral<br />
abnormalities, including impaired alertness, memory, processing speed <strong>an</strong>d motor skills,
attentional lapses, <strong>an</strong>d depressed mood. Mech<strong>an</strong>isms underlying <strong>the</strong> cognitive impairments are<br />
poorly understood. Using a mouse model of sleep apnea oxygenation patterns, we previously<br />
identified <strong>an</strong> upregulation of <strong>the</strong> unfolded protein response (UPR) in select motoneurons,<br />
evidenced by increased p-PERK, ATF-4, CHOP <strong>an</strong>d GADD35. We hypo<strong>the</strong>sized that<br />
oligodendrocytes would be susceptible to <strong>the</strong> frequent oxygen perturbations, as in sleep apnea.<br />
Young adult male B6 mice exposed to 8 wks of intermittent hypoxia (IH) modeling<br />
oxyhemoglobin desaturation patterns of moderate-severe obstructive sleep apnea evidenced<br />
increased immunoreactivity <strong>for</strong> p-PERK, ATF-4 <strong>an</strong>d CHOP throughout <strong>the</strong> corpus callosum,<br />
<strong>an</strong>terior commissure, optic chiasm, <strong>for</strong>nix, striatum, cerebral peduncle, cerebellar peduncle,<br />
sensory trigeminal root, facial nerve, <strong>an</strong>d spinocerebellar tract. Within white matter tracks,<br />
CHOP (a marker of uncompensated UPR) was evident in <strong>the</strong> nuclei of APC/CC-1 positive cells<br />
in IH mice. To more precisely localize CHOP, immunoelectron microscopy was implemented<br />
labeling CHOP with IgG conjugated to silver-enh<strong>an</strong>ced gold particles. Myelin pallor was<br />
observed <strong>for</strong> all myelinated axons within <strong>the</strong> facial nucleus in IH mice relative to sham-treated<br />
mice. In addition, increased ballooning of <strong>the</strong> mylein was observed in IH mice, 59±.06% of<br />
axons vs. 20±.03% of axons in sham treated mice, p
Authors: S. LIANG 1 , D. LIU 1 , Y. CHEN 1 , M.-L. T. MERCADO 1 , E. I. GRAZIANI 2 , P. H.<br />
REINHART 1 , M. N. PANGALOS 1 , A. WOOD 1 , *M. ZALESKA 1 ;<br />
1 Discovery Neurosci., Wyeth Res., Princeton, NJ; 2 Chem. Sci., Wyeth Res., Pearl River, NY<br />
Abstract: ILS-920 is a novel non-immunosuppressive immunophilin lig<strong>an</strong>d that binds to<br />
FKBP52 <strong>an</strong>d has potent neuroprotective <strong>an</strong>d neuroregenerative properties (Ru<strong>an</strong> et al., PNAS<br />
2008). In <strong>the</strong> present studies, we evaluated <strong>the</strong> effect of ILS-920 <strong>the</strong>rapy on stroke-induced<br />
impairment of a skilled-paw reaching task using a Montoya staircase protocol. Wistar rats were<br />
subjected to a perm<strong>an</strong>ent occlusion of <strong>the</strong> distal middle cerebral artery (pMCAO) by<br />
electrocoagulation through a cr<strong>an</strong>iotomy. ILS-920 treatment was initiated at 6 hours post-onset<br />
of ischemia <strong>an</strong>d continued <strong>for</strong> five days by daily iv bolus injections at a dose of 10 mg/kg.<br />
Beginning on <strong>the</strong> 4 th day post-stroke, rats were tested 3 times/week <strong>for</strong> 3 weeks <strong>an</strong>d <strong>the</strong> number<br />
of pellets retrieved by each <strong>for</strong>epaw counted. Results showed that ILS-920-treated <strong>an</strong>imals<br />
per<strong>for</strong>med signific<strong>an</strong>tly better with <strong>the</strong> contralateral (impaired) paw reaching <strong>an</strong>d grasping ability<br />
recovered almost to <strong>the</strong> level of <strong>the</strong> unimpaired (ipsilateral) paw per<strong>for</strong>m<strong>an</strong>ce at 3 weeks poststroke.<br />
Vehicle-treated <strong>an</strong>imals did not show <strong>an</strong>y recovery of skilled reaching function.<br />
We previously demonstrated that ILS-920 promotes neuroprotection that could contribute to<br />
<strong>the</strong>se beneficial in vivo outcomes after ischemic injury. In current studies we also examined<br />
whe<strong>the</strong>r ILS-920 c<strong>an</strong> protect o<strong>the</strong>r brain cell types <strong>from</strong> ischemic insult. Treatment of purified<br />
oligodendrocytes in culture with ILS-920 signific<strong>an</strong>tly increased cell viability following oxygenglucose<br />
deprivation. In addition, ILS-920 signific<strong>an</strong>tly increased <strong>the</strong> number of myelin-binding<br />
protein-positive oligodendrocytes in a cell culture model of myelination, suggesting ei<strong>the</strong>r<br />
preservation of myelin-competent oligodendrocytes or promotion of <strong>the</strong> maturation of<br />
oligodendrocytes. Finally, ILS-920 treatment after pMCAO signific<strong>an</strong>tly increased <strong>the</strong> number<br />
of GST-pi-positive oligodendrocytes at <strong>the</strong> infarct site up to 40 days following <strong>the</strong> ischemic<br />
insult. Thus, <strong>the</strong> enh<strong>an</strong>ced functional recovery observed following stroke in ILS-920-treated<br />
<strong>an</strong>imals might be due to both neuroprotection <strong>an</strong>d preservation of mature oligodendrocytes.<br />
Disclosures: S. Li<strong>an</strong>g, Wyeth Research, A. Employment (full or part-time); D. Liu, Wyeth<br />
Research, A. Employment (full or part-time); Y. Chen, Wyeth Research, A. Employment (full or<br />
part-time); M.T. Mercado, Wyeth Research, A. Employment (full or part-time); E.I. Grazi<strong>an</strong>i,<br />
Wyeth Research, A. Employment (full or part-time); P.H. Reinhart, Wyeth Research, A.<br />
Employment (full or part-time); M.N. P<strong>an</strong>galos, Wyeth Research, A. Employment (full or parttime);<br />
A. Wood, Wyeth Research, A. Employment (full or part-time); M. Zaleska, Wyeth<br />
Research, A. Employment (full or part-time).<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 541.1/R4<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: James S. McDonnell Foundation<br />
The Swartz Foundation<br />
Title: Fluctuating EEG coherence in chronic brain injury<br />
Authors: *J. D. VICTOR, S. T. WILLIAMS, M. M. CONTE, J. D. DROVER, N. D. SCHIFF;<br />
Dept Neurol & Neurosci, Weill Cornell Med. Col., New York, NY<br />
Abstract: Chronic, severe disturb<strong>an</strong>ces of consciousness <strong>an</strong>d cognitive function consequent to<br />
brain injury is a major public health concern with enormous economic <strong>an</strong>d social consequences.<br />
Affected patients typically have structural damage to cortex, subcortical regions, <strong>an</strong>d <strong>the</strong>ir<br />
interconnections. However, <strong>the</strong> presence of regions of <strong>an</strong>atomically spared cortex with preserved<br />
metabolic activity, <strong>an</strong>d episodic evidence of fragments of behavior appropriate to those regions,<br />
suggests that functional disturb<strong>an</strong>ces in thalamocortical <strong>an</strong>d corticocortical interactions likely<br />
play a key role. Our ability to assess <strong>the</strong>se interactions, especially over extended periods of time,<br />
is severely limited.<br />
Here, we present a new approach to this problem. Our strategy uses <strong>the</strong> st<strong>an</strong>dard, accessible,<br />
EEG, along with a novel approach to its <strong>an</strong>alysis. The approach is motivated by a rigorous<br />
dynamical-systems study of a population model of thalamocortical dynamics. The population<br />
model is <strong>an</strong> extension of <strong>the</strong> model of <strong>the</strong> thalamocortical module developed by Robinson <strong>an</strong>d<br />
coworkers. As Robinson <strong>an</strong>d coworkers showed, this model provides a concise account of <strong>the</strong><br />
normal EEG, but does not display spont<strong>an</strong>eous state tr<strong>an</strong>sitions. <strong>When</strong> two such modules are<br />
coupled toge<strong>the</strong>r by a shared reticular population, we found (Drover et al. CoSyNe, <strong>2009</strong>) that<br />
<strong>the</strong> combined system shows spont<strong>an</strong>eous tr<strong>an</strong>sitions between multiple dynamical modes,<br />
distinguished by <strong>the</strong> coherence of activity between <strong>the</strong> modeled cortical populations. This<br />
observation supports <strong>the</strong> notion that <strong>the</strong> reticular thalamus is key to coordinating <strong>the</strong> activity of<br />
multiple cortical regions, <strong>an</strong>d suggests that <strong>the</strong> existence of multiple modes of coherence<br />
constitutes a fingerprint of normal thalamocortical <strong>an</strong>d corticocortical interactions.<br />
Motivated by <strong>the</strong>se observations, we <strong>an</strong>alyzed EEG/CCTV recordings in a patient with severe<br />
diffuse axonal brain injury, with predomin<strong>an</strong>t injury to <strong>the</strong> frontal lobes compared to posterior<br />
cortical regions as evidenced by metabolic (resting PET) <strong>an</strong>d <strong>an</strong>atomical (MRI, DTI) studies. We<br />
calculated EEG spectra <strong>an</strong>d coherence <strong>from</strong> 30 segments of artifact-free EEG (total 300 sec), <strong>an</strong>d<br />
determined time-localized spectra <strong>an</strong>d coherences. For coherences calculated <strong>from</strong> pairs of<br />
ch<strong>an</strong>nels over <strong>the</strong> frontal lobes bilaterally, we found spont<strong>an</strong>eous alternation between two<br />
modes; <strong>for</strong> coherences calculated <strong>from</strong> pairs of ch<strong>an</strong>nels within one hemisphere, this dynamical<br />
feature was absent. These preliminary findings indicate that time-varying patterns of coherence<br />
c<strong>an</strong> be reliably identified in <strong>the</strong> brain-injured patients, <strong>an</strong>d suggest that <strong>the</strong>ir presence or absence<br />
may correlate with recovery of function.<br />
Disclosures: J.D. Victor, None; S.T. Williams, None; M.M. Conte, None; J.D. Drover,<br />
None; N.D. Schiff, None.
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.2/R5<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Title: Early prefrontal white matter <strong>an</strong>isotropy predicts subsequent executive dysfunction in mild<br />
traumatic brain injury<br />
Authors: *M. L. LIPTON 1 , E. GULKO 1 , A. M. FELDMAN 5 , M. E. ZIMMERMAN 2 , B. W.<br />
FRIEDMAN 6 , M. KIM 3 , K. SHIFTEH 5 , R. B. LIPTON 2 , C. A. BRANCH 4 ;<br />
1 Dept Radiology, 2 Dept. of Neurol., 3 Dept. of Epidemiology <strong>an</strong>d Population Hlth., 4 Dept of<br />
Radiology, Albert Einstein Coll Med., Bronx, NY; 5 Dept Radiology, 6 Dept. of Emergency Med.,<br />
Montefiore Med. Ctr., Bronx, NY<br />
Abstract: Mild traumatic brain injury (mTBI) affects 1.4 million Americ<strong>an</strong>s <strong>an</strong>nually. While<br />
most mTBI patients recover over several months, up to one third have enduring disability,<br />
particularly frontal executive dysfunction. Predicting poor outcome after mTBI might set <strong>the</strong><br />
stage <strong>for</strong> preventive intervention. We have identified white matter abnormalities in dorsolateral<br />
prefrontal cortex (DLPFC) at <strong>the</strong> time of mTBI using diffusion tensor imaging (DTI). This study<br />
investigated <strong>the</strong> relationship between DLPFC fractional <strong>an</strong>isotropy (FA) at baseline as a<br />
predictor of frontal executive dysfunction at 3 months after injury.<br />
Methods: Eligible patients presented to <strong>the</strong> Emergency Department within 24 hours of mTBI<br />
(LOC
per<strong>for</strong>m<strong>an</strong>ce, evolving white matter ch<strong>an</strong>ges may have additional predictive validity. These<br />
findings, <strong>an</strong>d <strong>the</strong> timing of follow-up imaging warr<strong>an</strong>t fur<strong>the</strong>r study.<br />
Disclosures: M.L. Lipton, None; E. Gulko, None; A.M. Feldm<strong>an</strong>, None; M.E. Zimmerm<strong>an</strong>,<br />
None; B.W. Friedm<strong>an</strong>, None; M. Kim, None; K. Shifteh, None; R.B. Lipton, None; C.A.<br />
Br<strong>an</strong>ch, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.3/R6<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH Gr<strong>an</strong>t R01NS021889<br />
NIH Gr<strong>an</strong>t T32HD049350<br />
Title: Neuroimaging correlates of parent reported executive dysfunction following pediatric<br />
traumatic brain injury or orthopedic injury: A preliminary investigation<br />
Authors: *A. T. SCHMIDT, E. A. WILDE, K. D. ORSTEN, X. LI, H. S. LEVIN;<br />
Baylor Col. of Med., Houston, TX<br />
Abstract: Executive dysfunction is common following pediatric traumatic brain injury (TBI).<br />
Studies demonstrate executive deficits on st<strong>an</strong>dard laboratory measures <strong>an</strong>d more “ecologically<br />
valid” parent measures of executive functioning. Additionally, TBI often results in<br />
structural/volumetric ch<strong>an</strong>ges in areas import<strong>an</strong>t <strong>for</strong> executive skills (i.e., frontal brain regions).<br />
The aim of <strong>the</strong> current study was to examine <strong>the</strong> relationship between grey <strong>an</strong>d white matter<br />
volumes in various frontal lobe regions <strong>an</strong>d parent reported scores on <strong>the</strong> Behavior Rating<br />
Inventory of Executive Function (BRIEF) three months following pediatric TBI or orthopedic<br />
injury (OI). Controlling <strong>for</strong> age <strong>an</strong>d total intracr<strong>an</strong>ial volume, preliminary <strong>an</strong>alyses yielded<br />
signific<strong>an</strong>t findings within <strong>the</strong> OI (control) group only. Specifically, we observed modest<br />
positive correlations between left cingulate gray matter volume <strong>an</strong>d scores on <strong>the</strong> Behavior<br />
Regulation Index (BRI) <strong>an</strong>d <strong>the</strong> Inhibition subscale of <strong>the</strong> BRIEF. Left cingulate white matter<br />
volume was also modestly positively correlated with BRI <strong>an</strong>d Emotional Control <strong>an</strong>d Inhibition<br />
subscales whereas left superior frontal gray <strong>an</strong>d white matter volumes were negatively correlated<br />
with <strong>the</strong> Initiate subscale. No signific<strong>an</strong>t relationships were observed in <strong>the</strong> TBI group. The<br />
reason <strong>for</strong> this null finding in children who have sustained a TBI is unknown but may have to do
with <strong>the</strong> lack of sensitivity of parental report to persistent behavioral difficulties at this early<br />
stage post-injury or relatively subtle ch<strong>an</strong>ges in frontal grey/white matter volumes three months<br />
post-injury. Overall, <strong>the</strong>se findings suggest modest relationships between parent reported<br />
executive skills <strong>an</strong>d volumetric measures of frontal brain areas.<br />
Disclosures: A.T. Schmidt, None; E.A. Wilde, None; K.D. Orsten, None; X. Li, None; H.S.<br />
Levin, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.4/R7<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: Medical Research Council UK Clinici<strong>an</strong> Scientist Fellowship<br />
The Hammersmith Hospital Trustees' Research Committee small project gr<strong>an</strong>t (70173)<br />
Title: Altered patterns of functional connectivity following traumatic brain injury identified in<br />
resting state functional MRI<br />
Authors: *D. J. SHARP 1 , C. F. BECKMANN 1 , K. KINNUNEN 2 , V. BONNELLE 1 , R.<br />
GREENWOOD 3 , F. TURKHEIMER 1 , X. DE BOISSEZON 4,3 , E. DYSON 1 , R. LEECH 1,4,2 ;<br />
1 Div. of Neurosci. <strong>an</strong>d Mental Hlth., Imperial Col. London, London, United Kingdom; 2 Dept. of<br />
Psychology, Goldsmiths Col., London, United Kingdom; 3 Inst. of Neurol., Univ. Col. London,<br />
London, United Kingdom; 4 Imagerie Cérébrale et H<strong>an</strong>dicaps Neurologiques, Inserm, Toulouse,<br />
Fr<strong>an</strong>ce<br />
Abstract: Background. Traumatic brain injury (TBI) often results in signific<strong>an</strong>t cognitive<br />
impairment that limits recovery. The key pathological predictors of recovery are uncertain, but<br />
<strong>the</strong> extent of diffuse axonal injury (DAI) is likely to play <strong>an</strong> import<strong>an</strong>t role. Here we hypo<strong>the</strong>size<br />
that cognitive impairment derives in part <strong>from</strong> TBI induced DAI that alters functional<br />
connectivity of distributed brain networks with ensuing disruption of <strong>the</strong> related neuronal<br />
dynamics. Moreover, as patient per<strong>for</strong>m<strong>an</strong>ce often confounds interpretation of task-based fMRI,<br />
we tested <strong>the</strong> hypo<strong>the</strong>sis using resting state functional MRI. Methods. We studied 17 patients<br />
after TBI, a subgroup (n=9) of which had microbleeds visible on T2* MR imaging providing<br />
evidence of DAI. Ten spatially distinct functional brain networks of resting state fMRI were<br />
identified using Independent Component Analysis (ICA). Ch<strong>an</strong>ges in functional connectivity
within <strong>the</strong>se networks between healthy control <strong>an</strong>d patient groups were determined by measuring<br />
<strong>the</strong> extent of regional co-activation across a network using a dual regression approach. Network<br />
dynamics were fur<strong>the</strong>r tested by calculating <strong>the</strong> Hurst exponent of each networks time-series,<br />
which provides a measure of <strong>the</strong> persistence of neuronal firing within a network. Results. In <strong>the</strong><br />
patient group a region within <strong>the</strong> left temporal lobe was less integrated within a larger distributed<br />
fronto-temporal network, a ch<strong>an</strong>ge also observed in <strong>the</strong> subgroup of patients with DAI. In<br />
contrast, investigation of <strong>the</strong> default mode network (DMN) showed that part of <strong>the</strong> medial frontal<br />
lobe including <strong>the</strong> <strong>an</strong>terior cingulate cortex (ACC) showed increased integration with <strong>the</strong> rest of<br />
<strong>the</strong> network, again a ch<strong>an</strong>ge present in <strong>the</strong> DAI subgroup. The patient group showed a reduced<br />
Hurst exponent, which correlated with cognitive function: a reduction in Hurst was associated<br />
with a behavioural measure of declining executive function in <strong>the</strong> patient group. Discussion.<br />
Taken toge<strong>the</strong>r our results demonstrate that patterns of functional connectivity may be altered<br />
following TBI <strong>an</strong>d <strong>the</strong>se ch<strong>an</strong>ges c<strong>an</strong> be identified in resting state fMRI data. Reduced cohesion<br />
within a fronto-temporal network may result <strong>from</strong> disconnection following diffuse axonal injury,<br />
whereas increased medial frontal integration with <strong>the</strong> rest of <strong>the</strong> network likely reflects attempted<br />
compensation <strong>for</strong> disruption elsewhere in <strong>the</strong> brain. Global ch<strong>an</strong>ges in connectivity are <strong>the</strong> likely<br />
cause of <strong>the</strong> observed reduction of persistence of neuronal firing which is associated with lower<br />
executive function in <strong>the</strong> patient group.<br />
Disclosures: D.J. Sharp, None; C.F. Beckm<strong>an</strong>n, None; K. Kinnunen, None; V. Bonnelle,<br />
None; R. Greenwood, None; F. Turkheimer, None; X. De Boissezon, None; E. Dyson,<br />
None; R. Leech, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.5/R8<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: The National Program <strong>for</strong> Integrated Clinical Specialist <strong>an</strong>d PhD-training <strong>for</strong><br />
Psychologists<br />
Title: Assessment of consciousness <strong>an</strong>d awareness in <strong>the</strong> minimally conscious state by using<br />
fMRI<br />
Authors: *P. M. ASLAKSEN 1,2 , C. SCHÄFER 2 , T. VANGBERG 3 ;<br />
1 Univ. Troms, Tromsø, Norway; 2 The Rehabil. Clin., Univ. Hosp. of North Norway, Tromsø,<br />
Norway; 3 Dept. of Clin. Med., Univ. of Tromsø, Tromsø, Norway
Abstract: Behavioral assessment of consciousness <strong>an</strong>d awareness could be difficult in patients<br />
with disorders of consciousness (DOC) due to limited verbal <strong>an</strong>d motor functioning.<br />
Conventional assessment based on overt responses has limited value in patients where goal<br />
directed verbal <strong>an</strong>d motor behavior is absent or ambiguous. The present study tested a fMRI<br />
paradigm that requires active cooperation by mental activity instead of overt behavioral<br />
responses in a female patient that had been evaluated to be in minimal conscious state (MCS)<br />
based on scores on <strong>the</strong> Coma Recovery Scale - Revised (CRS-R), 13 months after she sustained<br />
a traumatic brain injury. Ten age-matched healthy females volunteered as control group <strong>an</strong>d<br />
per<strong>for</strong>med <strong>the</strong> same tasks as <strong>the</strong> patient in <strong>the</strong> sc<strong>an</strong>ner. Written <strong>an</strong>d in<strong>for</strong>med consent was<br />
obtained <strong>from</strong> <strong>the</strong> patient’s parents <strong>an</strong>d <strong>from</strong> all <strong>the</strong> particip<strong>an</strong>ts in <strong>the</strong> control group.<br />
The fMRI paradigm employed in <strong>the</strong> present study relies on specific mental imagery tasks that<br />
were per<strong>for</strong>med after verbal instructions. The verbal instructions were recorded on a cd <strong>an</strong>d<br />
presented through earphones in <strong>the</strong> fMRI sc<strong>an</strong>ner. The tasks that were given to <strong>the</strong> patient <strong>an</strong>d<br />
<strong>the</strong> controls were similar to <strong>the</strong> tasks presented by Owen et al. (2006), <strong>an</strong>d consisted of verbal<br />
instructions to imagine playing a tennis game <strong>an</strong>d spatially navigate through <strong>the</strong> rooms of <strong>the</strong>ir<br />
homes. In addition, cortical speech processing was tested by measuring <strong>the</strong> cortical response to<br />
passive listening. The study was conducted at Tromsø University Hospital, <strong>an</strong>d a Phillips 1.5<br />
Tesla MR-sc<strong>an</strong>ner with fMRI capability was used.<br />
The patient displayed similar cortical activity in Wernickes area as <strong>the</strong> control group (p
Support: The Rockefeller University Center <strong>for</strong> Clinical <strong>an</strong>d Tr<strong>an</strong>slational Science, Medical<br />
Student Year Off Training Program<br />
Charles A. D<strong>an</strong>a Foundation<br />
James S. McDonnell Foundation<br />
Title: Qu<strong>an</strong>titative neurophysiologic characterization of a paradoxical response to zolpidem in a<br />
severely brain-injured hum<strong>an</strong> subject<br />
Authors: *S. T. WILLIAMS 1 , M. M. CONTE 1 , E. J. KOBYLARZ 1 , J. E. HERSH 2 , J. D.<br />
VICTOR 1 , N. D. SCHIFF 1 ;<br />
1 Dept. of Neurol. <strong>an</strong>d Neurosci., 2 Dept. of Publ. Hlth., Weill Cornell Med. Col., New York, NY<br />
Abstract: Each year, over 1.4 million people in <strong>the</strong> United States experience <strong>an</strong> injury to <strong>the</strong><br />
brain which results in temporary or sustained loss of consciousness. The neurophysiologic<br />
processes underlying <strong>the</strong>se disorders of consciousness are poorly understood, <strong>an</strong>d <strong>the</strong>re are no<br />
approved <strong>the</strong>rapeutic measures to ameliorate <strong>the</strong>m. A thorough underst<strong>an</strong>ding of <strong>the</strong> common<br />
circuit mech<strong>an</strong>isms underlying arousal <strong>an</strong>d cognitive dysfunction after severe brain injury is <strong>an</strong><br />
import<strong>an</strong>t step to underst<strong>an</strong>ding recovery <strong>an</strong>d developing targeted <strong>the</strong>rapeutic strategies to<br />
improve outcomes.<br />
Rare cases of unexpected cognitive improvement years after severe brain injury have been<br />
reported, some spont<strong>an</strong>eously <strong>an</strong>d o<strong>the</strong>rs in response to central nervous system medications,<br />
including paradoxically, depress<strong>an</strong>ts such as zolpidem. We present a qu<strong>an</strong>titative EEG <strong>an</strong>alysis<br />
of a 27 year old m<strong>an</strong> who remained in a minimally conscious state (MCS) <strong>for</strong> two years<br />
following a near-drowning hypoxic injury, <strong>an</strong>d whose cognitive function reproducibly improves<br />
following zolpidem administration.<br />
Video EEG was recorded using a modified longitudinal bipolar montage over a five day period.<br />
Zolpidem 10 mg PO was administered at intervals of three to five hours during <strong>the</strong> day. Artifactfree<br />
epochs (two to three seconds in duration) were selected <strong>from</strong> recordings at baseline (one<br />
hour prior to dosing) <strong>an</strong>d at intervals up to four hours following each dose. Power spectra <strong>an</strong>d<br />
coherences were evaluated using multitaper spectral <strong>an</strong>alysis techniques. Metabolic imaging was<br />
also conducted using 18 FDG-PET on <strong>an</strong>d off zolpidem.<br />
We observed EEG power increases in <strong>the</strong> beta (13-30Hz) <strong>an</strong>d gamma (30-50Hz) frequency<br />
r<strong>an</strong>ges <strong>an</strong>d concomit<strong>an</strong>t loss of aberr<strong>an</strong>t 7-8Hz peaks in <strong>the</strong> frontal regions bilaterally one to two<br />
hours after drug administration, compared to <strong>the</strong> pre-drug state. This corresponded to clinical<br />
improvements in behavioral responsiveness, communication <strong>an</strong>d motor control. PET imaging<br />
also revealed <strong>an</strong> increase in frontal, striatal <strong>an</strong>d thalamic metabolism after administration of <strong>the</strong><br />
drug. We interpret <strong>the</strong>se observations as indicating reactivation of <strong>the</strong> frontal systems underlying<br />
<strong>the</strong> paradoxical zolpidem response. Toge<strong>the</strong>r with o<strong>the</strong>r case reports, our observations support<br />
<strong>the</strong> hypo<strong>the</strong>sis that disinhibition of <strong>the</strong> central thalamus plays a key role in reactivation of<br />
cortico-striato-pallido-thalamo-cortical networks that are downregulated in <strong>the</strong> deafferented,<br />
injured brain. A possible mech<strong>an</strong>ism <strong>for</strong> <strong>the</strong> impact of zolpidem on <strong>the</strong>se networks is presented.<br />
Future studies will assess <strong>the</strong> generalizability of <strong>the</strong>se observations to patients with o<strong>the</strong>r types of<br />
brain injuries <strong>an</strong>d across stages of recovery.
Disclosures: S.T. Williams, None; M.M. Conte, None; E.J. Kobylarz, None; J.E. Hersh,<br />
None; J.D. Victor, None; N.D. Schiff, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.7/R10<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Title: Functional MRI <strong>an</strong>d DTI in mild traumatic brain injury<br />
Authors: *P. E. VOS 1 , T. M. J. C. ANDRIESSEN 1 , M. STULEMEIJER 1 , S. VAN DER<br />
WERF 2 , M. RIJPKEMA 3 , M. ZWIERS 3 , G. W. VAN DIJK 4 , D. NORRIS 3 , G. FERNANDEZ 3 ;<br />
1 Neurol., 2 Med. Psychology, Radboud Univ. Nijmegen Med. Ctr., Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 FC<br />
Donders Ctr., Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 Neurol., CWZ, Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: INTRODUCTION: Post concussive symptoms(PCS) <strong>an</strong>d memory deficits are<br />
frequently reported after Mild Traumatic Brain injury (MTBI). The nature of <strong>the</strong>se<br />
deficits is poorly understood as <strong>the</strong> brain usually appears intact using imaging techniques.<br />
Recent studies reveal altered brain activity with functional Magnetic Reson<strong>an</strong>ce Imaging<br />
(fMRI) <strong>an</strong>d white matter lesions using diffusion tensor imaging (DTI). In this study we<br />
investigate <strong>the</strong> relationship between memory impairments <strong>an</strong>d brain activity using fMRI<br />
<strong>an</strong>d post concussive symptoms, mood <strong>an</strong>d white matter integrity using DTI.<br />
METHODS: 48 MTBI patients were studied. Injury severity was graded according to <strong>the</strong><br />
length of post traumatic amnesia(PTA). At 6 weeks post injury fMRI probing prefrontal<br />
<strong>an</strong>d medial temporal functionality using <strong>the</strong> n-back task <strong>an</strong>d DTI(MRI1) <strong>an</strong>d<br />
neuropsychological testing was per<strong>for</strong>med. DTI(MRI2) was repeated after 6 months.<br />
Eighteen healthy controls were sc<strong>an</strong>ned once. Fractional Anisotropy (FA) maps were<br />
<strong>an</strong>alyzed using a whole brain voxel-by-voxel (VbV) <strong>an</strong>d a region of interest approach. PCS<br />
was measured with <strong>the</strong> Rivermead Post Concussion Questionnaire, mood with <strong>the</strong> Beck<br />
Depression Inventory <strong>an</strong>d <strong>an</strong>xiety with <strong>the</strong> Spielberger State-Trait Anxiety Inventory.<br />
RESULTS: Patients showed poorer declarative memory per<strong>for</strong>m<strong>an</strong>ce th<strong>an</strong> controls <strong>an</strong>d<br />
decreasing per<strong>for</strong>m<strong>an</strong>ce with increasing injury severity. Task per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> sc<strong>an</strong>ner<br />
was similar in patients <strong>an</strong>d controls. An inverse relationship between medio temporal<br />
lobe(MTL) activity <strong>an</strong>d PTA duration was observed. No difference in prefrontal<br />
activation(PFC) was found between patients <strong>an</strong>d controls nor a relation with injury<br />
severity.<br />
At MRI1 40% <strong>an</strong>d at MRI2 25% of <strong>the</strong> patients reported severe PCS. Whole brain VbV
<strong>an</strong>d ROI <strong>an</strong>alyses revealed no areas of signific<strong>an</strong>tly ch<strong>an</strong>ged FA when patients were<br />
compared to controls. At MRI1, patients with severe PCS were more depressed th<strong>an</strong> those<br />
without severe PCS (me<strong>an</strong> (SD): PCS+ = 9.8 (6.8), PCS- = 4.5 (3.5), p= .005). Around<br />
MRI2, severe PCS patients reported both higher levels of depression (PCS+ = 9.9 (4.9),<br />
PCS- = 1.8 (1.9) p
Abstract: We conducted a functional MRI study of visually guided saccades (VGS) to<br />
interrogate potential neurobiologic correlates of neurophysiologic dysfunction based on recent<br />
reports of impairments in oculomotor function in traumatic brain injury (TBI). The st<strong>an</strong>dard<br />
VGS test simply requires a subject to disengage <strong>from</strong> a central fixation <strong>an</strong>d complete a reflexive<br />
saccadic movement to a stimulus presented in <strong>the</strong> periphery. Although <strong>the</strong>re are direct<br />
connections between primary visual cortices <strong>an</strong>d <strong>the</strong> superior colliculus, <strong>the</strong>re are also indirect or<br />
cortical pathways which involve regions in <strong>the</strong> parietal lobes, <strong>the</strong> frontal <strong>an</strong>d supplementary eye<br />
fields, cerebellum, thalamus, <strong>an</strong>d brain stem.<br />
To investigate <strong>the</strong> effects of shearing injury on this network, a group of 36 healthy controls, 41<br />
patients with a history of mild TBI <strong>an</strong>d 26 patients with a history of moderate to severe TBI<br />
completed a visually guided saccade task both in <strong>the</strong> laboratory <strong>an</strong>d during fMRI data<br />
acquisition. The VGS task in <strong>the</strong> laboratory required subjects to fixate on a central target be<strong>for</strong>e<br />
looking toward peripheral targets presented unpredictably at 10, 20, or 30 degrees of visual <strong>an</strong>gle<br />
to <strong>the</strong> right or left of central fixation. Peripheral targets were presented r<strong>an</strong>domly ei<strong>the</strong>r 200 ms<br />
after <strong>the</strong> central fixation point was extinguished (gap condition) or 200 ms be<strong>for</strong>e <strong>the</strong> central<br />
fixation point was extinguished (overlap condition). During fMRI, subjects completed 6, 30<br />
second blocks of VGS trials intermixed with blocks of central fixation.<br />
The patients with a history of mild TBI were matched in age, education <strong>an</strong>d premorbid IQ to <strong>the</strong><br />
controls. The patients with a history of moderate to severe TBI were matched in age <strong>an</strong>d<br />
education to controls. Behaviorally, patients with a history of moderate to severe TBI showed<br />
increased latencies <strong>an</strong>d decreased accuracy relative to controls. The mild TBI also showed a<br />
signific<strong>an</strong>t reduction in accuracy relative to controls. Group-wise comparisons on <strong>the</strong> fMRI data<br />
between controls <strong>an</strong>d mild TBI showed increased thalamic activation <strong>for</strong> <strong>the</strong> mild TBI. The<br />
moderate to severe TBI also showed increased thalamic activation relative to controls, as well as<br />
increased prefrontal activation <strong>an</strong>d decreased activation in <strong>the</strong> frontal <strong>an</strong>d supplementary eye<br />
fields.<br />
In subjects with TBI, <strong>the</strong> decreased accuracy <strong>an</strong>d increased activation in prefrontalthalamocortical<br />
circuitry during visually guided saccades may be indicative of a greater need <strong>for</strong><br />
cortical control of responses relative to controls.<br />
Disclosures: D.M. Little, None; M.F. Kraus, None; S. Wojtowicz, None; M. Siroko,<br />
None; A. Alex<strong>an</strong>der, None; J.A. Sweeney, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.9/R12<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies
Support: JIEDDO-001<br />
Mid-Atl<strong>an</strong>tic MIRECC<br />
W.G. Hefner VAMC<br />
Title: Comparison of post-deployment veter<strong>an</strong>s divergent <strong>for</strong> exposure to primary blast <strong>for</strong>ces<br />
Authors: *K. H. TABER 1,2,3 , S. D. HURT 1 , R. A. HURLEY 1,2,4 ;<br />
1 W.G. Hefner VAMC, Salisbury, NC; 2 Mid-Atl<strong>an</strong>tic Mental Illness Research, Educ. <strong>an</strong>d Clin.<br />
Ctr., Durham, NC; 3 Edward Via Virginia Col. of Osteo. Med., Blacksburg, VA; 4 Wake Forest<br />
Univ. Sch. of Med., Winston-Salem, NC<br />
Abstract: Traumatic brain injury (TBI) has been termed a signature injury of <strong>the</strong> current<br />
conflicts. Exposure to <strong>an</strong> explosion (blast) is common during deployment, <strong>an</strong>d a major cause of<br />
injury. There is no question that secondary <strong>an</strong>d tertiary blast-related <strong>for</strong>ces c<strong>an</strong> injure <strong>the</strong> brain.<br />
The potential <strong>for</strong> brain injury due to primary blast <strong>for</strong>ces remains controversial. This study is part<br />
of a multi-institution project investigating <strong>the</strong> effects of primary blast <strong>for</strong>ces. The goal of <strong>the</strong><br />
present IRB-approved study is to identify a cohort of post-deployment veter<strong>an</strong>s who were free of<br />
<strong>an</strong>y conditions prior to deployment (e.g., head injury, concussion, psychiatric disorder) that<br />
could confound identification of TBI acquired during deployment. Exclusion criteria include a<br />
history of exposure to conditions during deployment likely to result in a TBI due to <strong>for</strong>ces o<strong>the</strong>r<br />
th<strong>an</strong> primary blast (e.g., secondary or tertiary blast-related injury, motor vehicle accident)<br />
because this study solely focuses on <strong>the</strong> effects of exposure to primary blast. Approximately 75%<br />
of potential particip<strong>an</strong>ts are excluded. Study groups: 1] no exposure during deployment to <strong>an</strong>y<br />
conditions likely to result in TBI, 2] exposure only to primary blast <strong>for</strong>ces with ei<strong>the</strong>r no<br />
symptoms at <strong>the</strong> time or symptoms that resolved quickly, 3] exposure only to primary blast<br />
<strong>for</strong>ces with symptoms at <strong>the</strong> time <strong>an</strong>d now. Two control groups are included (1 <strong>an</strong>d 2) to allow<br />
<strong>for</strong> <strong>the</strong> difficulties inherent in self-report of concussion experienced under combat conditions <strong>an</strong>d<br />
<strong>the</strong> possibility that exposure to primary blast <strong>for</strong>ces has previously unreported subtle long term<br />
effects. All subjects complete a multidisciplinary clinical evaluation <strong>an</strong>d neuroimaging<br />
(structural <strong>an</strong>d functional) examination.<br />
This initial stage of data <strong>an</strong>alysis focuses on data <strong>from</strong> groups 1 <strong>an</strong>d 2. Three sets of comparisons<br />
were made: 1] to age-matched civili<strong>an</strong> norms (where available), 2] between groups 3] across <strong>the</strong><br />
combined groups split by presence/absence of current PTSD <strong>an</strong>d/or depression.<br />
Results indicate: both groups per<strong>for</strong>m signific<strong>an</strong>tly better (average Z scores >0.5) th<strong>an</strong> agematched<br />
civili<strong>an</strong>s on multiple neurobehavioral measures; although Group 2 has a higher<br />
incidence of psychiatric diagnoses, <strong>the</strong>re were clear per<strong>for</strong>m<strong>an</strong>ce differences between groups 1<br />
<strong>an</strong>d 2 that were not attributable to this factor; <strong>the</strong>re were per<strong>for</strong>m<strong>an</strong>ce differences that tracked<br />
with psychiatric state but not group membership. These results indicate that comparison to<br />
civili<strong>an</strong> norms may be misleading when applied to this cohort, <strong>an</strong>d lead to underestimation of<br />
functional deficits. Presence of psychiatric conditions <strong>an</strong>d exposure to primary blast <strong>for</strong>ces may<br />
be separate risk factors in this cohort.<br />
Disclosures: K.H. Taber, None; S.D. Hurt, None; R.A. Hurley, None.
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.10/R13<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH 1R01 HD42385-01<br />
JSMF 220020082<br />
Title: Higher complexity predicts less BOLD activity in default mode regions after traumatic<br />
brain injury<br />
Authors: *A. C. RAJA, N. KOVACEVIC, D. TISSERAND, W. CHAU, A. R. MCINTOSH, B.<br />
LEVINE;<br />
Rotm<strong>an</strong> Res. Inst., Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Traumatic brain injury (TBI) causes diffuse damage that affects distributed<br />
in<strong>for</strong>mation processing, <strong>an</strong>d, in turn, behavior. Neuroimaging techniques emphasizing signal<br />
ch<strong>an</strong>ges may not fully capture <strong>the</strong> effects of such diffuse damage. Measures of complexity,<br />
which represent in<strong>for</strong>mation processing capacity, may be <strong>an</strong> improved me<strong>an</strong>s of detecting <strong>the</strong><br />
neural markers of TBI. Here we calculate multiscale entropy (MSE) <strong>for</strong> MEG signal during a<br />
visual feature-integration task. MSE is a recent method that qu<strong>an</strong>tifies complexity at varying<br />
time scales. We also measure fMRI BOLD activity during this task <strong>an</strong>d assess whe<strong>the</strong>r <strong>the</strong>re are<br />
regional patterns related to a subject’s neural complexity.<br />
Ten healthy <strong>an</strong>d seven TBI subjects participated in <strong>the</strong> task during MEG <strong>an</strong>d subsequent fMRI<br />
sc<strong>an</strong>ning. The feature-integration task consisted of single-feature, multi-feature, <strong>an</strong>d redund<strong>an</strong>t<br />
conditions. MSE, which determines non-linear temporal dependencies, was measured in <strong>the</strong><br />
signal <strong>from</strong> each MEG source <strong>an</strong>d averaged to represent a subject’s overall complexity. Brain<br />
regions where BOLD signal was reliably related to complexity were investigated using partial<br />
least squares (PLS).<br />
PLS identified one signific<strong>an</strong>t latent variable involving fusi<strong>for</strong>m gyri, <strong>an</strong>terior <strong>an</strong>d posterior<br />
cingulate, cerebellum, postcentral gyrus, inferior parietal lobule, <strong>an</strong>d several frontal clusters<br />
where greater BOLD signal amplitude was reliably related to lower MEG signal complexity<br />
across all three conditions in TBI patients (Fig. 1, error bars = 95% confidence intervals). Several<br />
of <strong>the</strong>se midline <strong>an</strong>d lateral parietal structures have been implicated as part of <strong>the</strong> default mode<br />
network. Separate <strong>an</strong>alyses revealed that <strong>the</strong>se relationships were mainly driven by <strong>the</strong> TBI<br />
group. This suggests that in TBI, enh<strong>an</strong>ced engagement of default mode areas is related to
diminished overall capacity to integrate task-related in<strong>for</strong>mation. The increased default mode<br />
activity may be a maladaptive consequence of <strong>the</strong> neuropathology of TBI or related to less<br />
attention being paid to <strong>the</strong> task.<br />
Disclosures: A.C. Raja, None; N. Kovacevic, None; D. Tisser<strong>an</strong>d, None; W. Chau,<br />
None; A.R. McIntosh, None; B. Levine, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.11/R14<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Title: The shape of <strong>the</strong> hemodynamic response remains intact in severe traumatic brain injury<br />
survivors<br />
Authors: *H. S. PALMER, B. GARZÓN, E. M. BERNTSEN, J. XU, A. HÅBERG;<br />
ISB/ NTNU, Trondheim, Norway<br />
Abstract: The st<strong>an</strong>dard method <strong>for</strong> inferring neuronal activation <strong>from</strong> functional magnetic<br />
reson<strong>an</strong>ce imaging (fMRI) is based on convolving <strong>the</strong> blood oxygen level dependent signal<br />
ch<strong>an</strong>ges with st<strong>an</strong>dardized models of <strong>the</strong> hemodynamic response (HDR). The reliability of <strong>the</strong>
esults depends on <strong>the</strong> match between <strong>the</strong> st<strong>an</strong>dardized HDR <strong>an</strong>d <strong>the</strong> individuals’ HDR. HDR is<br />
known to be influenced by neurological disorders such as ischemia <strong>an</strong>d multiple sclerosis. The<br />
effect of traumatic brain injury (TBI) on <strong>the</strong> HDR has not been investigated.<br />
Ten chronic severe TBI survivors (27 ± 1.7 yr, time since accident 4.4 ± 0.5 yr) <strong>an</strong>d 9 matched<br />
controls were recruited. All underwent fMRI whilst presented with visual stimuli lasting 700 ms<br />
(yellow Hs in a checkerboard configuration) at r<strong>an</strong>domized intervals. Subjects were instructed to<br />
press a response button each time <strong>the</strong> Hs were presented. Diffusion tensor images were acquired.<br />
FSL was used <strong>for</strong> region of interest (ROI) <strong>an</strong>alyses in V1 <strong>an</strong>d V2. BOLD signal ch<strong>an</strong>ges <strong>for</strong> each<br />
subject was estimated <strong>from</strong> temporally filtered, spatially smoo<strong>the</strong>d, normalized fMRI time series<br />
extracted <strong>from</strong> <strong>the</strong> ROI. The data were interpolated at points spaced TR/20 s <strong>an</strong>d <strong>the</strong> following<br />
parameters extracted: height, time-to-peak, rise rate, width <strong>an</strong>d area under <strong>the</strong> curve. A mixedmodel<br />
r<strong>an</strong>dom effects <strong>an</strong>alysis of between group differences of BOLD response was per<strong>for</strong>med.<br />
From <strong>the</strong> DTI sc<strong>an</strong>s <strong>the</strong> optical tracts were identified using diffusion tensor tractography in DTI<br />
studio. Fractional <strong>an</strong>isotropy (FA) <strong>an</strong>d apparent diffusion coefficient (ADC) values were<br />
calculated.<br />
TBI survivors were signific<strong>an</strong>tly slower to respond to <strong>the</strong> visual stimuli (427 ± 6 ms) compared<br />
to <strong>the</strong> controls (394 ± 4 ms) <strong>an</strong>d also less accurate (76% cf. 92%). HDR curves <strong>for</strong> TBI <strong>an</strong>d<br />
control groups were fully tr<strong>an</strong>sposable. There was no signific<strong>an</strong>t difference in <strong>an</strong>y of <strong>the</strong><br />
parameters tested. TBI survivors had signific<strong>an</strong>tly reduced FA. In controls <strong>the</strong>re was a signific<strong>an</strong>t<br />
correlation between <strong>the</strong> strength of neuronal activation in <strong>the</strong> ROI <strong>an</strong>d FA values (R 2 =0.52,<br />
p=0.04) but not in TBIs (R 2 =0.29, p=0.16). There was no correlation between FA values <strong>an</strong>d size<br />
of activation. Between group contrast showed TBI patients to have greater activation in V2<br />
during visual stimulation th<strong>an</strong> controls.<br />
Severe TBI with signific<strong>an</strong>t ch<strong>an</strong>ges in FA indicating axonal injury does not affect <strong>the</strong> HDR.<br />
Still TBI survivors have additional activity in V2, perhaps reflecting differences in local<br />
processing.<br />
Disclosures: H.S. Palmer, None; B. Garzón, None; E.M. Berntsen, None; J. Xu, None; A.<br />
Håberg, None.<br />
Poster<br />
541. Trauma: Brain- Hum<strong>an</strong> Studies<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 541.12/R15<br />
Topic: C.10.b. Brain: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH MH068787
DOD PT075675<br />
Marshall Goldberg Fund<br />
Title: Shearing of thalamic projection fibers following traumatic brain injury<br />
Authors: *E. K. GEARY, M. F. KRAUS, J. JOSEPH, X. J. ZHOU, P. B. GORELICK, D. M.<br />
LITTLE;<br />
Neurol., Univ. of Illinois Chicago, Chicago, IL<br />
Abstract: Traumatic brain injury (TBI) is commonly associated with impairments in executive<br />
function. The prevalence of such impairments following TBI has led to <strong>the</strong> hypo<strong>the</strong>sis that TBI<br />
compromises <strong>the</strong> structural integrity of <strong>the</strong> frontal lobe <strong>an</strong>d it is this structural damage which<br />
underlies functional disability. However, <strong>the</strong>re is often <strong>an</strong> absence of observable structural<br />
ch<strong>an</strong>ge in <strong>the</strong> frontal lobe that c<strong>an</strong> explain ei<strong>the</strong>r <strong>the</strong> extent or presence of observed cognitive<br />
impairment. This dissociation between observable structural damage in <strong>the</strong> frontal lobes <strong>an</strong>d<br />
functional impairment led us to test <strong>the</strong> hypo<strong>the</strong>sis that perhaps damage to <strong>the</strong> fibers projecting<br />
<strong>from</strong> <strong>the</strong> thalamus underlies executive dysfunction <strong>an</strong>d not damage to <strong>the</strong> frontal lobe per se.<br />
The effects of brain injury on fibers connecting to <strong>the</strong> thalamus was assessed using high<br />
resolution diffusion tensor imaging (DTI) <strong>an</strong>d neuropsychological testing in patients with a<br />
history of a single closed-head injury (n=12 mild TBI, 12 moderate severe TBI, 12 controls).<br />
Fractional <strong>an</strong>isotropy (FA) was extracted <strong>from</strong> 12 cortical regions of interest <strong>an</strong>d <strong>from</strong> 7 thalamic<br />
nuclei including <strong>the</strong> <strong>an</strong>terior (AN), ventral <strong>an</strong>terior (VA), ventral lateral (VL), dorsomedial<br />
(DM), ventral posterior lateral (VPL), ventral posterior medial (VPM), <strong>an</strong>d <strong>the</strong> pulvinar (PU).<br />
Patients with a history of TBI showed reduced FA in <strong>the</strong> <strong>an</strong>terior <strong>an</strong>d posterior corona radiata,<br />
<strong>for</strong>ceps major, <strong>an</strong>d <strong>the</strong> body of <strong>the</strong> corpus callosum. There was also <strong>an</strong> effect of subject group in<br />
fibers extracted <strong>from</strong> <strong>the</strong> AN <strong>an</strong>d VA seed voxels. For <strong>the</strong> TBI, <strong>the</strong>re were no correlations<br />
between <strong>an</strong>y cortical ROIs with executive function, attention, or memory. Executive function<br />
was, however, signific<strong>an</strong>tly correlated with FA extracted <strong>from</strong> seed voxels in <strong>the</strong> AN, VA, <strong>an</strong>d<br />
VL. Similar relationships were also found between <strong>the</strong> attention domain score <strong>an</strong>d integrity of<br />
fibers <strong>from</strong> <strong>the</strong> AN, VA, <strong>an</strong>d VL. In contrast, memory function was associated with integrity of<br />
VPL fibers. These findings provide initial evidence that damage to thalamic projection fibers,<br />
especially those involved in frontal-thalamic circuitry, is of great import<strong>an</strong>ce in underst<strong>an</strong>ding<br />
executive dysfunction following TBI.<br />
Disclosures: E.K. Geary, None; M.F. Kraus, None; J. Joseph, None; X.J. Zhou, None; P.B.<br />
Gorelick, None; D.M. Little, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.1/R16<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH Gr<strong>an</strong>t NS047718<br />
NIH-NINDS Supplement NS-47718<br />
Title: Assessment of <strong>the</strong> usefulness of CST-YFP mice <strong>for</strong> studies of corticospinal tract<br />
regeneration following spinal cord injury<br />
Authors: *R. WILLENBERG 1,2 , O. STEWARD 1,2,3,4 ;<br />
1 Reeve-Irvine Res. Ctr., 2 Dept. of Anat. & Neurobio., 3 Dept. of Neurobio. & Behavior, 4 Dept. of<br />
Neurosurg., Univ. of Cali<strong>for</strong>nia at Irvine Sch. of Med., Irvine, CA<br />
Abstract: A strain of mice has been genetically engineered to express yellow fluorescent protein<br />
(YFP) in <strong>the</strong> corticospinal tract (CST) (Bareyre et al. Nat Med 11(12): 1355, 2005). Here, we<br />
assess <strong>the</strong> degree to which <strong>the</strong>se mice provide a useful model system <strong>for</strong> assessing regenerative<br />
growth of <strong>the</strong> CST after spinal cord injury (SCI). In CST-YFP mice, YFP-expression is driven<br />
by requisite expression of Thy1 <strong>an</strong>d EMX in cortical neurons, including upper motoneurons in<br />
<strong>the</strong> sensorimotor cortex. YFP is delivered to CST axons <strong>an</strong>d pre-terminal arbors in <strong>the</strong> spinal<br />
cord. If <strong>the</strong>se mice are to be useful <strong>for</strong> studies of <strong>the</strong> CST after SCI, it is critical to determine<br />
whe<strong>the</strong>r YFP is expressed in only CST axons after spinal cord injury <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong> labeling<br />
with YFP is sufficient to allow visualization of individual CST axons. We assessed YFP-labeled<br />
axons in <strong>the</strong> spinal cords of intact CST-YFP mice <strong>an</strong>d mice with dorsal hemisection lesions at<br />
T6. Some mice received injections of biotinylated dextr<strong>an</strong> amine (BDA) into <strong>the</strong> sensorimotor<br />
cortex so as to compare labeling with YFP vs BDA tract tracing. As reported previously, YFP<br />
labeled axons were seen in <strong>the</strong> dorsal <strong>an</strong>d dorsolateral CST, <strong>an</strong>d in some mice, a few labeled<br />
axons were also seen in <strong>the</strong> ventral column in <strong>the</strong> position of <strong>the</strong> ventral CST. BDA-injected<br />
mice also had BDA-labeled axons in <strong>the</strong> dorsal <strong>an</strong>d dorsolateral CST that were co-labeled with<br />
YFP, but <strong>the</strong>re were no definitive examples of co-labeled axons extending longitudinally along<br />
<strong>the</strong> ventral column. In mice with dorsal hemisection lesions, YFP-labeled CST axons terminated<br />
in characteristic retraction bulbs; it was difficult, however, to visualize individual axons in <strong>the</strong><br />
densely packed dorsal CST. Mice with dorsal hemisection lesions also had YFP-labeled axons<br />
diffusely distributed in <strong>the</strong> lateral <strong>an</strong>d ventral white matter; <strong>the</strong>se ectopic axons had a distinctly<br />
different appear<strong>an</strong>ce th<strong>an</strong> <strong>the</strong> labeled CST axons in <strong>the</strong> main tracts in that <strong>the</strong> labeling appeared<br />
to be associated with <strong>the</strong> myelin sheath or axonal surface membr<strong>an</strong>e. Our results thus indicate<br />
that some YFP-labeled axons in <strong>the</strong> spinal cord of CST-YFP mice are not CST axons, <strong>an</strong>d that<br />
<strong>the</strong> labeling with YFP is not ideal <strong>for</strong> tracing individual CST axons in mice with spinal cord<br />
injuries. These caveats limit <strong>the</strong> usefulness of CST-YFP mice <strong>for</strong> studies of CST growth<br />
following SCI.<br />
Disclosures: R. Willenberg, None; O. Steward, None.
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.2/R17<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: NIH<br />
Veter<strong>an</strong>s Administration<br />
Wings <strong>for</strong> Life<br />
C<strong>an</strong>adi<strong>an</strong> Spinal Research Org<strong>an</strong>ization<br />
<strong>the</strong> Swiss Institute <strong>for</strong> Research into Paraplegia<br />
Title: Combinatorial <strong>the</strong>rapies promote motor axonal plasticity <strong>an</strong>d functional improvement<br />
when administered one year after spinal cord injury<br />
Authors: *P. P. LU 1,2 , A. BLESCH 1 , K. MCHALE 1 , L. GRAHAM 1 , W. WANG 1 , M.<br />
TUSZYNSKI 1,2 ;<br />
1 UCSD, La Jolla, CA; 2 VA Med. Ctr., S<strong>an</strong> Diego, CA<br />
Abstract: Previously we <strong>an</strong>d o<strong>the</strong>rs have reported that <strong>the</strong>rapies targeting multiple cellular <strong>an</strong>d<br />
extracellular mech<strong>an</strong>isms are required to achieve axonal regeneration into <strong>an</strong>d beyond sites of<br />
spinal cord injury, when <strong>the</strong>rapies are administered acutely after injury. In <strong>the</strong> present study we<br />
explored <strong>the</strong> hypo<strong>the</strong>sis that combinatorial <strong>the</strong>rapies would also support axonal sprouting or<br />
regeneration when administered at prolonged delays of more th<strong>an</strong> one year after <strong>the</strong> original SCI.<br />
Fischer 344 rats underwent C5 hemisection lesions. 15 months later <strong>the</strong>y received ei<strong>the</strong>r: Group<br />
1) cAMP injection into <strong>the</strong> reticular nucleus (to stimulate <strong>the</strong> endogenous growth state of<br />
neurons), plus impl<strong>an</strong>tation of syngenic marrow stromal cells (MSCs) into <strong>the</strong> lesion site that<br />
were genetically modified to secrete BDNF, plus injection of Lentiviral <strong>an</strong>d AAV vectors<br />
expressing BDNF 1.25 <strong>an</strong>d 2.5mm caudal to <strong>the</strong> lesion site; or Group 2) <strong>the</strong> same treatments as<br />
group 1 plus CHASE administration to <strong>the</strong> sites that also received viral injections below <strong>the</strong><br />
lesion; or Group 3) lesions but no cAMP, growth factor or CHASE. <strong>When</strong> assessed 6 weeks<br />
later, or a total of 16 months after <strong>the</strong> original injury, Group 2 showed signific<strong>an</strong>t improvement<br />
compared to Group 1 <strong>an</strong>d controls on recovery of grip strength (p
lesion site, <strong>an</strong>d potentially beyond. Qu<strong>an</strong>tification is underway. These findings indicate that<br />
motor axonal sprouting or regeneration c<strong>an</strong> be induced into <strong>an</strong>d beyond sites of chronic SCI<br />
following combinatorial treatment, <strong>an</strong>d that combinatorial treatment that addresses multiple<br />
plastic mech<strong>an</strong>isms may optimize functional outcomes.<br />
Disclosures: P.P. Lu, None; A. Blesch, None; K. McHale, None; L. Graham, None; W.<br />
W<strong>an</strong>g, None; M. Tuszynski, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.3/R18<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Title: Acupuncture inhibits apoptotic cell death <strong>an</strong>d improves functional <strong>an</strong>d pathological<br />
outcomes by attenuating microglial activation after spinal cord injury<br />
Authors: *D. C. CHOI 1,2 , J. Y. LEE 1,2 , Y. J. MOON 1,2 , S. W. KIM 1,2,3 , T. H. OH 1,2 , T. Y.<br />
YUNE 1,2,3 ;<br />
1 Age-Related <strong>an</strong>d Brain Dis. Res. Ctr., 2 Neurodegeneration Control Res. Ctr., 3 Dept. of<br />
Biochem. <strong>an</strong>d Mol. Biol., Kyung Hee Univ. Sch. of Med., Seoul, Republic of Korea<br />
Abstract: Inflammation has been known to play <strong>an</strong> import<strong>an</strong>t role in pathogenesis after spinal<br />
cord injury (SCI), which causes a perm<strong>an</strong>ent neurological impairment. In particular, microglia<br />
activated after SCI produce a variety of pro-inflammatory factors, leading to neuronal <strong>an</strong>d<br />
oligodendroglial apoptosis. In this study, we investigate <strong>the</strong> neuroprotective effects of<br />
acupuncture after SCI by examining whe<strong>the</strong>r <strong>the</strong> treatment contributes to inhibition of microglial<br />
activation following inflammatory reactions. Acupuncture was immediately applied to rats<br />
receiving a moderate contusion injury at <strong>the</strong> T9 spinal cord at two acupoints, GV26 <strong>an</strong>d GB34,<br />
followed by once a day <strong>for</strong> 2 weeks or until rats were sacrificed at <strong>the</strong> indicated time points.<br />
Acupuncture led to signific<strong>an</strong>t inhibition of apoptotic death of both neurons at 1 day <strong>an</strong>d<br />
oligodendrocytes at 5 days, <strong>an</strong>d improved functional recovery. The size of lesion cavity, <strong>an</strong>d<br />
extent of loss of axons <strong>an</strong>d myelins at 38 days after SCI were reduced following <strong>the</strong> treatment.<br />
Fur<strong>the</strong>rmore, acupuncture effectively attenuated microglial activation at 5 days <strong>an</strong>d expression of<br />
proinflammatory cytokines, such as TNF-α, IL-1β, <strong>an</strong>d IL-6, as well as inflammatory mediators,<br />
including iNOS <strong>an</strong>d COX-2, after injury. In particular, activation of p38MAPK following<br />
proNGF production, which is activated only in microglia <strong>an</strong>d involved in apoptotic death of<br />
oligodendrocytes after SCI, was markedly suppressed by acupuncture. Taken toge<strong>the</strong>r, our data
indicate that acupuncture signific<strong>an</strong>tly reduces apoptotic cell death, in part, by inhibiting<br />
microglial activation following inflammatory reactions after injury, <strong>an</strong>d support its use as a<br />
potential <strong>the</strong>rapeutic tool <strong>for</strong> treating spinal injury in hum<strong>an</strong>s.<br />
Disclosures: D.C. Choi, None; J.Y. Lee, None; Y.J. Moon, None; S.W. Kim, None; T.H. Oh,<br />
None; T.Y. Yune, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.4/S1<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: CAPES<br />
HCFMUSP<br />
Title: Specific training enh<strong>an</strong>ces functional recovery of Wistar rats with consistent spinal cord<br />
injury<br />
Authors: *T. A. MIRANADA 1,2 , J. M. Y. VICENTE 2 , M. SOARES 3 , R. M. MARCON 4 , A. C.<br />
VALLE 2 , E. MORYA 5 ;<br />
1 São Paulo, Brazil; 2 Program of Exptl. Pathophysiology, Med. Fac. São Paulo Univ., São Paulo,<br />
Brazil; 3 Lab. of Pathophysiology,, But<strong>an</strong>t<strong>an</strong> Inst., São Paulo, Brazil; 4 Orthopedy <strong>an</strong>d<br />
Traumatology Inst., São Paulo, Brazil; 5 Alberto S<strong>an</strong>tos Dumont Res. Support Association, Inst.<br />
of Teaching <strong>an</strong>d Res. Sírio Lib<strong>an</strong>ês Hosp., São Paulo, Brazil<br />
Abstract: Objective: The objective of this study was to investigate <strong>the</strong> functional <strong>an</strong>d<br />
histological recovery of Wistar rats with moderate contusive spinal cord injury (SCI), submitted<br />
to physical training.<br />
Methods: All procedures were approved by <strong>the</strong> local Ethical Committee <strong>an</strong>d per<strong>for</strong>med in<br />
accord<strong>an</strong>ce with Mutlicenter Animal SCI Study. 51 Wistar rats were r<strong>an</strong>domized into five<br />
groups: maze, ramp, runner, control <strong>an</strong>d sham. Rats were <strong>an</strong>es<strong>the</strong>tized with Diazepam (ip<br />
5mg/ml), Ketamine (ip 100mg/kg) <strong>an</strong>d Xylestesin (local), be<strong>for</strong>e <strong>the</strong>y underwent contusive SCI<br />
(NYU-Impactor) at T9-T10 levels, except <strong>for</strong> <strong>the</strong> sham group. After <strong>the</strong> SCI, Cefazolin (ip<br />
0,1ml/100g) was administered. Training in three apparatuses (runner trace, maze <strong>an</strong>d ramp) were<br />
per<strong>for</strong>med twice a week <strong>for</strong> two weeks be<strong>for</strong>e <strong>an</strong>d five after SCI. Control was not submitted to<br />
<strong>the</strong> training <strong>an</strong>d sham was trained in <strong>the</strong> maze. Motor recovery was assessed with Basso, Beattie
<strong>an</strong>d Bresnah<strong>an</strong> (BBB) Scale once a week during a five week period. The motor behaviors<br />
<strong>an</strong>alyzed were dorsal or pl<strong>an</strong>tar stepping <strong>an</strong>d <strong>for</strong>elimb-hindlimb coordination. Animals were<br />
perfused on <strong>the</strong> 45th postoperative day <strong>an</strong>d 1,5cm of <strong>the</strong> spinal cords including injury epicenters<br />
were carefully dissected, paraffin-embedded <strong>an</strong>d tr<strong>an</strong>sversely sliced at 7µm thickness. Spinal<br />
cords morphometry was carried out in 50 r<strong>an</strong>domly chosen slices of each group stained with<br />
cresyl-violet. The values obtained of BBB Scale <strong>an</strong>d histology were <strong>an</strong>alyzed using ANOVA<br />
considering p
einnervate <strong>the</strong>ir distal targets. To this end, in a previously published study, we examined <strong>an</strong>d<br />
demonstrated <strong>the</strong> potential use of electrospinning to engineer <strong>an</strong> impl<strong>an</strong>table directional matrix<br />
<strong>for</strong> axonal guid<strong>an</strong>ce. In view of <strong>the</strong> recognition that successful spinal cord injury (SCI) <strong>the</strong>rapies<br />
require a multifaceted tactic, we proposed a novel approach of incorporating into electrospun<br />
guid<strong>an</strong>ce matrices pharmacologic agents that c<strong>an</strong> promote neuronal survival <strong>an</strong>d neutralize<br />
inhibitory molecules associated with <strong>the</strong> gliotic scar. Specifically, nerve growth factor (NGF)<br />
was encapsulated into electrospun alginate microspheres <strong>an</strong>d <strong>the</strong> extent to which this protein<br />
remains bioactive was assessed with dorsal root g<strong>an</strong>glion (DRG) cultures. These microspheres<br />
were placed in Tr<strong>an</strong>swell membr<strong>an</strong>e inserts over <strong>the</strong> g<strong>an</strong>glia so as to provide <strong>the</strong> only source of<br />
NGF in <strong>the</strong> culture. Their presence supported vigorous neuritic outgrowth in contrast to control<br />
beads containing only albumin. The bioactivity of NGF was similarly preserved when NGFcontaining<br />
microspheres were incorporated into electrospun matrices. Fur<strong>the</strong>rmore, following 1<br />
week of incubation <strong>an</strong>d release, NGF availability in <strong>the</strong> remaining alginate microspheres was still<br />
sufficient to support growth. We next sought to investigate <strong>the</strong> incorporation of chondroitinase<br />
into <strong>the</strong> matrix as it has been previously shown that this enzyme c<strong>an</strong> reduce <strong>the</strong> axonal growth<br />
inhibition of various chondroitin sulfate proteoglyc<strong>an</strong>s (CSPG). Stripes of aggrec<strong>an</strong>, a CSPG<br />
found in <strong>the</strong> CNS, were placed on cover slips with DRGs pinned in between <strong>the</strong> inhibitory l<strong>an</strong>es.<br />
In control cultures, DRG neurites did not extend onto aggrec<strong>an</strong> l<strong>an</strong>es. However, upon exposure<br />
of <strong>the</strong> cultures to chondroitinase-incorporated matrices, <strong>the</strong> growth inhibition of aggrec<strong>an</strong> was<br />
neutralized <strong>an</strong>d neurites grew extensively across <strong>the</strong> stripes. Collectively, <strong>the</strong>se experiments<br />
point toward a novel SCI <strong>the</strong>rapeutic approach of generating electrospun scaffolds that c<strong>an</strong><br />
directionally guide axons across <strong>the</strong> lesion site while providing trophic <strong>an</strong>d inhibitor-neutralizing<br />
support. We are currently investigating <strong>the</strong> possibility of incorporating o<strong>the</strong>r compounds of<br />
interest into electrospun matrices as well as examining <strong>the</strong> efficacy of <strong>the</strong>se enh<strong>an</strong>ced matrices at<br />
inducing regeneration in rat models of SCI.<br />
Disclosures: W. Chow, None; D.G. Simpson, Issued <strong>an</strong>d pending U.S. <strong>an</strong>d international<br />
patents, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); K.M.<br />
Savia, None; J.R. Bowm<strong>an</strong>, None; J.W. Bigbee, None; K.D. Lee, None; R.J. Colello, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.6/S3<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: U.S. Department of Veter<strong>an</strong>s Affairs, Northport VAMC
NYS DOH<br />
CDRF<br />
Title: Qu<strong>an</strong>titative evaluation of behavioral <strong>an</strong>d physiological impairments after chronic<br />
hemisection of adult rat spinal cord<br />
Authors: L. M. COLLIER 1,2 , A. S. HUNANYAN 1,2 , V. A. ALESSI 1,2 , A. S. TOLPYGO 2 , K.<br />
LEESHA 3 , L. LOU 2 , L. M. MENDELL 2 , L. SCHNELL 4 , J. K. ROBINSON 3 , *V. L.<br />
ARVANIAN 1,2 ;<br />
1 Northport VAMC, Northport, NY; 2 Neurobio. <strong>an</strong>d Behavior, 3 Psychology, Stony Brook Univ.,<br />
Stony Brook, NY; 4 Brain Res. Inst., Univ. of Zurich, Zurich, Switzerl<strong>an</strong>d<br />
Abstract: There is growing interest in using <strong>the</strong> hemisection (HX) spinal cord injury model both<br />
as a model of partial damage resulting <strong>from</strong> combat wounds <strong>an</strong>d as a basic research model<br />
especially well-suited <strong>for</strong> <strong>the</strong> evaluation of conduction deficits in <strong>the</strong> adult rat. Our research has<br />
revealed that after a chronic HX preparation, <strong>the</strong> uninjured fibers contralateral to <strong>the</strong> HX exhibit<br />
reduced ability to tr<strong>an</strong>sfer signals to individual lumbar motoneurons through <strong>the</strong> white matter<br />
across <strong>from</strong> <strong>the</strong> HX. We have also found that <strong>the</strong>se conduction deficits develop during <strong>the</strong> first<br />
two weeks following HX, <strong>an</strong>d plateau at <strong>the</strong> diminished level after that. The goal of <strong>the</strong> present<br />
study was to per<strong>for</strong>m a comprehensive behavioral <strong>an</strong>alysis of this lesion to study <strong>the</strong> relationship<br />
between <strong>the</strong> developing deficit of tr<strong>an</strong>smission <strong>an</strong>d deficit of locomotor functions after chronic<br />
HX. We have compared <strong>the</strong> outcomes <strong>from</strong> behavioral testing, physiological recordings, <strong>an</strong>d<br />
<strong>an</strong>atomical reconstructions of <strong>the</strong> injury <strong>for</strong> each rat <strong>from</strong> two groups, sham laminectomy <strong>an</strong>d<br />
HX injury. Since <strong>the</strong>re is spont<strong>an</strong>eous recovery of locomotor function after chronic HX in rats,<br />
we assessed intercorrelations among measures in <strong>the</strong> Open-Field (BBB), Narrowing Beam,<br />
Irregular Ladder Crossing, <strong>an</strong>d <strong>the</strong> automated CatWalk gait <strong>an</strong>alyses measures, such as ipsilateral<br />
hindlimb intensity, hindlimb base of support <strong>an</strong>d phase lags (interlimb coordination). Deficits in<br />
<strong>the</strong>se tasks were most pronounced during <strong>the</strong> 7-14 dpo time period. Import<strong>an</strong>tly, slowing in<br />
spont<strong>an</strong>eous recovery of motor function coincided with <strong>the</strong> developing deficit in synaptic<br />
tr<strong>an</strong>smission after chronic HX injury. This work provides <strong>the</strong> most detailed study to date of <strong>the</strong><br />
ch<strong>an</strong>ges in weight support, gait <strong>an</strong>d o<strong>the</strong>r subcoments of motor function, over <strong>the</strong> course of<br />
recovery, <strong>an</strong>d <strong>the</strong> relationship between <strong>the</strong>se qu<strong>an</strong>titative ch<strong>an</strong>ges in motor competence <strong>an</strong>d<br />
conduction.<br />
Disclosures: L.M. Collier, None; A.S. Hun<strong>an</strong>y<strong>an</strong>, None; V.A. Alessi, None; A.S. Tolpygo,<br />
None; K. Leesha, None; L. Lou, None; L.M. Mendell, None; L. Schnell, None; J.K.<br />
Robinson, None; V.L. Arv<strong>an</strong>i<strong>an</strong>, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.7/S4<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: ISRT CLI004<br />
Title: Functional outcomes following robot assisted locomotor training in incomplete spinal cord<br />
injury<br />
Authors: *B. A. CONWAY 1 , C. CATTON 1 , S. GALEN 1 , D. B. ALLAN 2 , K. HUNT 3 ;<br />
1 Bioengineering Unit, Univ. Strathclyde, Glasgow, United Kingdom; 2 Queen Elizabeth Natl.<br />
Spinal Injuries Unit, Glasgow, United Kingdom; 3 Univ. of Glasgow, Glasgow, United Kingdom<br />
Abstract: Robot assisted locomotor training in subjects with Incomplete Spinal Cord Injury<br />
(ISCI) has been shown to be beneficial. These gains have been measured using functional<br />
outcome measures such as Walking Index in Spinal Cord Injury (WISCI II) <strong>an</strong>d timed tests such<br />
as <strong>the</strong> 10 meter walk test. Although <strong>the</strong>se outcome measures are sensitive to ch<strong>an</strong>ges in<br />
ambulatory status of a subject <strong>the</strong>y are not sensitive to ch<strong>an</strong>ges in per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d quality of<br />
gait, which may have been enh<strong>an</strong>ced by robot assisted locomotor training. In this study we<br />
propose a gait assessment tool that uses foot switches, is body worn, portable <strong>an</strong>d is designed <strong>for</strong><br />
use in a clinical environment. The gait assessment tool has <strong>the</strong> capability to assess <strong>the</strong><br />
ambulatory capacity, per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d quality of gait. Eighteen subjects with incomplete SCI<br />
participated in this study (Age r<strong>an</strong>ge: 26-63 years). Among <strong>the</strong> subjects who participated 5 were<br />
classified as chronic subjects (> 6 months <strong>from</strong> time of injury) <strong>an</strong>d 13 were classified as acute<br />
subjects (
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.8/S5<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: Craig H Nielsen Foundation<br />
Title: Superoxide dismutase 1 (SOD1)-overexpressing rats as a model to investigate oxidative<br />
stress in oligodendrocyte lineage after spinal cord injury<br />
Authors: *S. VEIGA 1,1 , K.-A. IRVINE 1 , A. LIN 1 , J. O. LY 1 , P. H. CHAN 2 , J. C.<br />
BRESNAHAN 1 , M. S. BEATTIE 1 ;<br />
1 2<br />
Neurolog. surgery, UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; Neurosurg., St<strong>an</strong><strong>for</strong>d Univ. Sch. of Med.,<br />
St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Oligodendrocyte (OL) death <strong>an</strong>d associated demyelination have been postulated to<br />
contribute to chronic deficits after spinal cord injury (SCI). Oligodendrocyte precursor cells<br />
(OPCs) respond rapidly to demyelination by increasing <strong>the</strong>ir proliferative rate, a behavior that is<br />
considered as <strong>an</strong> endogenous reparative mech<strong>an</strong>ism to replenish OLs lost after SCI. However,<br />
this response is hampered, resulting in a failure to differentiate into myelinating OLs <strong>an</strong>d/or<br />
death of OPCs one week post-injury. Previous work in our lab has shown that oxidative stress<br />
might be a key factor involved in <strong>the</strong> death of OLs <strong>an</strong>d OPCs following SCI. To test this<br />
hypo<strong>the</strong>sis, we used a tr<strong>an</strong>sgenic rat that overexpresses <strong>the</strong> <strong>an</strong>tioxid<strong>an</strong>t enzyme, Superoxide<br />
Dismutase Type-1 (SOD1). In vitro studies have shown that LDH release in OPC cultures<br />
exposed to different concentrations of <strong>the</strong> oxid<strong>an</strong>t agent tert-butyl hydroperoxide is signific<strong>an</strong>tly<br />
lower in OPCs derived <strong>from</strong> SOD1 overexpressors th<strong>an</strong> those derived <strong>from</strong> wild-type <strong>an</strong>imals<br />
(p
Disclosures: S. Veiga, None; K. Irvine, None; A. Lin, None; J.O. Ly, None; P.H. Ch<strong>an</strong>,<br />
None; J.C. Bresnah<strong>an</strong>, None; M.S. Beattie, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.9/S6<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Title: q-space MR imaging depict demyelination after spinal cord injury in non-hum<strong>an</strong> primate<br />
Authors: *K. FUJIYOSHI 1 , O. TSUJI 2 , K. HIKISHIMA 5 , M. YAMADA 6 , K. KITAMURA 2 ,<br />
S. MOMOSHIMA 3 , M. MATSUMOTO 2 , K. CHIBA 2 , H. OKANO 4 , Y. TOYAMA 2 , M.<br />
NAKAMURA 2 ;<br />
2 Orthopaedic Surgery, 3 Radiology, 4 Physiol., 1 Keio Univ., Tokyo, Jap<strong>an</strong>; 5 Central Inst. <strong>for</strong> Exptl.<br />
Animals, K<strong>an</strong>agawa, Jap<strong>an</strong>; 6 Fac. of Radiological technology, Sch. of Hlth. Sciences, Fujita<br />
Hlth. Univ., Aichi, Jap<strong>an</strong><br />
Abstract: Diffusion weighted MR imaging (DWI) gives us a lot of in<strong>for</strong>mation about biological<br />
structures. Recently, it is said that q-space MR imaging (QSI) which is one of <strong>the</strong> adv<strong>an</strong>ced<br />
technique of DWI is a powerful tool to evaluate micro-structural ch<strong>an</strong>ges in central nerves<br />
system. QSI allows <strong>for</strong> detecting ra<strong>the</strong>r smaller microstructure enh<strong>an</strong>ced by restricted diffusion.<br />
We have already reported that QSI enables in vivo imaging of micro-structural ch<strong>an</strong>ges in intact<br />
<strong>an</strong>d injured spinal cord of marmosets. In this study we focused on myelin a major restricted<br />
structure <strong>an</strong>d tried to QSI <strong>for</strong> visualizing demyelination after spinal cord injury.<br />
A total of eight adult common marmosets were used. One group (n=6) received a contusive SCI<br />
at <strong>the</strong> C5 level, <strong>an</strong>o<strong>the</strong>r group (n=2) was used as a non-operate naïve control. The spinal cord<br />
was imaged in a 7.0 Tesla MRI (Bruker Biospin) at pre-injury, 3day, 2, 4, 10 weeks after injury.<br />
Histological evaluations were conducted using Hematoxylin-Eosin <strong>an</strong>d Luxol fast blue staining.<br />
Spinal cord sections were also observed under <strong>an</strong> electron microscope <strong>for</strong> micro-structural<br />
examination. All procedures were per<strong>for</strong>med under general <strong>an</strong>es<strong>the</strong>sia. QSI was done by custom<br />
written codes in IDL (interactive data l<strong>an</strong>guage). We use two QSI parameter“Average<br />
displacement <strong>an</strong>d Kurtosis ”.<br />
The displacement map of <strong>the</strong> QSI clearly illustrated spinal cords histological ch<strong>an</strong>ges such as <strong>the</strong><br />
neuronal loss, inflammatory cell infiltrations <strong>an</strong>d cavity <strong>for</strong>mation. Fur<strong>the</strong>rmore, <strong>the</strong> myelin map<br />
which is modified kurtosis map reflected <strong>the</strong> spared myelin stained with LFB precisely.<br />
Histology of <strong>the</strong> spinal cords in both control <strong>an</strong>d injured groups were consistent with QSI<br />
findings, verifying <strong>the</strong> accuracy of QSI. Our results revealed that QSI could depict <strong>the</strong>
demyelination of <strong>the</strong> injured spinal cord in live <strong>an</strong>imals, demonstrating <strong>the</strong> possible contribution<br />
of QSI to <strong>the</strong> clinical studies of SCI <strong>the</strong>rapy.<br />
Disclosures: K. Fujiyoshi, None; O. Tsuji, None; K. Hikishima, None; M. Yamada,<br />
None; K. Kitamura, None; S. Momoshima, None; M. Matsumoto, None; K. Chiba, None; H.<br />
Ok<strong>an</strong>o, None; Y. Toyama, None; M. Nakamura, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.10/S7<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: Christopher <strong>an</strong>d D<strong>an</strong>a Reeve Foundation<br />
Craig H. Neilsen Foundation<br />
CIHR<br />
Title: Ketogenic diet initiated after SCI improves functional recovery in rats<br />
Authors: *F. STREIJGER, W. T. PLUNET, J. LIU, J. H. T. LEE, C. K. LAM, S. PARK, C.<br />
M. YUAN, J. LU, W. TETZLAFF;<br />
ICORD (International Collaboration On Repair Discoveries), V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: The ketogenic diet (KD) has been well recognized as <strong>an</strong> effective nonpharmacological<br />
treatment <strong>for</strong> different neurological disorders including epilepsy, Alzheimer’s<br />
disease, brain injury <strong>an</strong>d Parkinson’s disease. In this study, we investigated <strong>the</strong> preclinical use of<br />
a KD as a potential alternative <strong>the</strong>rapy <strong>for</strong> acute spinal cord injury. KDs contain a high content of<br />
fat, very few carbohydrates <strong>an</strong>d variable amount of proteins. Due to <strong>the</strong> minimal dietary sources<br />
of glucose in KDs, <strong>the</strong> energy supply of <strong>the</strong> brain primarily comes <strong>from</strong> ketone bodies produced<br />
in <strong>the</strong> liver via <strong>the</strong> metabolism of fats.<br />
Previously, we observed improved behavioral recovery after a unilateral crush of <strong>the</strong> cervical<br />
spinal cord in <strong>an</strong>imals fed a KD with <strong>an</strong> 7:1 ratio of fat to carbohydrate plus protein. However,<br />
<strong>the</strong> 7:1 ratio is higher th<strong>an</strong> <strong>the</strong> ratio used clinically, which r<strong>an</strong>ges <strong>from</strong> 2:1 to 4:1 due to a higher<br />
protein content. There<strong>for</strong>e, in <strong>the</strong> present study, we tested <strong>the</strong> effectiveness of <strong>the</strong> more clinically<br />
relev<strong>an</strong>t 3:1 KD <strong>an</strong>d compared it to a st<strong>an</strong>dard control diet (SD) <strong>an</strong>d <strong>the</strong> previously used 7:1 KD<br />
after a left unilateral contusion of <strong>the</strong> cervical spinal cord in adult male rats.
Blood β-hydroxybutyrate levels of <strong>an</strong>imals fed <strong>the</strong> 7:1 or 3:1 KD were similar between <strong>the</strong><br />
groups, reaching maximum levels during <strong>the</strong> first week after spinal cord injury. Although, daily<br />
caloric intake was similar between <strong>the</strong> three groups, <strong>the</strong> 7:1 KD group showed attenuated weight<br />
gain compared to <strong>the</strong> 3:1 KD <strong>an</strong>d <strong>the</strong> SD control group. Behaviorally, <strong>for</strong>elimb usage during<br />
vertical exploration was profoundly affected after cervical spinal cord contusion. The rats on <strong>the</strong><br />
SD used <strong>the</strong> ipsilateral <strong>for</strong>elimb less th<strong>an</strong> 3% while rearing. The <strong>an</strong>imals fed a 3:1 KD however<br />
showed a four to five fold increase in ipsilateral <strong>for</strong>elimb use to around 13% (p
<strong>an</strong>es<strong>the</strong>tized rats was developed.<br />
METHODS. In 24 female rats <strong>the</strong> spinal cord was tr<strong>an</strong>sected at T10. The H-reflex was tested<br />
be<strong>for</strong>e surgery <strong>an</strong>d <strong>the</strong>n weekly during <strong>the</strong> first 30 days <strong>an</strong>d biweekly <strong>for</strong> 60 days. Four groups<br />
were used (n=6 each): tr<strong>an</strong>section only (TX), motorized bicycle exercise training (MBET), Ldopa<br />
(LD), <strong>an</strong>d MBET <strong>an</strong>d L-dopa combination (LD+MBET). Treatments <strong>for</strong> <strong>the</strong> latter three<br />
groups was 60 days in duration starting 30 days after SCI. Percut<strong>an</strong>eous stimulation allowed Hreflexes<br />
to be tested in alert, non-<strong>an</strong>es<strong>the</strong>tized rats in a m<strong>an</strong>ner parallel to that used in hum<strong>an</strong>s.<br />
Results obtained using percut<strong>an</strong>eous stimulation was found to correlate with those obtained by<br />
<strong>the</strong> more widely used direct nerve stimulation method (ICC = .88). One electrode was inserted<br />
percut<strong>an</strong>eously to a site about 1 cm proximal of <strong>the</strong> <strong>an</strong>kle <strong>an</strong>d adjacent to <strong>the</strong> tibial nerve; <strong>the</strong><br />
o<strong>the</strong>r was placed about 1 cm more proximal. A gel coated recording electrode was taped to <strong>the</strong><br />
surface of <strong>the</strong> paw pad <strong>an</strong>d <strong>an</strong>o<strong>the</strong>r to <strong>the</strong> digits (reference). A strength series <strong>for</strong> <strong>the</strong> H-reflex<br />
was made over <strong>the</strong> r<strong>an</strong>ge of stimuli 1-4xT. For low frequency-dependent depression testing <strong>the</strong><br />
H-reflex was recorded at 0.2, 1, 5, <strong>an</strong>d 10 Hz using a stimulus of 1.6-1.8xT. After <strong>the</strong> first 5<br />
responses were discarded, <strong>an</strong> average of 10 responses was obtained.<br />
RESULTS. Low frequency-dependent depression (LFD) decreased signific<strong>an</strong>tly by seven days<br />
in a group consisting of all <strong>an</strong>imals (n=24) be<strong>for</strong>e treatments beg<strong>an</strong> at day 30. For <strong>the</strong> TX group,<br />
LFD continually decreased to reach signific<strong>an</strong>tly low levels by 90 days (<strong>an</strong> indication of<br />
adv<strong>an</strong>ced hyperreflexia). At 90 days, all treatment groups had signific<strong>an</strong>tly lower H-reflex<br />
amplitudes th<strong>an</strong> <strong>the</strong> TX group at both 5 <strong>an</strong>d 10 Hz <strong>an</strong>d were not different <strong>from</strong> <strong>the</strong>ir pre-SCI<br />
levels. At 90 days, <strong>the</strong> H-max/M-max ratio increased signific<strong>an</strong>tly in <strong>the</strong> TX group, but<br />
decreased signific<strong>an</strong>tly after treatment in <strong>the</strong> LD group.<br />
CONCLUSIONS. 1) Percut<strong>an</strong>eous tibial n. stimulation <strong>for</strong> H-reflex testing in alert rats was<br />
equivalent to direct tibial n. stimulation in <strong>an</strong>es<strong>the</strong>tized rats <strong>an</strong>d represents a method parallel to<br />
that used in hum<strong>an</strong> SCI investigation. 2) MBET <strong>an</strong>d L-dopa treatments were equally effective in<br />
restoring <strong>the</strong> H-reflex LFD to normal levels, with LD+MBET being <strong>the</strong> most effective.<br />
Disclosures: A. Arfaj, None; R.D. Skinner, None; C. Yates, None; K. Garrison, None; N.B.<br />
Reese, None; E. Garcia-Rill, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.12/S9<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH Gr<strong>an</strong>t NS38079
NIH Gr<strong>an</strong>t NS31193<br />
New York State CoRE CO19772<br />
Title: Irvine, Beattie <strong>an</strong>d Bresnah<strong>an</strong> (IBB) <strong>for</strong>elimb scale: A novel method <strong>for</strong> assessing distal<br />
<strong>for</strong>elimb functional recovery following cervical spinal cord injury<br />
Authors: *K. A. IRVINE, A. R. FERGUSON, A. LIN, S. B. BEATTIE, M. S. BEATTIE, J. C.<br />
BRESNAHAN;<br />
UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Spinal cord injury (SCI) is a devastating <strong>an</strong>d life-ch<strong>an</strong>ging event of which <strong>the</strong> level<br />
<strong>an</strong>d extent of injury c<strong>an</strong> drastically impact <strong>the</strong> degree of independence of <strong>the</strong> patient. Restoration<br />
of h<strong>an</strong>d/arm function has been repeatedly highlighted as a key priority <strong>for</strong> cervical injury<br />
patients. However, while most injuries in m<strong>an</strong> are at <strong>the</strong> cervical level, most studies of<br />
experimental SCI have used thoracic injuries <strong>an</strong>d examined locomotor function as <strong>the</strong> primary<br />
outcome. We <strong>an</strong>d o<strong>the</strong>rs have developed models of cervical SCI in rats to address this issue <strong>an</strong>d<br />
use several behavioural assays that are sensitive to different aspects of <strong>for</strong>elimb recovery <strong>an</strong>d<br />
repair such as, paw placement or grooming. However, <strong>the</strong>se a<strong>for</strong>ementioned behavioural tasks do<br />
not provide in<strong>for</strong>mation regarding <strong>the</strong> recovery of fine motor control <strong>an</strong>d digital movement<br />
following SCI. There<strong>for</strong>e, we have developed a new measure <strong>for</strong> assessing recovery of distal<br />
<strong>for</strong>elimb function following cervical contusion SCI, <strong>the</strong> Irvine, Beattie <strong>an</strong>d Bresnah<strong>an</strong> <strong>for</strong>elimb<br />
scale (IBB). Video footage of rats eating spherically-shaped cereals prior to <strong>an</strong>d after mild (75<br />
Kdyns, n=8) or moderate (100 kdyns, n=8) unilateral cervical contusions using <strong>the</strong> IH impactor<br />
device were <strong>an</strong>alyzed <strong>an</strong>d <strong>the</strong> features of recovery of <strong>for</strong>elimb use, such as postural <strong>an</strong>d joint<br />
position, object support, presence of abnormal mirror movements, digital extension, digital<br />
movement <strong>an</strong>d grasping technique, were assessed over time. The results indicate that rats with a<br />
mild contusion injury recover signific<strong>an</strong>tly better th<strong>an</strong> rats with moderate contusion injuries. The<br />
most critical difference between <strong>the</strong>se two groups is <strong>the</strong> return of independent digital<br />
movements, which do not recover following a moderate injury. The IBB scale also continues to<br />
detect improvements in <strong>for</strong>elimb recovery whilst o<strong>the</strong>r behavioural scores reach plateau. The<br />
IBB scale has a number of adv<strong>an</strong>tages: (1) rats do not need to be food deprived prior to <strong>the</strong> task<br />
in order <strong>for</strong> <strong>the</strong>m to per<strong>for</strong>m (2) it takes adv<strong>an</strong>tage of <strong>an</strong> innate behaviour <strong>the</strong>re<strong>for</strong>e <strong>the</strong> rats do<br />
not need to be extensively trained (3) <strong>the</strong> scale could potentially be adjusted to <strong>an</strong>alyze recovery<br />
of <strong>for</strong>elimb function in o<strong>the</strong>r rodents or species. (4) The scale explores elements of digital<br />
dexterity that are similar to those used by hum<strong>an</strong>s.<br />
Disclosures: K.A. Irvine, None; A.R. Ferguson, None; A. Lin, None; S.B. Beattie,<br />
None; M.S. Beattie, None; J.C. Bresnah<strong>an</strong>, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.13/S10<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: FWF Austri<strong>an</strong> Science Fund L512-N13<br />
Title: Motor control in spinal cord injured people below <strong>the</strong> level of <strong>the</strong> lesion<br />
Authors: J. LADENBAUER 1 , F. RATTAY 1 , B. FREUNDL 2 , H. BINDER 2 , *M. R.<br />
DIMITRIJEVIC 3,1 ;<br />
1<br />
Inst. of Analysis <strong>an</strong>d Scientific Computing, Vienna Univ. of Technol., Vienna, Austria;<br />
2 3<br />
Neurolog. Ctr., SMZ Baumgartner Hoehe, Otto Wagner Hosp., Vienna, Austria; Baylor Col.<br />
Med., Houston, TX<br />
Abstract: The activation of motor units of <strong>the</strong> lower trunk <strong>an</strong>d lower limbs of spinal cord injured<br />
people results in a variety of motor output features, depending on <strong>the</strong> spinal level <strong>an</strong>d degree of<br />
<strong>the</strong> lesion. Such activities of motor units c<strong>an</strong> be induced by reflexes <strong>an</strong>d by automatic <strong>an</strong>d<br />
volitional motor tasks. They c<strong>an</strong> be recorded by multich<strong>an</strong>nel surface electromyography (sEMG)<br />
<strong>from</strong> muscles below <strong>the</strong> injury.<br />
We <strong>an</strong>alyzed <strong>the</strong> features in sEMG outputs recorded during <strong>the</strong> attempt to per<strong>for</strong>m volitional<br />
single joint movements (unilateral <strong>an</strong>kle dorsi- <strong>an</strong>d pl<strong>an</strong>tar flexion) <strong>an</strong>d multijoint movements<br />
(unilateral hip <strong>an</strong>d knee flexion <strong>an</strong>d extension) in a population of 97 subjects with different<br />
degrees of paresis <strong>an</strong>d paralysis after spinal cord lesion.<br />
Data <strong>an</strong>alysis demonstrated that <strong>the</strong> ef<strong>for</strong>t to per<strong>for</strong>m single or multi-joint movements produced<br />
sEMG outputs that had characteristic features, regardless of whe<strong>the</strong>r <strong>the</strong> motor tasks resulted in<br />
partially present or clinically absent movements. These features were specified by on- <strong>an</strong>d offset<br />
<strong>an</strong>d amplitude of motor units-activity during <strong>the</strong> motor tasks, <strong>an</strong>d by <strong>the</strong> specific pattern of <strong>the</strong><br />
distribution of sEMG activity among <strong>the</strong> lower trunk, thigh <strong>an</strong>d leg muscle groups. These<br />
characteristic features of <strong>the</strong> spatiotemporal motor output patterns were consistent <strong>an</strong>d<br />
repeatable.<br />
Based on <strong>the</strong> characteristic motor outputs induced by a specific motor task, we identified 4<br />
groups within <strong>the</strong> population of patients who had different types of movement paralysis due to<br />
different severities of <strong>the</strong> spinal cord lesions. The sEMG of <strong>the</strong> 4 groups featured (i) localized<br />
activity corresponding to <strong>the</strong> motor task time windows, with expected motor output or (ii) with<br />
altered, weaker output <strong>an</strong>d co-activation of unrelated muscles; (iii) diffuse activity without clear<br />
on- <strong>an</strong>d offsets but increased motor unit discharge during task times; (iv) no activity. On <strong>the</strong><br />
basis of those groups, we describe to what extent <strong>the</strong> motor unit output characteristics depend on<br />
<strong>the</strong> underlying mech<strong>an</strong>isms of conduction through <strong>the</strong> spinal cord injury zone <strong>an</strong>d processing<br />
capabilities of <strong>the</strong> lumbar cord networks altered by <strong>the</strong> severity <strong>an</strong>d profile of <strong>the</strong> injury.<br />
Disclosures: J. Ladenbauer, None; F. Rattay, None; B. Freundl, None; H. Binder,<br />
None; M.R. Dimitrijevic, None.
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.14/S11<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: ISRT NSCG1D1R<br />
Title: Does acute treatment with docosahexaenoic or eicosapentaenoic acid affect inflammatory<br />
markers following compression spinal cord injury?<br />
Authors: *J. C. HALL 1 , J. V. PRIESTLEY 1 , V. H. PERRY 2 , A. T. MICHAEL-TITUS 1 ;<br />
1 Ctr. of Neurosci. & Trauma, Blizard Inst., London, United Kingdom; 2 Southampton Neurosci.<br />
Group, Sch. of Biol. Sci. <strong>an</strong>d Sch. of Med., University of Southampton, United Kingdom<br />
Abstract: We have shown that <strong>an</strong> acute i.v. bolus of docosahexaenoic (DHA) or<br />
eicosapentaenoic acid (EPA) is neuroprotective following rat hemisection (King et al., 2006) <strong>an</strong>d<br />
compression spinal cord injury (SCI) (Hu<strong>an</strong>g et al., 2007; Lim, et al., 2008). In vitro <strong>an</strong>d in vivo<br />
data in various models have shown that DHA <strong>an</strong>d EPA have potent <strong>an</strong>ti-inflammatory properties.<br />
Hu<strong>an</strong>g et al. (2007) demonstrated that acute DHA treatment reduced macrophage infiltration <strong>an</strong>d<br />
<strong>the</strong> cyclooxygenase-2 level at <strong>the</strong> injury epicentre. The effect on infiltration of o<strong>the</strong>r leukocytes<br />
such as neutrophils has not yet been characterised in <strong>the</strong> compression SCI model. Chemokines<br />
such as KC/GRO/CINC (<strong>the</strong> rat IL-8 counterpart), <strong>an</strong>d <strong>the</strong> cytokine IL-6 provide gradients <strong>for</strong><br />
chemotaxis of neutrophils <strong>an</strong>d macrophages to <strong>the</strong> injury epicentre. The aim of our study was to<br />
investigate whe<strong>the</strong>r DHA <strong>an</strong>d EPA could affect neutrophil infiltration <strong>an</strong>d tissue cytokines <strong>an</strong>d<br />
chemokines after injury. We qu<strong>an</strong>tified neutrophils <strong>an</strong>d measured a p<strong>an</strong>el of pro-inflammatory<br />
cytokines <strong>an</strong>d chemokines in <strong>the</strong> spinal cord, 4 <strong>an</strong>d 24 h following compression SCI at thoracic<br />
level 12. We assessed <strong>the</strong> effects of treatment of <strong>an</strong> i.v. bolus of ei<strong>the</strong>r saline, EPA (250<br />
nmol/kg) or DHA (250 nmol/kg) administered 30 min after SCI. Treatment with DHA<br />
signific<strong>an</strong>tly reduced <strong>the</strong> number of neutrophils (-55% vs. saline-treated) in <strong>the</strong> dorsal columns<br />
(DC) of <strong>the</strong> injured epicentre 4 h after SCI. The reduction in neutrophils in <strong>the</strong> DC (-31%),<br />
ventral horn (-51%) <strong>an</strong>d <strong>the</strong> ventrolateral white matter (-37%) at 1 day did not reach statistical<br />
signific<strong>an</strong>ce. EPA reduced <strong>the</strong> number of neutrophils (-55%) in <strong>the</strong> DC 1 day after SCI. In <strong>the</strong><br />
injury epicentre at 4 h <strong>the</strong>re was <strong>an</strong> increase in IL-6 (2169 ± 305 pg/µg), KC/GRO/CINC (672 ±<br />
89 pg/µg), IL-1beta (59 ± 6 pg/µg) <strong>an</strong>d TNF-alpha (65 ± 12 pg/µg) compared to laminectomy<br />
(27 ± 24 pg/µg; 140 ± 57 pg/µg; 0.4 ± 0.4 pg/µg <strong>an</strong>d 34 ± 12 pg/µg, respectively) <strong>an</strong>d naïve (0.6<br />
± 0.4 pg/µg; 4.2 ± 3 pg/µg; 0.2 ± 0.2 pg/µg <strong>an</strong>d 5.4 ± 2.0 pg/µg, respectively) levels. Cytokines<br />
<strong>an</strong>d chemokines returned to baseline levels at 24 h. Treatment with DHA or EPA did not affect
<strong>the</strong> levels of <strong>the</strong>se cytokines/chemokines or <strong>the</strong>ir ch<strong>an</strong>ges after SCI. Our data, in a clinically<br />
relev<strong>an</strong>t model of SCI, show that DHA <strong>an</strong>d EPA induced a slight reduction in neutrophil<br />
infiltration, but did not reduce <strong>the</strong> levels of <strong>the</strong> inflammatory markers assessed. Fur<strong>the</strong>r studies<br />
on are required to fully characterise <strong>the</strong> effects of <strong>the</strong>se compounds on <strong>the</strong> inflammatory<br />
response after SCI.<br />
Disclosures: J.C. Hall, None; J.V. Priestley, None; V.H. Perry, None; A.T. Michael-Titus,<br />
None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.15/S12<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH gr<strong>an</strong>t NS30226<br />
Craig Neilsen Foundation gr<strong>an</strong>t 83848<br />
The Miami Project to Cure Paralysis<br />
Title: Inter-rater reliability <strong>for</strong> classification of spasms in muscles paralyzed by spinal cord<br />
injury<br />
Authors: *C. K. THOMAS 1 , S. FERRELL 1 , M. DIDIDZE 1 , I. STEVENSON 2 , K. KORDING 2 ;<br />
1 Miami Proj Cure Paralysis, Univ. Miami Sch. Med., Miami, FL; 2 Physical Med. <strong>an</strong>d<br />
Rehabilitation, Physiol., Rehabil. Inst. of Chicago <strong>an</strong>d Northwestern Univ., Chicago, IL<br />
Abstract: Trauma to <strong>the</strong> spinal cord usually eliminates voluntary control of skeletal muscles<br />
innervated <strong>from</strong> spinal segments below <strong>the</strong> injury. Within a few weeks of injury, <strong>the</strong>se paralyzed<br />
muscles begin to contract involuntarily (spasm), often disrupting daily activities. To measure <strong>the</strong><br />
severity of <strong>the</strong>se contractions, people with spinal cord injury (SCI) are often asked to count <strong>the</strong><br />
number of spasms <strong>the</strong>y experience in <strong>an</strong> hour. The nature of <strong>the</strong> contractions <strong>the</strong>mselves may<br />
also dictate whe<strong>the</strong>r or not <strong>the</strong> person finds <strong>the</strong> contractions problematic. Our aim was to reliably<br />
classify spasms of paralyzed muscles into different types of contractions. In seven people with<br />
chronic cervical SCI, 24-hour EMG recordings were made <strong>from</strong> paralyzed leg muscles<br />
bilaterally (vastus lateralis, biceps femoris, medial gastrocnemius, tibialis <strong>an</strong>terior). One rater<br />
classified all of <strong>the</strong> spasm events as one of five types of contractions: spasms involving tonic
EMG, trains of motor unit potentials, clonus, myoclonus, or a mix of <strong>the</strong>se contraction types. A<br />
second, independent rater repeated <strong>the</strong> <strong>an</strong>alysis <strong>for</strong> 12 hours of data in four recordings (n=48<br />
hours). The two independent raters agreed on spasm events 75 % of <strong>the</strong> time. Classification<br />
agreement was less, largely due to classification of myoclonus as trains of unit potentials, or vice<br />
versa. These data provide invaluable in<strong>for</strong>mation <strong>for</strong> evaluating <strong>the</strong> success of automatic<br />
methods to detect <strong>an</strong>d classify muscle spasms.<br />
Disclosures: C.K. Thomas, None; S. Ferrell, None; M. Dididze, None; I. Stevenson,<br />
None; K. Kording, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.16/S13<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: NIH 60240B01<br />
Mission Connect, a project of TIRR Foundation<br />
Title: Amiloride improves locomotor recovery after spinal cord injury<br />
Authors: *J. C. LEE, V. U. L. MOKKAPATI, K. M. JOHNSON, O. NESIC;<br />
Biochem. & Mol. Biol, UTMB, Galveston, TX<br />
Abstract: Amiloride has shown beneficial effects in different neuropathological conditions, but<br />
its <strong>the</strong>rapeutic potential has not been tested in spinal cord injury (SCI). We tested amiloride using<br />
a clinically relev<strong>an</strong>t rat model of moderate contusion SCI inflicted at <strong>the</strong> thoracic segment T10<br />
using <strong>the</strong> IH device. Amiloride was administered daily by i.p. injections until perfusion of<br />
<strong>an</strong>imals on day 35. Hindlimb locomotor scoring (BBB scores) revealed a signific<strong>an</strong>t<br />
improvement by two BBB units in amiloride treated <strong>an</strong>imals that started at 21 days, <strong>an</strong>d lasted<br />
until 35 days after injury, when compared to vehicle treated SCI <strong>an</strong>imals. Fur<strong>the</strong>rmore, western<br />
blot <strong>an</strong>alysis showed that amiloride treatment induced a signific<strong>an</strong>t increase in <strong>an</strong>giopoietin-1<br />
(Ang-1) at both <strong>the</strong> site of injury (T10) <strong>an</strong>d <strong>the</strong> lumbar segments L4/5, when compared to vehicle<br />
treated SCI <strong>an</strong>imals, thus partially reversing <strong>the</strong> decrease of Ang-1 observed with injury. Ang-1<br />
is a critically import<strong>an</strong>t regulator of <strong>the</strong> maturation <strong>an</strong>d mainten<strong>an</strong>ce of <strong>the</strong> blood spinal cord<br />
barrier (BSCB). The signific<strong>an</strong>t decrease in Ang-1 induced by SCI is consistent with our findings<br />
that indicate <strong>the</strong> BSCB is persistently compromised <strong>an</strong>d has increased permeability in
chronically injured rats. Additionally, amiloride treatment partially, yet signific<strong>an</strong>tly, reversed<br />
<strong>the</strong> injury induced increases of de-phosphorylated neurofilament levels, which are indicators of<br />
axonal damage. Thus, amiloride treatment caused stabilization of <strong>the</strong> BSCB, consequently<br />
reducing axonal damage <strong>an</strong>d improving locomotor recovery after SCI, <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e c<strong>an</strong> be<br />
considered as a potential <strong>the</strong>rapeutic strategy <strong>for</strong> SCI patients.<br />
Disclosures: J.C. Lee, None; V.U.L. Mokkapati, None; K.M. Johnson, None; O. Nesic,<br />
None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.17/S14<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Title: Neurotrophic activities of tr<strong>an</strong>spl<strong>an</strong>ted neural progenitor cells expressing mut<strong>an</strong>t NT3 in<br />
injured spinal cord<br />
Authors: K. KUSANO 1 , *M. ENOMOTO 1 , P. TSOULFAS 2 , K. SHINOMIYA 1 , A. OKAWA 1 ;<br />
1 Orthopeadic & Spinal surgery, Tokyo Med. & Dent. Univ., Tokyo, Jap<strong>an</strong>; 2 Univ. of Miami,<br />
Miller Sch. of Medicine, The Miami Project to Cure Paralysis, Miami, FL<br />
Abstract: Neutrotrophin-3 (NT3) plays a protective role in injured CNS tissue through<br />
interaction with trk receptors. Multineurotrophin NT-3/D15A was a modified hum<strong>an</strong> NT-3 that<br />
c<strong>an</strong> bind <strong>an</strong>d activate both trkB <strong>an</strong>d trkC receptors (Urfer et al., EMBO J 13:5896-5909, 1994).<br />
Tr<strong>an</strong>spl<strong>an</strong>tation of NT-3/D15A-expressing glial progenitors in subacute phase leaded to increase<br />
myelin <strong>for</strong>mation <strong>an</strong>d recovery of hindlimb locomotion (Cao et al., J Neurosci 25: 6947-57<br />
2005). We have generated a new mut<strong>an</strong>t NT3 (NT3/D15A/-p75) with a reduced ability to bind to<br />
p75 receptor. We examined whe<strong>the</strong>r tr<strong>an</strong>spl<strong>an</strong>tation of neural progenitor cells (NPCs) expressing<br />
NT3/D15A/-p75 would be a successful <strong>the</strong>rapy <strong>for</strong> <strong>the</strong> chronic phase of spinal cord injury. NPCs<br />
derived <strong>from</strong> E16 rat hippocampi were cultured <strong>an</strong>d labeled with lentivirus vectors containing<br />
GFP (LV-GFP). After a passage <strong>the</strong> cells were infected with LV -NT3/D15A/-p75 <strong>an</strong>d prepared<br />
<strong>for</strong> <strong>the</strong> tr<strong>an</strong>spl<strong>an</strong>tation. Rats were received contusive spinal cord injuries at <strong>the</strong> level of Th9. The<br />
laminectomy site was reexposed 6 weeks after injury <strong>an</strong>d <strong>the</strong> cells with ei<strong>the</strong>r LV-GFP or LV-<br />
NT3/D15A/-p75 (total 1X10 6 cells/4µl) were tr<strong>an</strong>spl<strong>an</strong>ted into <strong>the</strong> contused spinal cord. We<br />
observed <strong>the</strong> rats <strong>for</strong> eight weeks after <strong>the</strong> tr<strong>an</strong>spl<strong>an</strong>tation <strong>an</strong>d scored <strong>the</strong>ir behavior using Basso-<br />
Beattie-Bresnah<strong>an</strong> (BBB) locomotor rating scale every week. Histological evaluation of tissue<br />
damage around <strong>the</strong> injury site employed <strong>the</strong> estimated spinal cord volume, <strong>the</strong> cavity area, <strong>an</strong>d
<strong>the</strong> area of residual myelin in <strong>the</strong> white matter. NT3/D15A/-p75 tr<strong>an</strong>spl<strong>an</strong>ts displayed better<br />
survival th<strong>an</strong> GFP tr<strong>an</strong>spl<strong>an</strong>ts. Moreover, rats with NT3/D15A/-p75 tr<strong>an</strong>spl<strong>an</strong>ts exhibited<br />
enh<strong>an</strong>ced myelin <strong>for</strong>mation <strong>an</strong>d partially improved recovery of hindlimb function. NT3-p75<br />
receptor signaling mediates neural apoptosis <strong>an</strong>d inhibition of axonal regeneration. We suggest<br />
that <strong>the</strong>se effects result <strong>from</strong> <strong>the</strong> absence of negative signaling by NT3-p75 receptor.<br />
Disclosures: K. Kus<strong>an</strong>o, None; M. Enomoto, None; P. Tsoulfas, None; K. Shinomiya,<br />
None; A. Okawa, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.18/S15<br />
Topic: C.10.d. Spinal cord: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: GAPDH mediated apoptosis after spinal cord injury<br />
Authors: *M. L. MATHER, S. CASHA;<br />
Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: S-nitrosylation <strong>an</strong>d nuclear tr<strong>an</strong>slocation of Glyceraldehyde-3-phosphate<br />
dehydrogenase (GAPDH) has been implicated in <strong>the</strong> apoptotic cell death seen in several<br />
neurodegenerative processes. In this study we examined <strong>the</strong> role of GAPDH mediated apoptosis<br />
after spinal cord injury (SCI). Adult female Wistar rats were spinal injured at T8 using <strong>the</strong><br />
Infinite Horizon impactor. Western blotting <strong>an</strong>d biotin switch assay (to isolated nitrosylated<br />
proteins) revealed a small decrease in GAPDH during <strong>the</strong> first 24 hours after SCI followed by a<br />
return to normal levels through 14 days. The proportion of nitrosylated GAPDH was elevated<br />
during <strong>the</strong> first 48 hours after injury with return to levels slightly above baseline subsequently.<br />
Deprenyl (10ug/Kg daily versus saline control) did not ch<strong>an</strong>ge overall GAPDH levels in <strong>the</strong><br />
spinal cord after injury however it reduced <strong>the</strong> nitrosylated portion. In addition, <strong>the</strong> early peak<br />
(24-48 hours) seen in caspase 3 activation (on western blot) was also diminished. TUNEL<br />
positive nuclei in <strong>the</strong> spinal cord counted at <strong>the</strong> injury epicenter <strong>an</strong>d at 200 um rostral <strong>an</strong>d caudal<br />
to <strong>the</strong> injury were also decreased in those <strong>an</strong>imal treated with Deprenyl. Locomotor recovery<br />
after SCI (by BBB locomotor scale <strong>for</strong> 6 weeks) was improved when <strong>an</strong>imals were treated with<br />
Deprenyl (10ug/Kg daily or 1ug/Kg daily x 7 days) versus saline control (P=0.044, repeated<br />
measures ANOVA). Histology examination using Eriochrome staining with Neutral Red counter<br />
stain revealed decreased lesion volume <strong>an</strong>d tissue sparing with 1ug/Kg Deprenyl treatment<br />
(P=0.075). A similar effect was less seen using <strong>the</strong> 10ug/Kg dose. Retrograde florogold tracing
to brainstem nuclei (to assess axonal survival) at 6 weeks showed increased counts of retrograde<br />
labeled neurons with both 1ug/Kg <strong>an</strong>d 10ug/Kg Deprenyl treatment although this was not<br />
statistically signific<strong>an</strong>t. These data support <strong>the</strong> hypo<strong>the</strong>sis that GAPDH nitrosylation is a<br />
secondary injury mech<strong>an</strong>ism that contributes to <strong>the</strong> pathophysiology of SCI which when<br />
inhibited results in neuroprotection.<br />
Disclosures: M.L. Ma<strong>the</strong>r, None; S. Casha, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.19/S16<br />
Topic: C.10.d. Spinal cord: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: New York State Spinal Cord Injury Research Board (CO20927)<br />
The Shrine Hospital <strong>for</strong> Children<br />
Title: Gene expression ch<strong>an</strong>ges in axotomized lateral vestibular neurons in rats<br />
Authors: *M. MURRAY 1 , J. HOULE 1 , K. OGATA 1 , L. SANTI 1 , R. LUTZ 1 , G. BARR 2,3 ;<br />
1 Drexel Univ. Col. Med., Philadelphia, PA; 2 Developmental Neurosci., Columbia U Med. Ctr.,<br />
New York, NY; 3 Anes<strong>the</strong>siol., Children's Hosp. of Philadelphia, Philadelphia, PA<br />
Abstract: Axotomized adult neurons normally do not regenerate but c<strong>an</strong> do so into a permissive<br />
environment (peripheral nerve graft, PNG). Immature neurons have greater potential <strong>for</strong><br />
plasticity/regeneration th<strong>an</strong> adult neurons yet are also more vulnerable to axotomy. After a C3<br />
hemisection (HX), we examined temporal patterns of gene expression in injured lateral<br />
vestibular neurons (LVN). To study <strong>the</strong> effects of <strong>the</strong> environment <strong>an</strong>d age, we lesioned adult<br />
(A-HX), 3-week old (Y-HX) <strong>an</strong>d adult rats that received <strong>an</strong> acute PNG (PNG-HX) to identify<br />
categories of genes associated with ch<strong>an</strong>ges in <strong>the</strong> neuronal response to injury <strong>an</strong>d attempted<br />
repair. LVN neurons were laser-micro dissected at 0 <strong>an</strong>d 4 hours, 1, 3, 7, 14, or 42 days post<br />
injury <strong>an</strong>d prepared <strong>for</strong> microarray <strong>an</strong>alysis. Following preprocessing that included vari<strong>an</strong>ce<br />
filtering, ch<strong>an</strong>ges in gene expression were detected <strong>from</strong> 24 hours to 42 days in all groups with<br />
<strong>the</strong> most pronounced ch<strong>an</strong>ges evident at 3-14 days postlesion. More genes were altered in Y-HX<br />
th<strong>an</strong> in A-HX <strong>an</strong>d PNG-HX <strong>an</strong>imals, but <strong>the</strong>re was greater agreement, with more overlap,<br />
between PNG-HX <strong>an</strong>d Y-HX compared to A- HX. In a first <strong>an</strong>alysis we used k-me<strong>an</strong>s clustering<br />
of signific<strong>an</strong>t genes (maSigPro) to identify coherent clusters. Clusters with signific<strong>an</strong>t
upregulation of genes in <strong>the</strong> A-HX group but not in <strong>the</strong> o<strong>the</strong>r two groups were represented by<br />
cytoskeletal, actin <strong>an</strong>d microtubule genes In <strong>the</strong> Y-HX group, developmental genes were down<br />
regulated after injury. One cluster showed signific<strong>an</strong>t upregulation of mitochondrial genes in<br />
PNG-HX but down regulation in <strong>the</strong> A-HX <strong>an</strong>d Y-HX <strong>an</strong>imals. In a second <strong>an</strong>alysis, we<br />
calculated <strong>the</strong> correlation of gene expression to time <strong>an</strong>d compared overlap among groups in<br />
which genes were up- or down regulated. In general, <strong>an</strong>ti apoptotic genes were upregulated <strong>an</strong>d<br />
genes related to learning <strong>an</strong>d/or memory were down regulated. More signific<strong>an</strong>tly altered genes<br />
were regulated in <strong>the</strong> same direction <strong>for</strong> <strong>the</strong> PNG-HX <strong>an</strong>d Y-HX groups whereas very few genes<br />
were congruent in <strong>the</strong> A-HX. Thus we conclude that axotomy induces a complex temporal<br />
pattern of ch<strong>an</strong>ge in neuronal gene expression <strong>an</strong>d that tr<strong>an</strong>spl<strong>an</strong>tation of peripheral nerve to<br />
promte regeneration results in a pattern of neuronal gene expession more similar to that of<br />
immature neurons.<br />
Disclosures: M. Murray, None; J. Houle, None; K. Ogata, None; L. s<strong>an</strong>ti, None; R. Lutz,<br />
None; G. Barr, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.20/S17<br />
Topic: C.10.d. Spinal cord: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: The Heptagon Award<br />
Title: Zinc distribution in rat spinal cord <strong>an</strong>d response to acute injury<br />
Authors: *S. A. AVERILL, M. ISMAJLI, V. KING, W. HUANG, A. MICHAEL-TITUS;<br />
Ctr. <strong>for</strong> Neurosci. & Trauma, Barts <strong>an</strong>d <strong>the</strong> London Med. Sch., London, United Kingdom<br />
Abstract: Zinc is <strong>the</strong> second most import<strong>an</strong>t trace element in <strong>the</strong> body after iron; it is essential<br />
<strong>for</strong> CNS development <strong>an</strong>d <strong>the</strong> structure <strong>an</strong>d function of a multitude of proteins. A small<br />
percentage of <strong>the</strong> total zinc (~10%) c<strong>an</strong> be found in synaptic vesicles <strong>an</strong>d it is this pool of ionic<br />
or loosely bound zinc that c<strong>an</strong> be visualised by autometallographic techniques.<br />
Traumatic spinal cord injury results in pathophysiological ch<strong>an</strong>ges that extend <strong>from</strong> minutes to<br />
years after <strong>the</strong> injury. In <strong>the</strong> acute phase <strong>the</strong>re are vascular <strong>an</strong>d biochemical alterations, such as<br />
oxidative damage, excitotoxicity <strong>an</strong>d inflammation. The aims of this study were: i) to visualise<br />
zinc in rat spinal cord <strong>an</strong>d dorsal root g<strong>an</strong>glia (DRG) using <strong>the</strong> autometallographic silver<br />
enh<strong>an</strong>cement technique, <strong>an</strong>d ii) to examine ch<strong>an</strong>ges in <strong>the</strong> dynamics of zinc after spinal cord
injury. Naive adult Wistar <strong>an</strong>d Sprague-Dawley male <strong>an</strong>d female rats were injected with sodium<br />
selenite (Na2SeO3) (10 mg/kg i.p.) at 2.5 hours, 6 hours <strong>an</strong>d 24 hours be<strong>for</strong>e sacrifice in order to<br />
visualise zinc pathways. At shorter time points (2.5 <strong>an</strong>d 6 hours), Na2SeO3 injection produced<br />
only neuropil staining in <strong>the</strong> spinal cord, with cellular staining as well as satellite/glial cell<br />
staining in <strong>the</strong> DRG (2.5 hours). 24 hours after Na2SeO3 injection, <strong>the</strong>re was neuronal zinc<br />
staining in all <strong>the</strong> tissues <strong>an</strong>alysed. At this time point, zinc staining was seen mainly in smallmedium<br />
sized IB4-positive DRG cells. After a hemisection or compression injury of <strong>the</strong> spinal<br />
cord at thoracic T12 level, between 3h <strong>an</strong>d 48h <strong>the</strong>re was <strong>an</strong> accumulation of zinc around <strong>the</strong> site<br />
of injury, <strong>an</strong>d zinc-containing axonal end bulbs were also observed. To validate <strong>the</strong> zinc staining<br />
methodology we injected clioquinol, a known chelator of zinc (50 mg/kg i.p.) 30 min after<br />
injury, <strong>an</strong>d this largely abolished <strong>an</strong>y zinc labelling. Our results confirm previous studies, that<br />
<strong>the</strong> autometallographic technique c<strong>an</strong> be used to visualise zinc pathways in <strong>the</strong> nervous system,<br />
<strong>an</strong>d show ch<strong>an</strong>ges in zinc distribution following acute mech<strong>an</strong>ical trauma of <strong>the</strong> spinal cord.<br />
Disclosures: S.A. Averill, None; M. Ismajli, None; V. King, None; W. Hu<strong>an</strong>g, None; A.<br />
Michael-Titus, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.21/S18<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NIH-NO1-NS-3-2353<br />
Rom<strong>an</strong> Reed Spinal Cord Injury Research Fund of Cali<strong>for</strong>nia<br />
Title: Bladder impairment varies with comparable spinal cord injuries at different spinal levels<br />
Authors: *B. T. DAVID, O. STEWARD;<br />
Univ. Cali<strong>for</strong>nia, Irvine, Irvine, CA<br />
Abstract: Loss of bladder function is regarded as one of <strong>the</strong> most import<strong>an</strong>t consequences of a<br />
spinal cord injury (SCI) but is rarely assessed in <strong>an</strong>imal studies of SCI. Here, we use a simple<br />
outcome measure (volume of retained urine) to assess bladder dysfunction over time following<br />
spinal cord injuries at 3 different thoracic levels. Three separate experiments (n=30) compared<br />
bladder function following moderate contusion lesions (200 kDyne <strong>for</strong>ce injuries produced with<br />
<strong>the</strong> Infinite Horizon device) at T1, T4, or T9. Two follow-up experiments (n=37) were <strong>the</strong>n
carried out to confirm differences in bladder function following lesions at <strong>the</strong> different levels. An<br />
additional experiment (n=14) was per<strong>for</strong>med to assess differences in bladder function following<br />
crush injuries at T1 or T9. The volume of urine retained in <strong>the</strong> bladder was measured by<br />
<strong>an</strong>es<strong>the</strong>tizing <strong>the</strong> <strong>an</strong>imals with isoflur<strong>an</strong>e, expressing <strong>the</strong> bladder, <strong>an</strong>d weighing <strong>the</strong> urine daily<br />
<strong>for</strong> fourteen days. To compare bladder deficits with <strong>the</strong> degree of impairment of hindlimb motor<br />
function, locomotion was assessed using <strong>the</strong> BBB open field rating scale. Rats with contusions at<br />
T4 <strong>an</strong>d T9 exhibited bladder impairments reflected by increased urine retention <strong>from</strong> 1-12 days<br />
post injury. In contrast, rats with contusions at T1 exhibited minimal deficits (smaller volumes of<br />
retained urine). Lesion size <strong>an</strong>d overall functional impairment was comparable between groups<br />
based on qu<strong>an</strong>titative assessments of lesion area at <strong>the</strong> epicenter <strong>an</strong>d BBB locomotor scores.<br />
Moreover, a radial <strong>an</strong>alysis of sparing of different portions of <strong>the</strong> white matter revealed no<br />
differences in sparing of different funiculi between <strong>the</strong> groups. Retrograde labeling with<br />
Fluorogold, however, did demonstrate greater labeling of <strong>the</strong> pontine micturition center (PMC)<br />
following T1 injury when compared to T4 or T9. Possible expl<strong>an</strong>ations <strong>for</strong> <strong>the</strong> relative sparing of<br />
bladder function following contusion injuries at T1 include sparing of a critical descending<br />
pathway or sparing of a spinal circuit between T1 <strong>an</strong>d T4 that is able to mediate reflex voiding in<br />
rats.<br />
Disclosures: B.T. David, None; O. Steward, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.22/T1<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: Swiss National Science Foundation (NCCR NO PBFRB--120920)<br />
Schweizerische Stiftung für Medizinisch-Biologische Stipendien (SSMBS)<br />
Swiss Paraplegic Research, Nottwill, Switzerl<strong>an</strong>d<br />
Wellcome Trust Centre <strong>for</strong> Neuroimaging, UCL Institute of Neurology<br />
Title: Chronic spinal cord injury results in cervical atrophy <strong>an</strong>d topographical reorg<strong>an</strong>ization of<br />
affected upper limb muscles during h<strong>an</strong>dgrib
Authors: *P. FREUND 1 , N. WARD 2 , O. CICCARELLI 1 , K. FRISTON 3 , M. CRAGGS 4 , N.<br />
WEISKOPF 3 , A. THOMPSON 1 ;<br />
1 Brain Repair <strong>an</strong>d Rehabil., 2 Sobell Dept. of Motor Neurosci., 3 Wellcome Trust Ctr. <strong>for</strong><br />
Neuroimaging, UCL, Institue of Neurol., London, United Kingdom; 4 Spinal Cord Injury Ctr.,<br />
Royal Natl. Orthopedic Hosp. St<strong>an</strong>more, UCL, United Kingdom<br />
Abstract: Background: After spinal cord injury (SCI), deafferentation of <strong>the</strong> body caudal to <strong>the</strong><br />
lesion c<strong>an</strong> induce ch<strong>an</strong>ges within central somatosensory <strong>an</strong>d motor body representation. The aim<br />
of this study was to explore functional brain reorg<strong>an</strong>isation <strong>an</strong>d structural spinal cord ch<strong>an</strong>ges in<br />
SCI patients with bilateral upper limb impairment, using functional <strong>an</strong>d structural MRI.<br />
Methods: We recruited nine volunteers with a chronic traumatic SCI with a me<strong>an</strong> history of<br />
injury of 13 years <strong>an</strong>d 13 healthy age matched control subjects. Upper limb impairment was<br />
assessed using <strong>the</strong> 9-hole-peg test (9HPT). In all particip<strong>an</strong>ts, <strong>the</strong> cross-sectional cord area was<br />
measured at cervical level C2, which was rostral to <strong>the</strong> injury in SCI subjects. During fMRI<br />
active blocks consisted of right sided (i) repetitive isometric h<strong>an</strong>dgrip, (ii) medi<strong>an</strong> or (iii) tibial<br />
nerve stimulation. Six repetitions of each 20s block were per<strong>for</strong>med in a r<strong>an</strong>domised order,<br />
alternating with rest blocks. Contrast images representing h<strong>an</strong>dgrip compared to rest were<br />
generated <strong>an</strong>d used in a second level two sample t-test (in SPM5) to identify regions of altered<br />
task-related brain activation in SCI subjects when compared with controls. Results: SCI subjects<br />
had reduced cross sectional cord area (45 mm² vs 74 mm², p< 0.001) <strong>an</strong>d impairment on <strong>the</strong><br />
9HPT using <strong>the</strong> grip h<strong>an</strong>d (119.3 sec vs. 16.6 sec, p=0.003) when compared to controls. In SCI<br />
subjects, <strong>the</strong> cord area correlated with 9HPT scores (r= - 0.91, p = 0.001). Analysis of <strong>the</strong><br />
imaging data revealed relative increased task-related brain activity during h<strong>an</strong>dgrip in primary<br />
motor cortex (defined as <strong>the</strong> leg area) as well as in cerebellum (lobule VI) bilaterally (cluster<br />
level, p< 0.05, corrected). There were no relative increases in activation in controls compared to<br />
SCI subjects.<br />
Discussion: The results indicate that a traumatic SCI induces severe cervical atrophy,<br />
presumably reflecting axonal loss. The reduced cortical output, triggered through axonal loss,<br />
may explain <strong>the</strong> task-related topographical reorg<strong>an</strong>ization of <strong>the</strong> representation of <strong>the</strong> impaired<br />
upper limb muscles. These results enable us to next characterise SCI-effects of peripheral<br />
stimulation on cortical responses in grip-related areas; both in terms of regional responses <strong>an</strong>d<br />
effective connectivity.<br />
Disclosures: P. Freund, None; N. Ward, None; O. Ciccarelli, None; K. Friston, None; M.<br />
Craggs, None; N. Weiskopf, None; A. Thompson, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 542.23/T2<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: Wings <strong>for</strong> Life Foundation<br />
Title: The evaluation of minocycline <strong>an</strong>d simvastatin in a rodent model of acute unilateral<br />
cervical spinal cord injury<br />
Authors: J. H. T. LEE 1 , J. LIU 1 , A. T. STAMMERS 1 , *E. B. OKON 3 , W. TETZLAFF 1 , B. K.<br />
KWON 1,2 ;<br />
1 ICORD (International Collaboration On Repair Discovery), 2 Orthopaedics, Univ. of British<br />
Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada; 3 Univ. British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: Background: Minocycline (Mino), a commonly prescribed <strong>an</strong>tibiotic, has shown<br />
promise as a potential <strong>the</strong>rapeutic agent in <strong>an</strong>imal models of a variety of neurologic disorders<br />
such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, Huntington’s<br />
disease, stroke, <strong>an</strong>d spinal cord injury (SCI). Simvastatin is one of a number of ,<br />
hydroxymethylglutaryl-coenzyme-A reductase inhibitors prescribed to lower cholesterol. These<br />
drugs are also known to be <strong>an</strong>ti-inflammatory, reduce oxidative stress, improve endo<strong>the</strong>lial<br />
function <strong>an</strong>d modulate <strong>the</strong> immune system in stroke, traumatic brain injury <strong>an</strong>d SCI. As both<br />
drugs have tr<strong>an</strong>slational potential, we evaluate <strong>the</strong>m here in a clinically relev<strong>an</strong>t model of<br />
contusive cervical spinal cord injury.<br />
Objective: To investigate <strong>the</strong> neuroprotective properties of simvastatin <strong>an</strong>d minocycline in acute<br />
traumatic cervical SCI.<br />
Methods: Sprague Dawley rats underwent a unilateral cervical contusion SCI at C5, <strong>an</strong>d <strong>the</strong>n<br />
were r<strong>an</strong>domized to receive: 1. Saline, 2. Simvastatin 20 mg/kg x 7 days, 3. Simvastatin 20<br />
mg/kg x 7 days <strong>the</strong>n 5 mg/kg x 35 days, 4. Minocycline 90 mg/kg x 3 days. Behavior recovery<br />
was assessed over 6 weeks using Cylinder rearing test, Horizontal Ladder test, Catwalk test,<br />
Montoya’s staircase <strong>an</strong>d grooming test. Forepaw sensitivity was assessed using <strong>the</strong> electronic<br />
von Frey Aes<strong>the</strong>siometer. For histology, <strong>the</strong> corticospinal <strong>an</strong>d rubrospinal tract was traced <strong>an</strong>d<br />
<strong>the</strong> spinal cords were harvested 2 months after injury. The extent of glial scarring, grey matter<br />
<strong>an</strong>d white matter sparing <strong>an</strong>d <strong>the</strong> number of motor neurons <strong>an</strong>d myelinated axons were evaluated<br />
in cross sections.<br />
Results: Behaviorally, <strong>the</strong>re were no signific<strong>an</strong>t differences between Saline, Mino, Sim 7 <strong>an</strong>d<br />
Sim 42 in <strong>the</strong> Cylinder rearing test, Horizontal ladder test <strong>an</strong>d sensory test. Sim 7 group<br />
signific<strong>an</strong>tly retrieved less pellets compared to Saline <strong>an</strong>d Mino groups in <strong>the</strong> staircase test<br />
(p=0.002); however, in <strong>the</strong> Grooming test Mino, Sim 7 <strong>an</strong>d Sim 42 groups scored signific<strong>an</strong>tly<br />
higher th<strong>an</strong> <strong>the</strong> Saline group at 2 weeks (p
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.24/T3<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: NS041548<br />
HD058412<br />
Title: Exercise <strong>the</strong>rapy implemented during <strong>the</strong> acute phase of SCI facilitates functional<br />
recovery<br />
Authors: A. K. BROWN 1 , S. A. WOLLER 2 , G. L. MORENO 2 , J. W. GRAU 2 , *M. HOOK 2 ;<br />
1 Emporia State Univ., Emporia, KS; 2 Texas A&M, College Station, TX<br />
Abstract: Behavioral training is one of <strong>the</strong> most successful strategies currently available <strong>for</strong><br />
facilitating recovery of function (motor <strong>an</strong>d sensory) after a spinal cord injury (SCI). In <strong>the</strong><br />
development of <strong>the</strong>se strategies, however, <strong>the</strong>re is still controversy regarding <strong>the</strong> timing of<br />
initiation of treatment <strong>for</strong> maximal recovery benefits. Studies of patients with SCI suggest that<br />
<strong>the</strong> longer <strong>the</strong> interval between injury <strong>an</strong>d <strong>the</strong> onset of treatment, <strong>the</strong> greater <strong>the</strong> motor gains.<br />
However, m<strong>an</strong>y laboratory studies with rats show that <strong>the</strong> spinal cord is quite plastic <strong>an</strong>d<br />
improvements in function are observed regardless of whe<strong>the</strong>r treatment begins immediately after<br />
<strong>an</strong> injury or several months later.<br />
The present study compares <strong>the</strong> effects of exercise initiated in <strong>the</strong> acute <strong>an</strong>d chronic phases of<br />
SCI. Subjects received a moderate spinal contusion injury <strong>an</strong>d beg<strong>an</strong> a voluntary exercise<br />
program 24 hours post-injury (D1) or 7 days later (D7). They were individually placed into<br />
tr<strong>an</strong>sparent exercise balls (22.86 cm to 29.21 cm in diameter) <strong>for</strong> 60 minutes of voluntary<br />
exercise. Exercise sessions occurred during <strong>the</strong> dark cycle (21:00-22:00 hrs) <strong>for</strong> 14 consecutive<br />
days. A control group was also placed into <strong>the</strong> exercise balls <strong>for</strong> 14 days post-injury, but <strong>the</strong>y<br />
were unable to roll around in <strong>the</strong> balls (balls were placed in a fixed position). Motor <strong>an</strong>d sensory<br />
recovery were assessed <strong>for</strong> 28 days after injury (including <strong>the</strong> exercise periods).<br />
The results indicate that D1 subjects recovered signific<strong>an</strong>tly more locomotor function (BBB<br />
scale) th<strong>an</strong> controls <strong>an</strong>d D7 subjects. Initiation of exercise 7 days after injury did not facilitate<br />
locomotor recovery: BBB scores were similar <strong>for</strong> <strong>the</strong> D7 <strong>an</strong>d control subjects. Analyses of tactile<br />
reactivity also revealed a main effect of exercise 21-days post-injury. Subjects in <strong>the</strong> D7 group<br />
showed signific<strong>an</strong>tly lower tactile reactivity thresholds th<strong>an</strong> both control <strong>an</strong>d D1 exercise<br />
subjects. Lower reactivity thresholds may be indicative of <strong>the</strong> development of allodynia in <strong>the</strong><br />
D7 subjects. Trend <strong>an</strong>alyses also revealed a signific<strong>an</strong>t linear relationship <strong>for</strong> measures of lesion<br />
size caudal to <strong>the</strong> injury center. Subjects in <strong>the</strong> D7 exercise group had signific<strong>an</strong>tly larger areas
of damage across spinal sections that were 600-2400 µm caudal to <strong>the</strong> injury center, compared<br />
with <strong>the</strong> D1 subjects. Overall, <strong>the</strong>se results indicate that implementing <strong>an</strong> exercise regimen in <strong>the</strong><br />
acute phase of SCI maximizes <strong>the</strong> potential <strong>for</strong> recovery of function.<br />
Disclosures: A.K. Brown, None; S.A. Woller, None; G.L. Moreno, None; J.W. Grau,<br />
None; M. Hook, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.25/T4<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: Mike Utley Foundation<br />
Naylor Family Foundation<br />
Title: Induced pluripotent stem cell-derived astrocytes as a resource <strong>for</strong> spinal cord lesion repair<br />
<strong>an</strong>d plasticity<br />
Authors: *S. NUTT 1 , S. SUHR 2 , Y. CHEN 2 , J. CIBELLI 2 , P. HORNER 1 ;<br />
1 Inst. For Stem Cell <strong>an</strong>d Regenerative Med., Seattle, WA; 2 Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing,<br />
MI<br />
Abstract: Astrocytes represent a morphologically <strong>an</strong>d functionally diverse population of cells in<br />
<strong>the</strong> central nervous system (CNS). A particular subset of <strong>the</strong>se cells, reactive astrocytes, <strong>for</strong>ms in<br />
response to all CNS insults, including spinal cord injury. Reactive astrocytes provide <strong>an</strong> innate<br />
defense mech<strong>an</strong>ism separating spared <strong>from</strong> damaged nervous tissue, <strong>the</strong>reby limiting <strong>the</strong> extent<br />
of injury. While <strong>the</strong> <strong>for</strong>mation of a scar is life preserving, <strong>the</strong> long-term mainten<strong>an</strong>ce of <strong>the</strong> glial<br />
scar is arguably <strong>the</strong> strongest impediment to axon regeneration <strong>an</strong>d functional recovery. In<br />
contrast to injury of <strong>the</strong> adult spinal cord, embryonic injuries have limited scarring <strong>an</strong>d hence<br />
increased regeneration. Increased regeneration in <strong>the</strong> embryonic spinal cord is attributed, in part,<br />
to astrocytes that not only react to injury but also provide a substrate <strong>for</strong> regeneration.<br />
Surprisingly little is known regarding <strong>the</strong> heterogeneity of astrocytes with respect to<br />
differentiation pathways <strong>an</strong>d function. Our lab has spent considerable time exploring <strong>the</strong><br />
subtypes of astrocytes <strong>an</strong>d <strong>the</strong> signals that direct astrocyte differentiation <strong>from</strong> adult neural<br />
progenitor cells (NPCs). In <strong>the</strong>ory, astrocytes c<strong>an</strong> be produced <strong>from</strong> adult stem cells that c<strong>an</strong><br />
serve as a tr<strong>an</strong>spl<strong>an</strong>table substrate <strong>for</strong> regeneration in <strong>the</strong> setting of hum<strong>an</strong> spinal cord injury.
Work has already been initiated in our laboratory in murine models to investigate this effect. The<br />
recent achievement of generating induced pluripotent stem cells (iPS) <strong>from</strong> hum<strong>an</strong> fibroblasts<br />
has created a unique opportunity to investigate post-injury astrocyte behavior in a m<strong>an</strong>ner more<br />
directly applicable to hum<strong>an</strong>s. Fibroblasts are reprogrammed into iPS cells via infection with<br />
lentiviral constructs encoding OCT4, LIN28, NANOG, <strong>an</strong>d SOX2. Our research tests <strong>the</strong> ability<br />
of astrocytes to be generated <strong>from</strong> iPS cells through multiple signaling pathways (BMP4 or<br />
CNTF <strong>an</strong>d LIF) <strong>an</strong>d that <strong>the</strong>y will differ both in vitro <strong>an</strong>d in vivo with respect to <strong>the</strong>ir abilities to<br />
support axon regeneration. In vitro, <strong>the</strong>se differences are demonstrated with respect to <strong>the</strong> ability<br />
of subtypes of astrocytes to support axonal growth in microfluidic compartmented neuron culture<br />
chambers. In vivo, we test <strong>the</strong> astrocytes’ differential capacity to support axon regeneration in a<br />
defined lesion in <strong>the</strong> rubrospinal tract, where histological observations are correlated with two<br />
behavioral assays: <strong>the</strong> pellet reaching task, <strong>an</strong>d <strong>the</strong> cylinder test.<br />
Disclosures: S. Nutt, None; S. Suhr, None; Y. Chen, None; J. Cibelli, None; P. Horner,<br />
None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.26/T5<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: CSN<br />
CIHR<br />
Title: Development of a chronic porcine model of spinal cord injury<br />
Authors: *J. E. HILLYER, C. JONES, J. LEE, R. HILDEBRANDT, G. GRAY, T. GODBEY,<br />
B. KWON, B. KWON;<br />
ICORD, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: In <strong>an</strong> ef<strong>for</strong>t to create a large-<strong>an</strong>imal, non-primate model to serve as <strong>an</strong> intermediary<br />
testing ground between rodent models <strong>an</strong>d hum<strong>an</strong> patients in <strong>the</strong> clinical tr<strong>an</strong>slation of promising<br />
new <strong>the</strong>rapies, we have begun developing a porcine model of spinal cord injury. For cell<br />
tr<strong>an</strong>spl<strong>an</strong>tation technologies in particular, <strong>the</strong> spatial considerations of a cellular graft within a<br />
hum<strong>an</strong> spinal cord are more closely resembled in a pig spinal cord th<strong>an</strong> in a rodent/mouse cord.<br />
Although pigs are commonly used in biomedical research because of <strong>the</strong> <strong>an</strong>atomic <strong>an</strong>d
physiologic characteristics <strong>the</strong>y share with hum<strong>an</strong>s, <strong>the</strong>re has been little work done using pigs <strong>for</strong><br />
investigations of recovery after spinal cord injury.<br />
The Yucat<strong>an</strong> pig was chosen as <strong>the</strong> optimal breeds due to size/weight constraints <strong>an</strong>d our ability<br />
to obtain <strong>the</strong>m in a disease-free state. Female Yucat<strong>an</strong>s weighing approximately 20-25 kg<br />
underwent ei<strong>the</strong>r a 50g or 100g weight drop injury at T10, <strong>from</strong> a height of 50 cm. Static<br />
compression was applied <strong>for</strong> 5 minutes after <strong>the</strong> actual impact.<br />
Bladder ca<strong>the</strong>terization was achieved pre-operatively <strong>an</strong>d maintained post-operatively until<br />
reflexive emptying was achieved. Post-operative morbidity in <strong>the</strong> first 24-48 hours required<br />
intensive <strong>an</strong>imal care, but <strong>the</strong>reafter <strong>the</strong> <strong>an</strong>imals were very m<strong>an</strong>ageable. Functional recovery<br />
measures included a gross locomotor scale (written by Zurita, et al, 2008), a step/ obstacle test,<br />
<strong>an</strong>d <strong>an</strong> inclined pl<strong>an</strong>e test. Additional health issues included delayed recovery of reflexive<br />
bladder, urinary tract infections, skin infections, <strong>an</strong>d pressure sores. Only one <strong>an</strong>imal was<br />
euth<strong>an</strong>ized prior to pl<strong>an</strong>ned end-point because of complications <strong>from</strong> surgery. Histological<br />
<strong>an</strong>alysis has shown that <strong>the</strong> injury spreads approximately 1cm both rostral <strong>an</strong>d caudal <strong>from</strong> <strong>the</strong><br />
epicenter <strong>an</strong>d produces several areas of hemorrhage along <strong>the</strong> cord.<br />
Our preliminary work indicates that a porcine model of SCI is indeed feasible, based on <strong>the</strong><br />
m<strong>an</strong>ageable morbidity/mortality in <strong>the</strong> acute post-injury phase. We <strong>an</strong>ticipate utilizing this model<br />
as a testing ground <strong>for</strong> <strong>the</strong>rapies that show promise in rodent models of SCI.<br />
Disclosures: J.E. Hillyer, None; C. Jones, None; J. Lee, None; R. Hildebr<strong>an</strong>dt, None; G.<br />
Gray, None; T. Godbey, None; B. Kwon, None; B. Kwon, C<strong>an</strong>adi<strong>an</strong> Stem Cell Network,<br />
CIHR, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts<br />
as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.27/T6<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: CIHR Gr<strong>an</strong>t MOP-84375<br />
NSERC Gr<strong>an</strong>t EQPEQ 360283-08<br />
Title: A novel pig model to measure cerebrospinal fluid pressures during a spinal cord injury<br />
event
Authors: C. F. JONES 1 , *B. K. KWON 2 , P. A. CRIPTON 1 ;<br />
1 ICORD Orthopaedic & Injury Biomech<strong>an</strong>ics Group, Departments of Mech<strong>an</strong>ical Engin. &<br />
Orthopaedics, Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada; 2 Orthopaedics, Univ. British<br />
Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: Introduction: The hum<strong>an</strong> spinal cord is surrounded by cerebrospinal fluid (CSF)<br />
which may influence how <strong>for</strong>ces are imparted to <strong>the</strong> spinal cord when <strong>the</strong> surrounding spinal<br />
column fractures/dislocates. However, current rodent, computational <strong>an</strong>d cadaver SCI models do<br />
not consider this fluid compartment <strong>an</strong>d how it may influence <strong>the</strong> <strong>for</strong>ces tr<strong>an</strong>sferred <strong>from</strong> <strong>the</strong><br />
spinal column to <strong>the</strong> spinal cord during traumatic SCI. We have developed a porcine model of<br />
SCI in which CSF pressure is directly measured at several locations in <strong>the</strong> intra<strong>the</strong>cal space<br />
during <strong>the</strong> contusion injury event.<br />
Methods: A contusion SCI was induced by dropping <strong>an</strong> instrumented 50 g (n=6) or 100 g (n=3)<br />
weight with 3/8” spherical tip <strong>from</strong> a height of 50 cm onto <strong>the</strong> exposed spinal cord (T10) of<br />
<strong>an</strong>es<strong>the</strong>tized Yucat<strong>an</strong> pigs (22-26 kg, female). A load cell mounted on <strong>the</strong> weight measured <strong>the</strong><br />
weight-to-spinal cord contact load, <strong>an</strong>d weight velocity <strong>an</strong>d cord displacement were determined<br />
<strong>from</strong> high speed video captured at 5 kHz. Miniature pressure sensors (Preclin 420LP, Samba<br />
Sensors, Sweden) were inserted into <strong>the</strong> intra<strong>the</strong>cal space cr<strong>an</strong>ial <strong>an</strong>d caudal to <strong>the</strong> injury site to<br />
measure CSF pressure during <strong>the</strong> injury. All data were sampled at 40 kHz. The pigs were<br />
<strong>an</strong>aes<strong>the</strong>tized <strong>an</strong>d received <strong>an</strong>algesics throughout. Four pigs were euth<strong>an</strong>ized post-procedure <strong>an</strong>d<br />
five were enrolled in a functional recovery study. The protocol was approved by <strong>the</strong> UBC<br />
Animal Care Committee.<br />
Results: Eight <strong>an</strong>imals were stable throughout <strong>an</strong>d survived to <strong>the</strong> prescribed endpoint. One<br />
<strong>an</strong>imal died during recovery. The me<strong>an</strong> impact velocity was 2.3 m/s (SD 0.4, n=6) <strong>an</strong>d 2.8 m/s<br />
(SD 0.1, n=2) <strong>for</strong> <strong>the</strong> 50g <strong>an</strong>d 100 g weights respectively. Maximum spinal cord/dura<br />
displacement was 7.6 mm (SD 1.4) <strong>an</strong>d 8.6 mm (SD 0.8) <strong>for</strong> <strong>the</strong> two injury severities, with<br />
corresponding peak loads of 44.8 N (SD 6.8, n=4) <strong>an</strong>d 101 N (n=1). CSF pressure data was<br />
obtained at 1, 2 or 3 sites in each <strong>an</strong>imal. The peak positive CSF pressure, zeroed to <strong>the</strong> preinjury<br />
pressure, was 69.4 mmHg (SD 59.4, 9 measurements) <strong>for</strong> 50 g <strong>an</strong>d 85.7 mmHg (SD 65.1,<br />
5 measurements) <strong>for</strong> 100 g. Negative relative pressure troughs of lower magnitude were also<br />
observed in most <strong>an</strong>imals.<br />
Discussion: This model is <strong>the</strong> first to directly measure intra<strong>the</strong>cal CSF pressure during a SCI<br />
event, with minimal alteration of <strong>the</strong> fluid space. Because <strong>the</strong> CSF distribution much more<br />
closely resembles <strong>the</strong> hum<strong>an</strong> condition, <strong>the</strong> Yucat<strong>an</strong> pig may be <strong>an</strong> appropriate experimental<br />
model of SCI. The pressures observed were of similar magnitude to that measured in a single cat<br />
by Hung et al. (1975), <strong>an</strong>d considerably higher th<strong>an</strong> baseline physiologic pressures. This data<br />
may be used to verify future SCI models that seek to incorporate <strong>the</strong> effects of <strong>the</strong> intra<strong>the</strong>cal<br />
space.<br />
Disclosures: C.F. Jones, None; B.K. Kwon, None; P.A. Cripton, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.28/T7<br />
Topic: C.10.e. Spinal cord: Animal models <strong>an</strong>d hum<strong>an</strong> studies<br />
Support: Taipei Veter<strong>an</strong>s General Hospital<br />
Title: Cortical neuronal reorg<strong>an</strong>ization after spinal cord injury in monkeys<br />
Authors: *J. HE, H. ZHANG, C. MA;<br />
Arizona State Univ., Tempe, AZ<br />
Abstract: In this study, we developed <strong>an</strong> effective approach <strong>for</strong> treating spinal cord injury (SCI)<br />
subjects <strong>an</strong>d discovered <strong>the</strong> cortical neuronal reorg<strong>an</strong>ization <strong>for</strong> voluntary bipedal walking after<br />
SCI in a nonhum<strong>an</strong> primate model. Two rhesus monkeys received unilateral surgical hemisection<br />
at <strong>the</strong> T8 spine level (3-5 mm gap), <strong>an</strong>d were tr<strong>an</strong>spl<strong>an</strong>ted with sural nerve segments as<br />
well as infused acidic fibroblast growth factor to injured spinal cord sites. Four 16-ch<strong>an</strong>nel<br />
microelectrode arrays were plugged into <strong>the</strong> primary motor cortical areas related to lower limb<br />
movement, <strong>an</strong>d cortical neuronal signals were recorded while <strong>the</strong>y per<strong>for</strong>med <strong>the</strong> bipedal<br />
walking on <strong>the</strong> treadmill. The cortical neuronal reorg<strong>an</strong>ization was measured by <strong>the</strong> synchrony<br />
ch<strong>an</strong>ge among <strong>the</strong> detected neuronal ensemble. The function of bipedal walking started to<br />
recover 4 weeks post surgery, <strong>an</strong>d continued to progress till 14 weeks post surgery when we<br />
stopped recording. Comparing <strong>the</strong> neural population activity patterns of monkeys be<strong>for</strong>e <strong>an</strong>d<br />
after SCI, we identified a signific<strong>an</strong>t increase in <strong>the</strong> synchrony among <strong>the</strong> recorded cortical<br />
neuronal population. The synchrony continued to increase till 14 weeks after SCI. This indicates<br />
<strong>the</strong> existence of cortical neuronal reorg<strong>an</strong>ization during <strong>the</strong> functional recovery. This in<strong>for</strong>mation<br />
would be critical <strong>for</strong> <strong>the</strong> development of effective <strong>an</strong>d practical techniques to harness <strong>the</strong> cortical<br />
adaptation or plasticity after spinal cord injury to promote voluntary control of lower limb<br />
functions or neuropros<strong>the</strong>tics <strong>for</strong> SCI victims who are reli<strong>an</strong>t on wheelchair.<br />
Disclosures: J. He, None; H. Zh<strong>an</strong>g, None; C. Ma, None.<br />
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.29/T8
Topic: C.10.d. Spinal cord: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t NS046724<br />
NIH Gr<strong>an</strong>t NS07144<br />
NIH Gr<strong>an</strong>t GM007108<br />
The International Foundation <strong>for</strong> Research on Paraplegia<br />
The Mary Gates Endowment<br />
ARCS<br />
Title: Delivery of Neuregulin-1 type III, but not type I, results in increased oligogenesis <strong>an</strong>d<br />
thicker myelin on spared axons after spinal cord injury<br />
Authors: *B. E. JACOBSON, J. LASIENE, B. M. LILLENESS, P. A. SETRAN, P. J.<br />
HORNER;<br />
Univ. Washington, Seattle, WA<br />
Abstract: Incomplete traumatic spinal cord injury (SCI) produces spared pathways containing<br />
fragmented or abnormally thin myelin. Physiological evidence indicates that inadequate<br />
remyelination after injury causes failed or sluggish conduction in spared pathways, making<br />
myelin production <strong>an</strong> import<strong>an</strong>t <strong>the</strong>rapeutic target. Neuregulin-1 (NRG1) type I <strong>an</strong>d type III are<br />
<strong>the</strong> only growth factors shown to specifically alter myelin thickness in development <strong>an</strong>d we<br />
hypo<strong>the</strong>sized that <strong>the</strong>y might also do so after adult SCI. In support of this concept, we found<br />
signific<strong>an</strong>tly decreased NRG1 type III gene expression <strong>an</strong>d early peaks followed by decreased<br />
NRG1 type I expression in mouse motor cortices <strong>an</strong>d lesion epicenters following SCI. To<br />
determine <strong>the</strong> <strong>an</strong>atomical effects of enh<strong>an</strong>cing NRG1 availability after injury, we impl<strong>an</strong>ted<br />
mini-osmotic pumps to intra<strong>the</strong>cally deliver ei<strong>the</strong>r NRG1 type I or type III hum<strong>an</strong> recombin<strong>an</strong>t<br />
protein <strong>for</strong> 14 days in a model of moderate thoracic spinal contusion injury. Infusion of NRG1<br />
type I led to increased cell proliferation at <strong>the</strong> lesion epicenter <strong>an</strong>d surprisingly, thinner myelin<br />
compared to controls 15 days post-injury (p.i.). In contrast, NRG1 type III infusion led to<br />
increased proliferation, a decreased percentage of newly generated NG2+ progenitor cells <strong>an</strong>d<br />
increased density of new Olig2+/CC1+ oligodendrocytes by 15 days p.i. Import<strong>an</strong>tly, NRG1 type<br />
III administration also resulted in thicker myelin on spared axons at both 15 <strong>an</strong>d 52 days p.i.<br />
Nei<strong>the</strong>r iso<strong>for</strong>m signific<strong>an</strong>tly altered gross locomotor behavior. Our results demonstrate that<br />
infused NRG1 type III, but not type I, supports generation of new cells committed to <strong>the</strong><br />
oligodendrocyte lineage <strong>an</strong>d thicker myelin after SCI.<br />
Disclosures: B.E. Jacobson, None; J. Lasiene, None; B.M. Lilleness, None; P.A. Setr<strong>an</strong>,<br />
None; P.J. Horner, None.
Poster<br />
542. Spinal Cord Injury: From Animals to Hum<strong>an</strong>s...<strong>an</strong>d Back Again II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 542.30/T9<br />
Topic: C.10.f. Spinal cord: Therapeutic strategies<br />
Support: NS 059776.<br />
Title: Administration of <strong>the</strong> PPAR-α agonist gemfibrozil deteriorates functional <strong>an</strong>d <strong>an</strong>atomical<br />
outcomes following spinal cord injury (SCI)<br />
Authors: *A. A. ALMAD 1 , Y. YANG 2 , A. LOVETT-RACKE 3 , D. MCTIGUE 4 ;<br />
1 Ohio State Univ., columbus, OH; 2 Dept. of Neurol., 3 Dept. of Mol. Virology, Immunology, <strong>an</strong>d<br />
Med. Genet., 4 Dept. of Neurosci., Ohio State Univ., Columbus, OH<br />
Abstract: Peroxisome proliferator activated receptors (PPAR) are members of <strong>the</strong> nuclear<br />
hormone receptor family known <strong>for</strong> <strong>the</strong>ir role in body metabolism. PPAR-α, <strong>an</strong> isotype of PPAR<br />
family involved in lipid metabolism, has been shown to modulate neuroinflammation. Agonists<br />
of PPAR-α inhibit pro-inflammatory cytokine production <strong>an</strong>d improve clinical scores in models<br />
of multiple sclerosis (experimental autoimmune encephalomyelitis) <strong>an</strong>d o<strong>the</strong>r neurodegenerative<br />
diseases such as Alzheimer’s disease. Spinal cord injury (SCI) afflicts 11,000 individuals<br />
<strong>an</strong>nually in United States with no accepted <strong>the</strong>rapeutic treatments available yet. SCI has a large<br />
inflammatory component involved in <strong>the</strong> progression of <strong>the</strong> lesion <strong>an</strong>d altering this response<br />
could lead to better functional recovery. Based on this hypo<strong>the</strong>sis, a T9 contusion injury was<br />
per<strong>for</strong>med on C57/BL6 mice <strong>an</strong>d <strong>the</strong> <strong>an</strong>imals were r<strong>an</strong>domly assigned to two groups. Oral<br />
administration of specific PPAR-α agonist gemfibrozil was given to one group while <strong>the</strong> o<strong>the</strong>r<br />
group received vehicle treatment; treatments started 3 days be<strong>for</strong>e injury <strong>an</strong>d continued through<br />
28 days post-injury (dpi). Locomotor assessment was per<strong>for</strong>med using <strong>the</strong> Basso Mouse Scale<br />
(BMS) <strong>an</strong>d <strong>the</strong> <strong>an</strong>imals were sacrificed at 28 dpi. The spinal cords of <strong>the</strong> <strong>an</strong>imals were sectioned<br />
at 10 µm <strong>an</strong>d processed <strong>for</strong> immunohistochemistry. The behavioral assessment of <strong>the</strong> <strong>an</strong>imals<br />
surprisingly revealed that <strong>the</strong> gemfibrozil group had severe behavioral deficits compared to <strong>the</strong><br />
vehicle group starting at 5 dpi continuing through <strong>the</strong> end of <strong>the</strong> study. In addition, <strong>an</strong>atomical<br />
<strong>an</strong>alysis of <strong>the</strong> tissue stained with eriochrome cy<strong>an</strong>ine <strong>an</strong>d neurofilament to delineate spared<br />
tissue showed decreased spared white matter in <strong>the</strong> gemfibrozil <strong>an</strong>imals versus controls. There<br />
was no ch<strong>an</strong>ge observed in activated macrophage immunoreactivity qu<strong>an</strong>tified by CD68 stain,<br />
while a decrease in astrocytic immunoreactivity (GFAP) <strong>an</strong>d a trend <strong>for</strong> decreased T-cell<br />
infiltration (CD3+ cells) was noted in <strong>the</strong> gemfibrozil <strong>an</strong>imals compared to <strong>the</strong> vehicle <strong>an</strong>imals.<br />
Thus, in stark contrast to o<strong>the</strong>r neuroinflammatory diseases where PPAR-α agonists are<br />
beneficial, gemfibrozil is particularly detrimental after SCI. This is <strong>an</strong> import<strong>an</strong>t finding <strong>from</strong> a<br />
clinical perspective where <strong>the</strong> same drug c<strong>an</strong> have contrasting effects in different disease models.
Disclosures: A.A. Almad, None; Y. Y<strong>an</strong>g, None; A. Lovett-Racke, None; D. McTigue, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.1/T10<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA 1 PO1 AG026572; Project 5<br />
F31NS059174-01A1 <strong>for</strong> JCC<br />
Title: Effects of progesterone treatment on expression <strong>an</strong>d function of estrogen receptors in<br />
rodent models<br />
Authors: *A. JAYARAMAN, J. C. CARROLL, T. E. MORGAN, S. LIN, L. ZHAO, W.<br />
MACK, C. J. PIKE;<br />
Univ. So Cali<strong>for</strong>nia, Los Angeles, CA<br />
Abstract: The Progesterone in Brain Aging <strong>an</strong>d Alzheimer’s Disease Program Project is<br />
designed to determine <strong>the</strong> neurobiology of progesterone (P4) in brain regions involved in<br />
cognition <strong>an</strong>d vulnerable to age-related cognitive decline <strong>an</strong>d Alzheimer’s disease. The depletion<br />
of <strong>the</strong> sex steroid hormones estrogen <strong>an</strong>d P4 during menopause is associated with increased risk<br />
<strong>for</strong> several disorders including Alzheimer’s disease (AD). Although abund<strong>an</strong>t research has linked<br />
hormone <strong>the</strong>rapy with prevention of age-related cognitive decline <strong>an</strong>d AD, recent clinical<br />
findings indicated adverse neural effects as well. These inconsistent observations underscore <strong>the</strong><br />
need <strong>for</strong> additional research to better underst<strong>an</strong>d of estrogen <strong>an</strong>d progesterone interactions in<br />
brain, particularly as <strong>the</strong>y relate to AD. One area of particular interest is <strong>the</strong> function of P4 as a<br />
negative regulator of neural estrogen actions. Several cell culture <strong>an</strong>d <strong>an</strong>imal model studies have<br />
shown that P4 is capable of repressing <strong>the</strong> beneficial functions of estrogen in neurons, although<br />
<strong>the</strong> mech<strong>an</strong>ism(s) underlying <strong>the</strong>se effects are unclear. Recent experimental evidence <strong>from</strong> our<br />
laboratory demonstrates that P4 c<strong>an</strong> inhibit estrogen action in primary neuron cultures by downregulating<br />
expression of estrogen receptors (ER), ERα <strong>an</strong>d ERβ. The decrease in ER expression<br />
by P4 affects both ER-dependent tr<strong>an</strong>scription as well as estrogen-mediated neuroprotection<br />
against apoptosis. In this study, we use rodent models to investigate <strong>the</strong> effects of P4 exposure on<br />
<strong>the</strong> expression <strong>an</strong>d function of ERs in vivo. The results of this study promise to provide<br />
import<strong>an</strong>t insight into estrogen <strong>an</strong>d P4 interactions relev<strong>an</strong>t to protective actions against AD.
Disclosures: A. Jayaram<strong>an</strong>, None; J.C. Carroll, None; T.E. Morg<strong>an</strong>, None; S. Lin, None; L.<br />
Zhao, None; W. Mack, None; C.J. Pike, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.2/T11<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA Gr<strong>an</strong>t U01AG031115 to RDB<br />
NIA Gr<strong>an</strong>t P01AG026572; Project 3 to RDB<br />
Title: Allopregn<strong>an</strong>olone promotes myelin <strong>for</strong>mation associated with decreased accumulation of<br />
Amyloid β in <strong>the</strong> triple tr<strong>an</strong>sgenic mouse model of Alzheimer's disease<br />
Authors: *S. CHEN 1 , J. WANG 1,3 , R. W. IRWIN 1 , J. YAO 1 , L. LIU 1 , R. T. HAMILTON 1 , J.<br />
LEMUS 1 , R. D. BRINTON 1,2 ;<br />
1 Pharmacol. <strong>an</strong>d Pharmaceut. Sci., 2 Program in Neurosci., USC, Los Angeles, CA; 3 Dept. of<br />
Pathology, Univ. of Mississippi, Med. Ctr., Jackson, MS<br />
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder accomp<strong>an</strong>ied<br />
by cognitive <strong>an</strong>d memory deficits. The neuropathology is defined by intr<strong>an</strong>euronal <strong>an</strong>d<br />
extracellular accumulation of <strong>the</strong> amyloid β (Aβ) peptide, particularly <strong>the</strong> 56kD soluble Aβ<br />
oligomer (Aβ*56), intr<strong>an</strong>euronal aggregates of <strong>the</strong> microtubule-associated protein tau, as well as<br />
synaptic dysfunction. Our previous study (Chen et al., 2007) showed that allopreg<strong>an</strong>olone (APα),<br />
a metabolite of progesterone, modified intracellular Aβ accumulation at <strong>an</strong> early age in <strong>the</strong> triple<br />
tr<strong>an</strong>sgenic mouse model (3xTg) of AD that closely mimics <strong>the</strong> disease progression in hum<strong>an</strong>s<br />
(Oddo et al., 2003). Behavioral <strong>an</strong>alyses indicated that APα signific<strong>an</strong>tly reversed <strong>the</strong> learning<br />
<strong>an</strong>d memory deficits of 3xTg-AD mice (W<strong>an</strong>g et al., 2007 <strong>an</strong>d Singh et al., 2007, 2008). We also<br />
found that chronic APα treatment decreased intracellular Aβ accumulation during <strong>the</strong><br />
development of pathology in 3xTg AD mouse. In 9-month old 3xTg-AD mice, treatment with<br />
APα reduced Aβ*56 level by 25 ± 4 % (n = 5, p=0.004 versus vehicle treatment). In 12-month<br />
old 3xTg-AD mice, Aβ*56 level was reduced by 15 ± 4% (p=0.05, n = 3-4) (Chen et al., 2008).<br />
We fur<strong>the</strong>r characterized <strong>the</strong> effects of APα treatment on myelination, microglia activation <strong>an</strong>d<br />
ABAD expression using biochemical <strong>an</strong>d immunohistochemical <strong>an</strong>alyses. Preliminary data <strong>from</strong><br />
Western Blot <strong>an</strong>alysis indicate that APα-treatment signific<strong>an</strong>tly increased <strong>the</strong> level of CNPase<br />
(cyclic nucleotide 3'-phosphodiesterase), a marker of oligodendrocytes <strong>an</strong>d myelin generation in
ain. This was fur<strong>the</strong>r confirmed by immunohistochemical <strong>an</strong>alysis. APα treatment reduced <strong>the</strong><br />
level of OX42, a microglia marker in 3xTg-AD mouse brain, as well as <strong>the</strong> level of ABAD, a<br />
mitochondrial protein overexpressed in AD. APα did not ch<strong>an</strong>ge <strong>the</strong> level of insulin-degrading<br />
enzyme (IDE), <strong>an</strong> Aβ degrading enzyme. Overall, <strong>the</strong>se results indicate that APα treatment<br />
promotes myelination, reduces Aβ accumulation, <strong>an</strong>d is paralleled with a reduction in microglia<br />
activation <strong>an</strong>d ABAD expression. Collectively, <strong>the</strong>se findings indicate that in a mouse model of<br />
AD, APα induces a profile consistent with reduction of <strong>an</strong>d or delay in progression of<br />
Alzheimer’s pathology. These findings have import<strong>an</strong>t implications <strong>for</strong> APα as a <strong>the</strong>rapeutic <strong>for</strong><br />
<strong>the</strong> treatment of Alzheimer’s disease pathology.<br />
Disclosures: S. Chen, None; J. W<strong>an</strong>g, None; R.W. Irwin, None; J. Yao, None; L. Liu,<br />
None; R.T. Hamilton, None; J. Lemus, None; R.D. Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.3/T12<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: AG10485<br />
AG22550<br />
AG027956<br />
Title: Estrogen attenuates inhibition of GluR1 phosphorylation by amyloid-beta oligomers<br />
Authors: *S. M. LOGAN, S. SARKAR, J. SIMPKINS;<br />
Univ. North Texas Hlth. Sci. Ctr., Fort Worth, TX<br />
Abstract: Neuromodulation of synaptic plasticity by 17β-estradiol (E2) is thought to influence<br />
in<strong>for</strong>mation processing <strong>an</strong>d storage in <strong>the</strong> cortex <strong>an</strong>d hippocampus. Because E2 rapidly affects<br />
cortical memory <strong>an</strong>d synaptic plasticity we examined its effects on phosphorylation <strong>an</strong>d<br />
trafficking of AMPAR (GluR1 subunit), a crucial mech<strong>an</strong>ism underlying activity-induced<br />
ch<strong>an</strong>ges in synaptic tr<strong>an</strong>smission. Using primary cortical neurons we show that E2 (10 nM)<br />
induced <strong>the</strong> phosphorylation of GluR1 within 30 min., <strong>an</strong>d this phosphorylation is specific to<br />
CaMKII at <strong>the</strong> serine 831 site. E2 treatment also increased GluR1 insertion into <strong>the</strong> membr<strong>an</strong>e of<br />
primary cortical neurons. Because soluble amyloid-beta (Aβ) oligomers inhibit synaptic
plasticity, we also tested E2’s ability to ameliorate Aβ-induced dysfunction of synaptic plasticity.<br />
We found that E2 (10 nM) ameliorated soluble Aβ1-42 (0.5 µM) oligomer-induced inhibition of<br />
GluR1 phosphorylation <strong>an</strong>d subsequent GluR1 insertion in primary cortical neurons. As this<br />
phosphorylation event mech<strong>an</strong>istically links increased dendritic spine growth <strong>an</strong>d spine density,<br />
we also examined E2-induced alterations in dendritic spine shape <strong>an</strong>d spine density in cortical<br />
neurons. E2 (10 nM) treatment increased <strong>the</strong> density of mushroom- <strong>an</strong>d long-thin-type spines.<br />
E2-induced spine density of mushroom- <strong>an</strong>d long-thin-type spines was inhibited in <strong>the</strong> presence<br />
of a CaMKII inhibitor (KN-93, 5 µM) <strong>an</strong>d a MEK inhibitor (U0126, 10 µM), respectively. E2<br />
(10 nM) also ameliorated Aβ-induced decreased spine density of mushroom-type spines in<br />
primary cortical neurons. Investigating E2-induced signaling molecules that target synaptic<br />
plasticity machinery will provide <strong>the</strong> molecular mech<strong>an</strong>isms of synaptic plasticity that correlate<br />
with memory, <strong>an</strong>d will also provide a <strong>the</strong>rapeutic strategy that may ameliorate age <strong>an</strong>d<br />
Alzheimer’s disease related dementia.<br />
Disclosures: S.M. Log<strong>an</strong>, None; S. Sarkar, None; J. Simpkins, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.4/T13<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH gr<strong>an</strong>t R01NS054687(Dr. Y<strong>an</strong>g)<br />
NIH gr<strong>an</strong>t R01NS054651(Dr .Y<strong>an</strong>g)<br />
An award <strong>from</strong> Americ<strong>an</strong> Heart Association, Inc. Texas Affiliate (Dr .Y<strong>an</strong>g)<br />
NIH gr<strong>an</strong>t P01AG22550(Dr. Simpkins)<br />
NIH gr<strong>an</strong>t P01AG10485(Dr. Sompkins)<br />
Activase by Genentech<br />
Title: Combination <strong>the</strong>rapy of 17β-estradiol <strong>an</strong>d recombin<strong>an</strong>t tissue plasminogen activator <strong>for</strong><br />
experimental ischemic stroke
Authors: *S. YANG, R. LIU, Q. LIU, S. HE, J. W. SIMPKINS;<br />
Univ. North Texas Hlth. Sci. Ctr., Fort Worth, TX<br />
Abstract: Thrombolysis with recombin<strong>an</strong>t tissue plasminogen activator (rtPA) in ischemic<br />
stroke is limited by its short <strong>the</strong>rapeutic window <strong>an</strong>d <strong>the</strong> increased risks of hemorrhage<br />
tr<strong>an</strong>s<strong>for</strong>mation. We determined <strong>the</strong> interaction of 17β-estradiol (E2) <strong>an</strong>d rtPA on <strong>the</strong> activation of<br />
plasminogen system <strong>an</strong>d matrix metalloproteinases (MMPs) in a tr<strong>an</strong>sient middle cerebral artery<br />
occlusion (MCAO) model. Ovariectomized female rats were subjected to 1 hour tr<strong>an</strong>sient focal<br />
cerebral ischemia using a suture MCAO model. Ischemic lesion volume was signific<strong>an</strong>tly<br />
reduced with acute treatment of E2 despite of exogenous administration of rtPA. The expression<br />
<strong>an</strong>d activation of uPA, MMP2, <strong>an</strong>d MMP9 were signific<strong>an</strong>tly increased in ischemic hemisphere<br />
after tr<strong>an</strong>sient cerebral ischemia. Exogenous rtPA administration fur<strong>the</strong>r enh<strong>an</strong>ced both<br />
expression <strong>an</strong>d activation of uPA, MMP2 <strong>an</strong>d MMP9, which was blocked by E2 treatment. We<br />
fur<strong>the</strong>r determined whe<strong>the</strong>r combination E2 treatment could expend <strong>the</strong> <strong>the</strong>rapeutic window of<br />
rtPA in <strong>an</strong> embolic MCAO model. While no protection was indicated upon acute treatment of E2<br />
alone, combination treatment of E2 expended <strong>the</strong> <strong>the</strong>rapeutic window of rtPA. Collectively, <strong>the</strong><br />
present study suggest that estrogen could be a c<strong>an</strong>didate <strong>for</strong> <strong>the</strong> combination <strong>the</strong>rapy with rtPA to<br />
attenuate its side effect, <strong>an</strong>d hence expend its short <strong>the</strong>rapeutic window <strong>for</strong> <strong>the</strong> treatment of<br />
ischemic stroke.<br />
Disclosures: S. Y<strong>an</strong>g, Activase received <strong>from</strong> Genentech, C. O<strong>the</strong>r Research Support (receipt<br />
of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); R. Liu, None; Q. Liu, None; S. He,<br />
None; J.W. Simpkins, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.5/T14<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA 1P01 AG026572; Project 4 to TEM & CEF<br />
NIA 2-T32 AG000037; to EMC<br />
Title: Role of glial progesterone receptors in neurite sprouting<br />
Authors: *J. M. ARIMOTO, N. BALI, N. IWATA, T. E. MORGAN, C. E. FINCH;<br />
Davis Sch. of Gerontology, Dept. of Biol. Sci., Univ. So. Cali<strong>for</strong>nia, Los Angeles, CA
Abstract: The 'Progesterone in Brain Aging <strong>an</strong>d Alzheimer’s Disease Program Project' is<br />
designed to determine <strong>the</strong> neurobiology of progesterone (P4) in brain regions involved in<br />
cognition <strong>an</strong>d vulnerable to age-related cognitive decline <strong>an</strong>d Alzheimer’s disease. Synaptic<br />
remodeling in <strong>the</strong> hippocampus shows <strong>an</strong>tagonistic synergies of estradiol (E2) <strong>an</strong>d P4 during<br />
fertility cycles <strong>an</strong>d in response to axotomy. With entorhinal cortex lesions (ECL) to induce<br />
compensatory sprouting in hippocampus, P4 <strong>an</strong>tagonized E2-dependent neurite outgrowth<br />
(Wong et al, Endocrinology <strong>2009</strong>, PMID18772232). The ’wounding-in-a dish’ model of gli<strong>an</strong>euron<br />
co-cultures was used to show that <strong>the</strong> E2-P4 <strong>an</strong>tagonism on neurite outgrowth involved<br />
microglia. Specifically, P4 induced neurite sprouting in co-cultures of E18 neurons with<br />
astrocytes (A, >97%), whereas P4 attenuated E2-induced sprouting in E18 neurons with mixed<br />
glia (MxG, 70% astrocytes + 30% microglia). This suggests a new role <strong>for</strong> microglia in<br />
mediating P4 activities in brain. A progesterone receptor (PR)-mediated mech<strong>an</strong>ism is implicated<br />
because <strong>the</strong> PR <strong>an</strong>tagonist ORG31710 partially reversed P4 inhibition of E2-induced neurite<br />
outgrowth. Because glial PRs have not been characterized in detail, we looked <strong>for</strong> PR sequences<br />
in primary cultures of rat MxG, enriched A, <strong>an</strong>d enriched microglia (Mc, >97%). The PCR<br />
primers were validated in o<strong>the</strong>r tissues <strong>for</strong> nuclear (nPR) <strong>an</strong>d membr<strong>an</strong>e PRs (Pgrmc1, mPRα,<br />
mPRβ <strong>an</strong>d mPRγ). We detected definitive nPR-specific amplicons (with sequence confirmation)<br />
in RNA <strong>from</strong> MxG <strong>an</strong>d A. However, Mc had negligible nPR expression, confirming Sierra et al<br />
(Glia 2008, PMID18286612). In contrast, all four mPRs were detected in Mc, A <strong>an</strong>d MxG. This<br />
suggests a membr<strong>an</strong>e PR-initiated pathway in microglial mediated P4 <strong>an</strong>tagonism of E2.<br />
Disclosures: J.M. Arimoto, None; N. Bali, None; N. Iwata, None; T.E. Morg<strong>an</strong>, None; C.E.<br />
Finch, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.6/T15<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: 5U01AG031115-02<br />
Title: Dysregulation of cholesterol homeostasis in <strong>the</strong> aging 3xTG mouse: Implication <strong>for</strong><br />
increased cholesterol turnover, catabolism, <strong>an</strong>d neurodegeneration<br />
Authors: *R. T. HAMILTON, J. LEMUS, E. AGBONWANETEN, A. M. PEDROZA, A. J.<br />
COMPADRE, J. YAO, R. W. IRWIN, S. CHEN, R. D. BRINTON;<br />
Pharmacol. & Pharmaceut. Sci., USC, Los Angeles, CA
Abstract: Dysregulation of cholesterol homeostasis is <strong>an</strong> <strong>an</strong>teced<strong>an</strong>t to <strong>the</strong> development of<br />
Alzheimer’s pathology. High neuronal cholesterol induces amyloid beta production implicating<br />
cholesterol in <strong>the</strong> development of amyloid pathology. We propose that cholesterol turnover,<br />
clear<strong>an</strong>ce <strong>an</strong>d trafficking are dysregulated in <strong>the</strong> triple tr<strong>an</strong>sgenic mouse leading to <strong>the</strong> observed<br />
cholesterol phenotype in AD. To test this hypo<strong>the</strong>sis, we utilized <strong>the</strong> normal aging nontr<strong>an</strong>sgenic<br />
(NTG) <strong>an</strong>d a 3xTgAD LaFerla mouse. Protein was isolated <strong>from</strong> <strong>the</strong> cortex of intact female NTG<br />
<strong>an</strong>d 3xTgAD mice at 3, 6 <strong>an</strong>d 9 months of age <strong>an</strong>d <strong>an</strong>alyzed by Western blot <strong>for</strong> cholesterol<br />
turnover, trafficking, uptake, catabolism, efflux <strong>an</strong>d myelin protein expression . Results of those<br />
<strong>an</strong>alyses indicate that cholesterol clear<strong>an</strong>ce (CYP46, CYP27a1, LXR) was unch<strong>an</strong>ged with aging<br />
in <strong>the</strong> NTG but was increased in <strong>the</strong> 3xTG mice (CYP46, CYP27a1, <strong>an</strong>d PXR) with age. It was<br />
fur<strong>the</strong>r observed that cholesterol clear<strong>an</strong>ce was increased (CYP46, CYP27a1 <strong>an</strong>d LXR) while<br />
PXR decreased in 3xTG mice versus NTG. Cholesterol trafficking was increased in NTG <strong>an</strong>d<br />
unch<strong>an</strong>ged in 3xTG (ApoE <strong>an</strong>d ApoF) while ABCA1 was unch<strong>an</strong>ged in <strong>the</strong> NTG <strong>an</strong>d decreased<br />
in 3xTgAD mice. Cholesterol <strong>an</strong>d lipid syn<strong>the</strong>sis was decreased at all ages in <strong>the</strong> NTG mice <strong>an</strong>d<br />
3xTgAD mice (HMG-CoA-R, ACC) while citrate lyase was unch<strong>an</strong>ged in <strong>the</strong> NTG <strong>an</strong>d<br />
increased in <strong>the</strong> 3xTG mice with age. Citrate lyase expression was increased in <strong>the</strong> 3xTG mice<br />
but HMG-CoA-R <strong>an</strong>d ACC were both signific<strong>an</strong>tly decreased in <strong>the</strong> 3xTG mice compared to<br />
NTG. Intracellular cholesterol trafficking was unch<strong>an</strong>ged in <strong>the</strong> NTG (NPC1, <strong>an</strong>d StAR)<br />
whereas both proteins signific<strong>an</strong>tly increased with age in <strong>the</strong> 3xTgAD mice. The 3xTgAD mice<br />
also had greater StAR protein expression while <strong>the</strong>y had decreased cholesterol trafficking <strong>from</strong><br />
<strong>the</strong> lysosomes (NPC1) as compared to age matched NTG. Lipid metabolism was unch<strong>an</strong>ged <strong>for</strong><br />
SChAD <strong>an</strong>d trifunctional protein in <strong>the</strong> NTG mice whereas in <strong>the</strong> 3xTgAD mice SChAD levels<br />
were signific<strong>an</strong>tly elevated <strong>an</strong>d trifunctional protein levels were unch<strong>an</strong>ged. It was fur<strong>the</strong>r<br />
observed that SChAD was signific<strong>an</strong>tly elevated in <strong>the</strong> 3xTgAD as compared to NTG while<br />
trifunctional protein was elevated at <strong>the</strong> oldest age only. Myelin basic protein expression was<br />
signific<strong>an</strong>tly lower in <strong>the</strong> 3xTgAD at nine months of age <strong>an</strong>d unch<strong>an</strong>ged in <strong>the</strong> non tr<strong>an</strong>sgenic<br />
mice. These data suggest that cholesterol <strong>an</strong>d lipid homeostasis in <strong>the</strong> 3xTgAD mice are<br />
decreased at <strong>the</strong> syn<strong>the</strong>sis, tr<strong>an</strong>sport. clear<strong>an</strong>ce, <strong>an</strong>d catabolism level ultimately leading to<br />
increased elimination of cholesterol that is necessary <strong>for</strong> myelin syn<strong>the</strong>sis, neurosteroid<br />
syn<strong>the</strong>sis, acetylcholine production, <strong>an</strong>d proper synaptic function.<br />
Disclosures: R.T. Hamilton, None; J. Lemus, None; E. Agbonw<strong>an</strong>eten, None; A.M.<br />
Pedroza, None; A.J. Compadre, None; J. Yao, None; R.W. Irwin, None; S. Chen, None; R.D.<br />
Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.7/T16
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIMH R01 MH67159<br />
Title: Estrogen increases maximal respiratory capacity of neuronal <strong>an</strong>d glia mitochondria as well<br />
as basal respiration in glia: Involvement of both ERbeta <strong>an</strong>d ERalpha<br />
Authors: *J. YAO 1 , J. NILSEN 1 , R. W. IRWIN 1 , S. CHEN 1 , R. D. BRINTON 1,2 ;<br />
1 2<br />
Pharmacol. <strong>an</strong>d Pharmaceut. Sci., Univ. of Sou<strong>the</strong>rn Cali<strong>for</strong>nia, Los Angeles, CA; Program in<br />
Neurosci., USC, Los Angeles, CA<br />
Abstract: Estrogen has been demonstrated to protect against a variety of neurotoxic insults by<br />
sustaining <strong>an</strong>d enh<strong>an</strong>cing mitochondrial function. Previous in vivo studies <strong>from</strong> our group have<br />
shown that 17beta-estradiol (E2) increased whole brain mitochondrial respiration coupled to<br />
increased expression <strong>an</strong>d activity of enzymes involved in respiratory function, such as pyruvate<br />
dehydrogenase (PDH) <strong>an</strong>d cytochrome c oxidase (COX). In this study, using <strong>the</strong> Seahorse<br />
metabolic flux <strong>an</strong>alyzer, we sought to determine <strong>the</strong> regulation of mitochondrial function in<br />
different neuronal cell types by estrogen. Metabolic profiling of primary rat embryonic<br />
hippocampal neurons <strong>an</strong>d mixed glial cells demonstrated that neurons exhibit higher basal<br />
aerobic mitochondrial respiratory activity (oxygen consumption rate (OCR)) relative to glia,<br />
which exhibit glycolytic activity as evidenced by higher extracellular acidification rate (ECAR).<br />
Neuronal energy production was predomin<strong>an</strong>tly driven by glucose or pyruvate. In contrast,<br />
energy production by mixed glial cultures was supported by a broader substrate profile including<br />
glucose/pyruvate <strong>an</strong>d fatty acid (palmitate).<br />
To determine <strong>the</strong> role of <strong>the</strong> different cell types in estrogen-induced regulation of brain<br />
metabolism, we investigated <strong>the</strong> E2-induced metabolic profile of neuronal <strong>an</strong>d mixed glial<br />
cultures. E2 differentially regulated metabolic activity in neurons <strong>an</strong>d glial cells. In neurons, E2<br />
increased <strong>the</strong> maximal respiratory capacity in neurons whereas E2 increased both basal<br />
respiration <strong>an</strong>d maximal respiratory capacity in mixed glial cells. Moreover, <strong>the</strong> ERβ selective<br />
agonist DPN increased mitochondrial respiration to a greater extent th<strong>an</strong> did <strong>the</strong> ERα selective<br />
agonist PPT, indicating a role <strong>for</strong> both ERα <strong>an</strong>d ERβ with ERβ making a greater contribution to<br />
E2-induced regulation of mitochondrial function in neurons <strong>an</strong>d glial cells.<br />
From a basic science perspective, <strong>the</strong>se data indicate that neurons <strong>an</strong>d glial cells exhibit unique<br />
metabolic profiles reflective of <strong>the</strong>ir different roles in supporting brain function. Fur<strong>the</strong>r, E2<br />
differentially regulates mitochondrial function in neurons to promote maximal capacity while<br />
increasing both basal <strong>an</strong>d maximal respiratory capacity of glia. Tr<strong>an</strong>slationally, <strong>the</strong>se findings<br />
have implications <strong>for</strong> sustaining bioenergetics of both neurons <strong>an</strong>d glia in <strong>the</strong> aging brain.<br />
Disclosures: J. Yao, None; J. Nilsen, None; R.W. Irwin, None; S. Chen, None; R.D. Brinton,<br />
None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.8/T17<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH gr<strong>an</strong>ts R01NS054687<br />
R01NS054651,<br />
P01AG22550<br />
P01AG10485<br />
<strong>an</strong> award <strong>from</strong> Americ<strong>an</strong> Heart Association, Inc. Texas Affiliate<br />
Title: Amyloid beta oligomers down-regulate MMPs activation in cultured rat astrocyte<br />
Authors: W. LI, E. POTEET, R. LIU, L. XIE, Y. WEN, *J. W. SIMPKINS, S.-H. YANG;<br />
Univ. N Texas Hlth. Sci. Ctr., Ft Worth, TX<br />
Abstract: Deposition of amyloid beta peptide (Aβ) in <strong>the</strong> brain has been recognized as one of<br />
<strong>the</strong> prominent features of Alzheimer’s disease (AD). There is increasing evidence that <strong>the</strong><br />
accumulation of Aβ is because of imbal<strong>an</strong>ce between <strong>the</strong> production of Aβ <strong>an</strong>d its degradation<br />
<strong>an</strong>d clear<strong>an</strong>ce. In <strong>the</strong> present study, we determined <strong>the</strong> effect of Aβ on primary astrocytes.<br />
Primary astrocytes were cultured <strong>from</strong> neonatal rat brains, <strong>an</strong>d verified by immunocytochemical<br />
staining of glial fibrillary acidic protein (GFAP) followed by flow cytometry. The primary<br />
astrocytes were treated with hum<strong>an</strong> Aβ 1-42 oligomers or control. Cell viability was determined<br />
by lactate dehydrogenase (LDH) assay. The culture media <strong>an</strong>d cells were collected <strong>an</strong>d <strong>an</strong>alyzed<br />
<strong>for</strong> Aβ <strong>an</strong>d activation of matrix metalloproteinases (MMPs) by Western blots <strong>an</strong>d zymography,<br />
respectively. No signific<strong>an</strong>t cell death was found upon Aβ oligomers treatment. A marked uptake<br />
of Aβ by astrocytes was indicated, evidenced by <strong>the</strong> reduction of Aβ in <strong>the</strong> culture media <strong>an</strong>d<br />
increase of Aβ oligomers in <strong>the</strong> astrocyte lyses. Fur<strong>the</strong>r, Aβ oligomers treatment down regulated<br />
MMPs activation. In summary, <strong>the</strong> present study suggested that primary astrocytes could uptake<br />
Aβ oligomers <strong>an</strong>d Aβ oligomers down regulate MMPs activation. (Supported partly by NIH<br />
gr<strong>an</strong>ts R01NS054687, R01NS054651, P01AG22550, P01AG10485, <strong>an</strong>d <strong>an</strong> award <strong>from</strong><br />
Americ<strong>an</strong> Heart Association, Inc. Texas Affiliate)<br />
Disclosures: W. Li, None; E. Poteet, None; R. Liu, None; L. Xie, None; Y. Wen, None; J.W.<br />
Simpkins, None; S. Y<strong>an</strong>g, None.<br />
Poster
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.9/T18<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH Gr<strong>an</strong>t P01 AG10285<br />
NIH Gr<strong>an</strong>t P01 AaG22550<br />
NIH Gr<strong>an</strong>t P01 AG27956<br />
Title: Estrogen receptor β colocalizes with mitochondria import protein Tom20 in hum<strong>an</strong> neural<br />
progenitor cells<br />
Authors: *V. P. PEARCE, J. SIMPKINS;<br />
UNT Hlth. Sci. Ctr., <strong>for</strong>t worth, TX<br />
Abstract: Estrogen receptor β (ERβ) may affect mitochondrial biogenesis <strong>an</strong>d/or activity, a<br />
requirement <strong>for</strong> stem cell differentiation. Since mitochondrial activity is dependent on <strong>the</strong> import<br />
of proteins, <strong>an</strong>d studies purport that ER functions in <strong>the</strong> mitochondria, <strong>the</strong>n ERβ may be<br />
imported into <strong>the</strong> mitochondria through interaction with a principal receptor of <strong>the</strong> outer<br />
mitochondria membr<strong>an</strong>e complex (Tom20). Tom20 recognizes a consensus motif or presequence<br />
represented by φXXφφ (where φ is a hydrophobic amino acid such as leucine, isoleucine,<br />
phenylal<strong>an</strong>ine, tryptoph<strong>an</strong>, valine, <strong>an</strong>d tyrosine, <strong>an</strong>d X is <strong>an</strong>y amino acid), with a hydrophobic<br />
preference <strong>for</strong> LXXLL (motifs). ERβ contains several in <strong>the</strong> lig<strong>an</strong>d binding domain, as well as a<br />
putative internal targeting sequence that includes a reverse LXXLL motif. M<strong>an</strong>y recognized<br />
mitochondrial proteins such as p53, Bcl2, CREB, hTERT, <strong>an</strong>d connexin 43 also have <strong>the</strong>ir own<br />
possible presequence-like motifs. Our studies in immortalized hum<strong>an</strong> neural progenitor cell line<br />
(ReNcell CX) including dual immunofluorescent staining, western <strong>an</strong>alysis, <strong>an</strong>d proximity<br />
ligation assays, indicates that ERβ colocalizes with Tom20 at <strong>the</strong> mitochondria. Fur<strong>the</strong>r, we<br />
indicate that o<strong>the</strong>r mitochondrial proteins that effect mitochondrial biogenesis, such as hTERT<br />
colocalize with Tom20. Identification of how proteins are target to <strong>the</strong> mitochondria will provide<br />
better stem cell-based <strong>the</strong>rapies based on m<strong>an</strong>ipulation of mitochondrial biogenesis.<br />
Disclosures: V.P. Pearce, None; J. Simpkins, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.10/U1<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA1PO1 AG026572<br />
NIH1PO1AG026572<br />
NIAT32AG000093-25<br />
Title: Progesterone inhibits estrogen-mediated neuroprotection against excitotoxicity by downregulating<br />
estrogen receptor-beta<br />
Authors: *C. C. AGUIRRE 1 , A. JAYARAMAN 1 , C. PIKE 2 , M. BAUDRY 2 ;<br />
1 U.S.C., Los Angeles, CA; 2 USC, Los Angeles, CA<br />
Abstract: The Progesterone in Brain Aging <strong>an</strong>d Alzheimer's Disease Program Project is<br />
designed to determine <strong>the</strong> neurobiology of progesterone (P4) in brain regions involved in<br />
cognition <strong>an</strong>d vulnerable to age-related cognitive decline <strong>an</strong>d Alzheimer's disease.While both<br />
17ß-estradiol (E2) <strong>an</strong>d progesterone (P4) have been found to be neuroprotective in a number of<br />
experimental paradigms, P4 has also been shown to inhibit <strong>the</strong> neuroprotective effects of E2. We<br />
recently reported that a relatively short treatment of cultured hippocampal slices with P4 <strong>for</strong> 4 h<br />
following a prolonged (20 h) treatment with E2 eliminated <strong>the</strong> neuroprotective effects of E2<br />
against NMDA toxicity. In addition, we provided evidence that this effect was due in part to <strong>the</strong><br />
prevention by P4 of E2-induced increased expression of BDNF. In <strong>the</strong> present study, we<br />
evaluated <strong>the</strong> effects of <strong>the</strong> treatment of cultured hippocampal slices with P4 following E2<br />
treatment on <strong>the</strong> expression <strong>an</strong>d levels of <strong>the</strong> estrogen receptors, ERα <strong>an</strong>d ERß, as well as of<br />
BDNF. Treatment with P4 reversed <strong>the</strong> E2-elicited increases in levels of ERß mRNA <strong>an</strong>d<br />
protein. In contrast, E2 treatment did not modify ERα mRNA, but increased ERα protein levels,<br />
which was also reversed by P4 treatment. Fur<strong>the</strong>rmore, E2 treatment produced <strong>an</strong> increase in<br />
BDNF mRNA, <strong>an</strong>d P4 treatment following E2 treatment eliminated this increase. Experiments<br />
with <strong>an</strong> ERß-specific <strong>an</strong>tagonist, PHTPP, indicated that E2-mediated neuroprotection against<br />
NMDA toxicity was mediated by <strong>the</strong> activation of ERß receptors. This conclusion was also<br />
verified by results indicating that E2 had no neuroprotective effects against NMDA toxicity in<br />
cultured hippocampal slices prepared <strong>from</strong> ERß-/- mice. Our results indicate that E2-mediated<br />
neuroprotection against NMDA toxicity is due to <strong>the</strong> activation of ERß receptors <strong>an</strong>d that P4<br />
treatment following E2 treatment results in <strong>the</strong> down-regulation of ERß mRNA <strong>an</strong>d protein; this<br />
mech<strong>an</strong>ism thus provides a potential expl<strong>an</strong>ation <strong>for</strong> P4-mediated inhibition of E2<br />
neuroprotective effects. Fur<strong>the</strong>rmore, <strong>the</strong> existence of such a mech<strong>an</strong>ism has import<strong>an</strong>t<br />
consequences <strong>for</strong> our underst<strong>an</strong>ding of <strong>the</strong> interactions between <strong>the</strong>se two import<strong>an</strong>t sex<br />
hormones.
Disclosures: C.C. Aguirre, None; A. Jayaram<strong>an</strong>, None; C. Pike, None; M. Baudry, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.11/U2<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA 1PO1 AG026572; Project 1 to EC <strong>an</strong>d RDB.<br />
Title: Impact of progestins on estradiol potentiation of mitochondrial function<br />
Authors: *R. W. IRWIN 1 , J. YAO 1 , S. S. AHMED 1 , R. T. HAMILTON 1 , E. CADENAS 1 , R.<br />
D. BRINTON 1,2 ;<br />
1 Pharmacol. <strong>an</strong>d Pharmaceut. Sci., 2 Programs in Neurosci., USC, Los Angeles, CA<br />
Abstract: The Progesterone in Brain Aging <strong>an</strong>d Alzheimer’s Disease Program Project is<br />
designed to determine <strong>the</strong> neurobiology of progesterone (P4) in brain regions involved in<br />
cognition <strong>an</strong>d vulnerable to age-related cognitive decline <strong>an</strong>d Alzheimer’s disease (AD). AD is<br />
associated with glucose hypometabolism that compromises <strong>the</strong> energetic dem<strong>an</strong>ds of neuronal<br />
activation. Underlying this metabolic deficiency is a disruption of <strong>the</strong> signaling pathways that<br />
converge upon mitochondrial function. Previously, we identified brain mitoproteomic ch<strong>an</strong>ges<br />
associated with acute estradiol (E2) replacement (Nilsen, Irwin, et al. J. Neurosci 2007). We<br />
extended <strong>the</strong>se findings to demonstrate E2 <strong>an</strong>d P4 improve mitochondrial function alone but not<br />
in combination (Irwin, et al. Endocrinology 2008). This current study aims to identify <strong>the</strong><br />
mech<strong>an</strong>ism responsible <strong>for</strong> estrogen/progestin interaction. We investigated brain mitochondrial<br />
function of OVX 5-month female rats (n=10) following treatment with <strong>the</strong> progestin<br />
medroxyprogesterone acetate (MPA), E2, E2+MPA, vehicle, ShamOVX. MPA alone <strong>an</strong>d<br />
MPA+E2 resulted in diminished mitochondrial respiration <strong>an</strong>d ATP generation. Treatment<br />
ch<strong>an</strong>ges in mitochondrial proteins relative to controls were determined via immunoblot, RT-PCR<br />
<strong>an</strong>d/or enzyme activity assay <strong>for</strong> lysates of hippocampus, cortex, cerebellum, <strong>an</strong>d purified whole<br />
brain mitochondria. Altered proteins included ATP synthase, MnSOD, <strong>an</strong>d Prdx. To determine<br />
neuron-specific effects, progestin-mediated alterations in oxygen utilization were measured via<br />
<strong>the</strong> Seahorse XF24 metabolic <strong>an</strong>alyzer in primary cultured neurons. The set of mitochondrial<br />
proteins altered by estradiol/progestin interactions included key enzymes of glucose metabolism,<br />
glutamate metabolism, acetylcholine metabolism, lipid peroxidation, <strong>an</strong>d energy production.<br />
Bioin<strong>for</strong>matic <strong>an</strong>alyses of altered mitochondrial proteins will help us to distinguish <strong>the</strong> negative<br />
<strong>from</strong> <strong>the</strong> positive actions of progestins in <strong>the</strong> brain. We are currently investigating mitochondrial
mech<strong>an</strong>isms of neuroprotection regulated by <strong>the</strong> most promising progestin lead molecules<br />
generated within <strong>the</strong> Progesterone Program Project.<br />
Disclosures: R.W. Irwin, None; J. Yao, None; S.S. Ahmed, None; R.T. Hamilton, None; E.<br />
Cadenas, None; R.D. Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.12/U3<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: Alzheimer Association Gr<strong>an</strong>t NIRG57698<br />
NIH gr<strong>an</strong>ts R01NS054687<br />
NIH gr<strong>an</strong>ts R01NS054651<br />
Title: Methylene blue improves mitochondrial functions <strong>an</strong>d attenuates mitochondrial complex I<br />
inhibition induced neurotoxicity<br />
Authors: *Y. WEN 1 , C. TAN 2 , P. PALACIOS 2 , E. POTEET 2 , W. LI 2 , L. XIE 2 , R. LIU 2 , S.<br />
YANG 2 ;<br />
2 Pharmacol., 1 UNTHSC, Fort Worth, TX<br />
Abstract: Oxidative stress has been implicated in <strong>the</strong> etiology of Parkinson's disease (PD) <strong>an</strong>d in<br />
m<strong>an</strong>y <strong>an</strong>imal models including <strong>the</strong> MPTP <strong>an</strong>d rotenone models, both of which are widely used<br />
<strong>an</strong>imal models <strong>for</strong> PD. Both compounds were shown to inhibit <strong>the</strong> complex I of mitochondrial<br />
electron tr<strong>an</strong>sport chain, <strong>the</strong>reby leading to oxidative stress <strong>an</strong>d neuronal cell death. These<br />
observations suggest mitochondrial dysfunction <strong>an</strong>d reduction of complex 1 activity in sporadic<br />
PD. Thus, dysfunctional mitochondrial complex I appears to be directly linked with alterations in<br />
m<strong>an</strong>y aspects of PD. In <strong>the</strong> preliminary studies, we observed that a century old drug, Methylene<br />
Blue (MB), is a substrate of mitochondrial complex I, but insensitive to rotenone <strong>an</strong>d <strong>an</strong>timycin<br />
inhibition. It c<strong>an</strong> function as <strong>an</strong> alternative re-dox electron tr<strong>an</strong>sporter to deliver electrons <strong>from</strong><br />
NADH to Cytochrome c bypassing complex I <strong>an</strong>d complex III inhibition. MB is a substrate <strong>for</strong><br />
complex I, <strong>an</strong>d c<strong>an</strong> be reduced to MBH2 by NADH dehydrogenase, <strong>an</strong>d <strong>the</strong> reduced MBH2 c<strong>an</strong><br />
function as a complex III substrate to reduce cytochrome c. Such activity confers very potent<br />
neuroprotective activity, almost eliminating <strong>the</strong> generation of ROS <strong>from</strong> mitochondria at very
low doses (Ec50 less th<strong>an</strong> 5nM), especially those induced by mitochondrial complex I <strong>an</strong>d<br />
complex III inhibition. MB is <strong>an</strong> FDA approved drug <strong>for</strong> m<strong>an</strong>y diseases including <strong>an</strong>tidote <strong>for</strong><br />
cy<strong>an</strong>ide, carbon monoxide poisoning, <strong>an</strong>d sepsis. It is a lipid soluble drug that was shown to<br />
cross blood brain barrier. MB under <strong>the</strong> trade-name TauRx is under clinical trial <strong>for</strong> treatment of<br />
Alzheirmer’s Disease <strong>an</strong>d may provide a way of halting or slowing <strong>the</strong> progression of<br />
Alzheimer's dementia.<br />
We examined <strong>the</strong> effects of MB against mitochondrial complex I inhibition in tr<strong>an</strong>s<strong>for</strong>med<br />
mouse hippocampal HT22 cell with rotenone. Rotenone induced mitochondrial dysfunction,<br />
ROS generation <strong>an</strong>d cell death in HT-22 cells. MB treatment showed strong neuroprotection, <strong>an</strong>d<br />
attenuated <strong>the</strong>se toxic events. We propose that MB will be strong neuroprotective agent that<br />
attenuates complex I inhibition induced neurodegenerative diseases, such as PD.<br />
Disclosures: Y. Wen, None; C. T<strong>an</strong>, None; P. Palacios, None; E. Poteet, None; W. Li,<br />
None; L. Xie, None; R. Liu, None; S. y<strong>an</strong>g, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.13/U4<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH Gr<strong>an</strong>t AG10485<br />
NIH Gr<strong>an</strong>t AG22550<br />
NIH Gr<strong>an</strong>t AG027956<br />
Title: Okadaic acid induces tau phosphorylation in <strong>an</strong> estrogen preventable m<strong>an</strong>ner<br />
Authors: *Z. ZHANG, J. SIMPKINS;<br />
UNTHSC, Fort Worth, TX<br />
Abstract: Epidemiological studies showed that AD is three times more prevalent th<strong>an</strong> men <strong>an</strong>d<br />
early estrogen treatment protects against AD. One of <strong>the</strong> major pathological hallmarks of AD is<br />
<strong>the</strong> presence of neurofibrillary t<strong>an</strong>gles (NFT) <strong>an</strong>d <strong>the</strong>ir constituents, paired helical filaments,<br />
consisting mainly of hyperphosphorylated tau. Clinical studies have proposed that <strong>the</strong> numbers<br />
of NFT correlate closely with <strong>the</strong> degree of dementia. It is proposed that <strong>an</strong> imbal<strong>an</strong>ce between<br />
tau phosphorylation <strong>an</strong>d dephosphorylation is critical to AD. Okadaic acid (OA) is a cell
permeable <strong>an</strong>d sensitive inhibitor of protein phosphatase 1 <strong>an</strong>d 2A (PP 1/2A). Inhibition of PP<br />
2A by OA c<strong>an</strong> induce a hyperphosphorylation <strong>an</strong>d accumulation of tau in vitro. It is proposed<br />
that inhibition of PP1/2A in vivo may be useful tauopathy model. In <strong>the</strong> present study, OA was<br />
chronically administered into unilateral dorsal hippocampus of OVX female SD rats via a brain<br />
infusion kit which was connected to a subcut<strong>an</strong>eously impl<strong>an</strong>ted osmotic pump. We found OA<br />
induces tau phosphorylation in both cortex <strong>an</strong>d hippocampus accomp<strong>an</strong>ied by <strong>an</strong> upregulation of<br />
cdk5. In vitro, we found that OA induces tau phosphorylation in SH-SY5Y cells in <strong>an</strong> estrogen<br />
reversible m<strong>an</strong>ner <strong>an</strong>d this effect could be partially blocked by ICI 182,780, <strong>an</strong> estrogen receptor<br />
<strong>an</strong>tagonist. Our data suggest that PP2A plays <strong>an</strong> import<strong>an</strong>t role in tau phosphorlation <strong>an</strong>d<br />
estrogen c<strong>an</strong> reverse tau hyperphosphorylation, which may be <strong>an</strong> estrogen receptor mediated<br />
neuroprotective effect.<br />
Disclosures: Z. Zh<strong>an</strong>g, None; J. Simpkins, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.14/U5<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH gr<strong>an</strong>t R01NS054687(Dr. Y<strong>an</strong>g)<br />
NIH gr<strong>an</strong>t R01NS054651(Dr .Y<strong>an</strong>g)<br />
An award <strong>from</strong> Americ<strong>an</strong> Heart Association, Inc. Texas Affiliate (Dr .Y<strong>an</strong>g)<br />
NIH gr<strong>an</strong>t P01AG22550(Dr. Simpkins)<br />
NIH gr<strong>an</strong>t P01AG10485(Dr. Sompkins)<br />
Activase by Genentech<br />
Title: 17β-estradial reduces tissue plasminogen activator-induced matrix metalloproteinases<br />
activation in rat primary astrocytes<br />
Authors: *R. LIU, W. LI, L. XIE, E. POTEET, H. QIAN, Y. WEN, J. W. SIMPKINS, S.<br />
YANG;<br />
Dept Pharmacol & Neurosci, UNT Hlth. Sci. Ctr. Fort Worth, Fort Worth, TX
Abstract: We reported be<strong>for</strong>e that 17β-estradiol (E2) protects blood brain barrier (BBB)<br />
disruption induced by tr<strong>an</strong>sient focal cerebral ischemia in part by inhibition of MMP2 <strong>an</strong>d<br />
MMP9 activation. Our recent in vivo works demonstrated that E2 c<strong>an</strong> attenuate recombin<strong>an</strong>t<br />
tissue plasminogen activator (rtPA)-induced activation of MMP2 <strong>an</strong>d MMP9, hence expend <strong>the</strong><br />
<strong>the</strong>rapeutic window of rtPA in <strong>an</strong> ischemic stroke models. In <strong>the</strong> present study, we tested <strong>the</strong><br />
hypo<strong>the</strong>sis that E2 c<strong>an</strong> directly reduce rtPA-induced MMPs level in rat primary astrocyte culture.<br />
Primary astrocyte cultures were prepared <strong>from</strong> neonatal rat brains. The primary astrocyte<br />
cultures were verified by immunocytochemistry followed by flow cytometry. Astrocyte cultures<br />
were treated with E2 overnight, <strong>an</strong>d <strong>the</strong>n were exposed to hum<strong>an</strong> rtPA <strong>for</strong> 24 hours at <strong>the</strong><br />
concentrations of 10 or 20µg/ml. For estrogen receptors assessment, <strong>the</strong> estrogen receptor<br />
<strong>an</strong>tagonist, ICI 178,820 (100 nM), was added 2 hours be<strong>for</strong>e E2 treatment. Gelatin zymography<br />
was used to qu<strong>an</strong>tify MMPs activation in conditioned media. rtPA signific<strong>an</strong>tly increases <strong>the</strong><br />
activation of MMP2 <strong>an</strong>d MMP9, which were markedly attenuated by E2 treatment. Fur<strong>the</strong>r, <strong>the</strong><br />
inhibitive action of E2 on rtPA-induced MMP2 <strong>an</strong>d MMP9 activation were not blocked by <strong>the</strong><br />
estrogen receptor <strong>an</strong>tagonist. Collectively, our data indicated that estrogen c<strong>an</strong> attenuate rtPAinduced<br />
MMPs activation through estrogen receptor-independent mech<strong>an</strong>ism. Our study suggests<br />
that estrogen could be used as a c<strong>an</strong>didate <strong>for</strong> <strong>the</strong> combined <strong>the</strong>rapy with rtPA to attenuate its<br />
side effects, <strong>an</strong>d hence to expends <strong>the</strong> short <strong>the</strong>rapeutic window <strong>for</strong> rtPA in <strong>the</strong> treatment of<br />
ischemic stroke.<br />
Disclosures: R. Liu, Activase received <strong>from</strong> Genentech, C. O<strong>the</strong>r Research Support (receipt of<br />
drugs, supplies, equipment or o<strong>the</strong>r in-kind support); W. Li, None; L. Xie, None; E. Poteet,<br />
None; H. Qi<strong>an</strong>, None; Y. Wen, None; J.W. Simpkins, None; S. Y<strong>an</strong>g, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.15/U6<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: PO1 AG02657<br />
U01AG031115<br />
Title: Allopregn<strong>an</strong>olone enh<strong>an</strong>ces learning <strong>an</strong>d memory in adult female triple tr<strong>an</strong>sgenic<br />
Alzheimer’s disease mice
Authors: *C. SINGH 1 , R. IRWIN 2 , J. YAO 2 , S. CHEN 2 , L. LIU 1 , R. F. THOMPSON 1 , R. D.<br />
BRINTON 2 ;<br />
1 Neurosci. Program, 2 Dept. of Pharmacol. <strong>an</strong>d Pharmaceut. Sci., USC, Los Angeles, CA<br />
Abstract: Our previous work has shown that allopregn<strong>an</strong>olone (APα), a metabolite of<br />
progesterone, promotes proliferation of neural progenitor cells derived <strong>from</strong> rat hippocampus <strong>an</strong>d<br />
cerebral cortex. Recently we have shown that APα induced neurogenesis reverses <strong>the</strong> neurogenic<br />
<strong>an</strong>d learning <strong>an</strong>d memory deficits of young 3xTgAD mice <strong>an</strong>d also enh<strong>an</strong>ces <strong>the</strong> learning <strong>an</strong>d<br />
memory of adult 3xTgAD <strong>an</strong>d old non-tr<strong>an</strong>sgenic mice. In our current study, we sought to<br />
investigate <strong>the</strong> efficacy of APα-induced neurogenesis on learning <strong>an</strong>d memory of adult female<br />
3xTgAD mice <strong>an</strong>d non-Tg mice. In accord with our previous studies, trace eyeblink<br />
conditioning, a hippocampal dependent cognitive function was used as a behavioral test. 6<br />
months old ovariectomized 3xTgAD <strong>an</strong>d <strong>the</strong>ir non-Tg counterparts were treated with ei<strong>the</strong>r<br />
vehicle or APα (10mg/kg, subcut<strong>an</strong>eously) <strong>an</strong>d 100mg/kg BrdU. Seven days later, mice were<br />
entered into a training process (250 ms tone followed by a 100 ms, 60Hz shock, 30 trials, 2<br />
sessions/day <strong>for</strong> 5 days, trace interval was 250ms). Results of our <strong>an</strong>alyses indicate that APα<br />
enh<strong>an</strong>ced <strong>the</strong> rate of learning in adult female 3xTgAD mice, while it has no effect on <strong>the</strong> agematched<br />
non-Tg mice. Studies with sham controls <strong>an</strong>d <strong>an</strong>alyses of neurogenesis are in progress.<br />
Results of <strong>the</strong>se <strong>an</strong>alyses indicate that APα works equally well as a potent cognitive enh<strong>an</strong>cer <strong>for</strong><br />
both male <strong>an</strong>d female 3xTgAD mice. Fur<strong>the</strong>r, <strong>the</strong>se data suggest that APα could be a potential<br />
<strong>the</strong>rapeutic to prevent or delay cognitive deficits associated with Alzheimer’s disease.<br />
Disclosures: C. Singh, None; R. Irwin, None; J. Yao, None; S. Chen, None; L. Liu,<br />
None; R.F. Thompson, None; R.D. Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.16/U7<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH Gr<strong>an</strong>t AG 10465<br />
NIH Gr<strong>an</strong>t AG 22550<br />
Title: Serine/threonine phosphatases in estrogen-mediated neuroprotection against cerebral<br />
ischemia
Authors: *K. D. YI, J. W. SIMPKINS;<br />
Pharmacol. & Neurosci., Univ. of North Texas HSC, Fort Worth, TX<br />
Abstract: 17β-estradiol is a potent neuroprotect<strong>an</strong>t against various cellular effects such as<br />
oxidative stress, excitotoxicity, inflammatory responses, mitochondrial dysfunction, <strong>an</strong>d<br />
apoptosis. There is considerable evidence that estrogens act on neurons through a variety of<br />
signal tr<strong>an</strong>sduction pathways to induce rapid, but acute phosphorylation <strong>an</strong>d dephosphorylation<br />
of signaling proteins. However, <strong>the</strong> exact mech<strong>an</strong>isms by which estrogens are neuroprotective<br />
remain unclear. There<strong>for</strong>e, this study investigated <strong>the</strong> role of serine/threonine phosphatases<br />
during estrogen mediated neuroprotection against cerebral ischemic <strong>an</strong>d reperfusion injury.<br />
Ovariectomized female rats were subjected to tr<strong>an</strong>sient middle cerebral artery occlusion <strong>for</strong> one<br />
hour <strong>an</strong>d <strong>the</strong> area allowed to reperfuse <strong>for</strong> 24 hr. The area of infarction was measured using a 2%<br />
solution of 2,3,5-triphenyltetrazolium chloride . Brain was also harvested <strong>for</strong> proteins <strong>an</strong>alysis by<br />
western blots. Ischemic insult decreased serine/threonine phosphatase expression in a time<br />
dependent m<strong>an</strong>ner, while persistently increased ERK <strong>an</strong>d p38 phosphorylation. Estrogen<br />
treatment attenuated this decrease in serine/threonine phosphatase expression as well as <strong>the</strong><br />
increased ERK <strong>an</strong>d p38 phosphorylation on <strong>the</strong> ischemic side. There was no ch<strong>an</strong>ge on <strong>the</strong> nonischemic<br />
side. One of <strong>the</strong> mech<strong>an</strong>ism by which 17β-estradiol protects neurons may be via<br />
preservation of phosphatase activities to dephosphorylate ERK <strong>an</strong>d p38.<br />
Disclosures: K.D. Yi, None; J.W. Simpkins, Patent on estrogens as neuroprotect<strong>an</strong>ts, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.17/U8<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH gr<strong>an</strong>t R01NS054687<br />
NIH gr<strong>an</strong>t R01NS054651<br />
NIH gr<strong>an</strong>t P01AG22550 JWS<br />
NIH gr<strong>an</strong>t P01AG10485<br />
Award <strong>from</strong> <strong>the</strong> Americ<strong>an</strong> Heart Association Texas Affiliate
Title: Dimebon, cell viability <strong>an</strong>d mitochondrial electron tr<strong>an</strong>sport complexes<br />
Authors: *E. C. POTEET, C. TAN, Y. WEN, S. YANG;<br />
UNT Hlth. Sci. Ctr., Fort Worth, TX<br />
Abstract: Dimebon, originally developed as <strong>an</strong> <strong>an</strong>tihistamine, has seen recently identified as a<br />
promising <strong>the</strong>rapy <strong>for</strong> Alzheimer’s disease (AD). In a double-blind placebo controlled study on<br />
AD patients, dimebon has been shown to signific<strong>an</strong>tly increase cognitive scores over a 52 week<br />
period. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>the</strong> mech<strong>an</strong>ism mediated dimebon action on AD has been unclear,<br />
although mitochondrial protection has been indicated. In <strong>the</strong> present study, we determined <strong>the</strong><br />
effect of dimebon on neuronal cell viability, <strong>an</strong>d its direct action on mitochondrial electron<br />
tr<strong>an</strong>sport complexes. For <strong>the</strong> cell viability assay, murine hippocampal cell line, HT-22 cells,<br />
were seeded in 96-well plates. Oxidative insult was induced by 10 µM glutamate. Cell viability<br />
was determined by calcium AM assay. For mitochondrial electron tr<strong>an</strong>sport complexes activity<br />
<strong>an</strong>alysis, mitochondria were prepared <strong>from</strong> rat hearts, <strong>an</strong>d <strong>the</strong> activity of each complex was<br />
determined by spetrophotometric <strong>an</strong>alysis using different substrates. No protective effect against<br />
glutamate cytotoxicity was observed <strong>for</strong> dimebon. Fur<strong>the</strong>rmore, no direct action of dimebon on<br />
mitochondria electron tr<strong>an</strong>sport complexes was identified. In summary, our study suggest that<br />
<strong>the</strong> protective effect of dimebon on AD is not mediated through neuroprotection or direct action<br />
on mitochondrial electron tr<strong>an</strong>sport complexes.<br />
Disclosures: E.C. Poteet, None; C. T<strong>an</strong>, None; Y. Wen, None; S. Y<strong>an</strong>g, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.18/U9<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH gr<strong>an</strong>t R01NS054687<br />
NIH gr<strong>an</strong>t R01NS054651<br />
<strong>an</strong> award <strong>from</strong> Americ<strong>an</strong> Heart Association, Inc. Texas Affiliate<br />
Title: Astrocytes modulate neutrophil functions through direct interaction
Authors: L. XIE, E. POTEET, W. LI, R. LIU, Y. WEN, *H. K. DAS, S. YANG;<br />
Univ. N. TX Hlth. Sci. Ctr. Ft. Worth, Fort Worth, TX<br />
Abstract: Neutrophils are <strong>the</strong> most abund<strong>an</strong>t cellular component in <strong>the</strong> peripheral blood <strong>an</strong>d are<br />
key players in <strong>the</strong> peripheral innate immune system. Neutrophils are also <strong>the</strong> most abund<strong>an</strong>t<br />
immune cells entering <strong>the</strong> brain parenchyma after ischemic stroke. However, <strong>the</strong> interaction of<br />
neutrophils with astrocytes, <strong>the</strong> major players in cerebral inflammatory response, is unclear. In<br />
<strong>the</strong> present study, we determined <strong>the</strong> interaction of neutrophils <strong>an</strong>d primary astrocytes.<br />
Neutrophils <strong>an</strong>d primary astrocytes were cultured <strong>from</strong> neonatal C57B/6 mouse peripheral blood<br />
<strong>an</strong>d brains, respectively. The neutrophil <strong>an</strong>d primary astrocyte cultures were verified by<br />
immunocytochemistry followed by flow cytometry. For cell-cell interaction assay, neutrophils<br />
were labeled with carboxy-fluorescein diacetate succinimidyl este (CFSE) <strong>an</strong>d co-cultured with<br />
primary astrocytes. A direct interaction of neutrophils with primary astrocytes was indicated,<br />
which was not influenced by treatment of LPS <strong>an</strong>d poly I:C. In summary, <strong>the</strong> present study<br />
demonstrated that astrocytes could affect neutrophil functions via direct interaction. The<br />
alterations of neutrophil functions by astrocytes may provide fur<strong>the</strong>r insight <strong>for</strong> <strong>the</strong> cerebral<br />
damage after infiltration of peripheral neutrophils under some pathological conditions, especially<br />
ischemic stroke.<br />
Disclosures: L. Xie, None; E. Poteet, None; W. Li, None; R. Liu, None; Y. Wen, None; H.K.<br />
Das, None; S. Y<strong>an</strong>g, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.19/U10<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIA 1 P01 AG026572; Project 3 to RDB<br />
Title: Progesterone <strong>an</strong>d clinical progestins differentially regulate proliferation of rat neural<br />
progenitor cells both in vitro <strong>an</strong>d in vivo<br />
Authors: *L. LIU 1 , K. E. MCCLURE 2 , L. ZHAO 2 , R. D. BRINTON 2 ;<br />
1 Dept Neurosci Prgm, 2 Pharmacol. <strong>an</strong>d Pharmaceut. Sci., USC, Los Angeles, CA<br />
Abstract: Our previous studies indicated that progesterone promoted rat neural progenitor cell<br />
(rNPC) proliferation <strong>an</strong>d concomit<strong>an</strong>t regulation of mitotic cell-cycle genes via a PGRMC/Erk
pathway mech<strong>an</strong>ism (Liu et al <strong>2009</strong>). To determine <strong>the</strong> effects of several clinical-relev<strong>an</strong>t<br />
progestins on rNPC proliferation, BrdU Chemilluminecense ELISA <strong>an</strong>alysis was used <strong>an</strong>d<br />
results indicated that Nestorone, NET, Norethynodrel, <strong>an</strong>d Levonorgestrel exhibited a dosedependent<br />
increases in neural progenitor cell proliferation; NETA <strong>an</strong>d ORG 31710 had no<br />
signific<strong>an</strong>t effect on cell proliferation <strong>an</strong>d medroxyprogesterone acetate (MPA) inhibited BrdU<br />
incorporation in rNPCs. Structural <strong>an</strong>alysis of <strong>the</strong>se progestins indicated that <strong>an</strong> acetyl or<br />
hydroxyl group at <strong>the</strong> 17S-position is positively associated with promoting rNPC proliferation,<br />
while a methyl group in <strong>the</strong> 6-position, especially in <strong>the</strong> S configuration, appears to be<br />
unfavorable <strong>for</strong> cell proliferation. The proliferative effect of those progestins was fur<strong>the</strong>r<br />
determined in vivo in <strong>the</strong> hippocampus <strong>from</strong> 3-month-old ovxed Sprague-Dawley rats. 24-hour<br />
exposure of progesterone signific<strong>an</strong>tly <strong>an</strong>d specifically increased protein expression of PCNA<br />
<strong>an</strong>d CDC2, two well documented mitotic markers, while clinical progestins exhibited varied<br />
effects on PCNA <strong>an</strong>d CDC2 expression level. These results indicated that despite <strong>the</strong>ir similar<br />
effect in <strong>the</strong> uterus when compared to P4, clinical progestins may have different impact on<br />
central nervous system plasticity, <strong>an</strong>d such effects should be taken into considerations when<br />
prescribed to patients.<br />
Disclosures: L. Liu, None; K.E. McClure, None; L. Zhao, None; R.D. Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.20/U11<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: NIH Gr<strong>an</strong>t AG 10465<br />
NIH Gr<strong>an</strong>t AG 22550<br />
Title: Beta-1 integrin involvement in estrogen-mediated neuroprotection<br />
Authors: *M. CHANDRA, K. D. YI, J. W. SIMPKINS;<br />
Pharmacol. & Neurosci., Univ. of North Texas HSC, Fort Worth, TX<br />
Abstract: Oxidative stress has been implicated in a variety of neurodegenerative diseases such<br />
as Alzheimer’s disease, Parkinson’s disease, <strong>an</strong>d ischemic stroke. Estrogens are gonadal steroid<br />
hormones that have been shown to be neuroprotective against numerous in vitro <strong>an</strong>d in vivo<br />
challenges. Evidence <strong>from</strong> epidemiological studies has shown <strong>the</strong> beneficial effects of estrogens
against cognitive decline <strong>an</strong>d memory loss. Integrins are cell surface receptors shown to have <strong>an</strong><br />
import<strong>an</strong>t role in neural development, synaptic plasticity, <strong>an</strong>d cell survival. The present study<br />
investigated whe<strong>the</strong>r integrins are involved in estrogen-mediated neuroprotection. HT-22 cells,<br />
<strong>an</strong> immortalized murine hippocampal cell line, were subjected to oxidative stress caused by<br />
H2O2 treatment. Hydrogen peroxide induced oxidative stress caused <strong>an</strong> increase in <strong>the</strong><br />
expression of β1-integrin protein in a time-dependent m<strong>an</strong>ner. Simult<strong>an</strong>eous 17β-estradiol (E2)<br />
treatment attenuated this increase in integrin expression. Moreover, n<strong>an</strong>omolar concentrations of<br />
E2 treatment increased <strong>the</strong> expression of integrins <strong>an</strong>d showed a time-dependent increase in<br />
expression of integrins. However, neuroprotective concentrations of E2 did not have <strong>an</strong>y effects<br />
on basal level expressions of β1-integrin proteins, which suggest a bimodal effect of estrogens.<br />
Oxidative stress induced increase in integrin expression is attenuated by estrogens; moreover,<br />
estrogens alone appear to have <strong>an</strong> effect on integrin expression. It is yet unknown <strong>the</strong> mech<strong>an</strong>ism<br />
by which estrogens influence integrin expression <strong>an</strong>d <strong>the</strong> resulting consequences.<br />
Disclosures: M. Ch<strong>an</strong>dra, None; K.D. Yi, None; J.W. Simpkins, James W. Simpkins has<br />
patent on estrogens as neuroprotect<strong>an</strong>ts, E. Ownership Interest (stock, stock options, patent or<br />
o<strong>the</strong>r intellectual property).<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.21/U12<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: PO1 AG026572; Project 5<br />
JCC supported by F31NS059174<br />
Title: Effects of continuous versus cyclic progesterone on estrogen regulation of neuropathology<br />
in 3xTg-AD mice<br />
Authors: *J. C. CARROLL 1 , E. R. ROSARIO 2 , A. VILLAMAGNA 2 , E. GENTZSCHEIN 3 , F.<br />
Z. STANCZYK 3 , C. J. PIKE 2 ;<br />
1 Dept Neurosci, 2 Gerontology, 3 Obstetrics <strong>an</strong>d Gynecology, USC, Los Angeles, CA<br />
Abstract: The Progesterone in Brain Aging <strong>an</strong>d Alzheimer’s Disease Program Project is<br />
designed to determine <strong>the</strong> neurobiology of progesterone (P4) in brain regions involved in<br />
cognition <strong>an</strong>d vulnerable to age-related cognitive decline <strong>an</strong>d Alzheimer’s disease. Estrogen-
ased hormone <strong>the</strong>rapy (HT) may reduce <strong>the</strong> risk of Alzheimer’s disease (AD) in<br />
postmenopausal women. However, <strong>the</strong> progestin component of HT is controversial, in part<br />
because recent evidence suggests that progestins c<strong>an</strong> inhibit beneficial estrogen actions. In this<br />
study, we utilized <strong>the</strong> 3xTg-AD mouse model of AD to compare <strong>the</strong> effects of continuous versus<br />
cyclic P4 treatments both alone <strong>an</strong>d in combination with estrogen on markers of AD-like<br />
neuropathology: Beta-amyloid (ABeta) accumulation, tau hyperphosphorylation, <strong>an</strong>d<br />
hippocampal-dependent behavioral impairments. We previously demonstrated that continuous<br />
P4 reduced tau phosphorylation but blocked <strong>the</strong> ABeta-lowering effect of estrogen in<br />
ovariectomized female 3xTg-AD mice. In this study, we use this same paradigm to compare <strong>the</strong><br />
effects of continuous versus cyclic progesterone, which more closely mimics natural hormone<br />
fluctuations. We observed that cyclic progesterone treatment complemented <strong>the</strong> ABeta-lowering<br />
actions of estrogen in female 3xTg-AD mice while continuous P4 attenuated <strong>the</strong> beneficial<br />
effects of estrogen. These results suggest that varying protocols of estrogen/progestin treatment<br />
differentially regulate indices of AD neuropathology <strong>an</strong>d indicate <strong>the</strong> need <strong>for</strong> clinical studies to<br />
optimize <strong>the</strong> paradigm of hormone <strong>the</strong>rapy.<br />
Disclosures: J.C. Carroll, None; E.R. Rosario, None; A. Villamagna, None; E. Gentzschein,<br />
None; F.Z. St<strong>an</strong>czyk, None; C.J. Pike, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.22/U13<br />
Topic: C.11.h. Neuroprotective mech<strong>an</strong>isms: Estrogens <strong>an</strong>d o<strong>the</strong>r hormones<br />
Support: RO1 MH067159<br />
Title: Ovariectomy induces hypometabolism in normal <strong>an</strong>d tr<strong>an</strong>sgenic Alzheimer’s mouse brain<br />
which is prevented by 17β- estradiol<br />
Authors: *F. DING 1 , R. IRWIN 1 , J. YAO 1 , J. MAO 1 , S. CHEN 1 , R. PARK 2 , G. DAGLIYAN 2 ,<br />
P. CONTI 2 , R. D. BRINTON 1,3 ;<br />
1 2 3<br />
Pharmacol. <strong>an</strong>d Pharmaceut. Sci., Mol. Imaging Ctr., Program in Neurosci., USC, Los<br />
Angeles, CA<br />
Abstract: We have previously demonstrated that loss of ovari<strong>an</strong> hormones results in a<br />
signific<strong>an</strong>t decline in glucose metabolism <strong>an</strong>d mitochondrial function which is prevented by<br />
treatment with 17β-estradiol (E2) (Brinton, TINS 2008). Because glucose uptake / metabolism
<strong>an</strong>d mitochondrial function are essential <strong>for</strong> brain metabolism, we conducted micro [18F]<br />
Fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-microPET) imaging to<br />
determine <strong>the</strong> impact of ovari<strong>an</strong> hormones on brain metabolism in ovariectomized (ovx), sham<br />
ovx <strong>an</strong>d ovx + E2 (20µg/kg) 6 month old non-tr<strong>an</strong>sgenic (nonTg) <strong>an</strong>d 3xTgAD female mice.<br />
FDG-microPET imaging commenced <strong>for</strong> all <strong>an</strong>imals when OVX <strong>an</strong>imals exhibited a signific<strong>an</strong>t<br />
rise in skin temperature (5-6 weeks post ovx). Results of <strong>the</strong>se <strong>an</strong>alyses indicate that 1) loss of<br />
ovari<strong>an</strong> hormones results in about 1 o rise in skin tail temperature 4-5 weeks post-ovx which is<br />
prevented by E2; 2) OVX-induced a signific<strong>an</strong>t decline in whole brain glucose uptake <strong>an</strong>d brain<br />
metabolism in both nonTg <strong>an</strong>d 3xTgAD mice; <strong>an</strong>d 3) administration of E2 at <strong>the</strong> time of OVX<br />
c<strong>an</strong> partially prevent OVX-induced hypometabolism in brain. These findings indicate that loss of<br />
ovari<strong>an</strong> hormones c<strong>an</strong> signific<strong>an</strong>tly decrease brain metabolism that is coincident with a rise in<br />
skin temperature. Fur<strong>the</strong>r, <strong>the</strong> OVX-induced decline in brain metabolism occurred in both<br />
normal <strong>an</strong>d 3xTgAD mice. E2 treatment at <strong>the</strong> time of OVX restored brain metabolism to 60%<br />
<strong>an</strong>d 59% of sham-OVX <strong>an</strong>imals in both nonTg <strong>an</strong>d 3xTgAD mice respectively. These findings<br />
support <strong>the</strong> functional signific<strong>an</strong>ce of OVX-induced decline in glucose metabolism <strong>an</strong>d<br />
mitochondrial function <strong>an</strong>d <strong>the</strong> prevention of this decline by E2. Fur<strong>the</strong>r, <strong>the</strong>y suggest a<br />
relationship between loss of ovari<strong>an</strong> hormones <strong>an</strong>d development of hypometabolism in brain<br />
predictive of incipient Alzheimer’s disease.<br />
Disclosures: F. Ding, None; R. Irwin, None; J. Yao, None; J. Mao, None; S. Chen, None; R.<br />
Park, None; G. Dagliy<strong>an</strong>, None; P. Conti, None; R.D. Brinton, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.23/U14<br />
Topic: C.11.a. Cell death mech<strong>an</strong>isms: Mitochondria<br />
Support: RC 2008 Itali<strong>an</strong> Minister of Health<br />
RF 2005 Itali<strong>an</strong> Minister of Health<br />
Title: Decreased energy capacity <strong>an</strong>d membr<strong>an</strong>e potential of hepatic mitochondria in<br />
parkinsoni<strong>an</strong> rats<br />
Authors: *M.-T. ARMENTERO 1 , A. FERRIGNO 2 , V. RIZZO 4 , G. AMBROSI 1 , P.<br />
RICHELMI 3 , F. BLANDINI 1 , M. VAIRETTI 3 ;<br />
1 Interdepartmental Res. Ctr. <strong>for</strong> Parkinson's Dis., IRCCS Neurol Inst. C Mondino, Pavia, Italy;
2 Dept. of Intrnl. Med. <strong>an</strong>d Therapeut., 3 Univ. of Pavia, Pavia, Italy; 4 Dept. of Biochemistry,<br />
IRCCS S<strong>an</strong> Matteo, Univ. of Pavia, Pavia, Italy<br />
Abstract: Parkinson’s disease (PD) is a neurodegenerative disease that principally targets<br />
dopaminergic (DAergic) neurons. Recent evidence, however, has indicated that <strong>the</strong> degenerative<br />
illness may affect o<strong>the</strong>r systems, <strong>an</strong>d micro-<strong>an</strong>atomic damage has also been encountered in <strong>the</strong><br />
limbic, autonomic <strong>an</strong>d neurosecretory systems. In this study we assessed whe<strong>the</strong>r <strong>the</strong> intrastriatal<br />
injection of <strong>the</strong> catecholaminergic toxin 6-hydroxydopamine (6-OHDA) induces hepatic<br />
mitochondrial alterations.<br />
Male Sprague Dawley rats received a unilateral intrastriatal injection of 6-OHDA or vehicle, <strong>an</strong>d<br />
were sacrificed 24 hours, 4 or 8 weeks after <strong>the</strong> neurotoxic insult. The extent of <strong>the</strong> nigrostriatal<br />
degeneration, correlated to <strong>the</strong> expression of tyrosine hydroxylase (TH), was evaluated in <strong>the</strong><br />
striatum <strong>an</strong>d subst<strong>an</strong>tia nigra. At sacrifice, livers were immediately removed, intact mitochondria<br />
isolated, <strong>an</strong>d mitochondrial ATP content <strong>an</strong>d production, as well as mitochondrial membr<strong>an</strong>e<br />
potential were measured. In addition, expression of TH <strong>an</strong>d dopamine tr<strong>an</strong>sporter (DAT) was<br />
evaluated on liver whole extract <strong>an</strong>d acute 6-OHDA toxicity was evaluated on isolated<br />
hepatocytes.<br />
Typical time-dependent 6-OHDA-induced nigrostriatal degeneration was observed with a<br />
discrete lesion present at <strong>the</strong> striatal but not nigral level, 24 h post-injection, that reached<br />
maximum level in both nuclei (84.4% <strong>an</strong>d 68.9%, respectively), at <strong>the</strong> fourth week, <strong>an</strong>d<br />
remained subst<strong>an</strong>tially unmodified at <strong>the</strong> last point considered (eighth week). Liver mitochondria<br />
obtained 4 or 8 weeks after 6-OHDA injection exhibited decreased ATP content <strong>an</strong>d production<br />
compared with those <strong>from</strong> control <strong>an</strong>imals. Mitochondrial membr<strong>an</strong>e potential was signific<strong>an</strong>tly<br />
decreased only 8 weeks after infusion of <strong>the</strong> toxin. No ch<strong>an</strong>ges were detected in hepatic<br />
mitochondria obtained 24 hours after <strong>the</strong> toxin injection. In addition, no TH or DAT expression<br />
was detected in liver extracts <strong>an</strong>d 6-OHDA had no direct toxic effect on isolated hepatocytes,<br />
excluding a possible direct systemic <strong>an</strong>d acute hepatic effect of <strong>the</strong> toxin<br />
Our results show <strong>for</strong> <strong>the</strong> first time a correlation between nigrostriatal degeneration <strong>an</strong>d hepatic<br />
mitochondrial dysfunctions. 6-OHDA-induced neurodegeneration is knowingly accomp<strong>an</strong>ied by<br />
a relev<strong>an</strong>t neuroinflammatory process <strong>an</strong>d has been shown to affect peripheral systems. CNS<br />
inflammation may <strong>the</strong>re<strong>for</strong>e evoke modifications of peripheral cytokine system <strong>an</strong>d depress liver<br />
function. Altered hepatic function could subsequently intervene in a vicious circle in which<br />
reduced liver metabolism <strong>an</strong>d detoxification function may increase <strong>the</strong> oxidative burden in <strong>the</strong><br />
CNS, <strong>the</strong>reby fur<strong>the</strong>r enh<strong>an</strong>cing neurodegenerative processes<br />
Disclosures: M. Armentero, None; A. Ferrigno, None; V. Rizzo, None; G. Ambrosi,<br />
None; P. Richelmi, None; F. Bl<strong>an</strong>dini, None; M. Vairetti, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.24/U15<br />
Topic: C.11.a. Cell death mech<strong>an</strong>isms: Mitochondria<br />
Support: BMBF gr<strong>an</strong>t RUS 04/004<br />
BMBF gr<strong>an</strong>t RUS 08/002<br />
RFBR 06-04-48763<br />
RFBR 08-04-00723<br />
Title: In brain mitochondria calcium-dependent permeability tr<strong>an</strong>sition is controlled by newly in<br />
mitochondria identified proteins, 2’,3’-CNP <strong>an</strong>d brain-specific protein p42(IP4) (centaurin-alpha<br />
1; ADAP1), <strong>an</strong>d <strong>the</strong>ir substrates/lig<strong>an</strong>ds<br />
Authors: *G. REISER 1 , A. GALVITA 1 , T. AZARASHVILI 2 , O. KRESTININA 2 , D.<br />
GRACHEV 2,1 ;<br />
1 Otto-von-Guericke Univ., D39120 Magdeburg, Germ<strong>an</strong>y; 2 Inst. of Theoretical <strong>an</strong>d Exptl.<br />
Biophysics, Russi<strong>an</strong> Acad. of Science, Pushchino, Pushchino, Moscow region, Russi<strong>an</strong><br />
Federation<br />
Abstract: The myelin <strong>an</strong>d oligodendrocytes marker enzyme, 2’,3’-cyclic nucleotide 3’phosphodiesterase<br />
(CNP) is also present in neural <strong>an</strong>d non-neural mitochondria, as found<br />
recently. However, its role in mitochondria is still unclear. We detected CNP in rat brain<br />
mitochondria. Then, we demonstrate <strong>for</strong> <strong>the</strong> first time p42(IP4) localisation in mitochondria <strong>from</strong><br />
rat brain <strong>an</strong>d <strong>from</strong> cells tr<strong>an</strong>sfected with p42(IP4). In brain, p42(IP4) (centaurin-alpha-1; recently<br />
named ADAP 1, within <strong>the</strong> family of Arf-GAP proteins) is mainly expressed in neurons. In<br />
mitochondria, p42(IP4) is associated with <strong>the</strong> intermembr<strong>an</strong>e space <strong>an</strong>d <strong>the</strong> inner membr<strong>an</strong>e,<br />
indicating a still unknown mitochondrial function <strong>for</strong> p42(IP4). We studied whe<strong>the</strong>r p42IP4 is<br />
involved in Ca2+-induced permeability tr<strong>an</strong>sition pore (PTP) opening. We found in mitochondria<br />
<strong>from</strong> p42IP4-tr<strong>an</strong>sfected N2a cells, signific<strong>an</strong>t decrease in Ca2+capacity <strong>an</strong>d lag time <strong>for</strong> Ca2+<br />
retention. Thus, we propose that p42(IP4) promotes Ca2+-induced PTP opening <strong>an</strong>d thus<br />
destabilizes mitochondria. In addition we studied effects of lig<strong>an</strong>ds of p42IP4 that is a dual<br />
receptor <strong>for</strong> two second messengers, soluble inositol(1,3,4,5)tetrakisphosphate (InsP4) <strong>an</strong>d<br />
phosphatidylinositol(3,4,5)trisphosphate (PIP3). Then we studied effects of CNP substrates,<br />
2’,3’-cyclic nucleotides, on functions of mitochondria. 2’,3’-cAMP <strong>an</strong>d 2’,3’-cNADP stimulated<br />
Ca2+ -induced Ca2+ release <strong>from</strong> mitochondrial matrix. This Ca2+ release under threshold Ca2+<br />
load correlated with membr<strong>an</strong>e potential dissipation <strong>an</strong>d mitochondrial swelling. The effects of<br />
2’,3’-cyclic nucleotides were suppressed by cyclosporin A, a PT inhibitor. 2’,3’-cAMP effects<br />
were observed on <strong>the</strong> functions of rat brain mitochondria only when PT was developed. This<br />
demonstrates involvement of 2’,3’-cAMP in PT regulation. Under PT development specific CNP<br />
enzymatic activity was reduced. Thus, suppression of CNP activity under threshold Ca2+ load<br />
leads to elevated 2’,3’-cAMP levels that promotes PTP in brain mitochondria. Involvement of
CNP in PT regulation was confirmed in experiments using mitochondria <strong>from</strong> CNP-knock-down<br />
oligodendrocytes (OLN93 cells). CNP reduction correlated with lowered threshold <strong>for</strong> Ca2+ -<br />
induced Ca2+ release. In summary, we reveal a new function <strong>for</strong> CNP <strong>an</strong>d 2’,3’-cAMP in<br />
mitochondria, being a regulator/promotor of mitochondrial PT. In rat brain mitochondria <strong>the</strong>re is<br />
interaction of p42IP4 with 2’,3’-CNP, shown by pull-down binding assay <strong>an</strong>d<br />
immunoprecipitation. Thus, CNP <strong>an</strong>d p42(IP4) are involved in regulation of calcium-induced<br />
PTP, <strong>an</strong> import<strong>an</strong>t stage of initiation of programmed cell death.<br />
Disclosures: G. Reiser, None; A. Galvita, None; T. Azarashvili, None; O. Krestinina,<br />
None; D. Grachev, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.25/U16<br />
Topic: C.11.a. Cell death mech<strong>an</strong>isms: Mitochondria<br />
Support: NIH/NICHD HD 44517<br />
Title: General <strong>an</strong>es<strong>the</strong>sia causes <strong>the</strong> impairment of mitochondrial morphogenesis <strong>an</strong>d function in<br />
<strong>the</strong> developing rat subiculum<br />
Authors: *V. SANCHEZ, N. LUNARDI, A. ERISIR, V. JEVTOVIC-TODOROVIC;<br />
Univ. Virginia, Charlottesville, VA<br />
Abstract: General <strong>an</strong>es<strong>the</strong>tics cause signific<strong>an</strong>t damage to developing neurons in <strong>the</strong> immature<br />
rats. Based on presently available evidence, it appears that mitochondria are likely to be <strong>the</strong> most<br />
vulnerable initial intracellular target of <strong>an</strong>es<strong>the</strong>sia neurotoxicity <strong>an</strong>d, as such, have <strong>an</strong> import<strong>an</strong>t<br />
role in initiating <strong>the</strong> intrinsic apoptotic pathway that leads to signific<strong>an</strong>t neuronal death. To<br />
fur<strong>the</strong>r study how general <strong>an</strong>es<strong>the</strong>sia affects mitochondrial morphogenesis <strong>an</strong>d function we<br />
exposed 7-day-old rat pups (P7), when <strong>the</strong>y are most vulnerable to <strong>an</strong>es<strong>the</strong>sia-induced neuronal<br />
damage to 6 hr of highly neurotoxic <strong>an</strong>es<strong>the</strong>sia cocktail containing isoflur<strong>an</strong>e, nitrous oxide <strong>an</strong>d<br />
midazolam (experimental rats) or mock <strong>an</strong>es<strong>the</strong>sia (air plus vehicle, 0.1% DMSO) (control rats).<br />
The mitochondria in subiculum, a part of hippocampal complex were examined on P8 (<strong>the</strong> peak<br />
of synaptogenesis) <strong>an</strong>d P21 (final stage of synaptogenesis). We found that <strong>the</strong> ultrastructural<br />
appear<strong>an</strong>ce of mitochondria was indicative of ongoing mitochondrial degeneration especially in<br />
P21 experimental <strong>an</strong>imals. Experimental mitochondria appeared swollen, with balloon-like<br />
cristae, but had normal-looking inner <strong>an</strong>d outer membr<strong>an</strong>es (early stage). O<strong>the</strong>rs showed large
vesicles, broken <strong>an</strong>d disorg<strong>an</strong>ized cristae with disrupted inner membr<strong>an</strong>e <strong>an</strong>d outer membr<strong>an</strong>e<br />
tear. There was also <strong>an</strong> abund<strong>an</strong>ce of dark, condensed, <strong>an</strong>d shrunken mitochondrial profiles<br />
having no clear outline between <strong>the</strong> inner <strong>an</strong>d <strong>the</strong> outer membr<strong>an</strong>e (late stage). To assess <strong>the</strong><br />
effects of general <strong>an</strong>es<strong>the</strong>sia on mitochondrial function we focused on complexes II (encoded<br />
exclusively by nDNA) <strong>an</strong>d IV (encoded in part by mtDNA thus making it particularly vulnerable<br />
to mitochondrial dysfunction). Their activity was measured in mitochondrial homogenate<br />
prepared <strong>from</strong> fresh frozen subicular tissue of P8 <strong>an</strong>d P21 rat pups <strong>an</strong>d expressed as a ratio (per<br />
<strong>the</strong> activity of citrate synthase since its activity is proportional to mitochondrial content). We<br />
found a signific<strong>an</strong>t increase in complex IV activity in P8 but not P21 rats (*, p
ecause <strong>the</strong>y are highly susceptible to NMDAR overactivation, excessive Ca 2+ loading <strong>an</strong>d<br />
mitochondrial damage. Exposure of hippocampal slices to chemical ischemia (NaCN in glucosefree<br />
saline, 30 min) or excitotoxic NMDA (200 µM, 30 min) induced rapid, large free Ca 2+<br />
elevations (monitored with low-affinity Ca 2+ -sensitive dye Fluo-4FF) <strong>an</strong>d delayed death in <strong>the</strong><br />
majority of CA1, but not in CA3 neurons in <strong>the</strong> same slice. Calcium accumulation following<br />
NMDA exposure, as measured by electron probe micro<strong>an</strong>alysis (EPMA), was also much higher<br />
in mitochondria of CA1 neurons th<strong>an</strong> in CA3. Dendritic <strong>an</strong>d somatic mitochondria in CA1<br />
neurons exhibited qu<strong>an</strong>titatively more extensive swelling <strong>an</strong>d damage as well as stronger<br />
depolarization of <strong>the</strong> mitochondrial membr<strong>an</strong>e potential (monitored with rhodamine-123).<br />
Mitochondrial depolarization was paralleled by a dramatic, CA1-specific increase in <strong>the</strong> level of<br />
reactive oxygen species (ROS) detected using superoxide-sensitive fluorescent indicator<br />
dihydroethidium (DHE). ROS must have been produced by mitochondria since it was abolished<br />
by blocking mitochondrial Ca 2+ uptake with <strong>the</strong> protonophore FCCP. Both NMDA-induced Ca 2+<br />
elevations <strong>an</strong>d ROS generation were abolished by <strong>the</strong> NMDAR <strong>an</strong>tagonist MK-801 or <strong>the</strong> Ca 2+<br />
chelator EGTA, but not by Zn 2+ chelator TPEN. These results indicate that selective<br />
vulnerability of CA1 neurons is at least partially accounted <strong>for</strong> by <strong>the</strong>ir excessive susceptibility<br />
to Ca 2+ overload-dependent mitochondrial dysfunction.<br />
Disclosures: R.I. St<strong>an</strong>ika, None; C.A. Winters, None; C.A. Br<strong>an</strong>tner, None; N.B.<br />
Pivovarova, None; B.S. Andrews, None.<br />
Poster<br />
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.27/U18<br />
Topic: C.11.a. Cell death mech<strong>an</strong>isms: Mitochondria<br />
Support: NSERC PGS3D<br />
OGS<br />
Title: Proteasome inhibition <strong>an</strong>d mitochondrial protein import: Their role in mitochondrial<br />
homeostasis<br />
Authors: *N. SHULYAKOVA, L. R. MILLS;<br />
Genet. <strong>an</strong>d Develop., TWRI, TWH, UHN, Toronto, ON, C<strong>an</strong>ada
Abstract: Increasing evidence suggests mitochondrial deficits contribute to <strong>the</strong> pathogenesis of<br />
neurodegenerative diseases. Since more <strong>the</strong>n 99% of mitochondrial proteins are nuclear encoded<br />
mitochondrial structure <strong>an</strong>d function depend upon mitochondrial protein import (MPI). Under<br />
conditions where mitochondrial protein import is inhibited mitochondrial proteins accumulate in<br />
cytoplasm. Evidence also implicates proteasome dysfunction <strong>an</strong>d <strong>the</strong> excessive <strong>an</strong>d/or “ectopic”<br />
accumulation of proteins in variety of neurodegenerative diseases. These findings link<br />
proteasome dysfunction <strong>an</strong>d mitochondrial protein import. We hypo<strong>the</strong>sized that inhibiting MPI<br />
causes <strong>the</strong> accumulation of mitochondrial proteins in <strong>the</strong> cytoplasm, which over time results in<br />
proteasome overload/dysfunction. Conversely, inhibiting proteasome function (<strong>an</strong>d <strong>the</strong><br />
consequent accumulation of mitochondrial proteins) triggers ch<strong>an</strong>ges in MPI <strong>an</strong>d activation of<br />
retrograde (mitochondria to nucleus) signalling. In differentiated PC12 cells sublethal CCCP<br />
(2µM) caused a decrease in <strong>the</strong> import of mitochondrially targeted GFP (mtGFP) <strong>an</strong>d a<br />
concomit<strong>an</strong>t accumulation of mtGFP in <strong>the</strong> cytoplasm. These findings indicate that inhibiting<br />
mitochondrial protein import creates conditions that favour proteasome overloading. Inhibition<br />
of proteasome function with MG115 (5µM) decreased mtGFP, mtHSP70, Tom20 <strong>an</strong>d mtTFA<br />
import to mitochondria in PC12 cells, <strong>an</strong>d import of mtHSP70, Tom20 <strong>an</strong>d mtTFA in primary<br />
cortical neurons. In conjunction with decreased MPI, a massive accumulation of mtGFP <strong>an</strong>d<br />
Tom20 was observed in cytoplasmic cellular fractions of PC12 cells. Proteasome inhibition<br />
induced non-reversible ch<strong>an</strong>ges in mitochondrial morphology: mitochondria were punctate, <strong>an</strong>d<br />
appeared to <strong>for</strong>m aggregates at one pole of <strong>the</strong> cell. These results indicate that proteasome<br />
inhibition is associated with deficits in MPI, <strong>an</strong>d suggest that proteasome dysfunction may<br />
disrupt mitochondrial to nuclear signalling pathways, that normally serve to coordinate ch<strong>an</strong>ges<br />
in <strong>the</strong> expression of nuclear-encoded mitochondrial proteins <strong>an</strong>d MPI.<br />
Disclosures: N. Shulyakova, None; L.R. Mills, None.<br />
Poster
543. Cell Death Mech<strong>an</strong>isms; Mitochondria I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 543.28/U19<br />
Topic: C.04.k. O<strong>the</strong>r neurodegenerative disorders<br />
Support: VA Merit Review Gr<strong>an</strong>t<br />
1R01NS062766 <strong>from</strong> NINDS<br />
Title: Mitochondrial oxidative stress is linked to microtubule degradation in neuronal cell death<br />
Authors: *N. A. KELSEY 1,2 , A. ZOMMER 2 , E. K. SCHROEDER 2 , H. M. WILKINS 2 , J.<br />
PHELPS 2 , R. J. BOUCHARD 3 , D. A. LINSEMAN 3,2 ;<br />
1 Res. 151, Denver, CO; 2 Dept. of Biol. Sci. <strong>an</strong>d Ele<strong>an</strong>or Roosevelt Inst., Univ. of Denver,<br />
Denver, CO; 3 Res. 151, VA Med. Ctr., Denver, CO<br />
Abstract: Mitochondrial oxidative stress (MOS) <strong>an</strong>d disassembly of <strong>the</strong> microtubule network<br />
are two hallmarks of neuronal death leading to neurodegeneration. In this study, we investigated<br />
<strong>the</strong> link between MOS <strong>an</strong>d microtubule degradation in rat cerebellar gr<strong>an</strong>ule neurons (CGNs).<br />
CGNs were exposed to a number of insults previously shown to induce cell death principally<br />
through MOS, such as Bcl-2 inhibition, complex I inhibition, <strong>an</strong>d generation of hydrogen<br />
peroxide, peroxynitrite, or nitric oxide. CGNs incubated with <strong>the</strong>se mitochondrial oxidative<br />
stressors exhibited nearly complete fragmentation of <strong>the</strong>ir microtubule networks. Additionally,<br />
using a differential cell lysis protocol, MOS induced <strong>an</strong> increase in soluble “monomeric” tubulin<br />
relative to insoluble “polymerized” tubulin. In contrast, CGNs induced to undergo apoptosis by<br />
insults that do not cause overt MOS, like removal of depolarizing extracellular potassium,<br />
excitotoxicity, <strong>an</strong>d endoplasmic reticulum stress, maintained a signific<strong>an</strong>t network of<br />
microtubules. As a result, <strong>the</strong>se CGNs did not show <strong>an</strong>y signific<strong>an</strong>t increase in soluble<br />
“monomeric” tubulin. These studies indicate that MOS selectively induces microtubule<br />
degradation in CGNs. Currently, we are examining molecular signals that may be tr<strong>an</strong>sduced<br />
<strong>from</strong> oxidatively-stressed mitochondria to trigger microtubule breakdown. Specifically, we are<br />
measuring <strong>the</strong> expression <strong>an</strong>d phosphorylation status of microtubule bundling proteins, such as<br />
tau <strong>an</strong>d stathmin, when CGN death is induced by MOS-dependent <strong>an</strong>d MOS-independent<br />
mech<strong>an</strong>isms. Finally, we are using live cell confocal fluorescence microscopy to establish <strong>the</strong><br />
kinetics of MOS, microtubule disassembly, <strong>an</strong>d mitochondrial fragmentation in real time. These<br />
studies will provide novel in<strong>for</strong>mation on <strong>the</strong> molecular mech<strong>an</strong>isms linking MOS to<br />
microtubule disruption in dying neurons. (Acknowledgments: Funding was provided by a VA<br />
merit review gr<strong>an</strong>t <strong>an</strong>d 1R01NS062766 <strong>from</strong> NINDS)<br />
Disclosures: N.A. Kelsey, None; A. Zommer, None; E.K. Schroeder, None; H.M. Wilkins,<br />
None; J. Phelps, None; R.J. Bouchard, None; D.A. Linsem<strong>an</strong>, None.
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.1/U20<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: Fondazione B<strong>an</strong>ca del Monte di Lombardia<br />
Title: Org<strong>an</strong>ic cation tr<strong>an</strong>sporters <strong>an</strong>d cisplatin-induced peripheral neurotoxicity<br />
Authors: *C. CERESA 1 , G. NICOLINI 2 , A. CANTA 2 , V. A. CAROZZI 2 , G. TREDICI 2 , G.<br />
CAVALETTI 2 ;<br />
1 DNTB, Univ. of Mil<strong>an</strong>-Bicocca, Monza, Mi, Italy; 2 DNTB, Univ. of Mil<strong>an</strong>-Bicocca, Monza,<br />
Italy<br />
Abstract: Cisplatin (CDDP) is a widely used chemo<strong>the</strong>rapeutic agent <strong>for</strong> <strong>the</strong> treatment of<br />
several solid tumors. CDDP induces a severe <strong>an</strong>d dose-limiting sensory neuropathy, due to<br />
damage of <strong>the</strong> primary sensory neurons of <strong>the</strong> dorsal root g<strong>an</strong>glia (DRG) where it <strong>for</strong>ms DNA<br />
adducts.<br />
Previous in vivo <strong>an</strong>d in vitro studies demonstrated that org<strong>an</strong>ic cation tr<strong>an</strong>sporters (OCTs) are<br />
implicated in CDDP nephrotoxicity but <strong>the</strong> involvement of OCTs in CDDP neurotoxicity has<br />
never been examined.<br />
The present study was aimed to investigate <strong>the</strong> putative role of OCT-1, OCT-2, OCTN-1 <strong>an</strong>d<br />
OCTN-2 in CDDP induced neuropathy.<br />
Female Wistar rats were treated with CDDP administration 2 mg/Kg i.p. 2qw x4. Untreated rats<br />
were used as control. The effect of CDDP on OCTs expression in rat DRG was studied by<br />
TaqM<strong>an</strong> Real Time PCR <strong>an</strong>d it was demonstrated that CDDP administration reduced OCT2<br />
mRNA levels with respect to <strong>the</strong> control.<br />
Electrophisiological <strong>an</strong>alysis demonstrated a nerve conduction velocity (NCV) impairment due<br />
to <strong>the</strong> chronic administration of CDDP. DRG morphometric <strong>an</strong>alysis showed that neuronal soma,<br />
nucleus <strong>an</strong>d nucleolus size were signific<strong>an</strong>tly reduced.<br />
We, <strong>the</strong>re<strong>for</strong>e, investigated <strong>the</strong> correlation existing between <strong>the</strong> neurophysiologic/morphometric<br />
parameters <strong>an</strong>d <strong>the</strong> OCT2 mRNA levels. This <strong>an</strong>alysis evidenced that <strong>the</strong> reduction of OCT2<br />
expression correlated with <strong>the</strong> decrease of NCV as well as with <strong>the</strong> reduction of soma, nucleus<br />
<strong>an</strong>d nucleolus size.<br />
To obtain mech<strong>an</strong>istic in<strong>for</strong>mation about <strong>the</strong> role of OCT2 in CDDP neurotoxicity we used <strong>an</strong> in<br />
vitro model of DRG neurons obtained <strong>from</strong> embryonic rats E15. In order to verify <strong>the</strong><br />
involvement of OCT2 in <strong>the</strong> neuronal uptake of CDDP, nerve growth factor (NGF) differentiated
neurons were exposed to CDDP 7 µg/ml alone or in combination with <strong>the</strong> OCT2 inhibitor<br />
cimetidine (CMT) 20, 50 <strong>an</strong>d 100 µM <strong>for</strong> 24 <strong>an</strong>d 48 hours. After 24 <strong>an</strong>d 48 hours of co-treatment<br />
with CDDP <strong>an</strong>d CMT 100 µM we observed a signific<strong>an</strong>t increase of neuronal survival with<br />
respect to CDDP treated cells.<br />
Based on <strong>the</strong>se results, a pilot in vivo study was per<strong>for</strong>med that also confirmed a partial<br />
neuroprotective effect of CMT. Female Wistar rats were r<strong>an</strong>domized in 3 different groups <strong>an</strong>d<br />
treated as follows: CDDP 2 mg/Kg i.p. 2qw x4; CDDP 2 mg/Kg i.p. 2qw x4 + CMT 60 mg/kg os<br />
daily <strong>for</strong> 4 weeks; un-treated control rats. CMT 60 mg/Kg could not revert <strong>the</strong> CDDP-induced<br />
NVC impairment, although it signific<strong>an</strong>tly ameliorated morphometric parameters.<br />
Our data suggests <strong>an</strong> affinity of CDDP <strong>for</strong> cellular uptake via rat OCT2 in DRG neurons. The<br />
hypo<strong>the</strong>sis, to be fur<strong>the</strong>r validated, is that CDDP treatment could determine a decrease in OCT2<br />
expression as a neuronal defense response against subsequent exposure <strong>an</strong>d neuronal uptake of<br />
CDDP.<br />
Disclosures: C. ceresa, None; G. nicolini, None; A. c<strong>an</strong>ta, None; V.A. Carozzi, None; G.<br />
tredici, None; G. cavaletti, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.2/U21<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: INSERM,<br />
Neuropole (NeRF)<br />
Title: N<strong>an</strong>oparticle tr<strong>an</strong>slocation <strong>from</strong> muscle to brain<br />
Authors: *J. CADUSSEAU 1,2 , Z. KHAN 1 , V. ITIER 1,2 , F. CHRÉTIEN 1 , M. BRIGITTE 1 , R. K.<br />
GHERARDI 1 ;<br />
1 Unite INSERM 955, Creteil, Fr<strong>an</strong>ce; 2 Univ. Paris12-Paris Est, Creteil, Fr<strong>an</strong>ce<br />
Abstract: Defining biodistribution of n<strong>an</strong>oparticles is a crucial issue <strong>for</strong> both medical<br />
applications <strong>an</strong>d neurotoxicology. Vaccines containing Al(OH)3 n<strong>an</strong>oparticles as <strong>an</strong> adjuv<strong>an</strong>t<br />
may cause local «macrophagic myofasciitis» <strong>an</strong>d cognitive dysfunction in susceptible<br />
individuals. We developped several experimental approaches to determine <strong>the</strong> fate of Al(OH)3<br />
or surrogate fluorescent n<strong>an</strong>oparticles, <strong>the</strong>ir biodistribution <strong>an</strong>d modalities of <strong>the</strong>ir delivery to <strong>the</strong>
ain after i.m. injection.<br />
The highly specific atomic micro<strong>an</strong>alysis PIXE approach on 20um thick sections carefully<br />
protected <strong>from</strong> <strong>the</strong> environmental Al, revealed Al signal in murine muscle at D4 post vaccine<br />
injection, <strong>an</strong>d spleen (D21). In <strong>the</strong> brain, Al spots were detected with a frequency of 31% (at<br />
D21, D180, D365) tested fields (0,25sqmm, n=73) a much higher proportion th<strong>an</strong> in agematched<br />
controls (7,5% n=94). Fluorescent 500nm sized beads (fb) confirmed particle<br />
tr<strong>an</strong>slocation <strong>from</strong> <strong>the</strong> injected muscle to <strong>the</strong> brain. In <strong>the</strong> injected muscle, <strong>the</strong>y were found<br />
within macrophages. They were detected in <strong>the</strong> draining lymph nodes as soon as 1h, <strong>an</strong>d<br />
<strong>the</strong>reafter in <strong>the</strong> spleen <strong>an</strong>d <strong>the</strong> liver implicating <strong>the</strong>ir circulation in <strong>the</strong> blood stream. In <strong>the</strong> brain<br />
<strong>the</strong>y were detected at D21, D180 <strong>an</strong>d D365 in <strong>the</strong> parenchyma (87%) mostly in <strong>the</strong> grey matter,<br />
<strong>the</strong> choroid plexus (2%) or <strong>the</strong> meninges (11%). Similar experiments with i.m. injected fb were<br />
per<strong>for</strong>med in CX3CR1 GFP+/- mice. At D90 fb were observed in GFP+ microglial cells. Finally<br />
fb were i.m. injected in a mouse model of blood brain barrier (BBB) rupture (mdx). At D21 <strong>an</strong>d<br />
D90 post-injection, brains were removed, hemispheres separated <strong>an</strong>d treated ei<strong>the</strong>r <strong>for</strong><br />
macrophage/microglia (MM) extraction or histology. MM were extracted using CD45-coupled<br />
magnetic beads (Miltenyi) <strong>an</strong>d sorted according to <strong>the</strong>ir CD11b expression. Sorted cells were<br />
<strong>the</strong>reafter microscopically <strong>an</strong>alyzed on cytospins <strong>for</strong> <strong>the</strong>ir fb content. At D21 <strong>the</strong> number of fb<br />
within MM was sixfold higher in mdx compared to C57 (49,5 fb per 50.103 cells versus 8,3 per<br />
50.103). At D90 <strong>the</strong>re was a fur<strong>the</strong>r twofold increase of fb incorporation in mdx mice (96 fb per<br />
50.103 cells). These fb were clearly located in ramified perivascular microglia.<br />
These results indicate a tr<strong>an</strong>slocation of inert n<strong>an</strong>osized particle, ei<strong>the</strong>r aluminic or surrogate<br />
fluorescent ones, <strong>from</strong> <strong>the</strong> muscle to <strong>the</strong> brain, where <strong>the</strong>y may be incorporated by microglial<br />
cells. These observations suggest a troj<strong>an</strong> horse hypo<strong>the</strong>sis <strong>for</strong> <strong>the</strong> tr<strong>an</strong>slocation of n<strong>an</strong>oparticles<br />
<strong>from</strong> <strong>the</strong> muscle to <strong>the</strong> brain involving macrophagic/monocytes cells, blood stream <strong>an</strong>d<br />
physiologial renewal of brain microglia. This process is increased in chronically altered BBB.<br />
Disclosures: J. Cadusseau, None; Z. Kh<strong>an</strong>, None; V. Itier, None; F. Chrétien, None; M.<br />
Brigitte, None; R.K. Gherardi, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.3/U22<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: NIH Gr<strong>an</strong>t ES016931
Title: Development of a new assay <strong>for</strong> assessing m<strong>an</strong>g<strong>an</strong>ese tr<strong>an</strong>sport kinetics in a striatal cell<br />
model of Huntington’s disease<br />
Authors: *G. F. KWAKYE 1 , D. D. LI 2 , M. WEGRZYNOWICZ 3 , B. B. WILLIAMS 4 , B. K.<br />
VADODARIA 2 , A. B. BOWMAN 5 ;<br />
2 Neurosci. Undergraduate Program, 3 Neurol., 4 Neurosci. Grad. Program, 5 V<strong>an</strong>derbilt Kennedy<br />
Center, Dept. of Neurology, V<strong>an</strong>derbilt Ctr. <strong>for</strong> Mol. Toxicology, 1 V<strong>an</strong>derbilt Univ., Nashville,<br />
TN<br />
Abstract: M<strong>an</strong>g<strong>an</strong>ese (Mn) is <strong>an</strong> essential trace element necessary <strong>for</strong> proper functioning of a<br />
variety of physiological processes including cellular energy metabolism <strong>an</strong>d also acts as a<br />
cofactor <strong>for</strong> several m<strong>an</strong>g<strong>an</strong>ese dependent enzymes. Despite its essentiality, Mn in excess acts a<br />
neurotoxic<strong>an</strong>t in several neurodegenerative diseases including Huntington’s disease (HD),<br />
wherein; <strong>the</strong>re is selective degeneration of medium spiny neurons (MSNs) in <strong>the</strong> corpus striatum,<br />
which is a major target of m<strong>an</strong>g<strong>an</strong>ese (Mn) accumulation. The neurotoxicity observed <strong>from</strong> Mn<br />
accumulation in <strong>the</strong> brain has been associated with deficits in cellular energy metabolism <strong>an</strong>d<br />
alterations in iron homeostasis. Data <strong>from</strong> our lab provide evidence <strong>for</strong> <strong>the</strong> discovery of a<br />
pathophysiological gene-environment interaction between polyglutamine-exp<strong>an</strong>ded mouse<br />
huntingtin (htt) protein <strong>an</strong>d <strong>the</strong> neurotoxic<strong>an</strong>t metal ion, Mn. We find that <strong>the</strong> disease-causing<br />
protein <strong>an</strong>d Mn suppress <strong>the</strong> neurotoxic activities of each o<strong>the</strong>r. We hypo<strong>the</strong>size a geneenvironment<br />
interaction, in which, mut<strong>an</strong>t htt alters net Mn tr<strong>an</strong>sport <strong>an</strong>d storage. In order to<br />
assess dynamic Mn tr<strong>an</strong>sport kinetics, we have developed a new assay to measure <strong>the</strong> rate of<br />
import, export, <strong>an</strong>d total Mn levels in striatal cells. This fluorometric high-throughput assay takes<br />
adv<strong>an</strong>tage of <strong>the</strong> metal binding fluorescent dye, Fura-2, to measure total extracted cellular Mn<br />
content. We have now used this assay to detect time <strong>an</strong>d concentration dependent ch<strong>an</strong>ges in<br />
intracellular Mn levels in a striatal cell line model of HD. We find that mut<strong>an</strong>t (STHdh Q111/Q111 )<br />
striatal cells have decrease Mn uptake in a time <strong>an</strong>d concentration-dependent m<strong>an</strong>ner relative to<br />
wild-type (STHdh Q7/Q7 ) cells. This assay provides a me<strong>an</strong>s to assess <strong>the</strong> dynamic Mn tr<strong>an</strong>sport<br />
kinetics underlying HD-Mn disease toxic<strong>an</strong>t interaction.<br />
Disclosures: G.F. Kwakye, None; D.D. Li, None; M. Wegrzynowicz, None; B.B. Williams,<br />
None; B.K. Vadodaria, None; A.B. Bowm<strong>an</strong>, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.4/U23<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration
Support: FCT– Gr<strong>an</strong>t POCI/SAU-FCF/58330/2004<br />
Institute <strong>for</strong> Interdisciplinary Research, University of Coimbra, Gr<strong>an</strong>t III/34/2008<br />
FCT Fellowship (TCO) SFRH/BPD/34711/2007<br />
Title: Neurotoxicity of cocaine-opioid combinations in rat cortical neurons<br />
Authors: *T. CUNHA-OLIVEIRA 1 , A. REGO 1,2 , J. GARRIDO 3 , F. BORGES 4 , T.<br />
MACEDO 5 , C. R. OLIVEIRA 1,2 ;<br />
1 Ctr. For Neurosci. <strong>an</strong>d Cell Biol., Coimbra, Portugal; 2 Inst. of Biochem., Fac. of Medicine,<br />
Univ. of Coimbra, Coimbra, Portugal; 3 Chem. Engin. Dept., Sch. of Engineering, ISEP,<br />
Polytechnic Inst. of Porto, Porto, Portugal; 4 Org<strong>an</strong>ic Chem. Dept., Fac. of Pharmacy, Univ. of<br />
Porto, Porto, Portugal; 5 AIBILI, Coimbra, Portugal<br />
Abstract: Cocaine <strong>an</strong>d heroin are frequently co-abused by hum<strong>an</strong>s, in a combination known as<br />
speedball. The co-abuse of <strong>the</strong>se two drugs may represent <strong>an</strong> increased neurotoxic risk, since<br />
speedball abusers exhibit a more severe psychopathology as compared to o<strong>the</strong>r cocaine addicts,<br />
<strong>an</strong>d are more likely to fail in drug abuse treatment. Recently, chemical interactions between <strong>the</strong><br />
heroin (Her) metabolite morphine (Mor) <strong>an</strong>d cocaine (Coc) were described, resulting in <strong>the</strong><br />
<strong>for</strong>mation of Mor-Coc adducts (1). In this work, we evaluated whe<strong>the</strong>r chemical interactions<br />
between cocaine <strong>an</strong>d opioids affect <strong>the</strong> neurotoxicity of <strong>the</strong>se drugs. For this purpose, rat cortical<br />
neurons were incubated with Coc, Her, Her followed by Coc <strong>an</strong>d Her-Coc (combined 1:1).<br />
Neurons exposed to Her, Her plus Coc <strong>an</strong>d Her-Coc exhibited a decrease in cell viability, which<br />
was more pronounced in neurons exposed to Her <strong>an</strong>d Her plus Coc, in comparison with neurons<br />
exposed to <strong>the</strong> combined mixture. Cells exposed to <strong>the</strong> drug combination containing adducts<br />
(Her-Coc) showed increased intracellular calcium levels <strong>an</strong>d mitochondrial dysfunction, as<br />
determined by a decrease in intracellular ATP levels, mitochondrial membr<strong>an</strong>e potential <strong>an</strong>d<br />
mitochondrial cytochrome c levels, but decreased caspase-dependent apoptosis <strong>an</strong>d increased<br />
metabolic viability, when compared to cells sequentially exposed to Her <strong>an</strong>d Coc. Besides<br />
presenting apoptotic characteristics, neurons exposed to <strong>the</strong> combination containing adducts<br />
(Her-Coc) also displayed loss of membr<strong>an</strong>e integrity, associated with necrosis. Thus, <strong>the</strong><br />
combination of drugs containing adducts <strong>an</strong>d Her plus Coc induce cell death through different<br />
intracellular pathways, indicating that adduct <strong>for</strong>mation shifts cell death mech<strong>an</strong>isms towards<br />
necrosis. These effects may contribute to increase failure in drug abuse treatment, <strong>an</strong>d suggest<br />
that improvement of mitochondrial function is a promising target <strong>for</strong> neuroprotective strategies<br />
in poly-drug abusers.<br />
(1) Garrido et al. Anal.Bio<strong>an</strong>al.Chem. 388, 11799-1808 (2007).<br />
Disclosures: T. Cunha-Oliveira, None; A. Rego, None; J. Garrido, None; F. Borges,<br />
None; T. Macedo, None; C.R. Oliveira, None.<br />
Poster
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.5/U24<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: CICYT gr<strong>an</strong>t: AGL2005-07924-C04-03/ALI to M.T. Fernández-Sánchez<br />
CICYT gr<strong>an</strong>t: CTQ2008-06754-C04-03/PPQ to A. Novelli.<br />
Title: Neurodegeneration by domoic acid <strong>an</strong>d palytoxin<br />
Authors: A. PÉREZ-GÓMEZ 1 , A. NOVELLI 2 , *M. T. FERNANDEZ-SANCHEZ 1 ;<br />
1 2<br />
Dept. Biochem. <strong>an</strong>d Inst. of Biotech., Dept. Psychology <strong>an</strong>d Inst. of Biotech., Univ. of Oviedo,<br />
Oviedo, Spain<br />
Abstract: Enh<strong>an</strong>cement of domoic acid toxicity by polye<strong>the</strong>r compounds related to<br />
clupeotoxism.<br />
Clupeotoxism, a hum<strong>an</strong> syndrome due to clupeoid fish poisoning that may include neurological<br />
problems <strong>an</strong>d death, has been originally associated with <strong>the</strong> polye<strong>the</strong>r molecule palytoxin. The<br />
toxic action of palytoxin has been attributed to its capability to tr<strong>an</strong>s<strong>for</strong>m <strong>the</strong> Na+/K+ pump in a<br />
non selective cation ch<strong>an</strong>nel, inducing a progressive depolarization of cell membr<strong>an</strong>e potential<br />
<strong>an</strong>d ultimately cell death. We have used cultured cerebellar neurons to determinate <strong>the</strong><br />
neurotoxicity of palytoxin <strong>an</strong>d to explore <strong>the</strong> possibility of interaction between palytoxin <strong>an</strong>d <strong>the</strong><br />
amnesic seafood excitotoxin domoic acid, which toxicity has been already characterized by our<br />
group in <strong>the</strong>se neurons. Exposure of neuronal cultures to high picomolar concentrations of<br />
palytoxin caused a slow neurodegeneration characterized by initial swelling of <strong>the</strong> cell bodies<br />
<strong>an</strong>d followed by both neurite <strong>an</strong>d cell body degeneration after 24 h. Only 10 % neuronal survival<br />
was observed 24 h after exposure to 500 pM palytoxin. Intracellular Ca 2+ concentration<br />
following 2 h exposure to palytoxin increased 2-3 times over basal levels. Exposure of cultures<br />
to subtoxic concentrations of both palytoxin <strong>an</strong>d domoic acid resulted in massive<br />
neurodegeneration without <strong>an</strong>y fur<strong>the</strong>r increase in intracellular Ca 2+ concentration. The<br />
synergism between palytoxin <strong>an</strong>d domoic acid in producing neurodegeneration was prevented by<br />
<strong>the</strong> non-NMDA receptor <strong>an</strong>tagonist CNQX, but nei<strong>the</strong>r by NMDA receptor <strong>an</strong>tagonists nor<br />
VSCC blockers.<br />
These results provide fur<strong>the</strong>r insight on <strong>the</strong> excitotoxic process <strong>an</strong>d suggest <strong>the</strong> need to establish<br />
new safety limits <strong>for</strong> <strong>the</strong> presence of palytoxin <strong>an</strong>d domoic acid in seafood.<br />
Disclosures: A. Pérez-Gómez , None; A. Novelli, None; M.T. Fern<strong>an</strong>dez-S<strong>an</strong>chez, None.
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.6/U25<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Title: Mech<strong>an</strong>ism of thimerosal neurotoxicity in cortical culture<br />
Authors: T. RUSH, *J. A. HJELMHAUG, D. LOBNER;<br />
Biomed. Sci., Marquette Univ., Milwaukee, WI<br />
Abstract: Thimerosal is a well-known neurotoxic<strong>an</strong>t used as a preservative of vaccines.<br />
Previous studies have demonstrated that thimerosal-induced neurotoxicity involves oxidative<br />
stress <strong>an</strong>d c<strong>an</strong> be protected against by glutathione supplementation. Here we aim to elucidate <strong>the</strong><br />
mech<strong>an</strong>ism of thimerosal neurotoxicity by targeting <strong>the</strong> protection af<strong>for</strong>ded by <strong>the</strong> glutathione<br />
system. Exposure to 5 µM thimerosal was found to be toxic in murine mixed cortical cultures.<br />
Additionally, we posit that <strong>the</strong> ethyl mercury component of thimerosal is <strong>the</strong> toxic molecule as 5<br />
µM ethyl mercury induced a toxicity level similar to thimerosal, whereas <strong>the</strong> thiosalicyclic acid<br />
component was not toxic up to 100 µM. Oxidative stress was observed by DCF fluorescence<br />
following thimerosal exposure <strong>an</strong>d <strong>the</strong> free radical scavenger trolox (100 µM) decreased <strong>the</strong><br />
oxidative stress. However, trolox was unable to protect against <strong>the</strong> thimerosal insult. These data<br />
suggests that oxidative stress is secondary to <strong>the</strong> neurotoxic mech<strong>an</strong>ism(s) of thimerosal.<br />
Glutathione supplementation, with reduced glutathione (GSH) or <strong>the</strong> cell permeable glutathione<br />
monoethylester (GSHME) restored neuronal viability as well as decreased oxidative stress.<br />
Using methods <strong>for</strong> detecting reduced glutathione, oxidized/disulfide glutathione (GSSG) <strong>an</strong>d<br />
total glutathione (e.g. GSH + GSSG) we <strong>an</strong>alyzed thimerosal effects on glutathione cycling <strong>an</strong>d<br />
metabolism. In brief, thimerosal decreased total glutathione <strong>an</strong>d GSSG, without decreasing levels<br />
of reduced GSH available within <strong>the</strong> cell. These effects were observed <strong>from</strong> samples of <strong>the</strong><br />
intracellular content as well as <strong>from</strong> <strong>the</strong> extracellular media. Taking <strong>the</strong>se data toge<strong>the</strong>r, we<br />
speculate that endogenous glutathione primarily acts to limit thimerosal toxicity by conjugating<br />
<strong>the</strong> ethyl mercury <strong>an</strong>d subsequently shuttling it out of <strong>the</strong> cell via <strong>the</strong> multi-drug resist<strong>an</strong>ce<br />
protein (MRP) family of tr<strong>an</strong>sporters. Our speculation is supported by our measures of<br />
glutathione <strong>an</strong>d corroborated by our finding that inhibition of MRP with MK571 (10 <strong>an</strong>d 50µM)<br />
greatly enh<strong>an</strong>ces thimerosal neurotoxicity. Neuronal sensitivity to thimerosal toxicity is likely<br />
due to <strong>the</strong> reli<strong>an</strong>ce of neurons on <strong>the</strong> glial production <strong>an</strong>d provision of glutathione. To assess this<br />
we are <strong>an</strong>alyzing thimerosal-induced glutathione alterations in cultures containing only neurons<br />
or only glia.<br />
Disclosures: T. Rush, None; J.A. Hjelmhaug, None; D. Lobner, None.
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.7/U26<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: DA11389<br />
DA04222<br />
DA00869<br />
DA19447<br />
DA00378<br />
DA13367<br />
PhRMA foundation<br />
Title: Methamphetamine-induced dopamine tr<strong>an</strong>sporter complex <strong>for</strong>mation, microglial<br />
activation, <strong>an</strong>d neurotoxicity<br />
Authors: *G. C. HADLOCK 1 , J. W. GIBB 2 , G. R. HANSON 2 , A. E. FLECKENSTEIN 2 ;<br />
1 Univ. Utah, salt lake city, UT; 2 Univ. Utah, Salt lake city, UT<br />
Abstract: Methamphetamine (METH) is a widely abused psychostimul<strong>an</strong>t. In hum<strong>an</strong>s, longterm<br />
METH abuse c<strong>an</strong> cause persistent deficits in dopaminergic systems which may be<br />
associated with cognitive impairment in abstinent METH abusers. In a rodent model, multiple,<br />
high-dose administrations of METH cause persistent dopaminergic deficits. Multiple, high-dose<br />
administrations of METH also cause microglial activation <strong>an</strong>d METH-induced dopamine<br />
tr<strong>an</strong>sporter (DAT) complex <strong>for</strong>mation. Previous studies have suggested a role <strong>for</strong> oxidative stress<br />
in DAT complex <strong>for</strong>mation. Fur<strong>the</strong>r, microglia are activated during neurotoxic events <strong>an</strong>d c<strong>an</strong><br />
also contribute to <strong>the</strong> <strong>for</strong>mation of reactive species. However, <strong>the</strong> time-course <strong>an</strong>d relationship of<br />
microglial activation <strong>an</strong>d DAT complex <strong>for</strong>mation following METH treatment have not been<br />
reported. The present study investigated <strong>the</strong> time-course of DAT complex <strong>for</strong>mation, microglial<br />
activation, <strong>an</strong>d <strong>the</strong> loss of a striatal monoaminergic terminal marker, vesicular monoamine<br />
tr<strong>an</strong>sporter 2 (VMAT2). In all studies, hyper<strong>the</strong>rmia was maintained in METH-treated rats.
Results revealed DAT complexes present at 24, 48, <strong>an</strong>d 72 h following METH-treatment.<br />
Additionally, VMAT2 <strong>an</strong>d DAT monomer immunoreactivity were signific<strong>an</strong>tly decreased at<br />
<strong>the</strong>se time-points. In contrast, microglial activation, as determined by increased glial fibrillary<br />
acidic protein immunoreactivity, was signific<strong>an</strong>tly increased only at 48 <strong>an</strong>d 72 h. Consistent with<br />
previous studies showing a role <strong>for</strong> dopamine D2 receptor activation in DAT complex <strong>for</strong>mation,<br />
pretreatment with <strong>the</strong> dopamine D2 receptor <strong>an</strong>tagonist, eticlopride, attenuated <strong>the</strong> loss of<br />
VMAT2 24 h following multiple METH administration. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest<br />
that <strong>the</strong> mech<strong>an</strong>isms underlying DAT complex <strong>for</strong>mation within 24 h following multiple METH<br />
administration are not related to microglial activation. However, 48-72 h after multiple METH,<br />
DAT complexes are present <strong>an</strong>d <strong>the</strong>re is signific<strong>an</strong>t microglial activation. These results allow <strong>for</strong><br />
<strong>the</strong> possibility that microglial activation may contribute to <strong>the</strong> presence of DAT complexes<br />
within this period. Additionally, <strong>the</strong>se data provide evidence <strong>for</strong> dopamine D2 receptor activation<br />
in <strong>the</strong> acute (within 24 h) ch<strong>an</strong>ges in DAT <strong>an</strong>d VMAT2 immunoreactivity. These results <strong>an</strong>d<br />
<strong>the</strong>ir implications on METH-induced persistent dopaminergic deficits will be discussed.<br />
Disclosures: G.C. Hadlock, None; J.W. Gibb, None; G.R. H<strong>an</strong>son, None; A.E. Fleckenstein,<br />
None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.8/U27<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: NIEHS gr<strong>an</strong>t RO1ES015295 (J.Schneider)<br />
P30 ES014443 (K. Ramos)<br />
Title: Influence of gender <strong>an</strong>d lead exposure on gene expression profiles in hippocampus<br />
Authors: *D. W. ANDERSON 1 , N. G. F. COOPER 2 , S. DATTA 3 , A. CAMBON 3 , J. S.<br />
SCHNEIDER 1 ;<br />
1 Dept Pathol, Anat & Cell Biol, Thomas Jefferson Univ., Philadelphia, PA; 2 Dept. of Anatom.<br />
Sci. <strong>an</strong>d Neurobiology, Sch. of Med., 3 Dept. of Bioin<strong>for</strong>matics <strong>an</strong>d Biostatistics, Sch. of Publ.<br />
Hlth. <strong>an</strong>d In<strong>for</strong>mation Sci., Univ. of Louisville, Louisville, KY<br />
Abstract: Despite initiatives to reduce environmental sources of exposure, childhood lead<br />
poisoning remains <strong>an</strong> import<strong>an</strong>t public health problem in <strong>the</strong> United States <strong>an</strong>d around <strong>the</strong> world.
Recent clinical data suggest that gender may be a modifier of <strong>the</strong> association between lead <strong>an</strong>d<br />
brain development/function <strong>an</strong>d neuropsychological development. However, <strong>the</strong> influence of<br />
gender on outcomes <strong>from</strong> developmental lead exposure has received little attention<br />
experimentally. In <strong>an</strong> attempt to begin to fill this void, <strong>the</strong> present study has been per<strong>for</strong>med to<br />
examine <strong>the</strong> influence of gender on gene expression profiles in <strong>the</strong> hippocampus in rats exposed<br />
to lead developmentally. At we<strong>an</strong>ing (25 days of age), male <strong>an</strong>d female Long-Ev<strong>an</strong>s rats, housed<br />
4 per cage, were fed diets containing lead acetate (1,500 ppm) or control diet without lead <strong>for</strong> 30<br />
days (N = 4 per group). O<strong>the</strong>r th<strong>an</strong> diet, all <strong>an</strong>imals were housed <strong>an</strong>d h<strong>an</strong>dled in exactly <strong>the</strong> same<br />
m<strong>an</strong>ner during <strong>the</strong> study. Animals were <strong>the</strong>n euth<strong>an</strong>ized <strong>an</strong>d hippocampi were removed <strong>an</strong>d<br />
RNA extracted. Lead exposed <strong>an</strong>imals had me<strong>an</strong> blood Pb levels of approximately 25 µg/dl<br />
while control <strong>an</strong>imals had blood Pb levels
Title: D. mel<strong>an</strong>ogaster: A new model system <strong>for</strong> chemo<strong>the</strong>rapy induced neurotoxicity<br />
Authors: *J. PODRATZ, D. FROEMEL, A. TANG, A. J. WINDEBANK;<br />
Mayo Clin. Col. of Med., Rochester, MN<br />
Abstract: The platinum family of chemo<strong>the</strong>rapeutic drugs, cisplatin, oxaliplatin <strong>an</strong>d carboplatin,<br />
are used to treat various c<strong>an</strong>cers such as testicular, ovari<strong>an</strong>, breast <strong>an</strong>d metastatic colon c<strong>an</strong>cers.<br />
Peripheral neuropathy is <strong>the</strong> dose limiting effect <strong>for</strong> all of <strong>the</strong>se drugs; it limits chemo<strong>the</strong>rapy<br />
treatment in 20-30% of patients. Cisplatin <strong>an</strong>d Oxaliplatin bind both nuclear <strong>an</strong>d mitochondrial<br />
DNA (1) inducing DNA damage <strong>an</strong>d apoptosis by bax dependent <strong>an</strong>d independent pathways in rat<br />
DRG in vitro <strong>an</strong>d in vivo. M<strong>an</strong>y cell death pathways are conserved in eukaryotes. D.<br />
Mel<strong>an</strong>ogaster have homologous DNA damage-apoptosis pathways to mammals. We exposed<br />
adult drosophila to 10, 25, 50, 100, 200 <strong>an</strong>d 400µg/ml cisplatin in 20% sucrose <strong>for</strong> three days<br />
followed by tr<strong>an</strong>sfer to normal food vials. A geotaxis climbing assay was per<strong>for</strong>med at 0, 3 <strong>an</strong>d 6<br />
days following cisplatin treatment. At 50µg/ml, <strong>the</strong> most effective concentration, <strong>the</strong> number of<br />
flies able to climb 2 cm decreased 43% immediately after treatment ended, 34% after a fur<strong>the</strong>r 3<br />
days <strong>an</strong>d 28% after 6 days. Fly survival was 92% at 0 days, 89% at 3 days <strong>an</strong>d 79% at 6 days<br />
following cisplatin treatment. Brains harvested <strong>from</strong> cisplatin treated flies were positive <strong>for</strong><br />
active caspase staining. Electron microscopy showed both apoptosis <strong>an</strong>d mitochondrial<br />
degradation similar to <strong>the</strong> ch<strong>an</strong>ges seen in cisplatin treated rat dorsal root g<strong>an</strong>glia. Developing<br />
oocytes harvested <strong>from</strong> cisplatin treated adult female flies also showed positive active caspase<br />
staining <strong>an</strong>d DNA fragmentation. The germ line was lost at 100µg/ml cisplatin treatment. Using<br />
a combination of behavioral assays, immunostaining <strong>an</strong>d a defined phenotype, we are completing<br />
mutation experiments to look <strong>for</strong> resist<strong>an</strong>ce to or enh<strong>an</strong>cement of <strong>the</strong> effects of cisplatin. We will<br />
<strong>the</strong>n exp<strong>an</strong>d <strong>the</strong> assays to screen o<strong>the</strong>r chemo<strong>the</strong>rapy drugs that induce neuropathy. These<br />
experiments will help us develop targeted <strong>the</strong>rapies to eliminate chemo<strong>the</strong>rapy induced<br />
neurotoxicity.<br />
Disclosures: J. Podratz, None; D. Froemel, None; A. T<strong>an</strong>g, None; A.J. Windeb<strong>an</strong>k, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.10/U29<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: RO1 DA020142
G12-RR-003037<br />
Title: A neurotoxic dose of methamphetamine induces two distinct phases of cell death in <strong>the</strong><br />
striatum<br />
Authors: *I. K. TULLOCH 1 , L. AFANADOR 2 , J. A. ANGULO 2 ;<br />
2 Biol. Sci., 1 CUNY-Hunter Col., New York City, NY<br />
Abstract: Our laboratory has been investigating <strong>the</strong> impact of a neurotoxic exposure to<br />
methamphetamine (METH) on cellular components of <strong>the</strong> striatum post-synaptic to <strong>the</strong><br />
dopaminergic terminals. A systemic bolus injection of METH (30 mg/kg) induces <strong>the</strong> apoptosis<br />
of 25% of striatal neurons 24 hours after <strong>the</strong> injection. No additional cell loss is observed at 2 or<br />
3 days post-METH. However, 36 hours post-METH new cells are generated throughout <strong>the</strong><br />
striatum by mitosis. The duration of <strong>the</strong> METH-induced genesis of cells is <strong>from</strong> 36-72 hours<br />
post-METH. The newly generated cells arise <strong>from</strong> dorm<strong>an</strong>t striatal progenitors <strong>an</strong>d not <strong>from</strong> <strong>the</strong><br />
subventricular zone. A percentage of <strong>the</strong> newly generated cells display glial phenotypes <strong>an</strong>d<br />
begin to die after <strong>the</strong> third day post-METH. The protracted phase of cell death lasts <strong>for</strong> at least<br />
two months post-METH. Interestingly, not all <strong>the</strong> new cells die. Some cells display neuronal<br />
phenotype between 1 <strong>an</strong>d 2 months post-METH. In conclusion, a neurotoxic dose of METH<br />
induces two types of cell death in <strong>the</strong> striatum of mice: <strong>the</strong> first wave involves some of <strong>the</strong><br />
striatal neurons <strong>an</strong>d is of short duration, <strong>the</strong> second wave lasting several weeks involves <strong>the</strong><br />
newly generated cells.<br />
Disclosures: I.K. Tulloch, CUNY-Hunter College, A. Employment (full or part-time);<br />
RCMI/NIH G12-RR-003037, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); L. Af<strong>an</strong>ador, CUNY hunter<br />
college, A. Employment (full or part-time); R01 DA020142 NIDA/NIH, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); J.A. Angulo, CUNY hunter College, A. Employment (full or part-time); R01<br />
DA020142 NIDA/NIH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.11/U30<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration
Support: NIH Gr<strong>an</strong>t P20 RR16454<br />
USAMRMC Contract#W81XWH-07-2-0078<br />
Title: Neuroprotective strategies against neurodegeneration in <strong>an</strong> in vitro model involving<br />
m<strong>an</strong>g<strong>an</strong>ese toxicity<br />
Authors: *N. RIZVI 1 , A. O. ISAAC 1 , V. V. DUKHANDE 1 , I. KAWIKOVA 2 , A. L. M.<br />
BOTHWELL 2 , J. C. K. LAI 1 ;<br />
1 Idaho State Univ., Pocatello, ID; 2 Immuobiology, Yale Univ. Sch. of Med., New Haven, CT<br />
Abstract: Oxidative stress, mitochondrial/proteosomal dysfunction, <strong>an</strong>d inflammation constitute<br />
import<strong>an</strong>t pathogenetic factors leading in neurodegenerative diseases (e.g., Alzheimer’s (AD)<br />
<strong>an</strong>d Parkinson’s disease (PD)). Among recent strategies to retard this neuronal cell death <strong>an</strong>d<br />
restore function, one target is a member of nuclear receptors superfamily _ peroxisome<br />
proliferator-activated receptor gamma (PPAR γ), involved in inflammatory pathways purportedly<br />
activated in neurodegeneration. We have demonstrated ch<strong>an</strong>ges in expression <strong>an</strong>d subcellular<br />
distribution of PPAR isotypes (α, β/δ, <strong>an</strong>d γ) when U87 (astrocytes-like) glioma cells are treated<br />
with m<strong>an</strong>g<strong>an</strong>ese. Moreover, numerous studies have demonstrated brain m<strong>an</strong>g<strong>an</strong>ese accumulation<br />
<strong>an</strong>d development of ‘m<strong>an</strong>g<strong>an</strong>ism’ in PD <strong>an</strong>d AD patients. These observations prompted us to<br />
develop <strong>an</strong> in vitro model wherein U87 cells have been tr<strong>an</strong>sfected with PPARγ cDNA to<br />
overexpress <strong>the</strong> gene. We <strong>the</strong>re<strong>for</strong>e investigated <strong>the</strong> possibility that m<strong>an</strong>g<strong>an</strong>ese may induce<br />
differential neurotoxicity on <strong>the</strong> tr<strong>an</strong>sfected versus <strong>the</strong> wild-type (U87-WT) cells. In ongoing<br />
studies, we observe that PPARγ overexpression does not offer <strong>an</strong>y signific<strong>an</strong>t protection against<br />
m<strong>an</strong>g<strong>an</strong>ese-induced cell death. However, <strong>the</strong> dose-related effects of m<strong>an</strong>g<strong>an</strong>ese on U87 cells<br />
overexpressing Bcl-2 were less marked th<strong>an</strong> those on <strong>the</strong> U87 cells overexpressing PPARγ. We<br />
are continuing to determine <strong>the</strong> molecular mech<strong>an</strong>isms underlying <strong>the</strong>se differential roles of<br />
PPARγ <strong>an</strong>d Bcl-2 in light of <strong>the</strong>ir neuroprotective potential. Thus our findings may have<br />
pathophysiological implications regarding <strong>the</strong> roles of PPARγ <strong>an</strong>d Bcl-2 in neuroprotective<br />
mech<strong>an</strong>isms.<br />
Disclosures: N. Rizvi, None; A.O. Isaac, None; V.V. Dukh<strong>an</strong>de, None; I. Kawikova,<br />
None; A.L.M. Bothwell, None; J.C.K. Lai, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.12/U31
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Title: Selective neuronal toxicity of β-N-methylamino-L-al<strong>an</strong>ine<br />
Authors: X. LIU, J. CISKE, J. HJELMHAUG, *D. C. LOBNER;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: β-N-methylamino-L-al<strong>an</strong>ine (BMAA) is a non-protein amino acid that originally<br />
attracted attention because it is a potential cause of <strong>the</strong> neurodegenerative disease amyotrophic<br />
lateral sclerosis/Parkinson-dementia complex (ALS/PDC) found on Guam. However, recent<br />
evidence indicates that it may be involved in neurodegenerative diseases elsewhere. BMAA is<br />
produced by a large variety of cy<strong>an</strong>obacteria around <strong>the</strong> world (Proc. Natl. Acad. Sci., 102:<br />
5074-5078) <strong>an</strong>d is found at high levels in Alzheimer’s disease <strong>an</strong>d ALS patient brains (Acta.<br />
Neurol. Sc<strong>an</strong>d., 110: 267-269).<br />
Previous studies using cortical cultures have indicated that BMAA neurotoxicity involves at least<br />
three mech<strong>an</strong>isms: it c<strong>an</strong> act directly on <strong>the</strong> NMDA receptor to cause excitotoxicity, it c<strong>an</strong> cause<br />
activation of mGluR5 receptors <strong>an</strong>d it c<strong>an</strong> cause oxidative stress by acting on <strong>the</strong> cystineglutamate<br />
<strong>an</strong>tiporter (system xc-), leading to decreased glutathione levels (Neurobiol. Dis., 25:<br />
360-366; Exp. Neurol., 217: 429-433). BMAA has also been shown to cause selective toxicity of<br />
certain neurons. NAPDH-positive cells found in cortical cultures were found to be selectively<br />
sensitive to BMAA toxicity (Brain Res., 497: 64-71) <strong>an</strong>d motor neurons are highly sensitive to<br />
BMAA toxicity (Exp. Neurol., 201: 244-252). Since basal <strong>for</strong>ebrain cholinergic neurons are<br />
highly vulnerable in Alzheimer’s disease <strong>an</strong>d midbrain dopaminergic neurons are lost in<br />
Parkinson’s disease, we used basal <strong>for</strong>ebrain septal cultures <strong>an</strong>d mesencephalic cultures to test if<br />
cholinergic neurons <strong>an</strong>d dopaminergic neurons are selectively sensitive to BMAA toxicity. Our<br />
results indicate that cholinergic neurons are selectively vulnerable to BMAA toxicity. Ongoing<br />
experiments are designed to determine <strong>the</strong> mech<strong>an</strong>ism underlining <strong>the</strong> selective sensitivity of<br />
cholinergic neurons <strong>an</strong>d to determine whe<strong>the</strong>r dopaminergic neurons are also selectively<br />
sensitive to BMAA toxicity.<br />
Disclosures: X. Liu, None; J. Ciske, None; J. Hjelmhaug, None; D.C. Lobner, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.13/U32<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration
Support: Department of Chemistry WSU<br />
Title: Accumulation of m<strong>an</strong>g<strong>an</strong>ese in dopamine rich brain regions upon intermittent subcut<strong>an</strong>eous<br />
injection of MnCl2<br />
Authors: *R. A. AOUN 1 , T. A. MATHEWS 2 ;<br />
1 Dearborn, MI; 2 Chem., Wayne State Univ., Detroit, MI<br />
Abstract: M<strong>an</strong>g<strong>an</strong>ese (Mn), <strong>an</strong> abund<strong>an</strong>t metal found in soil, air, <strong>an</strong>d m<strong>an</strong>y foods, is essential<br />
<strong>for</strong> normal body development <strong>an</strong>d functioning. However, overexposure to Mn results in a<br />
neurological disorder, called m<strong>an</strong>g<strong>an</strong>ism, whose symptoms are very similar to Parkinson’s<br />
disease. The similarities between <strong>the</strong> two disorders are most likely due to <strong>the</strong>ir site of action in<br />
dopamine-rich areas of <strong>the</strong> basal g<strong>an</strong>glia (e.g. caudate-putamen (CPu) <strong>an</strong>d subst<strong>an</strong>tia nigra).<br />
Thus, research on <strong>the</strong> neurobiological consequences of Mn toxicity has largely focused on <strong>the</strong><br />
biochemical <strong>an</strong>d behavioral alterations associated with dopamine. Following a previously<br />
established protocol described by Dodd et al., (Int. J. Toxicol., 2005), MnCl2 (50 mg/kg, s.c.)<br />
was administered to C57/Bl6 mice (20-25 g) on days 1, 4, <strong>an</strong>d 7. This Mn administration<br />
protocol was modified to simult<strong>an</strong>eously measure Mn <strong>an</strong>d monogenic amines levels. Brain, liver,<br />
<strong>an</strong>d kidney homogenates were evaluated <strong>for</strong> Mn accumulation using graphite atomic absorption<br />
24 hrs after <strong>the</strong> last injection. Additionally, levels of monoamines (DA, NE, 5-HT) <strong>an</strong>d <strong>the</strong>ir<br />
metabolites were evaluated in <strong>the</strong> CPu, midbrain, cortex, <strong>an</strong>d hippocampus using HPLC coupled<br />
to electrochemical detection. Although, elevated levels of Mn were observed in <strong>the</strong> kidney <strong>an</strong>d<br />
liver, in <strong>the</strong> brain, only dopamine rich regions such as <strong>the</strong> striatum, midbrain, <strong>an</strong>d cortex showed<br />
increased levels. Despite <strong>an</strong> accumulation of Mn in certain brain regions, no ch<strong>an</strong>ges were<br />
observed in <strong>the</strong> tissue content levels of all <strong>the</strong> monoamines. This study indicates that while <strong>the</strong><br />
Mn paradigm used results in a signific<strong>an</strong>t elevation of Mn throughout <strong>the</strong> body, longer time<br />
points, such as 7 <strong>an</strong>d 14 days after Mn administration, may be needed to measure ch<strong>an</strong>ges in<br />
monoamine tissue content.<br />
Disclosures: R.A. Aoun, None; T.A. Ma<strong>the</strong>ws, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.14/U33<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration
Title: Midazolam administered <strong>from</strong> P7 to P4 in <strong>the</strong> mouse induced histological cerebellar<br />
cortex alterations<br />
Authors: *M. MARQUEZ-OROZCO, M. GAZCA-RAMIREZ, G. DE LA FUENTE-<br />
JUAREZ, A. MARQUEZ-OROZCO;<br />
Dept Embryol, Univ. of Mexico (UNAM), Mexivo, Mexico<br />
Abstract: The exposure of P7 to P14-day-old mouse pups to midazolam (MDZ) produces of<br />
histological alterations in <strong>the</strong> cerebellar cortex produces <strong>the</strong> same type of histological alterations<br />
in <strong>the</strong> cerebral cortex as those caused in mice by exposure to MDZ <strong>from</strong> day P8 to P29 of<br />
gestationTwo groups of ICR strain male mice were s.c. injected: <strong>the</strong> first (MDZ) with a single<br />
daily MDZ dose (2.0 mg/kg/bw) at <strong>the</strong> age of P7 to P14 days, <strong>an</strong>d <strong>the</strong> second, control group (C),<br />
received saline solution. All mice were killed with a CO2 atmosphere at <strong>the</strong> P15. The cerebellum<br />
was fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, <strong>an</strong>d embedded in epoxy resin. The<br />
semifine sections were stained with toluidine blue <strong>an</strong>d observed under <strong>the</strong> light microscope. In<br />
<strong>the</strong> MDZ group, <strong>the</strong> cerebellar cortex was thinner th<strong>an</strong> in C group, <strong>an</strong>d show delayed<br />
differentiation. in gr<strong>an</strong>ular layers <strong>an</strong>d int<strong>an</strong>d higher nuclear density per area. The number of<br />
Purkinje cells was reduced in relation to <strong>the</strong> C group (p
Title: The striatal neurokinin-1 <strong>an</strong>d npy2 receptors modulate <strong>the</strong> methamphetamine-induced<br />
production of nitric oxide but <strong>the</strong> kappa opioid receptor is without effect in mice<br />
Authors: *L. AFANADOR, J. A. ANGULO;<br />
Biol. Dept., CUNY Hunter Col., New York, NY<br />
Abstract: One of <strong>the</strong> cellular responses to methamphetamine (METH) investigated by our<br />
laboratory involves <strong>the</strong> role of striatal neuropeptides on <strong>the</strong> METH-induced production of nitric<br />
oxide. Recent findings <strong>from</strong> our lab connect <strong>the</strong> METH-induced production of nitric oxide with<br />
<strong>the</strong> apoptosis of some striatal neurons after a single high dose systemic injection of METH (30<br />
mg/kg). We have found that <strong>the</strong> striatal subst<strong>an</strong>ce P receptor (neurokinin-1 receptor) synergizes<br />
with METH to augment nitric oxide production but <strong>the</strong> NPY2 receptor <strong>an</strong>tagonizes this effect of<br />
METH. In contrast to subst<strong>an</strong>ce P <strong>an</strong>d NPY, <strong>the</strong> striatal kappa receptor has no effect on <strong>the</strong><br />
METH-induced production of nitric oxide. Dynorphin is <strong>the</strong> natural lig<strong>an</strong>d of <strong>the</strong> kappa opioid<br />
receptor <strong>an</strong>d is colocalized with subst<strong>an</strong>ce P in striatonigral projection neurons of <strong>the</strong> striatum.<br />
Work in progress is assessing <strong>the</strong> impact of kappa agonists on <strong>the</strong> METH-induced apoptosis of<br />
some striatal neurons.<br />
Disclosures: L. Af<strong>an</strong>ador, None; J.A. Angulo, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.16/U35<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: NIDA Gr<strong>an</strong>t R01 DA020142<br />
Title: Antagonistic effects of <strong>the</strong> neuropeptides subst<strong>an</strong>ce P <strong>an</strong>d NPY on <strong>the</strong> methamphetamineinduced<br />
production of nitric oxide in <strong>the</strong> striatum of mice<br />
Authors: *H. YAROSH, J. ANGULO;<br />
Hunter Col., New York, NY<br />
Abstract: Our laboratory has been investigating <strong>the</strong> cellular responses to methamphetamine<br />
(METH). A single bolus systemic injection of METH (30 mg/kg) induces <strong>the</strong> apoptosis of<br />
approximately 25% of striatal neurons. Agents that attenuate <strong>the</strong> METH-induced production of<br />
nitric oxide also decrease <strong>the</strong> METH-induced apoptosis of some striatal neurons. Recently we
found that some striatal neuropeptides modulate this METH-induced apoptosis <strong>an</strong>d nitric oxide<br />
production. For example, pharmacological blockade of <strong>the</strong> subst<strong>an</strong>ce P receptor (neurokinin-1<br />
receptor) attenuates <strong>the</strong> METH-induced production of nitric oxide while <strong>an</strong> agonist of <strong>the</strong><br />
neurokinin-1 receptor has <strong>the</strong> opposite effect. Thus subst<strong>an</strong>ce P synergizes with METH to<br />
augment <strong>the</strong> levels of nitric oxide in <strong>the</strong> striatum. Conversely, <strong>an</strong> agonist of <strong>the</strong> NPY2 receptor<br />
attenuates <strong>the</strong> METH-induced elevation of striatal nitric oxide. Our results are consistent with <strong>the</strong><br />
hypo<strong>the</strong>sis that <strong>the</strong> neuropeptides subst<strong>an</strong>ce P <strong>an</strong>d NPY exert push-pull activity on nitric oxide<br />
production in <strong>the</strong> presence of METH in <strong>the</strong> striatum of mice.<br />
Disclosures: H. Yarosh, Hunter College, A. Employment (full or part-time); R01<br />
DA020142/NIDA, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Angulo, Hunter College, A. Employment<br />
(full or part-time); R01 DA020142/NIDA, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.17/U36<br />
Topic: C.11.l. Neurotoxicity <strong>an</strong>d inflammation: Stem cells <strong>an</strong>d glia<br />
Support: NIH/NICHD HD 44517<br />
Harold Carron endowment<br />
Title: Isoflur<strong>an</strong>e causes <strong>the</strong> impairment of actin polymerization <strong>an</strong>d org<strong>an</strong>ization in very young<br />
astrocytes in culture<br />
Authors: *N. LUNARDI, C. HUCKLENBRUCH, V. JEVTOVIC-TODOROVIC;<br />
Anes<strong>the</strong>siol., Univ. Virginia, Charlottesville, VA<br />
Abstract: Astroglia plays <strong>an</strong> import<strong>an</strong>t role in modulating neuronal function in <strong>the</strong> developing<br />
brain. We have previously shown that <strong>an</strong> inhalational <strong>an</strong>es<strong>the</strong>tic, isoflur<strong>an</strong>e, signific<strong>an</strong>tly impairs<br />
proliferation of very young cultured astrocytes [day-in-vitro (DIV) 4]. Because proper actin<br />
polymerization <strong>an</strong>d its intracellular org<strong>an</strong>ization/distribution play a crucial role in regulating<br />
astroglial proliferation we set out to <strong>an</strong>alyze whe<strong>the</strong>r observed effect of isoflur<strong>an</strong>e on<br />
proliferation is caused by <strong>the</strong> impairment in actin polymerization <strong>an</strong>d org<strong>an</strong>ization. Primary<br />
astroglial cultures were exposed to 3% isoflur<strong>an</strong>e on DIV 4 <strong>for</strong> 24 h. Polymerized (F) actin was
visualized using fluorescent phalloidin staining <strong>an</strong>d <strong>the</strong> data <strong>an</strong>alyses were per<strong>for</strong>med on DIV 6<br />
<strong>an</strong>d 7 (when <strong>the</strong> effect on proliferation was most profound) using Image J software. The <strong>an</strong>alysis<br />
of <strong>the</strong> amount of F-actin (digitally qu<strong>an</strong>tified based on <strong>the</strong> degree of fluorescence) in <strong>the</strong><br />
experimental <strong>an</strong>d control astrocytes revealed no signific<strong>an</strong>t differences between <strong>the</strong> experimental<br />
<strong>an</strong>d control astrocytes on ei<strong>the</strong>r DIV 6 or 7. However, when <strong>the</strong> subcellular distribution <strong>an</strong>d<br />
org<strong>an</strong>ization of F-actin were examined we found a signific<strong>an</strong>t loss of actin stress fibers (ASF-<br />
bundles of actin filaments running through <strong>the</strong> cytoplasm) <strong>an</strong>d higher presence of depolymerized<br />
actin in <strong>the</strong> experimental astrocytes. For example, at DIV 6, 23.9 ±4.8% of control astrocytes<br />
(N=511) expressed ASF compared to 10.42±2.7% of <strong>the</strong> experimental ones (N=639), (p
Authors: *A. PADILLA 1,2 , A. ALMEYDA 2 , K. PAYNE 2,3 , M. DE LEON 2,4 ;<br />
2 Ctr. <strong>for</strong> Hlth. Disparities <strong>an</strong>d Mol. Med., 3 Anat. <strong>an</strong>d Hum<strong>an</strong> Patholgy, 4 Pathology <strong>an</strong>d Hum<strong>an</strong><br />
Anatomy, Physiol. <strong>an</strong>d Pharmacol., 1 Loma Linda Univ., Loma Linda, CA<br />
Abstract: Our laboratory is interested in underst<strong>an</strong>ding <strong>the</strong> process of lipotoxicity (LTx) <strong>an</strong>d its<br />
effects on nervous system. The present study explores <strong>the</strong> consequences of palmitic acid<br />
lipotoxicity (PA-LTx) on immortalized Schw<strong>an</strong>n cell (iSC) viability. ISC cells were cultured in<br />
two different glucose concentrations (17mM <strong>an</strong>d 50mM) <strong>an</strong>d exposed to PA:Bovine Serum<br />
Albumin (PA:BSA) molar ratios 2:1 (300uM/150uM)}. Hoescht staining <strong>an</strong>d Annexin V /7AAD<br />
assays were per<strong>for</strong>med to test <strong>for</strong> apoptotic cell death. Flow Cytometric measurements using 2’,<br />
7’-dichlorofluorescein diacetate (DCF) were per<strong>for</strong>med to assess <strong>the</strong> levels of reactive oxidative<br />
species (ROS) within <strong>the</strong> cell. Cultures were treated with <strong>the</strong> <strong>an</strong>tioxid<strong>an</strong>t MCI-186 (100uM or<br />
1mM) <strong>an</strong>d crystal violet <strong>an</strong>d WST-1 assays were per<strong>for</strong>med to assess cell viability following<br />
ROS reduction. Mitochondrial membr<strong>an</strong>e depolarization was <strong>an</strong>alyzed using JC-1. Our data<br />
show that PA-LTx decreases iSC cell viability in a dose <strong>an</strong>d time dependent m<strong>an</strong>ner. Hoescht<br />
<strong>an</strong>d Annexin V/ 7AAD staining show that <strong>the</strong> observed decrease in cell viability was caused by<br />
<strong>an</strong> increased in apoptotic cell death. Fur<strong>the</strong>rmore, <strong>an</strong> increase in ROS levels was observed in PA-<br />
LTx cells, with a greater induction of ROS at 48hrs. The increase in ROS was fur<strong>the</strong>r augmented<br />
in cells cultured in 50mM glucose. Treatment of cultures undergoing PA-LTx with MCI-186<br />
decreased levels of ROS <strong>an</strong>d increased cell viability at 24hrs only. JC-1 assays demonstrated<br />
mitochondrial membr<strong>an</strong>e depolarization be<strong>for</strong>e signific<strong>an</strong>t ROS was detected. Our data is<br />
consistent with <strong>the</strong> hypo<strong>the</strong>sis that elevated levels of saturated FFA are toxic to Schw<strong>an</strong>n cells<br />
<strong>an</strong>d <strong>the</strong>ir effects are mediated through <strong>an</strong> increase in ROS within <strong>the</strong> cell.<br />
Disclosures: A. Padilla, None; A. Almeyda, None; K. Payne, None; M. De Leon, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.19/V1<br />
Topic: C.11.k. Neurotoxicity <strong>an</strong>d neurodegeneration<br />
Support: PAPIIT IN214908<br />
Title: Exploring <strong>the</strong> toxicological consequences of CYP2E1 induction in cerebellar gr<strong>an</strong>ule<br />
neurons
Authors: *A. C. VALENCIA 1 , J. MORÁN 2 , J. J. ESPINOSA-AGUIRRE 1 ;<br />
1 Inst. De Investigaciones Biomédicas UNAM, Distrito Federal, Mexico; 2 Dept. de<br />
Neurociencias, Inst. de Fisiología Celular, UNAM, Distrito Federal, Mexico<br />
Abstract: Cytochrome P450 (CYP) is a well known family of enzymes responsible of <strong>the</strong><br />
oxidative metabolism of endogenous <strong>an</strong>d exogenous compounds. Although <strong>the</strong> CYP content in<br />
brain is low compared with hepatic levels, <strong>the</strong> bioactivation of xenobiotics into oxid<strong>an</strong>ts or<br />
carcinogens within <strong>the</strong> brain is of signific<strong>an</strong>ce, considering <strong>the</strong> limited regenerative capacity of<br />
<strong>the</strong> nervous cells <strong>an</strong>d its vulnerability to oxidative damage. CYP2E1 is considered as <strong>the</strong> most<br />
import<strong>an</strong>t iso<strong>for</strong>m involved in <strong>the</strong> generation of reactive oxygen species leading to lipid<br />
peroxidation <strong>an</strong>d cell death. There are some evidences that suggest <strong>the</strong> possible participation of<br />
CYP2E1 in eth<strong>an</strong>ol-induced brain toxicity but direct evidence involving cellular damage <strong>an</strong>d <strong>the</strong><br />
presence of elevated CYP2E1 activity is missing. We aimed to determine if CYP2E1 was<br />
involved in brain oxidative-toxicity by exposing <strong>the</strong> primary neuron culture to different inducers<br />
of CYP2E1. Eth<strong>an</strong>ol, isoniazid <strong>an</strong>d nicotine treatment clearly increased <strong>the</strong> CYP2E1 content<br />
with all <strong>the</strong> concentrations used, as determined by immunocitochemistry studies. In addition, in<br />
preliminary experiments, eth<strong>an</strong>ol treatment provoked <strong>the</strong> <strong>for</strong>mation of reactive oxygen species<br />
without compromising cell viability after 96 hours of exposure. Fur<strong>the</strong>r experiments are pl<strong>an</strong>ned<br />
to elucidate <strong>the</strong> participation CYP2E1 in oxidative brain toxicity.<br />
.<br />
Disclosures: A.C. Valencia, CONACyT, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Morán,<br />
None; J.J. Espinosa-Aguirre, None.<br />
Poster<br />
544. Neurotoxicity: Toxins II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 544.20/V2<br />
Topic: C.11.l. Neurotoxicity <strong>an</strong>d inflammation: Stem cells <strong>an</strong>d glia<br />
Support: NIH Gr<strong>an</strong>t 5P50AG025711-04<br />
Title: Effects of ochratoxin-A on <strong>the</strong> proliferation <strong>an</strong>d differentiation of neural progenitor cells<br />
in hippocampus
Authors: *V. M. SAVA 1 , I. RAFALOVICH 2 , J. SANCHEZ-RAMOS 3,4 ;<br />
1 Univ. South Florida, Tampa, FL; 2 Univesity of South Florida, Tampa, FL; 3 Univ. of South<br />
Florida, Tampa, FL; 4 James Haley VA Hosp. Res. Service, Tampa, FL<br />
Abstract: Ochratoxin A (OTA) is a toxic metabolite of several fungal species contaminating<br />
food including grapes <strong>an</strong>d wine, rice, corn, coffee etc. Our earlier findings suggested OTA as a<br />
possible etiological factor <strong>for</strong> neurodegeneration (Sava et al., 2004). The present study aimed to<br />
examine <strong>the</strong> effects of OTA on adult neurogenesis using BrdU <strong>an</strong>d NeuN markers detectable<br />
with flow cytometry <strong>an</strong>d immunohistochemistry. For <strong>the</strong> in vivo studies, male ICR mice were<br />
injected with saline or OTA (8 mg/kg, i.p.). BrdU was injected on <strong>the</strong> 2nd <strong>an</strong>d 3rd days (100<br />
mg/kg, i.p.). Animals were sacrificed 2 weeks after last injection of OTA. Brains were removed,<br />
hippocampus dissected <strong>an</strong>d dissociated into a single cell suspension. After appropriate fixation,<br />
cells were labeled with fluorescently conjugated <strong>an</strong>ti-Brdu <strong>an</strong>d <strong>an</strong>ti-NeuN <strong>an</strong>tibodies. Cells were<br />
also labeled with propidium iodide be<strong>for</strong>e <strong>an</strong>alysis on flow cytometry. OTA increased<br />
proliferation of neural/stem progenitors in hippocampus. In particular, BrdU incorporation<br />
revealed a 156% increase of BrdU+ cells in <strong>an</strong>imals acutely exposed to OTA as compared to<br />
control mice. Cell proliferation was also assessed by immunohistochemical stereologic <strong>an</strong>alysis.<br />
The results <strong>from</strong> <strong>the</strong> both methods demonstrated stimulation of hippocampal proliferation.<br />
Animals allowed surviving <strong>for</strong> 14 days after <strong>the</strong> last Brdu injection showed 60% loss in number<br />
of BrdU+ cells. OTA also decreased differentation of BrdU+ cells into neurons. Namely, in <strong>the</strong><br />
control group 82±9% of <strong>the</strong> BrdU+ cells expressed NeuN while in <strong>the</strong> OTA-treated group only<br />
64±2% cells were NeuN+ (p-value is 0.0091). Thus, OTA caused opposing effects on<br />
proliferation <strong>an</strong>d differentiation stages of neurogenesis that resulted in <strong>an</strong> insignific<strong>an</strong>t difference<br />
between <strong>the</strong> total numbers of newly <strong>for</strong>med neurons. These results are in agreement with our<br />
previous studies of neurogenesis in vitro (Sava et al, 2007) which disclosed differences in<br />
vulnerability of proliferating adult hippocampal neural progenitor cells <strong>an</strong>d differentiated<br />
neurons to <strong>the</strong> toxin. In light of <strong>the</strong> import<strong>an</strong>ce of hippocampal neurogenesis <strong>for</strong> some aspects of<br />
cognitive function, <strong>the</strong> impact of OTA on neurogenesis is relev<strong>an</strong>t <strong>from</strong> both molecular<br />
pathogenetic <strong>an</strong>d clinical perspectives.<br />
Disclosures: V.M. Sava, None; I. Rafalovich, None; J. S<strong>an</strong>chez-Ramos, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.1/V3<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms
Support: NIH/NIA/IRP<br />
FAPESP Brazil<br />
Title: Curcumin treatment alleviates cognitive impairment elicited by inflammation<br />
Authors: *E. M. KAWAMOTO 1 , S. CAMANDOLA 1 , M. MUGHAL 1 , C. SCAVONE 2 , M.<br />
MATTSON 1 ;<br />
1 Lab. of Neurosciences, NATIONAL INSTITUTE ON AGING/ NATIONAL INSTITUTES OF<br />
HEALTH, BALTIMORE, MD; 2 Pharmacol., Univ. of Sao Paolo, Sao Paolo, Brazil<br />
Abstract: Infectious agents <strong>an</strong>d brain injuries that activate innate immune responses in brain<br />
cells c<strong>an</strong> cause inflammation <strong>an</strong>d cognitive dysfunction, but effective treatments <strong>for</strong> such<br />
disorders are lacking. Because treatment with <strong>the</strong> phytochemical curcumin has been reported to<br />
protect neurons in several <strong>an</strong>imal models of neurodegenerative disease, we evaluated <strong>the</strong> effects<br />
of curcumin on behavioral deficits <strong>an</strong>d associated inflammatory processes in a mouse model of<br />
sepsis induced by systemic bacterial endotoxin. C57/BL6 mice were treated with ei<strong>the</strong>r vehicle<br />
or curcumin (50 mg/kg, ip) daily <strong>for</strong> 4 days <strong>an</strong>d were <strong>the</strong>n injected with LPS (250 µg/kg, ip).<br />
Learning <strong>an</strong>d memory were evaluated using Morris water maze <strong>an</strong>d novel object recognition<br />
tests, <strong>an</strong>d <strong>the</strong> open field test was used to measure <strong>an</strong>xiety/exploratory behaviors. LPS-treated<br />
mice were impaired in memory retention (probe trial in <strong>the</strong> water maze) <strong>an</strong>d curcumin treatment<br />
ameliorated this adverse effect of LPS. Curcumin also suppressed LPS-induced expression of <strong>the</strong><br />
pro-inflammatory markers TNF-alpha <strong>an</strong>d iNOS in <strong>the</strong> hippocampus of C57/BL6 mice. These<br />
results suggest that curcumin c<strong>an</strong> effectively suppress innate immune responses <strong>an</strong>d associated<br />
inflammation, <strong>the</strong>reby ameliorating <strong>the</strong> resulting cognitive deficits.<br />
Disclosures: E.M. Kawamoto, None; S. Cam<strong>an</strong>dola, None; M. Mughal, None; C. Scavone,<br />
None; M. Mattson, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.2/V4<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: NIH/NINDS Gr<strong>an</strong>t NS51445-04 (S.J.H.)
Title: Enh<strong>an</strong>ced astrocytic system xc - expression <strong>an</strong>d activity mediate <strong>the</strong> neurotoxic effect of<br />
interleukin-1β<br />
Authors: *N. A. JACKMAN, J. A. HEWETT, S. J. HEWETT;<br />
Neurosci., Univ. of Connecticut Hlth. Ctr., Farmington, CT<br />
Abstract: System xc - is <strong>an</strong> obligate exch<strong>an</strong>ger that links <strong>the</strong> import of L-cystine with <strong>the</strong> export<br />
of L-glutamate. While cystine import is critical to glutathione syn<strong>the</strong>sis, <strong>the</strong> efflux <strong>an</strong>d<br />
subsequent extracellular accumulation of glutamate has been demonstrated to contribute to<br />
neuropathology <strong>an</strong>d disease progression in a number of CNS disorders. Indeed, we recently<br />
demonstrated <strong>the</strong> import<strong>an</strong>ce of IL-1β-mediated increases in system xc - activity as a novel<br />
contributor to inflammatory hypoxic neuronal injury, a model of <strong>the</strong> ischemic penumbra (J<br />
Neurosci 27: 10094, 2007). By utilizing cells derived <strong>from</strong> <strong>an</strong>imals harboring a mutation in <strong>the</strong><br />
Slc7a11 gene (sut mice) that encodes <strong>for</strong> <strong>the</strong> system xc - light chain, xCT, we now provide<br />
definitive evidence that alterations in system xc - activity in astrocytes exclusively mediate this<br />
effect. Treatment of cultures with IL-1β (1 ng/ml, 24 hr)-- while not toxic alone-- increases<br />
hypoxic neuronal injury in mixed cortical cell cultures containing both neurons <strong>an</strong>d astrocytes.<br />
This enh<strong>an</strong>cement in hypoxic neuronal injury is prevented in chimeric cultures consisting of<br />
neurons derived <strong>from</strong> wild-type mice plated on top of astrocytes derived <strong>from</strong> sut mice,<br />
indicating that astrocytic system xc - is essential <strong>for</strong> <strong>the</strong> deleterious effects of IL-1β. While<br />
neurons <strong>an</strong>d astrocytes both express system xc - subunits, qu<strong>an</strong>titative real time PCR <strong>an</strong>alyses<br />
demonstrate that xCT mRNA increases in a time-dependent m<strong>an</strong>ner in astrocytes following IL-<br />
1β stimulation, while neuronal xCT expression is unch<strong>an</strong>ged. Additionally, astrocytes show <strong>an</strong><br />
increase in system xc - -mediated cystine uptake following IL-1β treatment (3 ng/ml; 20-24 hr),<br />
while neurons do not. Finally, this IL-1β-mediated increase in cystine uptake found in wild-type<br />
astrocytes is not observed in astrocytes derived <strong>from</strong> sut mice. These results identify system xc -<br />
as a novel cellular <strong>an</strong>d molecular target of IL-1β <strong>an</strong>d also suggest that altering astrocyte function<br />
may be a viable <strong>the</strong>rapeutic strategy <strong>for</strong> modifying hypoxic/excitotoxic neuronal injury.<br />
Disclosures: N.A. Jackm<strong>an</strong>, None; J.A. Hewett, None; S.J. Hewett, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.3/V5<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: FONDECYT Gr<strong>an</strong>t 1040831
FONDECYT Gr<strong>an</strong>t 1090353<br />
Title: Differential interplay of TGFβ1 with ERK-MAPK activation in hippocampal <strong>an</strong>d astrocyte<br />
cultures under inflammatory conditions<br />
Authors: *R. VON BERNHARDI, R. URIBE-SAN MARTÍN, R. HERRERA-MOLINA, J.<br />
TICHAUER, M. VALLEJOS;<br />
Dept Neurol Fac Med., Pontificia Univ. Catolica de Chile, S<strong>an</strong>tiago, Chile<br />
Abstract: TGFβ1 is a cytokine that modulates cellular reactivity <strong>an</strong>d has been associated to<br />
neuroprotection in several injury models. Subst<strong>an</strong>tial evidence shows a differential induction of<br />
TGFβ1 expression by various stimuli in neurons <strong>an</strong>d astrocytes. However, <strong>the</strong> mech<strong>an</strong>isms that<br />
ultimately explain <strong>the</strong>se differences are still unknown. Because <strong>the</strong> ERK signal tr<strong>an</strong>sduction<br />
pathway plays <strong>an</strong> import<strong>an</strong>t role on TGFβ1 syn<strong>the</strong>sis in glial cells, we evaluated <strong>the</strong> association<br />
of hippocampal (neuron-astrocyte) <strong>an</strong>d astrocyte production of TGFβ1 in response to<br />
inflammatory stimuli with <strong>the</strong> activation of ERK. On hippocampal cultures, proinflammatory<br />
conditions (LI: 1 µg/ml LPS + 10 ng/ml IFNγ) resulted in a 2.5-fold increase of <strong>the</strong> ERK<br />
pathway activation that peaked at approximately 6 h. ERK activation was observed prior to <strong>the</strong><br />
induction of TGFβ1 activity, which reached a 6-fold increase after 48 h of inflammatory<br />
stimulation (p
Program#/Poster#: 545.4/V6<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: MRC doctoral training gr<strong>an</strong>t G0501384<br />
Title: The voltage gated sodium ch<strong>an</strong>nel <strong>an</strong>d related proteins at <strong>the</strong> node of R<strong>an</strong>vier are targeted<br />
in a mouse model of <strong>the</strong> peripheral neuropathy Guillain-Barré syndrome<br />
Authors: *R. MCGONIGAL, S. K. HALSTEAD, H. J. WILLISON;<br />
Clin. Neuroimmunology, Univ. of Glasgow|910005876|0, Glasgow, United Kingdom<br />
Abstract: Guillain-Barré syndrome (GBS) is a peripheral neuropathy characterised by acute<br />
flaccid paralysis. The axonal vari<strong>an</strong>t is associated with <strong>an</strong>ti-GD1a g<strong>an</strong>glioside <strong>an</strong>tibodydependent,<br />
complement-mediated injury to <strong>the</strong> peripheral axon with conduction block. The<br />
blood-nerve barrier (BNB) relatively protects axons <strong>from</strong> factors in <strong>the</strong> extra-neural<br />
environment; however, it does not extend over <strong>the</strong> neuromuscular junction, leaving this terminal<br />
portion of <strong>the</strong> axon unprotected. It is here that we have previously demonstrated susceptibility to<br />
<strong>an</strong>tibody attack in a mouse model of GBS. We are now determining to what extent more<br />
proximal portions of <strong>the</strong> distal axon are at risk <strong>from</strong> circulating <strong>an</strong>tibody <strong>an</strong>d what exogenous<br />
protection c<strong>an</strong> be provided <strong>the</strong>rapeutically.<br />
GD1a is expressed at <strong>the</strong> nodes of R<strong>an</strong>vier (NoR) of intramuscular axons. To investigate <strong>the</strong><br />
injury caused by <strong>an</strong>ti-GD1a <strong>an</strong>tibodies in relation to BNB permeability, we applied <strong>an</strong>ti-GD1a<br />
<strong>an</strong>tibodies to mice genetically engineered both to over-express GD1a <strong>an</strong>d to express cy<strong>an</strong><br />
fluorescent protein (CFP) in <strong>the</strong> cytoplasm of axons. Endogenous fluorescence allowed<br />
identification of intramuscular nerve bundles <strong>an</strong>d <strong>the</strong>ir terminal br<strong>an</strong>ches, which were<br />
categorized depending on bundle size. Within <strong>the</strong>se categories, IgG <strong>an</strong>d <strong>the</strong> final product of <strong>the</strong><br />
complement pathway (membr<strong>an</strong>e attack complex, MAC) deposition were qu<strong>an</strong>tified after <strong>an</strong><br />
acute injury, alongside <strong>the</strong> deleterious effects on NoR proteins. Nerve conduction studies were<br />
also per<strong>for</strong>med to better elucidate <strong>the</strong> pathological pathway.<br />
IgG <strong>an</strong>d MAC were localized in a gradient-dependent m<strong>an</strong>ner, with signific<strong>an</strong>tly more<br />
deposition at NoR as <strong>the</strong> bundles progressively br<strong>an</strong>ch to a single terminating fibre. Fur<strong>the</strong>rmore,<br />
MAC deposition was associated with <strong>the</strong> loss or disruption to immunostaining <strong>for</strong> nodal proteins<br />
including voltage gated sodium ch<strong>an</strong>nel (VGSC) <strong>an</strong>d <strong>an</strong>kyrin G. This is indicative of targeted<br />
injury to this region of <strong>the</strong> distal axon in <strong>an</strong> acute model.<br />
Our studies are suggestive of a resilient proximal barrier that becomes more permeable towards<br />
<strong>the</strong> nerve terminal. There<strong>for</strong>e, it is not only <strong>the</strong> axon at <strong>the</strong> terminal that c<strong>an</strong> be a target of injury,<br />
but also <strong>the</strong> distal axons at <strong>the</strong>ir nodes of R<strong>an</strong>vier, resulting in disruption at this site.<br />
Disclosures: R. McGonigal, None; S.K. Halstead, None; H.J. Willison, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.5/V7<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: Harbor Br<strong>an</strong>ch Oce<strong>an</strong>ographic Institute, Florida Atl<strong>an</strong>tic University <strong>for</strong> providing<br />
MZA<br />
ORSP, Midwestern University <strong>for</strong> fin<strong>an</strong>cial support<br />
Title: Differential effect of <strong>the</strong> <strong>an</strong>tineuroinflammatory marine m<strong>an</strong>zamine A on p90 ribosomal<br />
S6 kinase 1 <strong>an</strong>d 2<br />
Authors: *A. M. MAYER 1 , M. L. HALL 2 ;<br />
2 Pharmacol., 1 Midwestern Univ., Downers Grove, IL<br />
Abstract: M<strong>an</strong>zamine A (MZA) is a β-carboline alkaloid first reported <strong>from</strong> <strong>the</strong> Okinaw<strong>an</strong><br />
marine sponge genus Haliclona in 1986. We have reported that in vitro M<strong>an</strong>zamine A (MZA)<br />
potently inhibited LPS-activated rat microglia TXB2 (IC50 < 0.016 µM) <strong>an</strong>d O2 - (IC50 = 0.1 µM)<br />
generation, (BioMedCentral Pharmacology 5(1)6, 2005), as well as LPS-activated hum<strong>an</strong><br />
microglia TXB2 (apparent IC50 < 0.7 µM) generation (Inflammation Research 53(S3): S217,<br />
2004). Fur<strong>the</strong>rmore, mech<strong>an</strong>ism of action studies revealed that MZA does not appear to inhibit<br />
ei<strong>the</strong>r <strong>the</strong> thrombox<strong>an</strong>e synthase (Inflammation Research 51(2):S130, 2002) or cyclooxygenase 1<br />
enzymes (Inflammation Research 52 (S2): S123, 2003). Interestingly, MZA showed selective<br />
inhibition of rat MAPK-activated protein kinase 1a (p90 ribosomal S6 kinase 1 or RSK1;<br />
Genb<strong>an</strong>k M99169) when tested against a 30 protein kinase screening array (Kinase Profiling<br />
Screening Service, University of Dundee, 2004). The purpose of this investigation was to<br />
confirm <strong>the</strong> inhibition of RSK1 by MZA in our laboratory, <strong>an</strong>d determine its effect on MAPKactivated<br />
protein kinase-1b (RSK2) (hum<strong>an</strong>, Genb<strong>an</strong>k NM_004586) using a radioisotope-based<br />
protein kinase assay protocol (Biochem J. 401, 29-38, 2007). Concentration-dependent studies<br />
with MZA showed a greater inhibition of RSK1 [IC50= 15 µM] versus RSK2 [IC50= 108.4 µM]<br />
(n=3 experiments). In conclusion, our current findings confirm that MZA inhibits RSK1, <strong>an</strong>d<br />
fur<strong>the</strong>rmore, that MZA appears to have a greater selectivity towards inhibition of RSK1 th<strong>an</strong><br />
RSK2. Because RSK1 is expressed in microglia (Molecular Brain Research 136: 134, 2005), we<br />
hypo<strong>the</strong>size that a potential mech<strong>an</strong>ism by which MZA inhibits LPS-activated rat <strong>an</strong>d hum<strong>an</strong><br />
brain microglia PMA-stimulated TXB2 <strong>an</strong>d O2 - release in vitro may involve <strong>the</strong> cellular<br />
inhibition of RSK1. Experimental studies to test this hypo<strong>the</strong>sis are currently being pl<strong>an</strong>ned in<br />
our laboratory. (We th<strong>an</strong>k Harbor Br<strong>an</strong>ch Oce<strong>an</strong>ographic Institute, Florida Atl<strong>an</strong>tic University<br />
<strong>for</strong> providing MZA <strong>an</strong>d Midwestern University <strong>for</strong> fin<strong>an</strong>cial support).<br />
Disclosures: A.M. Mayer, None; M.L. Hall, None.
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.6/V8<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: NIH-2RO1 HL51614<br />
NIH-RO1 NS43284<br />
NIH-RO1 NS38195<br />
Title: A novel dynamic in vitro model to study tr<strong>an</strong>sendo<strong>the</strong>lial cell migration at <strong>the</strong> BBB<br />
Authors: M. HOSSAIN 1 , N. MARCHI 1 , E. RAPP 2 , D. JANIGRO 1 , *L. CUCULLO 3,1 ;<br />
1 Clevel<strong>an</strong>d Clin. Lerner Col. of Medicine, Cerebrovascular Res., Clevel<strong>an</strong>d, OH; 2 Flocel Inc,<br />
Clevel<strong>an</strong>d, OH; 3 CCLCM, Clevel<strong>an</strong>d, OH<br />
Abstract: Although <strong>the</strong>re is signific<strong>an</strong>t evidence correlating peripheral inflammation, <strong>the</strong> bloodbrain<br />
barrier (BBB) <strong>an</strong>d <strong>the</strong> pathogenesis of neuroinflammatory diseases, <strong>the</strong> precise<br />
mech<strong>an</strong>isms by which <strong>the</strong> immune cells interact with <strong>the</strong> BBB <strong>an</strong>d enter <strong>the</strong> brain are largely<br />
unknown. Studies per<strong>for</strong>med on viable in vitro BBB models are set to facilitate <strong>the</strong> resolution of<br />
this problem. For this purpose we have developed a novel hum<strong>an</strong>ized dynamic in vitro BBB<br />
model (nDIV-BBB) which represents a signific<strong>an</strong>t adv<strong>an</strong>cement <strong>from</strong> <strong>the</strong> original DIV-BBB<br />
system. This new model not only reproduces a quasi-physiological environment where<br />
endo<strong>the</strong>lial cells <strong>an</strong>d astrocytes establish a functional BBB but also, by me<strong>an</strong>s of larger<br />
tr<strong>an</strong>scapillary pores (> 3µm), permits <strong>the</strong> trafficking of <strong>the</strong> immune cells into <strong>the</strong> CNS. Our data<br />
show that <strong>the</strong> larger pores did not affect <strong>the</strong> <strong>for</strong>mation of a tight barrier as assessed by tr<strong>an</strong>sendo<strong>the</strong>lial<br />
electrical resist<strong>an</strong>ce (TEER) (> 700 Ohm cm 2 ) <strong>an</strong>d by sucrose permeability<br />
measurements (4x10 -6 cm/sec). Flow-cessation <strong>for</strong> 1 hour followed by reperfusion (Fc/Rp) in<br />
modules with intraluminally circulating THP-1 monocytic cells caused a biphasic opening of <strong>the</strong><br />
BBB. The loss of BBB integrity was paralleled by <strong>the</strong> release of <strong>the</strong> pro-inflammatory cytokines<br />
interleukin-6 (IL-6) <strong>an</strong>d tumor necrosis factor alpha (TNF-α) as well as <strong>the</strong> release <strong>an</strong>d activation<br />
of matrix-metalloproteinases 2 <strong>an</strong>d 9. By contrast to <strong>the</strong> previous DIV-BBB, this new model<br />
allowed <strong>the</strong> tr<strong>an</strong>sendo<strong>the</strong>lial passage of THP-1 cells into <strong>the</strong> extraluminal space. Import<strong>an</strong>tly, this<br />
was observed only in systems subjected to Fc/Rp. In summary, our recent development in <strong>the</strong><br />
model design has allowed <strong>the</strong> DIV-BBB to become permissive <strong>for</strong> tr<strong>an</strong>sendo<strong>the</strong>lial migration of<br />
peripheral blood mononuclear cells (PBMNs) into <strong>the</strong> extra capillary space (ECS), thus<br />
providing a valuable tool to dissect <strong>the</strong> modulatory mech<strong>an</strong>isms of immune cell trafficking into<br />
<strong>the</strong> brain <strong>an</strong>d unveil <strong>the</strong> mech<strong>an</strong>isms that regulate <strong>the</strong> multi-step paradigms of leukocyte-
endo<strong>the</strong>lial interactions. This will greatly favor <strong>the</strong> development of novel <strong>the</strong>rapeutic strategies<br />
aimed at specifically inhibiting key subsets of inflammatory processes that drive <strong>the</strong><br />
pathogenesis <strong>an</strong>d progression of neuroinflammatory pathologies (such as Multiple Sclerosis <strong>an</strong>d<br />
Alzheimer’s disease) without affecting <strong>the</strong> migration <strong>an</strong>d function of leukocytes required <strong>for</strong><br />
protective immunity.<br />
Disclosures: M. Hossain, None; N. Marchi, None; E. Rapp, Flocel Inc, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); D. J<strong>an</strong>igro, Flocel Inc., E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); L. Cucullo ,<br />
Flocel Inc, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.7/V9<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: Peripheral activation of <strong>the</strong> receptor <strong>for</strong> adv<strong>an</strong>ced glycation endproducts (RAGE) triggers<br />
nuclear tr<strong>an</strong>slocation of NFκB in mouse brain<br />
Authors: *N. I. POZDNYAKOV, C. E. NOLAN, A. E. ROBSHAW, K. A. WELCH, L. F.<br />
LANYON, R. A. NOLAN, B. G. SAHAGAN, D. T. STEPHENSON, B. TATE, R. B. NELSON;<br />
CNS Biol, Pfizer Inc, Groton, CT<br />
Abstract: The multi-lig<strong>an</strong>d tr<strong>an</strong>s-membr<strong>an</strong>e receptor RAGE is known to be a major modulator<br />
<strong>for</strong> inflammatory <strong>an</strong>d immune responses in <strong>the</strong> body. Activation of RAGE leads to nuclear<br />
tr<strong>an</strong>slocation of <strong>the</strong> oxidative stress responsive tr<strong>an</strong>scription factor NFkB <strong>an</strong>d upregulation of its<br />
target genes. We report here that peripheral activation of RAGE by its lig<strong>an</strong>d AGE-BSA caused<br />
acute increase of NFkB p65 subunit in <strong>the</strong> nuclear fraction prepared <strong>from</strong> mouse brain. We<br />
sought to first induce RAGE expression in wild-type C57Blk6/J mice by peripherally<br />
administering <strong>the</strong> non-specific pro-inflammatory agent silver nitrate. RAGE expression increased<br />
in a time-dependent fashion <strong>an</strong>d peaked 4 days after silver nitrate administration as determined<br />
by Western blot <strong>an</strong>alysis. After “priming” <strong>the</strong> mice with silver nitrate to upregulate RAGE, we<br />
<strong>the</strong>n challenged <strong>the</strong> mice at 4 days with <strong>an</strong> acute intravenous administration of <strong>the</strong> RAGE lig<strong>an</strong>d<br />
AGE-BSA. This treatment induced <strong>an</strong> elevation of <strong>the</strong> p65 subunit of NFκB within <strong>the</strong> nuclear<br />
fraction obtained <strong>from</strong> <strong>the</strong> brain. Induction of p65 in <strong>the</strong> brain nuclear fraction was rapid,<br />
peaking by 45 min post AGE-BSA administration. Import<strong>an</strong>tly, no increase of p65 was detected<br />
in <strong>the</strong> brain nuclear fraction collected <strong>from</strong> identically treated RAGE knockout mice. There was
also no elevation in p65 signal measured at this timepoint in <strong>the</strong> nuclear fraction collected <strong>from</strong><br />
whole blood, indicating that <strong>the</strong> signal did not originate <strong>from</strong> <strong>the</strong> blood. The present findings<br />
indicate that activation of RAGE in <strong>the</strong> periphery activates NFkB in brain <strong>an</strong>d suggests a<br />
mech<strong>an</strong>ism <strong>for</strong> RAGE mediated signal tr<strong>an</strong>sduction across <strong>the</strong> blood-brain barrier.<br />
Disclosures: N.I. Pozdnyakov , Pfizer Inc., A. Employment (full or part-time); C.E. Nol<strong>an</strong>,<br />
Pfizer Inc., A. Employment (full or part-time); A.E. Robshaw, Pfizer Inc., A. Employment (full<br />
or part-time); K.A. Welch, Pfizer Inc., A. Employment (full or part-time); L.F. L<strong>an</strong>yon, Pfizer<br />
Inc., A. Employment (full or part-time); R.A. Nol<strong>an</strong>, Pfizer Inc., A. Employment (full or parttime);<br />
B.G. Sahag<strong>an</strong>, Pfizer Inc., A. Employment (full or part-time); D.T. Stephenson, Pfizer<br />
Inc., A. Employment (full or part-time); B. Tate, Pfizer Inc., A. Employment (full or part-time);<br />
R.B. Nelson, Pfizer Inc., A. Employment (full or part-time).<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.8/V10<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: A CD200 fusion protein attenuates LPS-induced ch<strong>an</strong>ges in vitro <strong>an</strong>d in vivo<br />
Authors: *F. COX, A.-M. MILLER, A. LYONS, M. A. LYNCH;<br />
Physiol. Dept., Trinity Col. Dublin, Dublin, Irel<strong>an</strong>d<br />
Abstract: Deficits in cognitive function are associated with neuroinflammatory ch<strong>an</strong>ges, typified<br />
by activation of glial cells <strong>an</strong>d <strong>an</strong> alteration of <strong>the</strong> pro- <strong>an</strong>d <strong>an</strong>ti-inflammatory cytokine bal<strong>an</strong>ce<br />
in <strong>the</strong> brain. CD200 is a membr<strong>an</strong>e-bound glycoprotein expressed on neurons <strong>an</strong>d endo<strong>the</strong>lial<br />
cells, with its cogn<strong>an</strong>t receptor expressed on microglia. Previous evidence <strong>from</strong> this laboratory<br />
has suggested that interactions between CD200 <strong>an</strong>d its receptor c<strong>an</strong> maintain microglia in a<br />
quiescent state, shifting <strong>the</strong> bal<strong>an</strong>ce towards <strong>an</strong> <strong>an</strong>ti-inflammatory profile. Here we report that<br />
mixed glia prepared <strong>from</strong> neonatal mice deficient in CD200 (CD200 -/- ) displayed <strong>an</strong> exaggerated<br />
response to LPS compared with cells <strong>from</strong> wild-type (WT) mice assessed by expression of <strong>the</strong><br />
pro-inflammatory cytokines IL-1β, IL-6 <strong>an</strong>d TNF-α at mRNA <strong>an</strong>d protein level. This was<br />
paralleled by enh<strong>an</strong>ced expression of surfaces markers of microglial activation, namely CD40<br />
<strong>an</strong>d CD11b. Consistent with this, pretreatment of mixed glia prepared <strong>from</strong> WT mice in <strong>the</strong><br />
presence of a CD200 fusion protein (CD200Fc) attenuated <strong>the</strong> LPS-induced ch<strong>an</strong>ges.<br />
Additionally, intrahippocampal administration of CD200Fc had <strong>the</strong> ability to attenuate <strong>the</strong> LPSinduced<br />
impairment in LTP in young Wistar rats. The data presented here reveal <strong>the</strong> import<strong>an</strong>ce
of CD200 in immune-regulation <strong>an</strong>d demonstrate <strong>the</strong> ability of CD200Fc to attenuate LPSinduced<br />
ch<strong>an</strong>ges both in vitro <strong>an</strong>d in vivo.<br />
Disclosures: F. Cox, None; A. Miller, None; A. Lyons, None; M.A. Lynch, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.9/V11<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: Marie Curie Actions<br />
UCD Seed Funding<br />
PRTLI Cycle 3<br />
Title: Modulatory effects of TNF-α <strong>an</strong>d glutamate pretreatment on calcium homeostasis <strong>an</strong>d<br />
glutamate-induced calcium influx in rat org<strong>an</strong>otypic hippocampal cultures<br />
Authors: O. WATTERS, *J. J. O'CONNOR;<br />
UCD Sch. of Biomol & Biomed Sci., UCD, Dublin 4, Irel<strong>an</strong>d<br />
Abstract: Glutamate-induced excitotoxicity contributes to neuronal damage during a cerebral<br />
ischaemic event such as stroke. Physiological levels of glutamate <strong>an</strong>d pro-inflammatory<br />
cytokines, such as TNF-α, play a role in <strong>the</strong> regulation of synaptic plasticity within <strong>the</strong><br />
hippocampus. Pathophysiological levels, which occur during stroke, cause dysregulation of <strong>the</strong>se<br />
processes, enh<strong>an</strong>cing vulnerability of <strong>the</strong>se cells to <strong>the</strong> ischaemic insult. However, previous<br />
studies have shown that a mild tr<strong>an</strong>sient ischaemic attack (TIA) within 72 hr of a stroke may<br />
attenuate its clinical severity (Castillo et al. Ann Neurol., 54,811, 2003). In this study we<br />
developed <strong>an</strong> in vitro model of TIA using org<strong>an</strong>otypic hippocampal cultures. Hippocampal slices<br />
(400 µm) were cultured <strong>from</strong> male Wistar rats at post-natal day 7 (hum<strong>an</strong>ely killed -<br />
decapitated). At 6 days in vitro (DIV), cultures were exposed to a mild acute dose of glutamate<br />
(30 µM) or TNF-α (5 ng/ml) <strong>for</strong> 30 min (mimic TIA condition), <strong>the</strong>n allowed to recover <strong>for</strong> 24<br />
hr be<strong>for</strong>e a second insult of glutamate (30 µM). As NMDA receptors are permeable to Ca 2+ ions,<br />
<strong>an</strong>d Ca 2+ is a well-established mediator of glutamate excitotoxicity, we investigated whe<strong>the</strong>r<br />
glutamate/TNF-α preconditioning alters glutamate-induced Ca 2+ influx after <strong>the</strong> recovery period.<br />
Intracellular Ca 2+ dynamics were visualised using Fluo-4 dye (3 µM) <strong>an</strong>d Zeiss LSM Pascal
confocal microscope. The effect of TNF-α/glutamate pretreatment on basal Ca 2+ homeostasis<br />
was examined using <strong>the</strong> ratiometric dye, Indo-1 (5 µM) <strong>an</strong>d <strong>the</strong> ratio of bound to unbound dye<br />
was <strong>an</strong>alysed over 20 s using a Zeiss LSM 510 META confocal microscope. Assessment of LDH<br />
levels showed that <strong>the</strong> concentrations of glutamate/TNF-α are non-toxic to <strong>the</strong> cultures.<br />
Pretreatment with glutamate/TNF-α resulted in signific<strong>an</strong>t reduction in glutamate-induced Ca 2+<br />
influx compared to controls. It is possible that <strong>the</strong>se pretreatments conditioned <strong>the</strong> cultures to<br />
become less responsive to <strong>the</strong> second insult, preventing a large influx of Ca 2+ into <strong>the</strong> cells which<br />
could lead to initiation of apoptotic pathways <strong>an</strong>d excitotoxicity. These pretreatments also<br />
resulted in a small but signific<strong>an</strong>t lowering of baseline Ca 2+ compared to controls. Preliminary<br />
data shows that <strong>the</strong> preconditioning effect of glutamate is not mediated by NMDAR’s, as D-AP5<br />
(100 µM) in <strong>the</strong> presence of glutamate exposure is unable to reverse <strong>the</strong> effect. Also TNF-α<br />
preconditioning does not appear to be mediated by mGluR5 activation, as MPEP (10 µM) in <strong>the</strong><br />
presence of TNF-α does not alter this effect. We conclude that glutamate/TNF-α exposure alters<br />
both basal Ca 2+ within <strong>the</strong> cells <strong>an</strong>d <strong>the</strong> responsiveness of cells to glutamate-induced Ca 2+ influx.<br />
Disclosures: O. Watters, None; J.J. O'Connor, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.10/V12<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: DTRA I10001_04_RC_C<br />
Title: The acute phase response <strong>an</strong>d som<strong>an</strong>-induced status epilepticus: Temporal <strong>an</strong>d regional<br />
ch<strong>an</strong>ges in brain cytokine concentrations <strong>an</strong>d neural cell expression in rat<br />
Authors: *E. A. JOHNSON, T. L. DAO, A. I. KOEMETER-COX, D. M. KNIFFIN, C. P.<br />
TOMPKINS, T. A. HAMILTON, R. K. KAN;<br />
US Army Med. Res. Inst. of Chem. Def., Aberdeen Proving Ground, MD<br />
Abstract: Neuroinflammation occurs following most brain injuries, including status epilepticus<br />
(SE) induced by <strong>the</strong> acetylcholinesterase inhibitor som<strong>an</strong> (GD). This intense inflammatory<br />
response may contribute to loss of neural tissue <strong>an</strong>d behavioral dysfunction, though little is<br />
known about this pathological process in <strong>the</strong> brain following GD exposure. A key component of<br />
neuroinflammation is <strong>the</strong> early induction of <strong>the</strong> acute phase response (APR) which is governed<br />
by specific cytokines, IL-1α, IL-1β, IL-6, <strong>an</strong>d TNF-α. This study qu<strong>an</strong>tifies <strong>the</strong> regional <strong>an</strong>d
temporal concentration increases of <strong>the</strong> APR cytokines in vulnerable brain regions (piri<strong>for</strong>m<br />
cortex, hippocampus <strong>an</strong>d thalamus) up to 72 hours following acute GD exposure <strong>an</strong>d SE onset.<br />
Using multiplex bead array immunoassays, we observed signific<strong>an</strong>t concentration increases in all<br />
four APR cytokines. Additionally, each cytokine was localized to resident neural cells (neurons,<br />
astrocytes, microglia <strong>an</strong>d endo<strong>the</strong>lial cells) in regions of injury using fluorescent<br />
immunohistochemistry. This neural expression suggests that GD-induced SE triggers a local<br />
brain APR that is distinct <strong>from</strong> <strong>the</strong> previously observed systemic APR response found with GD<br />
exposure. The temporal increase of <strong>the</strong>se cytokines in <strong>the</strong> brain suggests that <strong>an</strong>ti-inflammatory<br />
<strong>the</strong>rapies directed at <strong>the</strong> central nervous system may be effective <strong>for</strong> reducing SE-induced brain<br />
damage following GD poisoning.<br />
Disclosures: E.A. Johnson, None; T.L. Dao, None; A.I. Koemeter-Cox, None; D.M. Kniffin,<br />
None; C.P. Tompkins, None; T.A. Hamilton, None; R.K. K<strong>an</strong>, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.11/V13<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: CICYT Gr<strong>an</strong>t SAF2006-10243<br />
MEC Fellowship BES-2007-16588 to EJ<br />
IDIBAPS Fellowship to CS<br />
Title: Variations of PDE4B mRNA splice <strong>for</strong>ms in mouse brain after LPS administration<br />
Authors: C. SANABRA, E. JOHANSSON, R. MARTIN, *M. VILARO, G. MENGOD;<br />
IIBB-CSIC-IDIBAPS-CIBERNED, Barcelona, Spain<br />
Abstract: Cyclic nucleotide phosphodiesterases (PDEs) are involved in <strong>the</strong> regulation of cellular<br />
processes by modulating intracellular cyclic AMP <strong>an</strong>d GMP levels. Ten different families of<br />
PDEs have been described. The PDE4 family is composed by four enzymes (PDE4A, PDE4B,<br />
PDE4C <strong>an</strong>d PDE4D) encoded by different gene loci, <strong>an</strong>d each of <strong>the</strong>m has been shown to<br />
produce several mRNAs by alternative splicing. They are characterized by a low Km, Ca 2+ -<br />
insensitivity, specificity <strong>for</strong> cAMP as a substrate, <strong>an</strong>d sensitivity to <strong>the</strong> specific inhibitor<br />
rolipram. PDE4 genes produce <strong>the</strong> so-called long, short <strong>an</strong>d super-short iso<strong>for</strong>ms. PDE4A, 4B
<strong>an</strong>d 4D are expressed in brain <strong>an</strong>d, interestingly, <strong>an</strong> increase in <strong>the</strong> PDE4B2 tr<strong>an</strong>script, a short<strong>for</strong>m<br />
product of <strong>the</strong> PDE4B gene was detected in <strong>the</strong> cellular infiltrates in <strong>the</strong> brain of EAE <strong>an</strong>d<br />
LPS-treated rats (Reyes-Irisarri et al., 2007, JNEN 66:923; 2008, Synapse 62:74).<br />
M<strong>an</strong>y inflammatory responses involve cAMP. Pharmacological control of cAMP levels by PDE<br />
inhibitors provoke profound <strong>an</strong>ti-inflammatory response. Lipopolysacharide (LPS) is a potent<br />
inflammatory agent used to characterize <strong>the</strong> acute inflammatory process in <strong>the</strong> brain <strong>an</strong>d in<br />
periphery <strong>an</strong>d as a model of <strong>the</strong> acute phase response to inflammation. We have <strong>an</strong>alyzed <strong>the</strong><br />
influence of LPS administration (10mg/kg, i.p.) on <strong>the</strong> distribution pattern <strong>an</strong>d expression levels<br />
of <strong>the</strong> four PDE4B mRNA splice vari<strong>an</strong>ts toge<strong>the</strong>r with <strong>the</strong> inflammatory markers COX-2, IL-1β<br />
<strong>an</strong>d TNF-α mRNAs in mouse brain. We have characterized <strong>the</strong> cell populations involved in <strong>the</strong><br />
PDE alterations by double in situ hybridization histochemistry <strong>an</strong>d immunohistochemistry. By<br />
semi-qu<strong>an</strong>titative <strong>an</strong>alyses of <strong>the</strong> autoradiograms of <strong>the</strong> hybridized sections we observed that<br />
PDE4B2 <strong>an</strong>d PDE4B3 were <strong>the</strong> only mRNA splice vari<strong>an</strong>ts exhibiting <strong>an</strong> altered response to<br />
LPS administration. Whereas PDE4B2 presented <strong>an</strong> increase in expression levels at both 3 <strong>an</strong>d<br />
24h after LPS administration, PDE4B3 mRNA levels showed a reduction reaching minimal<br />
levels 8h after treatment. Qu<strong>an</strong>tification of <strong>the</strong> presence of PDE4B2 or PDE4B3 mRNA in<br />
different cellular populations in leptomeninges, cingulate cortex <strong>an</strong>d inferior colliculus of mouse<br />
brain, 3 <strong>an</strong>d 8h after LPS administration, revealed a clear expression of PDE4B2 mRNA in m<strong>an</strong>y<br />
endo<strong>the</strong>lial cells <strong>an</strong>d microglia whereas PDE4B3 mRNA was observed principally in<br />
oligodendrocytes <strong>an</strong>d in neurons. Knowledge about PDE4B mRNAs expression in mouse brain<br />
<strong>an</strong>d <strong>the</strong> alterations provoked by LPS administration might help to clarify <strong>the</strong>rapeutical<br />
possibilities with specific PDE4B inhibitors in neuroinflammatory diseases.<br />
Disclosures: C. S<strong>an</strong>abra, None; E. Joh<strong>an</strong>sson, None; R. Martin, None; M. Vilaro, None; G.<br />
Mengod, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.12/V14<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: <strong>the</strong> Americ<strong>an</strong> Federation <strong>for</strong> Aging Research (KA)<br />
Department of Defense PR043148 (KA)<br />
NIA R21AG033914
Alzheimer’s Association (KA)<br />
AG24011 (TJM)<br />
AG05136 (TJM)<br />
Title: The prostagl<strong>an</strong>din E2 EP4 receptor exerts <strong>an</strong>ti-inflammatory effects in brain innate<br />
immunity<br />
Authors: *J. SHI 1 , J. JOHANSSON 1 , Q. WANG 1 , T. J. MONTINE 2 , K. ANDREASSON 1 ;<br />
1 St<strong>an</strong><strong>for</strong>d Univ., St<strong>an</strong><strong>for</strong>d, CA; 2 Univ. of Washington, Seattle, WA<br />
Abstract: Peripheral inflammation leads to immune responses in brain characterized by<br />
microglial activation, elaboration of pro-inflammatory cytokines <strong>an</strong>d reactive oxygen species,<br />
<strong>an</strong>d secondary neuronal injury. The inducible cyclooxygenase COX-2 mediates a signific<strong>an</strong>t<br />
component of this response in brain via downstream prostagl<strong>an</strong>din signaling pathways, notably<br />
PGE2 which signals through a class of four EP (<strong>for</strong> E-prost<strong>an</strong>oid) receptors, termed EP1-EP4.<br />
Studies have indicated that <strong>the</strong> PGE2 EP2 receptor mediates a signific<strong>an</strong>t inflammatory response<br />
in a broad r<strong>an</strong>ge of neurodegenerative models, including <strong>the</strong> bacterial endotoxin<br />
lipopolysaccharide (LPS) model of innate immunity, <strong>the</strong> APPSwe-PS1∆E9 model of Familial<br />
AD, <strong>an</strong>d <strong>the</strong> G93ASOD model of Familial ALS. The PGE2 EP4 receptor shares certain<br />
characteristics with <strong>the</strong> EP2 receptor in that it is positively coupled to cAMP <strong>an</strong>d its expression is<br />
strongly induced in brain with systemic bacterial endotoxin lipopolysaccharide (LPS)<br />
administration. However, <strong>the</strong> function of <strong>the</strong> EP4 receptor in vivo in CNS inflammation in not<br />
known. Here, we investigated <strong>the</strong> function of <strong>the</strong> PGE2 EP4 receptor in <strong>the</strong> CNS innate immune<br />
response to LPS. We report that <strong>the</strong> EP4 receptor exerts signific<strong>an</strong>t <strong>an</strong>ti-inflammatory effects<br />
both in vitro <strong>an</strong>d in vivo. In vitro, activation of EP4 receptor signaling suppresses proinflammatory<br />
gene expression in response to LPS in microglial cells, <strong>an</strong>d this is associated with<br />
decreases in AKT <strong>an</strong>d IKK phosphorylation <strong>an</strong>d reduced nuclear tr<strong>an</strong>slocation of NF-kappa B<br />
p65 <strong>an</strong>d p50 subunits. In vivo, conditional deletion of <strong>the</strong> EP4 receptor in macrophage/microglial<br />
cells exacerbates <strong>the</strong> inflammatory response in hippocampus in response to systemic LPS;<br />
conversely, EP4 selective agonist AE1-329 decreased LPS-induced pro-inflammatory gene<br />
expression in hippocampus. In plasma, EP4 agonist AE1-329 signific<strong>an</strong>tly reduced levels of proinflammatory<br />
cytokines <strong>an</strong>d chemokines, indicating that peripheral EP4 activation protects <strong>the</strong><br />
brain <strong>from</strong> systemic inflammation. These findings indicate that <strong>the</strong> EP4 receptor functions as a<br />
beneficial <strong>an</strong>d <strong>an</strong>ti-inflammatory prostagl<strong>an</strong>din receptor in brain inflammation.<br />
Disclosures: J. Shi, None; J. Joh<strong>an</strong>sson, None; Q. W<strong>an</strong>g, None; T.J. Montine, None; K.<br />
Andreasson, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.13/V15<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: Chronic Inflammatory Disease Research Center KOSEF R13-2003-019-02004-0<br />
Title: Glycosylation is necessary <strong>for</strong> brain COX-2 maturation<br />
Authors: J.-I. CHUNG 1,4,5 , J.-Y. PARK 1 , Y.-S. JUNG 1 , S. LEE 1 , C.-H. MOON 1 , E.-H. JOE 2,4 ,<br />
S. TACCONELLI 6 , P. PATRIGNANI 6 , *E. J. BAIK 3,4,5 ;<br />
1 Dept Physiol, 2 Dept Pharmacol, Ajou Univ., Suwon, Republic of Korea; 3 Dept Physiol, Ajou<br />
Univ., Suwon, ; 4 Chronic Inflammatory Dis. Res. Ctr., Suwon, Republic of Korea; 5 Brain Korea<br />
21 <strong>for</strong> Med. Sci., Suwon, Republic of Korea; 6 G. d'Annunzio Univ., Chieti, Italy<br />
Abstract: Cyclooxygenases is known as 70-72kDa protein <strong>an</strong>d key enzyme <strong>for</strong> prost<strong>an</strong>oids<br />
syn<strong>the</strong>sis. In <strong>the</strong> present study, in vitro primary neurons <strong>an</strong>d astrocytes culture expressed about<br />
68kDa low molecular weight COX-2 in resting condition. Neurons expressed about 70kDa<br />
COX-1 same size as astrocytes, however <strong>the</strong> expression was very weak. N-methyl D-aspartate<br />
(NMDA) induced 72kDa COX-2 expression in astrocytes, which was missing in neurons.<br />
Neurons did not increased COX-2 protein in western blot even though mRNA was increased.<br />
NMDA promoted PGE2 release only in astrocytes through COXs activity. The 68kDa COX-2<br />
was demonstrated as unglycosylated COX-2 <strong>for</strong>m using endoglycosidase H (Endo H), cleaves<br />
high m<strong>an</strong>nose oligosaccharide to glycoprotein, <strong>an</strong>d tunicamycin, <strong>the</strong> blocker <strong>for</strong> new <strong>for</strong>mation<br />
of protein N-glycosidic linkages. In <strong>the</strong> astrocytes, <strong>the</strong> 72kDa COX-2 induced by glutamate, <strong>an</strong>d<br />
sometimes in resting conditions, <strong>an</strong>d glutamate-induced PGE2 release was blocked by<br />
tunicamycin. However, neurons lack <strong>the</strong> 72 kD COX-2 <strong>an</strong>d glutamate did not increase PGE2<br />
release. To explore <strong>the</strong> COX activity, exogenous arachidonic acid (AA) was treated <strong>for</strong> 1 hr.<br />
Neurons induced PGE2 release however did not reduced by several potent COX inhibitors such<br />
as indomethacin, NS-398, aspirin <strong>an</strong>d ketoprofen. These data suggest that glycosylation is<br />
necessary <strong>for</strong> COXs maturation in <strong>the</strong> brain. Unglycosylated COX-2 in neurons did not produce<br />
COX-dependent PGE2.<br />
Disclosures: J. Chung, None; J. Park, None; Y. Jung, None; S. Lee, None; C. Moon,<br />
None; E. Joe, None; S. Tacconelli, None; P. Patrign<strong>an</strong>i, None; E.J. Baik, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.14/V16<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: The National Research Centre <strong>for</strong> Growth <strong>an</strong>d Development<br />
The Lynette Sulliv<strong>an</strong> Research Fund<br />
Title: Valproic acid induces microglial dysfunction, but not apoptosis, in adult hum<strong>an</strong> mixed<br />
glial cultures<br />
Authors: *H. M. GIBBONS 1 , A. M. SMITH 1 , H. H. TEOH 2 , E. W. MEE 2 , R. L. M. FAULL 1 ,<br />
M. DRAGUNOW 1 ;<br />
1 Univ. Auckl<strong>an</strong>d, Auckl<strong>an</strong>d, New Zeal<strong>an</strong>d; 2 Auckl<strong>an</strong>d Hosp., Auckl<strong>an</strong>d, New Zeal<strong>an</strong>d<br />
Abstract: Valproic acid (VPA) is widely used <strong>for</strong> <strong>the</strong> treatment of mood disorders <strong>an</strong>d epilepsy,<br />
but its mech<strong>an</strong>ism of action is unclear. In vivo <strong>an</strong>d in vitro studies using rodent models have<br />
demonstrated that VPA has both neuroprotective <strong>an</strong>d neurotrophic effects. These beneficial<br />
effects are, in part, through modulation of glial cell function. Recently, we <strong>an</strong>d o<strong>the</strong>rs have<br />
shown that VPA selectively induces caspase 3-mediated apoptosis in a murine microglial cell<br />
line, in primary rat microglial cultures <strong>an</strong>d in a rat MCAO model. However, <strong>the</strong> effect of VPA on<br />
hum<strong>an</strong> microglia has not been tested. In this study, using microglia derived <strong>from</strong> adult hum<strong>an</strong><br />
brains, we demonstrate that VPA treatment does not induce microglial apoptosis as determined<br />
by <strong>the</strong> absence of caspase-3 cleavage <strong>an</strong>d TUNEL labelling. However, VPA does partially<br />
decrease <strong>the</strong> expression of <strong>the</strong> microglial markers PU.1 <strong>an</strong>d cell surface CD45 as well as<br />
reducing <strong>the</strong>ir phagocytic ability. Due to <strong>the</strong> m<strong>an</strong>y roles of microglia in <strong>the</strong> healthy <strong>an</strong>d diseased<br />
brain, <strong>the</strong>se VPA-induced alterations in microglial phenotype could potentially have damaging<br />
effects on <strong>the</strong>ir ability to become activated in response to insults. Although reduced microglial<br />
activation may be beneficial in neurodegenerative disorders where neuroinflammation plays a<br />
role, suppression of microglial activation in <strong>the</strong> healthy brain could lead to a reduced response to<br />
injury or disease.<br />
Disclosures: H.M. Gibbons, None; A.M. Smith, None; H.H. Teoh, None; E.W. Mee,<br />
None; R.L.M. Faull, None; M. Dragunow, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 545.15/V17<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: Gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> DANA foundation.<br />
Title: The choroid plexus response to a repeated peripheral inflammatory stimulus<br />
Authors: *F. MARQUES 1 , J. A. PALHA 1 , J. C. SOUSA 1 , G. COPPOLA 2 , D. H.<br />
GESCHWIND 2 , N. SOUSA 1 , M. CORREIA-NEVES 1 ;<br />
1 Sch. of Hlth. Sciences, Univ. of Minho, Braga, Portugal; 2 Program in Neurogenetics, Dept. of<br />
Neurology, David Geffen Sch. of Medicine-UCLA, Los Angeles, CA<br />
Abstract: Chronic systemic inflammation might trigger alterations in <strong>the</strong> central nervous system<br />
(CNS). Several illnesses of <strong>the</strong> CNS, including multiple sclerosis <strong>an</strong>d Alzheimer’s disease have<br />
<strong>an</strong> underlying chronic inflammatory component. Whe<strong>the</strong>r <strong>an</strong>d how peripheral chronic<br />
inflammation contributes in triggering <strong>the</strong> brain response in <strong>the</strong> course of such illnesses is far<br />
<strong>from</strong> understood. In this context increased attention has been paid to <strong>the</strong> involvement of <strong>the</strong><br />
blood- cerebrospinal fluid (CSF) barrier (BCSFB) (<strong>for</strong>med by epi<strong>the</strong>lial cells of <strong>the</strong> choroid<br />
plexus). In addition, <strong>the</strong> choroid plexus (CP) assumes <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> communication<br />
with <strong>the</strong> brain given its function as <strong>the</strong> major producer of <strong>the</strong> CSF. In <strong>the</strong> present study we<br />
investigated <strong>the</strong> CP gene expression profile 3 <strong>an</strong>d 15 days after a three month period of repeated<br />
inflammation, induced by <strong>the</strong> intraperitoneal administration of lipopolysaccharide every two<br />
weeks. The data shows that <strong>the</strong> CP displays a sustained response to <strong>the</strong> chronic inflammatory<br />
stimulus by altering <strong>the</strong> expression of several genes even 15 days after <strong>the</strong> last injection. Despite<br />
<strong>the</strong> number of genes whose expression is found statistically altered, not m<strong>an</strong>y display a fold<br />
ch<strong>an</strong>ge higher th<strong>an</strong> five times <strong>an</strong>d three times 3 <strong>an</strong>d 15 days <strong>from</strong> last injection, respectively.<br />
Major altered pathways include those facilitating cells entry into <strong>the</strong> brain, <strong>an</strong>d o<strong>the</strong>rs that<br />
participate in <strong>the</strong> innate immune response to infection by microorg<strong>an</strong>isms. Whe<strong>the</strong>r <strong>the</strong> final<br />
bal<strong>an</strong>ce of <strong>the</strong> CP response is protective or deleterious <strong>for</strong> <strong>the</strong> CNS remains to be clarified <strong>an</strong>d<br />
should be considered in <strong>the</strong> context of <strong>the</strong> brain response to inflammation.<br />
Disclosures: F. Marques, None; J.A. Palha, None; J.C. Sousa, None; G. Coppola,<br />
None; D.H. Geschwind, None; N. Sousa, None; M. Correia-Neves, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.16/V18
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t R01NS42168<br />
Sigma Xi GIAR Gr<strong>an</strong>t<br />
Title: Cleavage of monocyte chemoattract<strong>an</strong>t protein-1 (MCP1) by plasmin enh<strong>an</strong>ces its activity<br />
to compromise blood-brain barrier (BBB)<br />
Authors: *Y. YAO, C. ZHOU, S. TSIRKA;<br />
Dept Pharmacol, Stony Brook Univ., Stony Brook, NY<br />
Abstract: Blood-brain barrier (BBB) becomes compromised in m<strong>an</strong>y CNS diseases, including<br />
acute brain injury <strong>an</strong>d chronic neurodegenerative diseases. Monocyte chemoattract<strong>an</strong>t protein-1<br />
(MCP1, CCL2), a CC chemokine that is secreted by astrocytes <strong>an</strong>d neurons, has been found to<br />
disrupt <strong>the</strong> integrity of BBB. We have previously shown that MCP1 c<strong>an</strong> be cleaved by plasmin<br />
<strong>an</strong>d this cleavage increased its chemotactic activity. However, whe<strong>the</strong>r plasmin-mediated<br />
cleavage of MCP1 affects <strong>the</strong> integrity of BBB is not clear. Recent evidence indicated that tissue<br />
plasminogen activator (tPA), which converts plasminogen to plasmin, promotes BBB<br />
breakdown, suggesting that plasmin may be involved in MCP1-induced BBB compromise. Here<br />
we investigated <strong>the</strong> role of plasmin on MCP1-induced BBB compromise using recombin<strong>an</strong>t<br />
MCP1 proteins. We report that while full length (FL) MCP1 decreased <strong>the</strong> tr<strong>an</strong>s-endo<strong>the</strong>lial<br />
electric resist<strong>an</strong>ce (TEER) <strong>an</strong>d increased <strong>the</strong> permeability of BBB, <strong>the</strong> ‘constitutively active’,<br />
truncated MCP1 behaved similar to FL-MCP1, but with a higher potency. A mut<strong>an</strong>t MCP1 that<br />
c<strong>an</strong>’t be cleaved by plasmin, on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, did not ch<strong>an</strong>ge <strong>the</strong> TEER values <strong>an</strong>d BBB<br />
permeability. The subcellular localization of tight junction proteins (occludin <strong>an</strong>d ZO-1) <strong>an</strong>d <strong>the</strong><br />
reorg<strong>an</strong>ization of stress fiber F-actin were affected more by <strong>the</strong> truncated MCP1 <strong>an</strong>d least by <strong>the</strong><br />
non-cleavable mut<strong>an</strong>t. Our results indicate that plasmin-mediated cleavage of MCP1 plays <strong>an</strong><br />
import<strong>an</strong>t role on MCP1-induced BBB compromise, suggesting that plasmin inhibitor could<br />
alleviate BBB leakage.<br />
Disclosures: Y. Yao, None; C. Zhou, None; S. Tsirka, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.17/V19<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms
Support: FCT-POCTI/SAU/MMO/55955/2004<br />
FCT-PTDC/SAU-NEU/64385/2006<br />
FEDER<br />
Title: Microglia: New players in bilirubin neurotoxicity<br />
Authors: *R. F. SILVA, S. L. SILVA, C. OSÓRIO, A. R. VAZ, A. BARATEIRO, A. S.<br />
FALCÃO, A. FERNANDES, M. A. BRITO, D. BRITES;<br />
CPM, iMed.Ul, Fac Farmacia, Univ. Lisbon, Lisbon, Portugal<br />
Abstract: Microglia are <strong>the</strong> main brain cells associated to <strong>an</strong> inflammatory response upon insult.<br />
This response is initially adaptive but if increased may cause secondary damage to neurons <strong>an</strong>d<br />
o<strong>the</strong>r glial cells. Microglial activation involving ei<strong>the</strong>r phagocytosis of cellular debris or<br />
propagation of immune response(1), c<strong>an</strong> be modulated by astrocytes <strong>an</strong>d even neurons(2).<br />
Unconjugated bilirubin (UCB) is a neurotoxic compound that triggers <strong>the</strong> secretion of<br />
proinflammatory cytokines (TNF-alpha, IL-1beta <strong>an</strong>d IL-6) by astrocytes <strong>an</strong>d microglia(3). In<br />
this study we intent to investigate microglia reactivity to: (i) UCB; (ii) media conditioned by<br />
astrocytes <strong>an</strong>d neurons after exposure to UCB.<br />
Primary cortical cultures of rat microglia were incubated <strong>from</strong> 15 min to 24 h with 50 microM<br />
UCB plus 100 microM hum<strong>an</strong> serum albumin, at 37ºC. At selected time points we evaluated <strong>the</strong><br />
release of TNF-alpha, IL-1beta <strong>an</strong>d IL-6, cyclooxigenase-2 (COX-2), phagocytosis, MAPKs<br />
activation <strong>an</strong>d NF-κB nuclear tr<strong>an</strong>slocation. Apoptosis was estimated by caspase -3, -8 <strong>an</strong>d -9<br />
activities. Microglia were also incubated <strong>for</strong> 24 h with astrocyte- or neuron-conditioned medium<br />
exposed to 50 microM UCB <strong>for</strong> 12 h, <strong>an</strong>d nitric oxide (NO) release, LDH leakage, <strong>an</strong>d cytokine<br />
secretion were assessed.<br />
UCB led to <strong>the</strong> activation of p38 <strong>an</strong>d ERK1/2, with peak values at 15 <strong>an</strong>d 30 min, followed by<br />
NF-κB tr<strong>an</strong>slocation at 1 to 2 h <strong>an</strong>d by <strong>the</strong> release of TNF-alpha, IL-1beta <strong>an</strong>d IL-6 at maximum<br />
levels between 4 <strong>an</strong>d 12 h. Interestingly, phagocytic properties of UCB-exposed microglia only<br />
occurred at 4 h. From this point on, caspase activity was maintained with a slight increase at 12 h<br />
<strong>an</strong>d a decrease at 24 h. Upregulation of COX-2 occurred at 12 <strong>an</strong>d 24 h. Fur<strong>the</strong>rmore,<br />
preliminary results suggest that soluble factors <strong>from</strong> UCB-treated astrocytes decreased microglia<br />
viability, toge<strong>the</strong>r with a reduction in <strong>the</strong> release of NO, IL-1beta <strong>an</strong>d IL-6. Cause-effect relation<br />
of those events still needs fur<strong>the</strong>r investigation. In contrast, conditioned media <strong>from</strong> UCB-treated<br />
neurons revealed to intensify <strong>the</strong> NO production by microglia.<br />
These results indicate that microglia play <strong>an</strong> active role in nerve cell toxicity by UCB, engaging<br />
both phagocytic <strong>an</strong>d proinflammatory response. Moreover, <strong>the</strong> influence of damaged astrocytes<br />
or neurons might differently modulate microglial activation, even though cause-effect<br />
relationships need to be fur<strong>the</strong>r investigated. Thus, underst<strong>an</strong>ding <strong>an</strong>d modulating microglia<br />
activation may be a promising target in prevention <strong>an</strong>d treatment of UCB brain damage.<br />
(1)Chew et al, Ment Retard Dev Disabil Res Rev 2006;12:105-12.<br />
(2)Biber et al. Trends Neurosci 2007;30:596-602.<br />
(3)Brites et al. J Perinatol <strong>2009</strong>;29:S8-S13.
Disclosures: R.F. Silva, None; S.L. Silva, None; C. Osório, None; A.R. Vaz, None; A.<br />
Barateiro, None; A.S. Falcão, None; A. Fern<strong>an</strong>des, None; M.A. Brito, None; D. Brites, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.18/V20<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: Role of sphingolipids in central nervous system dysfunction<br />
Authors: *N. M. GRINKINA, E. KARNABI, S. WADGAONKAR, R. WADGAONKAR;<br />
R&D (151), VA Med. Ctr. New York Harbor Healthcare System/ SUNY Downstate Med. Cent,<br />
Brooklyn, NY<br />
Abstract: Background: The pathogenesis of central nervous system (CNS) dysfunction, which<br />
results in encephalopathy is poorly understood. Regulation of sphingolipid metabolism plays a<br />
crucial role in maintaining <strong>the</strong> cellular process in <strong>the</strong> CNS. Sphingosine kinase is <strong>an</strong> enzyme<br />
which converts sphingosine into sphingosine-1-phosphate; a pro-survival product. Here we<br />
characterize sphingosine kinases expression, tissue distribution <strong>an</strong>d regulation during CNS<br />
dysfunction. Using bacterial lipopolysaccharide (LPS) induced in-vivo brain injury model, we<br />
have established <strong>the</strong> parameters to study <strong>the</strong> regulation of <strong>the</strong>se kinases.<br />
Methods: Wild-type (WT) <strong>an</strong>d SphK1-/- mice were <strong>an</strong>es<strong>the</strong>tized, incision was made <strong>an</strong>d LPS or<br />
saline was instilled intracerebrally. After 4 hours, <strong>the</strong> brain was removed <strong>an</strong>d <strong>the</strong> extracted<br />
proteins were used in Western blotting <strong>an</strong>d ELISA, <strong>an</strong>d total RNA was isolated <strong>an</strong>d used <strong>for</strong><br />
Real-time PCR.<br />
Sphingosine kinase 1 upstream sequences containing 1Kb promoter region were PCR amplified<br />
<strong>an</strong>d cloned into Firefly Luciferase containing pGL2 basic vector <strong>an</strong>d tested into tsA201.<br />
Tr<strong>an</strong>sfected cells were treated with TGFβ 5ng/ml or TNFα 10ng/ml <strong>for</strong> 12 hours followed by<br />
measuring <strong>the</strong> luciferase activity.<br />
Results: H&E staining as well as <strong>an</strong>ti-O4 <strong>an</strong>d <strong>an</strong>ti-GFAP immunofluorescence of brain sections<br />
showed more severe injury in SphK1-/- mice as compared to WT mice. Western blotting, RT-<br />
PCR <strong>an</strong>d ELISA showed increased levels of GFAP, TNFα <strong>an</strong>d IL-6 after LPS instillation.<br />
Promoter <strong>an</strong>alysis revealed several tr<strong>an</strong>scription factor consensus sites in a 1Kb upstream<br />
fragment. A 2 fold increase of luciferase activity was determined by TGFβ <strong>an</strong>d TNFα.<br />
Conclusion: These preliminary data demonstrated <strong>the</strong> role of sphingosine kinases during LPS<br />
induced injury. Fur<strong>the</strong>r experiments will elucidate <strong>the</strong> possible involvement of sphingolipid<br />
signaling in modulating <strong>the</strong> pathophysiology of sepsis induced brain injury.
Disclosures: N.M. Grinkina, None; E. Karnabi, None; S. Wadgaonkar, None; R.<br />
Wadgaonkar, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.19/V21<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: The involvement of toll-like receptors in <strong>the</strong> neonatal brain after hypoxia-ischemia<br />
Authors: *L. STRIDH, X. WANG, C. MALLARD;<br />
Inst. of Neurosci. <strong>an</strong>d Physiol., Go<strong>the</strong>nburg, Sweden<br />
Abstract: Perinatal hypoxia-ischemia (HI) has high mortality <strong>an</strong>d morbidity <strong>an</strong>d is associated<br />
with <strong>an</strong> increased rate of major h<strong>an</strong>dicap, including cerebral palsy <strong>an</strong>d epilepsy. The precise<br />
etiology of perinatal brain injury remains unclear; however, recent evidences suggest that<br />
infection/inflammation is <strong>an</strong> import<strong>an</strong>t contributing factor. Toll-like receptors (TLRs) play a key<br />
role in <strong>the</strong> innate immune system by recognising a wide variety of pathogen-associated<br />
molecular patterns, such as lipopolysaccharide (LPS), bacterial DNA <strong>an</strong>d double-str<strong>an</strong>ded RNA.<br />
Recently, host-endogenous molecules associated with damaged cells <strong>an</strong>d tissues have been<br />
shown to activate TLRs. TLRs are constitutively expressed in <strong>the</strong> adult brain <strong>an</strong>d have been<br />
suggested to initiate <strong>the</strong> inflammatory response to ischemic injury. However, <strong>the</strong> role of TLRs in<br />
neonatal HI is unknown. The purpose of this study was to examine <strong>the</strong> expression of TLRs in <strong>the</strong><br />
neonatal brain after HI.<br />
Wild type C57/Bl6 mice were subjected to left carotid artery ligation <strong>an</strong>d 10% O2 <strong>for</strong> 50min (HI)<br />
on PND9. The <strong>an</strong>imals were sacrificed at 30min, 6h <strong>an</strong>d 24h after HI (n= 5-6/group) <strong>an</strong>d<br />
compared to mice not subjected to HI (n= 5). The brains were dissected out <strong>an</strong>d snap frozen or<br />
prepared <strong>for</strong> immunohistochemical <strong>an</strong>alysis. The mRNA expression was determined by a TLR<br />
pathway-specific PCR array (SABiosciences).<br />
It was found that after HI, TLR1 mRNA was down-regulated at 30min (fold regulation -1.83, p<<br />
0.001) <strong>the</strong>n up-regulated at 6h (fold regulation 2.56, p
vessels in <strong>the</strong> neonatal mouse brain.<br />
Conclusion: TLRs are regulated after HI in <strong>the</strong> neonatal brain, indicating that TLRs may play a<br />
role in HI induced inflammation in <strong>the</strong> neonatal brain.<br />
Disclosures: L. Stridh, None; X. W<strong>an</strong>g, None; C. Mallard, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.20/V22<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: CICYT Gr<strong>an</strong>t SAF2006-10243<br />
MEC Fellowship BES-2007-16588 to EJ<br />
IDIBAPS Fellowship to CS<br />
Title: Sex-related variations of PDE4B mRNA splicing <strong>for</strong>ms in mouse brain after LPS<br />
administration<br />
Authors: E. JOHANSSON, C. SANABRA, R. MARTIN, R. CORTES, *G. MENGOD;<br />
IIBB-CSIC-IDIBAPS-CIBERNED, Barcelona, Spain<br />
Abstract: Intracellular cAMP levels are regulated by a bal<strong>an</strong>ce between <strong>the</strong> activities of two<br />
types of enzymes, adenylyl cyclase <strong>an</strong>d cyclic nucleotide phosphodiesterase (PDE). M<strong>an</strong>y<br />
inflammatory responses involve cAMP <strong>an</strong>d pharmacological m<strong>an</strong>ipulation of cAMP levels using<br />
specific PDE inhibitors provoke profound <strong>an</strong>ti-inflammatory response.<br />
PDE4 represents a family of cAMP-specific PDEs consisting of four paralog genes (PDE4A-D)<br />
each distinguishable by its unique N-terminal sequence. Selective inhibition of PDE4 activity,<br />
both in vitro <strong>an</strong>d in vivo, has several proven <strong>an</strong>ti-inflammatory effects. Fur<strong>the</strong>rmore, recent<br />
publications indicate that <strong>the</strong> PDE4B gene is <strong>the</strong> predomin<strong>an</strong>t subtype involved in inflammatory<br />
induction by lipopolysaccaride (LPS) in mouse monocytes <strong>an</strong>d macrophages.<br />
Administration of LPS is a well known model of <strong>the</strong> acute phase response to inflammation. We<br />
have <strong>an</strong>alyzed <strong>the</strong> influence of LPS administration (10mg/kg, i.p.) on <strong>the</strong> distribution pattern <strong>an</strong>d<br />
expression levels of <strong>the</strong> four PDE4B mRNA splicing vari<strong>an</strong>ts toge<strong>the</strong>r with <strong>the</strong> inflammatory<br />
markers, COX-2, IL-1β <strong>an</strong>d TNF-α mRNAs in both male <strong>an</strong>d female mice brain.<br />
Characterization of <strong>the</strong> cell populations involved in <strong>the</strong> PDE alterations was established by
double in situ hybridization histochemistry <strong>an</strong>d immunohistochemistry. By semi-qu<strong>an</strong>titative<br />
<strong>an</strong>alyses of <strong>the</strong> autoradiograms we observed that PDE4B2 <strong>an</strong>d PDE4B3 were <strong>the</strong> only mRNA<br />
splice vari<strong>an</strong>ts that exhibited <strong>an</strong> altered response to LPS treatment, <strong>an</strong>d this response was<br />
fur<strong>the</strong>rmore associated with <strong>the</strong> sex. Whereas in female brain PDE4B2 presented <strong>an</strong> increase in<br />
mRNA levels at both 3 <strong>an</strong>d 24h after LPS treatment, in male brain <strong>the</strong> expression ch<strong>an</strong>ged at 3h<br />
but remained unaltered at 24h. PDE4B3 mRNA levels showed clear ch<strong>an</strong>ges in females 24h<br />
post-injection, whereas, in male <strong>the</strong> altered expression was less evident <strong>an</strong>d peaked 8h after<br />
treatment. Knowledge about PDE4B mRNAs expression in mouse brain in both sexes <strong>an</strong>d <strong>the</strong><br />
alterations provoked by LPS administration might help us to clarify sex-related differences in <strong>the</strong><br />
susceptibility to autoimmune diseases.<br />
Disclosures: E. Joh<strong>an</strong>sson, None; C. S<strong>an</strong>abra, None; R. Martin, None; R. Cortes, None; G.<br />
Mengod, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.21/V23<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: MRC Capacity Building PhD Studentship<br />
Title: Fibrin <strong>an</strong>d fibrinogen cause neuron non-cell autonomous degeneration<br />
Authors: *T. M. PIERS, E. EAST, J. M. POCOCK;<br />
Dept. of Neuroinflam., Inst. of Neurology, UCL, London, United Kingdom<br />
Abstract: Microglia are <strong>the</strong> resident immune cells of <strong>the</strong> central nervous system (CNS),<br />
patrolling <strong>the</strong> parenchyma returning homeostasis to imbal<strong>an</strong>ced areas via phagocytosis. It has<br />
been suggested, a chronically activated microglial phenotype has <strong>the</strong> potential to cause<br />
neuroinflammation <strong>an</strong>d neurodegeneration via persistent release of pro-inflammatory factors<br />
(Gao et al. 2003). In a r<strong>an</strong>ge of chronic neurodegenerative disorders blood brain barrier (BBB)<br />
disruption has been observed, permitting normally absent blood borne proteins, including<br />
fibrinogen, to enter <strong>the</strong> CNS parenchyma. This results in fibrin deposition that has been<br />
implicated in neuroinflammation (Paul et al. 2007). Recently, o<strong>the</strong>r blood proteins entering <strong>the</strong><br />
CNS has been targeted as a potential cause of excessive activation of microglia leading to<br />
neurodegenerative signalling cascades <strong>an</strong>d disease progression (Hooper et al. <strong>2009</strong>). Here direct<br />
exposure of fibrinogen to cerebellar gr<strong>an</strong>ule cell (CGC) cultures caused signific<strong>an</strong>t neuronal
death within 24 hours. This characteristic was not shared with fibrin, where a signific<strong>an</strong>t increase<br />
in neuronal death was observed only after 48 hours, <strong>an</strong>d was similar to <strong>the</strong> response observed<br />
after addition of <strong>the</strong> classic microglial activator lipopolysaccharide (LPS). Fibrinogen was also<br />
shown to have potential as <strong>an</strong> indirect neurotoxin, producing <strong>an</strong> activated microglia phenotype.<br />
Supporting this, media <strong>from</strong> microglia cultures conditioned with fibrinogen caused extensive<br />
CGC death to a level comparable with fibrin or LPS. The induction of pro-inflammatory factors<br />
<strong>from</strong> both primary microglia <strong>an</strong>d CGC cultures after exposure to fibrin or fibrinogen also raises<br />
<strong>the</strong> possibility that <strong>the</strong>se proteins could promote neuroinflammation. The increase of proinflammatory<br />
factors in CGC cultures was attributed to <strong>the</strong>ir production <strong>from</strong> microglia because<br />
leucine methyl ester (LME) depletion of microglia signific<strong>an</strong>tly attenuated <strong>the</strong>se increases. These<br />
observations lead us to propose that both fibrin <strong>an</strong>d fibrinogen have <strong>the</strong> potential to cause neuron<br />
non-cell autonomous degeneration via a microglia mediated pathway involving proinflammatory<br />
factors, but only fibrinogen is directly neurotoxic.<br />
Disclosures: T.M. Piers, None; E. East, None; J.M. Pocock, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.22/V24<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: National Natural Science Foundation of China, No. 30328015 .<br />
National Natural Science Foundation of China, No. 30671918.<br />
Title: Selective inactivation of adenosine A2A receptors in bone marrow cells attenuates<br />
traumatic brain injury in <strong>the</strong> mouse cortical impact model<br />
Authors: *Y. ZHOU 1 , S. DAI 1 , W. LI 1 , J. AN 1 , P. ZHU 1 , J.-F. CHEN 2 ;<br />
1 2<br />
Mol. Bio Ctr., Res. Inst. Surg <strong>an</strong>d Daping Hosp. ,TMMU, Chongqing, China; Dept. Neurol,<br />
Boston Uni. Sch. Med., Boston, MA<br />
Abstract: Adenosine A2A receptors (A2ARs) on bone marrow-derived cells (BMDCs) exert<br />
different effects in peripheral <strong>an</strong>d CNS injuries, but <strong>the</strong>ir relative contribution in traumatic brain<br />
injury (TBI) is unknown. In this study, we investigated <strong>the</strong> role of A2ARs on BMDCs in a<br />
mouse cortical impact model in which A2ARs on BMDCs were selectively inactivated or<br />
reconstituted by bone marrow tr<strong>an</strong>spl<strong>an</strong>tation. Following TBI, comparing with <strong>the</strong> wild type
control injured group, <strong>the</strong> neurological deficits, cerebral edema <strong>an</strong>d cell apoptosis were all<br />
signific<strong>an</strong>tly attenuated in all of <strong>the</strong> selective inactivation, reconstitution of A2ARs on BMDCs<br />
<strong>an</strong>d global A2ARs inactivation. However, <strong>the</strong> neurological deficit scores of selective inactivation<br />
of A2ARs on BMDCs are signific<strong>an</strong>t lower th<strong>an</strong> that of selective reconstitution of A2ARs on<br />
BMDCs <strong>an</strong>d <strong>the</strong> total A2ARs knock-out TBI mice. Fur<strong>the</strong>r investigation showed <strong>the</strong> beneficial<br />
effects of selective reconstitution of A2ARs on BMDCs like <strong>the</strong> global A2ARs inactivation<br />
resulted <strong>from</strong> suppressing <strong>the</strong> release of excitotoxic glutamate, but <strong>the</strong> protective role of selective<br />
inactivation of A2ARs on BMDCs were attributed to inhibiting <strong>the</strong> inflammatory cytokine TNFα<br />
<strong>an</strong>d IL-1β expressions <strong>an</strong>d decreasing release of glutamate reduced by inflammatory injury.<br />
These results indicate that <strong>the</strong> A2AR-stimulated cascade in BMDCs is <strong>an</strong> import<strong>an</strong>t modulator of<br />
TBI <strong>an</strong>d <strong>the</strong> brain damages are regulated by selective inactivation <strong>an</strong>d reconstitution of A2ARs<br />
on BMDCs with distinct mech<strong>an</strong>ism. (Supported by <strong>the</strong> National Natural Science Foundation of<br />
China, No. 30328015 <strong>an</strong>d No. 30671918. )<br />
Disclosures: Y. Zhou, None; S. Dai, None; W. Li, None; J. An, None; P. Zhu, None; J. Chen,<br />
None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.23/V25<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: Woodin Dendritics, LLC<br />
Title: Interferon-gamma unmasks functional brain-resident dendritic cells<br />
Authors: *A. C. GOTTFRIED-BLACKMORE 1,2 , U. KAUNZNER 2 , J. IDOYAGA 3 , J.<br />
FELGER 2 , B. S. MCEWEN 2 , K. BULLOCH 3 ;<br />
2 Lab. of Neuroendocrinology, 3 Lab. of Physiol. <strong>an</strong>d Immunol., 1 Rockefeller Univ., New York,<br />
NY<br />
Abstract: Dendritic cells (DC) are key regulators of immune function <strong>an</strong>d comprise a<br />
heterogeneous cell group. In <strong>the</strong> steady state, immature DC induce toler<strong>an</strong>ce in circulating T<br />
cells, whereas in <strong>an</strong> activated or mature state, <strong>the</strong>y behave as <strong>the</strong> professional <strong>an</strong>tigen presenting<br />
cells (APC) that bridge <strong>the</strong> innate <strong>an</strong>d adaptive immune system. Recently, using a CD11c_EYFP<br />
tr<strong>an</strong>sgenic mouse model developed to study DC functions, we described a population of EYFP+<br />
CD11b+ cells in various regions of <strong>the</strong> developing <strong>an</strong>d adult brain, termed brain dendritic cells
(bDC). These bDCs share a remarkable morphologic <strong>an</strong>d phenotypic similarity with <strong>the</strong> brain<br />
resident microglia (MG), raising <strong>the</strong> question whe<strong>the</strong>r <strong>the</strong>se cells could be derived <strong>from</strong> <strong>the</strong> MG<br />
population. Using <strong>the</strong> CD11c_EYFP tr<strong>an</strong>sgenic mouse model, chimeric mice, <strong>an</strong>d <strong>an</strong> acute<br />
stimulus of IFNgamma or IL-4, we have directly addressed in vivo whe<strong>the</strong>r bDC c<strong>an</strong> be induced<br />
to become mature APC <strong>an</strong>d stimulate <strong>an</strong>tigen specific CD4 or CD8 T-cell activation. Our results<br />
clearly show that acute administration of IFNγ potently generates bDCs <strong>from</strong> a subgroup of<br />
resident MG, <strong>an</strong>d that <strong>the</strong>se bDCs are functionally distinct <strong>from</strong> <strong>the</strong> remaining MG.<br />
Disclosures: A.C. Gottfried-Blackmore, Woodin Dentritics, LLC, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); U. Kaunzner, Woodin Dentritics, LLC, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Idoyaga,<br />
None; J. Felger, Woodin Dentritics, LLC, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); B.S. McEwen,<br />
Woodin Dentritics, LLC, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t<br />
<strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); K. Bulloch, Woodin Dentritics, LLC, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received).<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.24/V26<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: DTRA I10001_04_RC_C<br />
Title: Expression <strong>an</strong>d localization of <strong>the</strong> chemokines MCP-1 <strong>an</strong>d MIP-1α following som<strong>an</strong><br />
induced status epilepticus in rats<br />
Authors: T. L. DAO, E. A. JOHNSON, D. M. KNIFFIN, C. P. TOMPKINS, T. A.<br />
HAMILTON, *K. D. SPRADLING, R. K. KAN;<br />
USAMRICD, Aberdeen Proving Ground, MD<br />
Abstract: Som<strong>an</strong> (GD) irreversibly inhibits acetylcholinesterase (AChE), causing <strong>an</strong> excess<br />
accumulation of <strong>the</strong> neurotr<strong>an</strong>smitter acetylcholine (ACh) in both central <strong>an</strong>d peripheral nervous<br />
tissue. Increased ACh in <strong>the</strong> brain leads to seizures that rapidly progress to status epilepticus<br />
(SE) <strong>an</strong>d brain injury. Subsequently, <strong>the</strong> brain initiates a proinflammatory response to release
mediators, such as chemokines, at <strong>the</strong> site of injury. Monocyte chemotactic protein-1 (MCP-1)<br />
<strong>an</strong>d macrophage inflammatory protein 1α (MIP-1α) are chemokines that help activate <strong>an</strong>d guide<br />
microglial cells <strong>an</strong>d macrophages to damaged areas. These cells help clear cellular debris though<br />
prolonged activation of macrophages may exacerbate neural injury. However, very little is<br />
known about <strong>the</strong> production <strong>an</strong>d release of <strong>the</strong>se <strong>an</strong>d m<strong>an</strong>y o<strong>the</strong>r inflammatory factors in <strong>the</strong><br />
brain following GD-induced SE. There<strong>for</strong>e, we investigated <strong>the</strong> protein up-regulation of MCP-1<br />
<strong>an</strong>d MIP-1α, <strong>the</strong> neural cell expression of <strong>the</strong>se chemokines <strong>an</strong>d microglia/macrophage reactions<br />
following GD-induced SE neuronal damage. Brain tissue was collected up to 72 hrs after GDinduced<br />
seizures to determine <strong>the</strong> concentration of both chemokines in <strong>the</strong> hippocampus,<br />
piri<strong>for</strong>m cortex <strong>an</strong>d thalamus, regions vulnerable to GD-induced SE. Multiplex bead assay data<br />
showed a signific<strong>an</strong>t increase in both MCP-1 <strong>an</strong>d MIP-1α over <strong>the</strong> time course of <strong>the</strong> experiment<br />
in each region. MCP-1 <strong>an</strong>d MIP-1α expression was localized to m<strong>an</strong>y neural cell types including<br />
neurons, microglia, astrocytes, <strong>an</strong>d/or endo<strong>the</strong>lial cells by fluorescent immunohistochemistry.<br />
These data suggest that chemokines are part of <strong>the</strong> early proinflammatory response to neuronal<br />
injury after GD-induced SE, that <strong>the</strong>y likely participate in guiding microglia/monocytes to injury<br />
sites <strong>an</strong>d that <strong>the</strong>y help <strong>the</strong>m to activate into macrophages.<br />
Disclosures: T.L. Dao, None; E.A. Johnson, None; D.M. Kniffin, None; C.P. Tompkins,<br />
None; T.A. Hamilton, None; K.D. Spradling, None; R.K. K<strong>an</strong>, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.25/V27<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Title: Involvement of mTOR kinase in cytokine dependent microglial activation <strong>an</strong>d cell<br />
proliferation<br />
Authors: L. LISI, G. TRINGALI, P. NAVARRA, *C. DELLO RUSSO;<br />
Inst. Pharmacol., Univ. Cattolica S.Cuore, Rome, Italy<br />
Abstract: The mammali<strong>an</strong> target of rapamycin (mTOR) is a serin/threonin protein kinase with a<br />
central role in <strong>the</strong> regulation of cell growth <strong>an</strong>d proliferation, <strong>an</strong>d of physiological processes such<br />
as tr<strong>an</strong>scription, mRNA turnover <strong>an</strong>d tr<strong>an</strong>slation, autophagy <strong>an</strong>d cytoskeletal org<strong>an</strong>ization.<br />
mTOR is essential <strong>for</strong> <strong>the</strong> proper activation <strong>an</strong>d proliferation of effector T cells, yet restrains <strong>the</strong><br />
development of regulatory T cells. Second generation immunosuppress<strong>an</strong>ts, rapamycin (RAPA)<br />
<strong>an</strong>d RAD001 (RAD) induce immune suppression mainly by blocking cytokine-driven T cell
proliferation. It is now evident that mTOR also contributes to <strong>the</strong> regulation of <strong>the</strong> innate<br />
immune cells. In freshly isolated hum<strong>an</strong> monocytes <strong>an</strong>d dendritic cells mTOR activation inhibits<br />
proinflammatory cytokine production while enh<strong>an</strong>ces <strong>the</strong> release of IL-10. In contrast, mTOR<br />
inhibition decreases <strong>the</strong> global inflammatory response of in vitro generated dendritic cells <strong>an</strong>d<br />
nitric oxide (NO) production in LPS-stimulated macrophages. More recently, mTOR has been<br />
found to enh<strong>an</strong>ce <strong>the</strong> survival of EOC2 microglial cells during oxygen-glucose deprivation <strong>an</strong>d<br />
mediate <strong>the</strong> induction of NO synthase 2 (NOS2) during hypoxia in <strong>the</strong> BV2 microglial cell line.<br />
In this study we investigated <strong>the</strong> role of mTOR in <strong>the</strong> regulation of microglial proinflammatory<br />
activation. Using primary cultures of rat cortical microglia, we detected mTOR activation<br />
(measured by phosphorylation of <strong>the</strong> protein at ser 2448) in response to two different stimuli:<br />
LPS <strong>an</strong>d a mixture of pro-inflammatory cytokines. However pharmacological inhibition of<br />
mTOR while reduced cytokine-dependent NOS2 activity <strong>an</strong>d expression, did not affect LPS<br />
induced NOS activity <strong>an</strong>d enh<strong>an</strong>ced NOS2 mRNA levels. RAD reduced cytokine dependent<br />
PGE2 production, albeit not signific<strong>an</strong>tly, <strong>an</strong>d cyclooxygenase intracellular levels. Moreover,<br />
RAD inhibited microglial proliferation, alone or in combination with cytokines; <strong>an</strong>d under basal<br />
conditions signific<strong>an</strong>tly affected microglial viability. Interestingly, mTOR inhibitors did not<br />
display <strong>an</strong>y relev<strong>an</strong>t effect on astrocyte NOS2 activity as well as cell viability. Taken toge<strong>the</strong>r<br />
<strong>the</strong>se findings suggest that mTOR selectively controls microglial activation in response to<br />
proinflammatory cytokines, <strong>an</strong>d it is crucial <strong>for</strong> maintaining <strong>the</strong>ir viability.<br />
Disclosures: L. Lisi, None; G. Tringali, None; P. Navarra, None; C. Dello Russo, None.<br />
Poster<br />
545. Neuroinflammation: Cellular <strong>an</strong>d Molecular Mech<strong>an</strong>isms I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 545.26/V28<br />
Topic: C.11.m. Neuroinflammation: Cellular <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
Support: NIH gr<strong>an</strong>t NS065167<br />
NIH gr<strong>an</strong>t ES010586,<br />
NIH gr<strong>an</strong>t NS038644<br />
Title: A novel regulatory mech<strong>an</strong>ism of protein kinase cdelta (PKCδ) dependent<br />
proinflammatory cytokine TNF-alpha release <strong>from</strong> microglia: Relev<strong>an</strong>ce to neuroinflammation<br />
<strong>an</strong>d progression of Parkinson’s disease
Authors: *R. GORDON, V. ANANTHARAM, A. G. KANTHASAMY, A. KANTHASAMY;<br />
Biomed Sci, Iowa Ctr. <strong>for</strong> Adv<strong>an</strong>ced Neurotoxicology, Iowa State Univ., Ames, IA<br />
Abstract: Microglia-mediated neuroinflammation is increasingly recognized as a crucial factor<br />
in dopaminergic degeneration <strong>an</strong>d thus contributes to <strong>the</strong> progression of Parkinson’s disease<br />
(PD) after initiation of dopaminergic neuronal damage by idiopathic neurotoxic insults.<br />
Identifying <strong>the</strong> specific molecular signaling pathway that underlies microglia-mediated<br />
neurotoxicity could provide mech<strong>an</strong>istic insights into <strong>the</strong> progression of PD. Although<br />
proinflammatory cytokine release <strong>from</strong> microglia acts as a key contributor to neuroinflammatory<br />
processes in nigral dopaminergic degeneration, <strong>the</strong> cellular mech<strong>an</strong>isms underlying cytokine<br />
release <strong>from</strong> microglia have not yet been defined. Herein, we report that PKCδ, a member of <strong>the</strong><br />
novel PKC family, c<strong>an</strong> regulate release of proinflammatory cytokine TNFα <strong>from</strong> microglia<br />
following LPS as well as MMP-3 treatment. First, we observed a signific<strong>an</strong>t upregulation <strong>an</strong>d<br />
membr<strong>an</strong>e tr<strong>an</strong>slocation of PKCδ in <strong>the</strong> microglia BV2 cell model during MMP-3 <strong>an</strong>d LPS<br />
treatment. Interestingly, <strong>the</strong> PKCδ inhibitor rottlerin blocked LPS <strong>an</strong>d MMP-3-induced TNFα<br />
release <strong>from</strong> both BV2 cells <strong>an</strong>d primary microglia in a dose-dependent m<strong>an</strong>ner. Inhibition of<br />
PKCδ also attenuated basal TNFα release in microglial cells. Fur<strong>the</strong>rmore, knockdown of PKCδ<br />
by siRNA recapitulated <strong>the</strong> effect of rottlerin, indicating that PKCδ regulates cytokine release in<br />
microglia. Collectively, our results demonstrate, <strong>for</strong> <strong>the</strong> first time, that <strong>the</strong> Protein Kinase Cδ<br />
(PKCδ) signaling pathway could play <strong>an</strong> import<strong>an</strong>t role in sustaining microglial activation <strong>an</strong>d<br />
proinflammatory cytokine release <strong>an</strong>d that targeting <strong>the</strong> kinase could have <strong>the</strong>rapeutic<br />
implications <strong>for</strong> <strong>the</strong> progression of PD<br />
Disclosures: R. Gordon, None; V. An<strong>an</strong>tharam, None; A.G. K<strong>an</strong>thasamy, None; A.<br />
K<strong>an</strong>thasamy, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.1/V29<br />
Topic: C.15.d. Animal models<br />
Title: Progressive ratio schedule of rein<strong>for</strong>cement in rats: Investigation of its use as a potential<br />
model of negative symptoms of schizophrenia<br />
Authors: *K. B. GALLAGHER 1 , S. R. FRANKLIN 2 , L. E. RUETER 2 , A. M. BASSO 2 ;<br />
1 2<br />
Abbott Lab., Abbott Park, IL; Neurosci. Research, Global Pharmaceut. Res. <strong>an</strong>d Develop.,<br />
Abbott Labs., Abbott Park, IL
Abstract: Negative symptoms (avolition, affective flattening, alogia <strong>an</strong>d <strong>an</strong>hedonia) pose a<br />
major <strong>the</strong>rapeutic challenge in <strong>the</strong> treatment of schizophrenia. Even clozapine, considered <strong>the</strong><br />
most broadly efficacious <strong>an</strong>tipsychotic currently available, only has modest efficacy against<br />
negative symptoms. In <strong>the</strong> preclinical setting, negative symptoms are particularly challenging to<br />
model since only avolition <strong>an</strong>d <strong>an</strong>hedonia are possible to address in nonverbal species <strong>an</strong>d <strong>the</strong>re<br />
is not a truly efficacious clinical treatment to use in assessing <strong>the</strong> predictive validity of <strong>the</strong><br />
model. A number of studies have been published demonstrating <strong>the</strong> progressive ratio procedure<br />
as a useful technique <strong>for</strong> evaluating altered motivation <strong>for</strong> naturally rein<strong>for</strong>cing stimuli.<br />
Experimental m<strong>an</strong>ipulations that induce <strong>an</strong>hedonia <strong>an</strong>d/or avolition result in <strong>an</strong>imals having<br />
reduced breakpoints. Per<strong>for</strong>m<strong>an</strong>ce on progressive ratio has also been utilized to assess <strong>the</strong><br />
motivational effects of psychotropic drugs. We examined <strong>the</strong> effects of a conventional<br />
<strong>an</strong>tipsychotic (haloperidol), <strong>an</strong> atypical <strong>an</strong>tipsychotic (clozapine), <strong>an</strong>d d-amphetamine in <strong>the</strong><br />
model. Nei<strong>the</strong>r clozapine (0.3, 1, <strong>an</strong>d 3 mg/kg, ip) nor d-amphetamine (0.1, 0.5, <strong>an</strong>d 1 mg/kg, ip)<br />
altered <strong>the</strong> breakpoint. Haloperidol (0.01, 0.03, <strong>an</strong>d 0.1 mg/kg, ip) signific<strong>an</strong>tly decreased <strong>the</strong><br />
breakpoint at 0.1 mg/kg. However, while nei<strong>the</strong>r d-amphetamine nor haloperidol altered<br />
response rate, clozapine reduced <strong>the</strong> rate at <strong>the</strong> dose of 3 mg/kg. Using a 4-day escalating dose<br />
regimen of amphetamine (1-10 mg/kg, ip) <strong>an</strong>d subsequent withdrawal as a model of <strong>an</strong>hedonia,<br />
we fur<strong>the</strong>r assessed motivation in progressive ratio (Phillips, et al 1999). In agreement with<br />
previously published studies in rats exposed to amphetamine, breakpoint <strong>an</strong>d rates of responding<br />
were signific<strong>an</strong>tly reduced <strong>for</strong> 24 hours <strong>an</strong>d showed a trend in reduction <strong>for</strong> up to 3 days of<br />
amphetamine withdrawal. In order to assess <strong>the</strong> relative predictive validity of <strong>the</strong> model, a study<br />
was per<strong>for</strong>med to investigate <strong>the</strong> effect of clozapine on <strong>the</strong> amphetamine withdrawal-induced<br />
decreased breakpoint <strong>an</strong>d rate. Treatment with clozapine (3 mg/kg, ip) had no effect on its own;<br />
however, it was unable to reverse <strong>the</strong> deficits caused by amphetamine withdrawal. These results<br />
mirror clinical data in which clozapine is often ineffective in treating negative symptoms. A<br />
great need exists <strong>for</strong> a pre-clinical model to assess negative symptoms of schizophrenia,<br />
progressive ratio may serve as a potential model <strong>for</strong> fur<strong>the</strong>r evaluation.<br />
Funded by Abbott Laboratories<br />
Disclosures: K.B. Gallagher, Abbott Laboratories, A. Employment (full or part-time); S.R.<br />
Fr<strong>an</strong>klin, Abbott Laboratories, A. Employment (full or part-time); L.E. Rueter, Abbott<br />
Laboratories, A. Employment (full or part-time); A.M. Basso, Abbott Laboratories, A.<br />
Employment (full or part-time).<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.2/V30
Topic: C.15.d. Animal models<br />
Support: NIAAA-IRP<br />
Title: Assessment of AMPA GluA1 receptor knockout mice <strong>for</strong> schizoaffective-related<br />
abnormalities<br />
Authors: *P. J. FITZGERALD 1 , M. T. FEYDER 1 , L. M. WIEDHOLZ 1 , Y.-C. CHEN 1 , T. L.<br />
WRIGHT 1 , R.-M. KARLSSON 1 , A. IZQUIERDO 1 , C. GRAYBEAL 1 , L. M. SAKSIDA 2 , T. J.<br />
BUSSEY 2 , J. HARVEY-WHITE 3 , R. SPRENGEL 4 , R. M. COSTA 5 , A. HOLMES 1 ;<br />
1 BSG/LIN/NIAAA/NIH, Rockville, MD; 2 Univ. of Cambridge, Cambridge, United Kingdom;<br />
3 Section on Neuroendocrinology/LPS/NIAAA/NIH, Rockville, MD; 4 Max-Pl<strong>an</strong>ck Inst. <strong>for</strong> Med.<br />
Res., Heidelberg, Germ<strong>an</strong>y; 5 IVNF/LIN/NIAAA/NIH, Rockville, MD<br />
Abstract: Mice with targeted gene deletion of <strong>the</strong> AMPA GluA1 receptor subunit (GluA1<br />
knockout, KO) have previously been shown to exhibit schizophrenia-related behaviors, including<br />
locomotor hyperactivity, disorg<strong>an</strong>ized social interaction, impaired sensory gating <strong>an</strong>d cognition,<br />
but also a depression-like phenotype in certain tests. Schizophrenia <strong>an</strong>d bipolar disorder are<br />
increasingly recognized as sharing symptomatic <strong>an</strong>d etiological features, <strong>an</strong>d a high degree of<br />
comorbidity is recognized clinically as schizoaffective disorder. Here we assessed KO <strong>for</strong><br />
various m<strong>an</strong>ia-related phenotypes. Results showed that KO exhibited exaggerated open field<br />
locomotor responses to brief stress <strong>an</strong>d reduced immobility in <strong>the</strong> <strong>for</strong>ced swim test after a single<br />
but not, or less so, after repeated exposure, relative to WT. KO showed increased open arm<br />
exploration in <strong>the</strong> elevated plus-maze, increased open arena exploration in <strong>the</strong> dark/light<br />
emergence test <strong>an</strong>d increased stress-induced hyper<strong>the</strong>rmia, relative to WT. KO also showed<br />
impaired extinction of <strong>an</strong> oper<strong>an</strong>t response <strong>for</strong> food reward in a touchscreen-based apparatus, but<br />
showed normal pairwise visual discrimination <strong>an</strong>d reversal learning. Acute treatment with<br />
amphetamine or methylphenidate produced a greater locomotor hyperactivity response in KO<br />
th<strong>an</strong> WT. The dopamine depleting drug, alpha-methyl-para-tyrosine (AMPT), augmented<br />
locomotor hyperactivity in KO despite reducing striatal dopamine content to a similar extent as<br />
in WT. Chronic treatment with <strong>the</strong> mood stabilizing drug, lithium, rescued <strong>the</strong> elevated plusmaze<br />
open arm exploration but not open field hyperactivity phenotype in KO. In <strong>an</strong> ongoing<br />
experiment, we are per<strong>for</strong>ming in vivo neural recordings in <strong>the</strong> dorsal striatum of KO <strong>an</strong>d WT<br />
during open field testing, at baseline <strong>an</strong>d after various drug treatments. These findings<br />
demonstrate that targeted deletion of GluA1 produces a complex array of phenotypic<br />
abnormalities that may model symptomatic features of schizoaffective disorder. Research<br />
supported by NIAAA-IRP.<br />
Disclosures: P.J. Fitzgerald, None; M.T. Feyder, None; L.M. Wiedholz, None; Y. Chen,<br />
None; T.L. Wright, None; R. Karlsson, None; A. Izquierdo, None; C. Graybeal, None; L.M.<br />
Saksida, None; T.J. Bussey, None; J. Harvey-White, None; R. Sprengel, None; R.M. Costa,<br />
None; A. Holmes, None.<br />
Poster
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.3/V31<br />
Topic: C.15.d. Animal models<br />
Support: MH 075980<br />
Title: Role of norepinephrine receptors in central versus basolateral amygdala in <strong>the</strong> regulation<br />
of prepulse inhibition<br />
Authors: *A. K. RAJBHANDARI, S. ROZZI, V. P. BAKSHI;<br />
Univ. Wisconsin, Madison, WI<br />
Abstract: Prepulse inhibition (PPI) is a phenomenon by which a weak stimulus (prepulse)<br />
reduces <strong>the</strong> startle response to a subsequent stimulus (pulse), <strong>an</strong>d is <strong>an</strong> operational measure of<br />
sensorimotor gating, which is deficient in several psychiatric illnesses including schizophrenia.<br />
The amygdala previously has been implicated in <strong>the</strong> regulation of PPI through dopaminergic or<br />
glutamatergic substrates. Never<strong>the</strong>less, <strong>the</strong> role of NE tr<strong>an</strong>smission within <strong>the</strong> amygdaloid<br />
complex in <strong>the</strong> modulation of PPI has not been determined. The amygdala consists of several<br />
nuclei including <strong>the</strong> basolateral complex (BLA) <strong>an</strong>d <strong>the</strong> central nucleus (CeA), which are known<br />
to differ <strong>from</strong> each o<strong>the</strong>r <strong>an</strong>atomically <strong>an</strong>d functionally, but both of which receive heavy NE<br />
innervation <strong>from</strong> <strong>the</strong> locus coeruleus (LC). Given our previous finding that stimulation of LCoriginating<br />
NE-containing pathways c<strong>an</strong> disrupt PPI, <strong>the</strong> present experiments tested <strong>the</strong><br />
hypo<strong>the</strong>sis that increasing NE tr<strong>an</strong>smission within distinct amygdala subregions might disrupt<br />
PPI. Separate groups of male Sprague-Dawley rats were prepared with chronic indwelling<br />
c<strong>an</strong>nulae aimed at ei<strong>the</strong>r <strong>the</strong> CeA or <strong>the</strong> BLA. Rats received a cocktail solution of <strong>the</strong> alpha1<br />
receptor agonist phenylephrine plus <strong>the</strong> beta receptor agonist isoproterenol (equal parts of each<br />
agonist; 0, 3, 10, 30 µg/0.5µl/side) in a counterbal<strong>an</strong>ced order over several test days.<br />
Immediately after microinfusions, rats were tested in startle chambers <strong>for</strong> measuring PPI. In<br />
separate experiments, <strong>the</strong> effects of ei<strong>the</strong>r agonist alone were assessed. Infusion of <strong>the</strong> agonist<br />
cocktail into BLA caused a signific<strong>an</strong>t disruption of PPI in <strong>the</strong> absence of alterations in baseline<br />
startle, indicating a selective disruption of sensorimotor gating. No effects were seen when ei<strong>the</strong>r<br />
agonist was administered by itself, suggesting that coincident activation of alpha1 <strong>an</strong>d beta<br />
receptors may be necessary <strong>for</strong> this BLA-mediated deficit in PPI. In contrast, infusion of <strong>the</strong> NE<br />
receptor agonist cocktail into CeA had no effect on PPI, although it did tend to reduce baseline<br />
startle responses. Intra-CeA infusion of <strong>the</strong> dopamine agonist quinpirole, however, produced a<br />
trend towards a disruption in PPI in <strong>the</strong> same rats that failed to show NE agonist-mediated<br />
deficits. This trend was consistent with previous studies indicating that dopamine agonists in <strong>the</strong><br />
amygdala c<strong>an</strong> disrupt PPI (Swerdlow NR et al 1992). Taken toge<strong>the</strong>r, <strong>the</strong>se findings support a<br />
role <strong>for</strong> NE tr<strong>an</strong>smission within <strong>the</strong> amygdala in <strong>the</strong> regulation of PPI, but suggest that CeA <strong>an</strong>d
BLA are differentially involved, with alpha1 <strong>an</strong>d beta receptor stimulation selectively within<br />
BLA causing deficits in sensorimotor gating.<br />
Disclosures: A.K. Rajbh<strong>an</strong>dari, None; S. Rozzi, None; V.P. Bakshi, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.4/V32<br />
Topic: C.15.d. Animal models<br />
Title: Oxidative stress <strong>an</strong>d water-immersion restraint stress affect behaviors <strong>an</strong>d myelin in <strong>the</strong><br />
hippocampus of mice<br />
Authors: *M. TORITSUKA 1 , M. MAKINODAN 2 , T. YAMAUCHI 2 , K. TATSUMI 3 , H.<br />
OKUDA 3 , T. NAGASHIMA 2 , K. NAKAGAWA 2 , A. WANAKA 3 , T. KISHIMOTO 2 , S.<br />
KIMOTO 2 ;<br />
1 Nara Madical Univ., Nara Pref., Jap<strong>an</strong>; 2 Dept. of Psychiatry, Nara Madical Univ., Kashihara<br />
city, Jap<strong>an</strong>; 3 Dept. of Anat. <strong>an</strong>d Neurosci., Nara Med. Univ., Kashihara city, Jap<strong>an</strong><br />
Abstract: Several lines of evidence have shown a decrease of glutathione content in<br />
schizophrenic patients, which induces oxidative stress. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, mental stress <strong>from</strong><br />
puberty to adulthood could be involved in <strong>the</strong> pathogenesis. We hypo<strong>the</strong>sized oxidative stress<br />
<strong>an</strong>d mental stress implicate <strong>the</strong> onset of schizophrenia on <strong>the</strong> basis of two-hit hypo<strong>the</strong>sis.<br />
We treated mice with buthionine sulfoximine (BSO) first to decrease glutathione, <strong>an</strong>d 15 min<br />
later, added water-immersion restraint stress (WRS) to <strong>the</strong> mice every day <strong>for</strong> 21 days. Fifteen<br />
days after <strong>the</strong> treatments, behavioral tests were per<strong>for</strong>med. BSO or WRS treatment alone made<br />
no signific<strong>an</strong>t difference in all tests, but BSO+WRS treatment induced spatial memory deficits<br />
<strong>an</strong>d lower motivation compared with control mice. Additionally, we found myelin impairment in<br />
<strong>the</strong> hippocampus with BSO+WRS. These results could me<strong>an</strong> cognitive deficits <strong>an</strong>d negative<br />
symptoms of schizophrenia.<br />
Disclosures: M. Toritsuka, None; M. Makinod<strong>an</strong>, None; T. Yamauchi, None; K. Tatsumi,<br />
None; H. Okuda, None; T. Nagashima, None; K. Nakagawa, None; A. W<strong>an</strong>aka, None; T.<br />
Kishimoto, None; S. Kimoto, None.
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.5/V33<br />
Topic: C.15.d. Animal models<br />
Support: NIH Gr<strong>an</strong>t MH39415<br />
Title: Behavioral effects of viral vector overexpression of <strong>the</strong> neurotensin 1 receptor in <strong>the</strong><br />
nucleus accumbens <strong>an</strong>d ventral pallidum of Brown Norway rats<br />
Authors: *L. C. GUILLORY 1 , J. WANG 1 , B. KINKEAD 1 , R. CACEDA 1 , P. D. SHILLING 2 ,<br />
D. FEIFEL 2 , C. B. NEMEROFF 1 ;<br />
1 Dept of Psychiatry & Behavioral Sci., Emory Univ., Atl<strong>an</strong>ta, GA; 2 Psychiatry, UCSD, La Jolla,<br />
CA<br />
Abstract: Neurotensin (NT) is a 13 amino acid neuropeptide that has been shown to mediate <strong>the</strong><br />
behavioral effects of some <strong>an</strong>tipsychotic drugs, <strong>an</strong>d it has been hypo<strong>the</strong>sized that NT may be <strong>an</strong><br />
endogenous neuroleptic. Previously, we have demonstrated that viral vector overexpression of<br />
<strong>the</strong> neurotensin 1 receptor (NT1) in <strong>the</strong> nucleus accumbens (NAcc) of Long Ev<strong>an</strong>s rats prevents<br />
psychostimul<strong>an</strong>t-induced disruption of prepulse inhibition of <strong>the</strong> acoustic startle response (PPI)<br />
<strong>an</strong>d psychostimul<strong>an</strong>t-induced hyperactivity. The current studies were designed to examine<br />
whe<strong>the</strong>r genetic deficits in sensorimotor gating are similarly responsive to alterations in NT<br />
neurotr<strong>an</strong>smission using <strong>the</strong> Brown Norway rat model. The Brown Norway rat is <strong>an</strong> inbred strain<br />
that has been demonstrated to have impaired PPI at <strong>the</strong> 100 ms interstimulus interval (ISI)<br />
compared to o<strong>the</strong>r rat strains <strong>an</strong>d has been used to model <strong>the</strong> sensorimotor gating deficits seen in<br />
schizophrenia.<br />
Male Brown Norway rats received bilateral injections of lentiviral vectors expressing <strong>the</strong> NT1<br />
receptor or GFP in ei<strong>the</strong>r <strong>the</strong> NAcc or ventral pallidum (VP). Two to 8 weeks after surgery, rats<br />
were tested <strong>for</strong> PPI (100 ms ISI), latent inhibition (LI), <strong>an</strong>d locomotor behaviors. NT1<br />
overexpression in <strong>the</strong> NAcc increased baseline PPI <strong>an</strong>d decreased amphetamine-induced<br />
disruption of PPI in Brown Norway rats. In contrast, overexpression of <strong>the</strong> NT1 receptor in <strong>the</strong><br />
VP had no signific<strong>an</strong>t effect on baseline or amphetamine-induced disruption of PPI. In <strong>the</strong> LI<br />
paradigm, overexpression of NT1 receptors in <strong>the</strong> VP, but not NAcc, facilitated <strong>the</strong> LI following<br />
10 preexposures to <strong>the</strong> conditioned stimulus. Nei<strong>the</strong>r NT1 overexpression in <strong>the</strong> NAcc or VP had<br />
a signific<strong>an</strong>t effect on locomotor behavior.<br />
In contrast to Long Ev<strong>an</strong>s rats, baseline PPI in Brown Norway rats is increased by<br />
overexpression of NT1 receptors in <strong>the</strong> NAcc. In addition, endogenous NT in <strong>the</strong> VP, but not <strong>the</strong><br />
NAcc, modulates facilitation of LI. These data support a role of NT neurotr<strong>an</strong>smission in<br />
modulation of baseline PPI <strong>an</strong>d LI in Brown Norway rats <strong>an</strong>d provide evidence that compounds
targeting <strong>the</strong> NT system may be efficacious in normalizing genetic variation in sensorimotor<br />
gating.<br />
Disclosures: L.C. Guillory, NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. W<strong>an</strong>g, NIMH, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received); B. Kinkead, NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); R. Caceda, NIMH, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); P.D. Shilling, NIMH, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); D. Feifel,<br />
NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts<br />
as well as gr<strong>an</strong>ts already received); Argolyn Biosciences, Tenzia, E. Ownership Interest (stock,<br />
stock options, patent or o<strong>the</strong>r intellectual property); (Speaking, consulting or research funding):<br />
Abbot Laboratories, Arogyln Biosciences, Bristol Myers Sqibb, Otuska, Dainapoin-Sumitotomo,<br />
Org<strong>an</strong>on, Eli Lilly, Wyeth, Addrenex, Shire, J<strong>an</strong>seen, O<strong>the</strong>r; Astra Zeneca, Alexza, O<strong>the</strong>r; C.B.<br />
Nemeroff, NIH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Stockholder or Equity: Corcept, Revaax,<br />
NovaDel Pharma, CeNeRx, PharmaNeuroboost, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); Patent: Method <strong>an</strong>d devices <strong>for</strong> tr<strong>an</strong>sdermal delivery of<br />
lithium (US 6,375,990 B1), E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); Patent: Method to estimate serotonin <strong>an</strong>d norepinephrine tr<strong>an</strong>sporter<br />
occup<strong>an</strong>cy after drug treatment using patient or <strong>an</strong>imal serum (provisional filing April, 2001), E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); AFSP,<br />
AstraZeneca, NARSAD, Quintiles, PharmaNeuroboost, F. Consult<strong>an</strong>t/Advisory Board; Board of<br />
Directors: Americ<strong>an</strong> Foundation <strong>for</strong> Suicide Prevention (AFSP), George West Mental Health<br />
Foundation, NovaDel Pharma, Mt. Cook Pharma, Inc, F. Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.6/V34<br />
Topic: C.15.d. Animal models<br />
Title: Compulsive behavior <strong>an</strong>d orexin involvement in a rat model of psychotic polydipsia
Authors: *M. S. MILELLA, F. PASSARELLI, L. DE CAROLIS, C. SCHEPISI, P. NATIVIO,<br />
S. SCACCIANOCE, P. NENCINI;<br />
Sapienza, Univ. Rome, Rome, Italy<br />
Abstract: Despite being one of <strong>the</strong> main causes of medical complications <strong>an</strong>d death in<br />
schizophrenic inpatients, psychotic polydipsia remains a poorly understood phenomenon. We<br />
have shown that repeated treatment with D2/D3 dopaminergic receptor agonist quinpirole (QNP)<br />
in rats induces <strong>an</strong> excessive water-intake measured 5h after injections, <strong>an</strong> effect that may model<br />
hum<strong>an</strong> polydipsia.<br />
The goals of our study were to test <strong>the</strong> sensitivity of QNP polydipsia to <strong>the</strong> dopaminergic<br />
<strong>an</strong>tagonist haloperidol (Hal), both on free <strong>an</strong>d oper<strong>an</strong>t access to water, <strong>an</strong>d investigate whe<strong>the</strong>r<br />
<strong>the</strong> phenomenon may be stress-induced. Given that Modafinil, <strong>an</strong> orexin system activating drug,<br />
ameliorates cognitive per<strong>for</strong>m<strong>an</strong>ce in psychotics, <strong>an</strong>d that mutation in <strong>the</strong> orexin receptor 1 gene<br />
has been recently associated with susceptibility to polydipsia, we also inquired whe<strong>the</strong>r QNP<br />
treated rats would display higher expression of orexin protein in <strong>the</strong> cerebral cortex.<br />
In experiment 1, 72 Sprague-Dawley rats where divided in two groups, one where free waterintake<br />
was assessed daily 4 <strong>an</strong>d 5 hours after treatment in <strong>the</strong> home cage, <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r where<br />
rats, after 4 hours in <strong>the</strong> home cage, were moved to oper<strong>an</strong>t chambers to obtain water through<br />
lever pressing in <strong>the</strong> last hour. Rats were pretreated with Hal 0.1 or 0.2 mg/kg one hour be<strong>for</strong>e<br />
i.p. injection of QNP 0.5 mg/kg, <strong>for</strong> 9 consecutive days. In experiment 2, 25 rats were given<br />
acute or chronic QNP treatment <strong>an</strong>d <strong>the</strong>ir plasma <strong>an</strong>d brain collected <strong>for</strong> corticosterone<br />
determination <strong>an</strong>d immunohystochemical detection of orexin A per<strong>for</strong>med with <strong>the</strong> monoclonal<br />
<strong>an</strong>tibody.<br />
QNP treated rats exhibited <strong>an</strong> increase in water intake that was lowered to basal levels only by<br />
Hal 0.2 mg/kg. Hal alone did not influence water-intake at <strong>an</strong>y dose. A signific<strong>an</strong>t inverse<br />
correlation (r = 0.753) emerged in QNP treated rats between water drunk in <strong>the</strong> first 4h <strong>an</strong>d<br />
percentage of water consumed over water gained through oper<strong>an</strong>t responding in <strong>the</strong> last hour.<br />
This effect was prevented by <strong>the</strong> lower Hal dose. Corticosterone plasma levels were found<br />
depressed both after acute <strong>an</strong>d repeated QNP administration. Instead, orexin A expression in <strong>the</strong><br />
somatosensory cortex was signific<strong>an</strong>tly higher after chronic QNP treatment, <strong>an</strong> effect that was<br />
<strong>an</strong>tagonized by Hal.<br />
Apart <strong>from</strong> being exquisitely sensitive to D2 blockade, QNP induced polydipsia holds a<br />
compulsive feature, evidenced by <strong>the</strong> dissociation between appetitive <strong>an</strong>d consummatory<br />
behaviour in <strong>the</strong> oper<strong>an</strong>t setting. The high orexin level in <strong>the</strong> cortex may be interpreted as <strong>the</strong><br />
functional expression of a reduced cortical control over behavior, a hypo<strong>the</strong>sis that needs to be<br />
fur<strong>the</strong>r assessed.<br />
Disclosures: M.S. Milella, None; F. Passarelli, None; L. De Carolis, None; C. Schepisi,<br />
None; P. Nativio, None; S. Scacci<strong>an</strong>oce, None; P. Nencini, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.7/V35<br />
Topic: C.15.d. Animal models<br />
Support: NARSAD<br />
Title: Inhibition of fatty acid amide hydrolase by URB597 reverses PCP-induced social<br />
withdrawal<br />
Authors: *A. J. SEILLIER, A. GIUFFRIDA;<br />
UTHSCSA, S<strong>an</strong> Antonio, TX<br />
Abstract: Withdrawal <strong>from</strong> repeated PCP administration reduces social interaction, a behavioral<br />
phenotype used to model negative symptoms of schizophrenia in rats. Genetic <strong>an</strong>d experimental<br />
evidence indicate that <strong>the</strong> c<strong>an</strong>nabinoid CB1 receptor may be involved in <strong>the</strong> pathophysiology of<br />
schizophrenia, particularly in relation to negative symptoms.<br />
Previous studies <strong>from</strong> our lab showed that drug-naïve schizophrenic patients have increased CSF<br />
levels of <strong>the</strong> endoc<strong>an</strong>nabinoid <strong>an</strong><strong>an</strong>damide (AEA), which are negatively correlated to negative<br />
symptoms. To assess whe<strong>the</strong>r pharmacological enh<strong>an</strong>cement of endoc<strong>an</strong>nabinoid tone might<br />
reduce social withdrawal, we studied <strong>the</strong> effects of systemic administration of <strong>the</strong> FAAH<br />
inhibitor URB597 (a drug that elevates brain AEA by blocking its metabolism) on social<br />
interaction in rats undergoing withdrawal <strong>from</strong> sub-chronic PCP (5 mg/kg, twice a day <strong>for</strong> 1<br />
week). We also assessed whe<strong>the</strong>r <strong>the</strong> behavioral actions of URB597 were reversed by<br />
pharmacological <strong>an</strong>tagonism at CB1 <strong>an</strong>d TRPV1 receptors, <strong>the</strong> two major targets of AEA.<br />
URB597 reversed PCP-induced social withdrawal in PCP-treated rats, but reduced social<br />
interaction in saline-treated controls. Pretreatment with <strong>the</strong> CB1 <strong>an</strong>tagonist AM251 (1 mg/kg,<br />
i.p;) blocked <strong>the</strong> effect of URB597 in PCP- but in saline-treated rats. By contrast, <strong>the</strong> TRPV1<br />
<strong>an</strong>tagonist capsazepine (10 mg/kg, i.p;) blocked <strong>the</strong> effect of URB597 in saline- but not in PCPtreated<br />
rats. Preliminary data indicate that <strong>the</strong> PCP-induced social withdrawal is accomp<strong>an</strong>ied by<br />
a decrease in AEA levels in <strong>the</strong> amygdala, a brain area involved in social behavior, <strong>an</strong>d that this<br />
deficit is reversed by URB597.<br />
These findings suggest that <strong>the</strong> AEA deficit observed in <strong>the</strong> amygdala may underlie <strong>the</strong> social<br />
impairment exhibited in this <strong>an</strong>imal model of schizophrenia. The mech<strong>an</strong>isms involved in <strong>the</strong><br />
deleterious effects of URB597 in saline-treated controls <strong>an</strong>d <strong>the</strong> possible role of TRPV1 in<br />
schizophrenia are currently under investigation.<br />
Supported by NARSAD (A.G.).<br />
Disclosures: A.J. Seillier, None; A. Giuffrida, None.<br />
Poster
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.8/V36<br />
Topic: C.15.d. Animal models<br />
Support: NIH gr<strong>an</strong>t MH079991<br />
Research award <strong>from</strong> GlaxoSmithKline<br />
CIHR gr<strong>an</strong>t MOP68922<br />
Title: Effects of <strong>an</strong>tiepileptic drugs on neuronal survival <strong>an</strong>d behavior in a developmental <strong>an</strong>imal<br />
model of schizophrenia<br />
Authors: G. PALCHIK 1 , S. K. BHARDWAJ 4 , K. N. GALE 2 , *A. D. KONDRATYEV 3 , L. K.<br />
SRIVASTAVA 4 ;<br />
1 Dept Pediatrics, 2 Depts Pharmacol., 3 Depts Pediat & Pharmacol, Georgetown Univ. Med. Ctr.,<br />
Washington, DC; 4 Dept. of Psychiatry, Douglas Mental Hlth. Univ. Institute, McGill Univ.,<br />
Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Seizures are a signific<strong>an</strong>t risk factor <strong>for</strong> schizophrenia, but it is not known whe<strong>the</strong>r<br />
this is due to <strong>the</strong> seizures or to <strong>the</strong> <strong>an</strong>tiepileptic drugs (AEDs), such as phenobarbital (PB), used<br />
to treat <strong>the</strong>m (Vestergaard et al, Schizophr Res, 73, 343-349, 2005). The possibility that AEDs<br />
may be a contributing factor is suggested by <strong>the</strong> fact that exposure to certain AEDs such as PB,<br />
during a critical period of brain development, triggers enh<strong>an</strong>ced neuronal apoptosis in several<br />
brain regions. To test <strong>the</strong> hypo<strong>the</strong>sis that PB may increase <strong>the</strong> vulnerability to <strong>the</strong> expression of<br />
schizophrenia-like behaviors, we examined <strong>the</strong> effects of PB exposure in a developmental <strong>an</strong>imal<br />
model of schizophrenia, i.e., <strong>the</strong> neonatal ventral hippocampus (nVH) lesioned rat. Sprague-<br />
Dawley rat pups at postnatal day (P) 7 were injected i.p. with ei<strong>the</strong>r saline (1ml/kg) or PB (75<br />
mg/kg) 6 h be<strong>for</strong>e receiving a bilateral intra-VH microinjection of ibotenic acid (3µg/side) or<br />
saline (sham lesion). The 4 groups of <strong>an</strong>imals: saline-sham, saline-lesioned, PB-sham <strong>an</strong>d PBlesioned<br />
were tested as adults (after P50) in a test battery <strong>for</strong> schizophrenia-related behaviors.<br />
Both nVH lesions <strong>an</strong>d neonatal PB treatment alone led to disruption of prepulse inhibition of<br />
acoustic startle at prepulse intensities of 6-12 dB. Startle amplitude was not affected by <strong>an</strong>y<br />
treatment. Amphetamine (1.5 mg/kg)-evoked locomotor activity was signific<strong>an</strong>tly enh<strong>an</strong>ced in<br />
lesioned <strong>an</strong>imals as reported earlier. Neonatal PB exposure did not affect <strong>the</strong> behavior of sham<br />
<strong>an</strong>imals but it signific<strong>an</strong>tly enh<strong>an</strong>ced <strong>the</strong> lesion-induced increase in amphetamine-evoked<br />
locomotion. Novel object recognition memory was impaired only in <strong>the</strong> nVH lesioned <strong>an</strong>imals,<br />
with no signific<strong>an</strong>t interactive effect between lesion <strong>an</strong>d PB exposure. These results in a rodent<br />
model suggest that exposure to PB treatment during a critical period in brain maturation c<strong>an</strong><br />
increase <strong>the</strong> vulnerability to schizophrenia-like behavioral outcomes. Thus, iatrogenic factors
must be considered as contributing to <strong>the</strong> increased risk of schizophrenia associated with a<br />
history of seizures.<br />
Disclosures: G. Palchik, None; S.K. Bhardwaj, None; K.N. Gale, None; A.D. Kondratyev,<br />
Principal Investigator, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); L.K. Srivastava, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.9/V37<br />
Topic: C.15.d. Animal models<br />
Title: The effects of social isolation stress <strong>an</strong>d re-socialization on <strong>the</strong> cerebral expression levels<br />
of early growth response <strong>an</strong>d latent learning in mice<br />
Authors: Y. MURAKAMI, K. ONO, H. OUCHI, M. TOHDA, *K. MATSUMOTO;<br />
Inst. Natural Med. Univ. Toyama, Toyama, Jap<strong>an</strong><br />
Abstract: Social isolation stress (SIS) induces behavioral disturb<strong>an</strong>ces such as aggression,<br />
cognitive impairments, <strong>an</strong>d deficits in prepulse inhibition in mice. Social isolation mice have,<br />
<strong>the</strong>re<strong>for</strong>e, been studied as <strong>an</strong> <strong>an</strong>imal model of neuropsychiatric disorders such as schizophrenia.<br />
Recently, it was reported that <strong>the</strong> expression of Early growth response (EGR) gene is reduced in<br />
<strong>the</strong> postmortem brains of schizophrenia patients. Here, we evaluated <strong>the</strong> effects of social<br />
isolation on <strong>the</strong> expression levels of EGR genes <strong>an</strong>d protein in <strong>the</strong> frontal cortex <strong>an</strong>d cognitive<br />
deficits in mice. We also examined if re-socialized housing of isolated mice affects social<br />
isolation induced ch<strong>an</strong>ges of EGR gene expression <strong>an</strong>d learning behavior in socially isolated<br />
mice.<br />
Male ICR mice (4-weeks old) were housed in groups of five or socially isolated <strong>for</strong> 3-56 days<br />
be<strong>for</strong>e <strong>the</strong> experiments. In some experiments, <strong>the</strong> mouse socially isolated <strong>for</strong> 7 days was placed<br />
in <strong>the</strong> group cage <strong>for</strong> 7 days <strong>an</strong>d used as a re-socialized group. To evaluate <strong>the</strong> abilities of latent<br />
learning, working memory <strong>an</strong>d recognition memory in socially isolated <strong>an</strong>d re-socialized mice,<br />
water finding task, Y-maze test <strong>an</strong>d object recognition test were conducted. After <strong>the</strong> behavioural<br />
studies, <strong>the</strong> brains were dissected <strong>an</strong>d total RNA <strong>an</strong>d protein were extracted <strong>from</strong> <strong>the</strong> prefrontal<br />
cortex. The expression levels of EGR genes <strong>an</strong>d proteins were measured by real-time RT-PCR<br />
<strong>an</strong>d Western blotting, respectively.<br />
Expression of <strong>the</strong> EGR-1, -2, <strong>an</strong>d -4 but not -3 mRNA in <strong>the</strong> frontal cortex was signific<strong>an</strong>tly<br />
down-regulated in mice socially isolated <strong>for</strong> ≥7 days compared with that in <strong>the</strong> group housed
mice. EGR-1 protein levels were elevated by social isolation <strong>for</strong> 56 days. The down regulation of<br />
EGR mRNA expression induced by SIS was not reversed by re-socialization <strong>for</strong> 7 days. SIS<br />
impaired latent learning without affecting working memory <strong>an</strong>d object recognition memory. Resocialization<br />
failed to improve this deficit. These results suggest that (i) <strong>the</strong> tr<strong>an</strong>scriptional <strong>an</strong>d<br />
tr<strong>an</strong>slational mech<strong>an</strong>isms of EGR genes were disrupted in socially isolated mice, (ii)<br />
abnormalities of <strong>the</strong> EGR signaling systems impairs latent learning in socially isolated mice, <strong>an</strong>d<br />
(iii) re-socialization <strong>for</strong> 7 days has little effect on <strong>the</strong>se ch<strong>an</strong>ges. Social isolation mice may<br />
provide a useful epigenetic model <strong>for</strong> studying <strong>the</strong> schizophrenia pathogenesis.<br />
Disclosures: Y. Murakami, None; K. Ono, None; H. Ouchi, None; M. Tohda, None; K.<br />
Matsumoto , None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.10/V38<br />
Topic: C.15.d. Animal models<br />
Support: Wisconsin Alumni Research Foundation<br />
University of Wisconsin Graduate School<br />
Title: Identification of a novel mouse model <strong>for</strong> m<strong>an</strong>ia<br />
Authors: *S. C. GAMMIE, G. LEE, S. A. STEVENSON, M.-A. L. SCOTTI;<br />
Dept Zoology, Univ. Wisconsin, Madison, WI<br />
Abstract: Bipolar disorder is a devastating mental health disorder that affects approximately 1-<br />
2% of <strong>the</strong> population. M<strong>an</strong>ia is a critical component of bipolar disorder that leads to signific<strong>an</strong>t<br />
impairments in social functioning. Bipolar disorder is difficult to treat <strong>an</strong>d one of <strong>the</strong> difficulties<br />
in developing effective treatments has been <strong>the</strong> lack of appropriate <strong>an</strong>imal models <strong>for</strong> different<br />
aspects of bipolar disorder, including m<strong>an</strong>ia. Among <strong>the</strong> phenotypes considered import<strong>an</strong>t <strong>for</strong><br />
modeling of m<strong>an</strong>ia are: increased energy, activity or restlessness; heightened irritability; reduced<br />
sleep; provocative or aggressive behavior; increased sexual drive; elevated hedonistic tendencies;<br />
distractibility, <strong>an</strong>d reduced ability to concentrate. We present here a novel line of mice which<br />
exhibits at least four phenotypes <strong>for</strong> m<strong>an</strong>ia, including hyperactivity, elevated aggression,<br />
increased risk taking, <strong>an</strong>d decreased sleep. The line of mice was originally derived <strong>from</strong> outbred<br />
hsd:ICR mice as part of a study selecting <strong>for</strong> high wheel-running. We identified elevated
maternal <strong>an</strong>d intermale aggression in this one selected line (out of four). We found recently that<br />
relative to a control line with a similar hsd:ICR background history, that our m<strong>an</strong>ia model mice<br />
also exhibit decreased daytime sleep, elevated risk taking (as evidenced by higher time in <strong>the</strong><br />
light portion of <strong>the</strong> light/dark box), increased in cage hyperactivity, <strong>an</strong>d increased time<br />
swimming in <strong>the</strong> <strong>for</strong>ced swim test. In recent pilot tests, we found that valproate, <strong>the</strong> second most<br />
prescribed treatment <strong>for</strong> m<strong>an</strong>ia, signific<strong>an</strong>tly decreased risk taking in <strong>the</strong> m<strong>an</strong>ia male mice when<br />
administered <strong>for</strong> two weeks (200 mg/kg daily i.p.). Valproate also caused a signific<strong>an</strong>t increase<br />
in sleep (p
Abstract: α2 norepinephrine (NE) receptor agonists are used to treat <strong>the</strong> symptoms of patients<br />
with psychiatric disorders such as schizophrenia, Tourette’s syndrome, attention-deficit<br />
hyperactivity disorder <strong>an</strong>d post-traumatic stress disorder. Never<strong>the</strong>less, <strong>the</strong> exact neural<br />
mech<strong>an</strong>ism through which <strong>the</strong>se drugs derive <strong>the</strong>ir <strong>the</strong>rapeutic benefits is unknown. Since<br />
patients with <strong>the</strong>se disorders display deficits in prepulse inhibition (PPI), a model of<br />
sensorimotor gating, PPI may be utilized to identify potential neural substrates through which α2<br />
agonists are acting clinically. One potential site through which α2 NE receptors might regulate<br />
PPI is <strong>the</strong> medial prefrontal cortex (mPFC), as we have previously shown that stimulation of α1<br />
<strong>an</strong>d β NE receptors in this region disrupts PPI, indicating that <strong>the</strong> mPFC is a region through<br />
which increased NE tr<strong>an</strong>smission disrupts PPI. In <strong>the</strong> present study, we examined <strong>the</strong> role of α2<br />
receptors in <strong>the</strong> mPFC in modulating PPI by directly infusing ei<strong>the</strong>r <strong>the</strong> α2 agonist, gu<strong>an</strong>facine<br />
(N=8, 0, 0.01 µg, 0.1 µg, 1 µg/0.5 µL), or <strong>the</strong> α2 <strong>an</strong>tagonist, atipamezole, (N=9, 0, 0.025 µg, 0.25<br />
µg, 2.5 µg/0.5 µL) into <strong>the</strong> mPFC. We found no effect of ei<strong>the</strong>r drug, indicating that α2 receptors<br />
in this site do not modulate PPI. Next, we examined whe<strong>the</strong>r stimulation of α2 receptors would<br />
block deficits in PPI caused by increasing central NE levels. We have previously shown that<br />
pharmacological stimulation of <strong>the</strong> locus coeruleus (LC), <strong>the</strong> primary source of NE to <strong>the</strong><br />
<strong>for</strong>ebrain, with peri-LC infusions of <strong>the</strong> cholinergic agonist beth<strong>an</strong>echol (BET) disrupts PPI.<br />
There<strong>for</strong>e, we now examined whe<strong>the</strong>r clinically effective α2 agonists would prevent deficits in<br />
PPI induced by peri-LC infusion of BET. Rats were pretreated systemically with ei<strong>the</strong>r clonidine<br />
(N=7, 0, 0.01 mg/kg), or gu<strong>an</strong>facine (N=10, 0, 0.1 mg/kg) prior to peri-LC infusions of BET (0,<br />
500 ng/0.2µL). As in previous studies, we found that BET infusions into peri-LC disrupted PPI.<br />
Moreover, systemic injections of gu<strong>an</strong>facine were able to reverse <strong>the</strong> effects of LC stimulation<br />
on PPI, raising <strong>the</strong> possibility that stimulation of α2 receptors ei<strong>the</strong>r in <strong>the</strong> LC or in LC terminal<br />
regions underlies this effect. In a follow-up experiment (N=7), coinfusion of <strong>the</strong> α2 agonist,<br />
clonidine (0, 1.5 µg/0.5µL), directly into <strong>the</strong> peri-LC was able to reverse <strong>the</strong> effects of LC<br />
stimulation of PPI, suggesting that stimulation of α2 receptors in <strong>the</strong> LC may be <strong>the</strong> mech<strong>an</strong>ism<br />
through which systemic α2 agonists block LC-induced deficits in PPI. Taken toge<strong>the</strong>r, <strong>the</strong> results<br />
indicate that stimulating α2 receptors blocks <strong>the</strong> ability of LC stimulation to disrupt PPI, possibly<br />
due to decreasing NE activity through stimulation of presynaptic α2 receptors on <strong>the</strong> LC.<br />
Disclosures: K.M. Alsene, None; V.P. Bakshi, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.12/W2<br />
Topic: C.15.d. Animal models
Support: NIH DA016606<br />
NIH RR000168<br />
NIH DA022323<br />
NIH MH077995<br />
Shervert Frazier Research Institute<br />
McLe<strong>an</strong> Hospital Pope Fellowship<br />
Title: Mouse trace amine associated receptor 1 may mediate clozapine-induced sensory-motor<br />
gating improvement in vivo<br />
Authors: S. K. LYNN 1,2,3 , L. LYNCH 4,2 , *E. A. BUTTNER 1,2 , B. M. COHEN 1,2 , G. M.<br />
MILLER 4,2 ;<br />
1 McLe<strong>an</strong> Hosp., Belmont, MA; 2 Psychiatry, Harvard Med. Sch., Boston, MA; 3 Psychology,<br />
Boston Col., Chestnut Hill, MA; 4 Neurochemistry, New Engl<strong>an</strong>d Primate Res. Ctr.,<br />
Southborough, MA<br />
Abstract: Background. The unique <strong>an</strong>tipsychotic drug clozapine treats both <strong>the</strong> positive <strong>an</strong>d<br />
negative psychotic symptoms associated with schizophrenia with greater efficacy th<strong>an</strong> o<strong>the</strong>r<br />
<strong>an</strong>tipsychotic drugs, including o<strong>the</strong>r atypicals. In addition, clozapine improves <strong>the</strong> cognitive<br />
dysfunction associated with poor social <strong>an</strong>d occupational outcomes <strong>an</strong>d may stabilize mood.<br />
However, side effects limit clozapine's use to treatment-refractory psychosis in spite of its<br />
superior <strong>the</strong>rapeutic value. A challenge in adv<strong>an</strong>cing <strong>the</strong> treatment of schizophrenia is <strong>the</strong>re<strong>for</strong>e<br />
to identify <strong>the</strong> signaling pathways involved in both <strong>the</strong> <strong>the</strong>rapeutic <strong>an</strong>d harmful effects of<br />
clozapine. Rationale. Schizophrenia is associated with abnormal levels of trace amines,<br />
suggesting a role <strong>for</strong> trace amine signaling in this disease. Behavioral genetic studies, by our<br />
group, in <strong>the</strong> nematode Caenorhabditis eleg<strong>an</strong>s have recently implicated trace amine signaling in<br />
<strong>the</strong> biological effects of clozapine. Methods. We conducted behavioral studies in a trace amine<br />
associated receptor 1 (TAAR1) knockout mouse strain to determine whe<strong>the</strong>r this receptor<br />
mediates some of <strong>the</strong> biological effects of clozapine in mammals. We assayed prepulse inhibition<br />
(PPI) of <strong>the</strong> acoustic startle response, a behavior known to be abnormal in schizophrenia.<br />
Knockout <strong>an</strong>d wild-type mice received once-daily IP injection of 4 mg/kg clozapine or vehicle<br />
<strong>for</strong> three days. PPI was evaluated on day 1 prior to injection <strong>an</strong>d on day 3 one hour postinjection.<br />
Results. At n=25 per strain (12 clozapine-treated wild-type, 13 clozapine-treated<br />
knockout) <strong>an</strong> ANOVA (2 strain x 2 treatments x 2 repeated measures of prepulse intensity) of <strong>the</strong><br />
pre- vs. post-injection difference in percent PPI showed a trend to strain by treatment interaction<br />
(p=0.067). Follow-up <strong>an</strong>alysis within each strain showed that knockout mice receiving clozapine<br />
failed to exhibit elevated PPI after three days of treatment compared to vehicle-treated controls<br />
(p>0.6) whereas clozapine-treated wild-type mice exhibited signific<strong>an</strong>tly enh<strong>an</strong>ced PPI<br />
compared to vehicle-treated controls (p=0.004). Conclusion. Weak inhibition of <strong>the</strong> startle<br />
response by a prepulse implies that <strong>the</strong> functions recruited to process <strong>the</strong> prepulse are easily<br />
interrupted. Patients with schizophrenia exhibit weak PPI <strong>an</strong>d show signific<strong>an</strong>t association of
psychotic symptoms with PPI impairment. PPI improves in step with o<strong>the</strong>r symptoms in patients<br />
on <strong>an</strong>tipsychotic treatment. Our results thus implicate TAAR1 in <strong>the</strong> <strong>the</strong>rapeutic effects of<br />
clozapine.<br />
Disclosures: S.K. Lynn, None; L. Lynch, None; E.A. Buttner, None; B.M. Cohen,<br />
None; G.M. Miller, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.13/W3<br />
Topic: C.15.d. Animal models<br />
Support: Top Institute Pharma (Ne<strong>the</strong>rl<strong>an</strong>ds), gr<strong>an</strong>t T5-209-1<br />
Title: Neurobehavioural correlates of schizophrenia in <strong>the</strong> apomorphine susceptible (APO-SUS)<br />
rat<br />
Authors: *I. E. DE JONG 1 , K. F. HERRIK 1 , L. LERDRUP 1 , J. P. REDROBE 1 , G. J.<br />
MARTENS 2 , M. DIDRIKSEN 1 , P. KALLUNKI 1 ;<br />
1 H. Lundbeck A/S, Valby, Denmark; 2 Dept. of Mol. Animal Physiology, Fac. of Sci., Donders<br />
Ctr. <strong>for</strong> Neuroscience, Nijmegen Ctr. <strong>for</strong> Mol. Life Sci. (NCMLS), Radboud Univ., Nijmegen,<br />
Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: The apomorphine susceptible (APO-SUS) rat line has been pharmacogenetically<br />
selected <strong>an</strong>d bred <strong>from</strong> <strong>an</strong> outbred Wistar population based on high susceptibility to <strong>the</strong><br />
dopamine receptor agonist apomorphine (as measured by gnawing behaviour). Interestingly,<br />
APO-SUS rats display several phenotypic similarities in behavioural paradigms associated with<br />
schizophrenia. In <strong>the</strong> present studies, APO-SUS rats were fur<strong>the</strong>r characterised in assays<br />
correlated with positive <strong>an</strong>d cognitive symptomatology of schizophrenia. <strong>When</strong> compared to<br />
apomorphine unsusceptible (low-gnawing) rats, APO-SUS rats were hyper-responsive to <strong>the</strong><br />
locomotor stimul<strong>an</strong>t effects of <strong>the</strong> indirect dopamine receptor agonist amphetamine <strong>an</strong>d <strong>the</strong><br />
NMDA receptor <strong>an</strong>tagonist phencyclidine. Whereas <strong>the</strong>re were no differences in <strong>the</strong> firing<br />
characteristics of dopaminergic neurons in <strong>the</strong> ventral tegmental area, APO-SUS rats exhibited<br />
higher firing rates <strong>an</strong>d more irregular firing patterns in <strong>the</strong> subst<strong>an</strong>tia nigra pars compacta, when<br />
compared to low-gnawing controls. With respect to cognition, <strong>the</strong>re were no group differences in<br />
executive function, as measured in <strong>the</strong> attentional set shifting task, or long-term memory in <strong>the</strong><br />
fear conditioning paradigm. These results suggest that APO-SUS rats display phenotypic
characteristics correlated with positive, but not cognitive symptomatology related to <strong>the</strong><br />
attentional set shifting <strong>an</strong>d fear conditioning paradigms. Fur<strong>the</strong>r studies will address <strong>the</strong><br />
sensitivity to <strong>the</strong> disruptive effects of psychostimul<strong>an</strong>ts on cognition, as well as per<strong>for</strong>m<strong>an</strong>ce of<br />
APO-SUS rats in negative symptom assays.<br />
Disclosures: I.E. de Jong, H. Lundbeck A/S, Valby, Denmark, A. Employment (full or parttime);<br />
K.F. Herrik, H. Lundbeck A/S, Valby, Denmark, A. Employment (full or part-time); L.<br />
Lerdrup, H. Lundbeck A/S, Valby, Denmark, A. Employment (full or part-time); J.P. Redrobe,<br />
H. Lundbeck A/S, Valby, Denmark, A. Employment (full or part-time); G.J. Martens, None; M.<br />
Didriksen, H. Lundbeck A/S, Valby, Denmark, A. Employment (full or part-time); P. Kallunki,<br />
H. Lundbeck A/S, Valby, Denmark, A. Employment (full or part-time).<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.14/W4<br />
Topic: C.15.d. Animal models<br />
Support: NIMH MH-39415<br />
Title: Behavioral effects of neurotensin 1 receptor over-expression in ventral tegmental area,<br />
prefrontal cortex <strong>an</strong>d nucleus accumbens of Long Ev<strong>an</strong>s rats<br />
Authors: *J. WANG, R. CACEDA, B. KINKEAD, C. NEMEROFF;<br />
Psychiatry, Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: The mesolimbic dopamine pathway arises in <strong>the</strong> ventral tegmental area of <strong>the</strong><br />
midbrain <strong>an</strong>d projects to <strong>the</strong> nucleus accumbens, <strong>the</strong> amygdala, <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong> medial<br />
prefrontal cortex. Neurotensin (NT) has a close <strong>an</strong>atomical <strong>an</strong>d functional relationship with<br />
dopamine in this system. Several lines of evidence implicate NT in <strong>the</strong> pathogenesis of<br />
schizophrenia <strong>an</strong>d NT has been hypo<strong>the</strong>sized to function as <strong>an</strong> endogenous <strong>an</strong>tipsychotic.<br />
Previously we demonstrated that similar to <strong>an</strong>tipsychotic drugs (APDs), viral vector overexpression<br />
of <strong>the</strong> NT1 receptor (NT1) in <strong>the</strong> nucleus accumbens (NAcc) reduced <strong>the</strong> locomotor<br />
<strong>an</strong>d sensorimotor gating effects of <strong>the</strong> psychostimul<strong>an</strong>ts amphetamine <strong>an</strong>d dizocilpine. The<br />
current study was designed to examine <strong>the</strong> brain region specificity of <strong>the</strong>se behavioral effects.<br />
GFP or NT1 lentiviral vectors were bilaterally injected into <strong>the</strong> ventral tegmental area (VTA) or<br />
prefrontal cortex (PFC) of adult male Long Ev<strong>an</strong>s rats. The effects of amphetamine (2.0 mg/kg)<br />
or dizocilpine (0.1 mg/kg) on locomotor activity <strong>an</strong>d prepulse inhibition (PPI) of <strong>the</strong> acoustic
startle response were examined between 2 <strong>an</strong>d 8 weeks after viral vector injection. Similar to<br />
results in <strong>the</strong> NAcc, over-expression of NT1 in <strong>the</strong> PFC or VTA had no effect on baseline PPI,<br />
but reduced amphetamine-induced disruption of PPI. In contrast to <strong>the</strong> NAcc, over-expression of<br />
NT1 in <strong>the</strong> PFC or VTA did not effect dizocilpine-induced disruption of PPI. Over-expression of<br />
NT1 receptors in VTA <strong>an</strong>d PFC had no effect on baseline locomotor activity or amphetamineinduced<br />
hyperactivity. These data demonstrate that endogenous NT does not play a role in<br />
modulation of baseline locomotor activity or stimul<strong>an</strong>t-induced hyperlocomotion. The behavioral<br />
effects of NT1 receptor overexpression in <strong>the</strong> VTA <strong>an</strong>d PFC, may be due to receptor<br />
overexpression on non-dopaminergic neurons or glia. The ability of NT1 receptor overexpression<br />
in <strong>the</strong> NAcc, but not VTA or PFC, to block dizocilpine-induced disruption of PPI indicate that<br />
endogenous NT specifically in <strong>the</strong> NAcc, modulates <strong>the</strong> PPI disrupting effects of this NMDA<br />
receptor <strong>an</strong>tagonist.<br />
Disclosures: J. W<strong>an</strong>g, Emory University, A. Employment (full or part-time); NIMH, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); R. Caceda, None; B. Kinkead, Emory University, A. Employment<br />
(full or part-time); NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t<br />
<strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); C. Nemeroff, Emory University, A.<br />
Employment (full or part-time); NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator<br />
or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); AFSP; AstraZeneca;<br />
NARSAD; Quintiles; PharmaNeuroboost, D. Speakers Bureau/Honoraria (speakers bureau,<br />
symposia, <strong>an</strong>d expert witness); Corcept; Revaax; NovaDel Pharma; CeNeRx,<br />
PharmaNeuroboost, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); Americ<strong>an</strong> Foundation <strong>for</strong> Suicide Prevention (AFSP); George West Mental Health<br />
Foundation; NovaDel Pharma, Mt. Cook Pharma, Inc, F. Consult<strong>an</strong>t/Advisory Board; Method<br />
<strong>an</strong>d devices <strong>for</strong> tr<strong>an</strong>sdermal delivery of lithium (US 6,375,990 B1), O<strong>the</strong>r.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.15/W5<br />
Topic: C.15.d. Animal models<br />
Support: NARSAD Young Investigator Award<br />
Title: Behavioral effects of increased endogenous kynurenic acid concentration: Implications <strong>for</strong><br />
cognitive dysfunction in schizophrenia
Authors: L. GUO 1 , G. C. EVANS 2 , *D. J. BUCCI 2 ;<br />
1 2<br />
Program in Exptl. <strong>an</strong>d Mol. Med., Dartmouth Med. Sch., H<strong>an</strong>over, NH; Dartmouth Col.,<br />
H<strong>an</strong>over, NH<br />
Abstract: Concentrations of kynurenic acid (KYNA), a metabolite of tryptoph<strong>an</strong>, are chronically<br />
elevated in <strong>the</strong> brains of persons with schizophrenia. KYNA is syn<strong>the</strong>sized <strong>an</strong>d released solely<br />
by astrocytes <strong>an</strong>d acts as a potent <strong>an</strong>tagonist of alpha-7 nicotinic acetylcholine receptors <strong>an</strong>d also<br />
<strong>the</strong> glycine site of NMDA glutamate receptors. Both of <strong>the</strong>se receptors have well-established<br />
roles in cognitive function, such as learning, attention, <strong>an</strong>d memory, suggesting that high levels<br />
of KYNA may contribute to cognitive <strong>an</strong>d behavioral deficits typically associated with<br />
schizophrenia. Our previous studies support this idea <strong>an</strong>d indicate that spatial <strong>an</strong>d contextual<br />
learning are impaired in rats when endogenous KYNA levels are increased to concentrations<br />
observed in <strong>the</strong> brains of persons with schizophrenia (Chess et al, 2007; <strong>2009</strong>). In contrast,<br />
learning about a specific cue (i.e., fear conditioning to a tone) was not impacted by KYNA in<br />
those studies. In <strong>the</strong> present series of studies, we exp<strong>an</strong>ded this research to examine <strong>the</strong> effects of<br />
KYNA on additional <strong>for</strong>ms of cue-specific learning. To elevate levels of KYNA, we<br />
administered its precursor L-kynurenine (L-KYN; 100mg/kg), which is converted into KYNA by<br />
astrocytes <strong>an</strong>d results in physiologically-relev<strong>an</strong>t increases in KYNA. Control <strong>an</strong>d L-KYNtreated<br />
rats were tested in a novel object recognition task to test <strong>the</strong> hypo<strong>the</strong>sis that KYNA might<br />
produce specific impairments in contextual <strong>an</strong>d configural learning systems but not learning<br />
about specific cues. Consistent with this hypo<strong>the</strong>sis, increased KYNA concentration did not alter<br />
novel object recognition. A second study examined <strong>the</strong> effect of increased KYNA concentration<br />
on social behavior. Indeed, persons with schizophrenia often exhibit abnormal social behavior<br />
(lack of motivation, social withdraw), leading to <strong>the</strong> hypo<strong>the</strong>sis that rats with increased levels of<br />
KYNA would interact less with <strong>an</strong> unfamiliar rat in a social interaction task compared with<br />
saline-treated controls. Elevation of KYNA concentration did not have a signific<strong>an</strong>t effect on<br />
social interaction, a result that may fur<strong>the</strong>r support <strong>the</strong> notion that KYNA does not affect<br />
learning about discrete stimuli. Moreover, <strong>the</strong> lack of effects on KYNA on some learning<br />
memory tasks known to depend on NMDA receptor activation (fear learning, social interaction)<br />
suggests that <strong>the</strong> behavioral effects of KYNA may be mediated primarily by its effects on alpha-<br />
7 nicotinic acetylcholine receptors.<br />
Disclosures: L. Guo, None; G.C. Ev<strong>an</strong>s, None; D.J. Bucci, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.16/W6
Topic: C.15.d. Animal models<br />
Support: NIH Gr<strong>an</strong>t 5R01MH074672-02<br />
Title: Behavioral <strong>an</strong>d molecular evidence <strong>for</strong> benefits of continuous impl<strong>an</strong>t risperidone in mice<br />
Authors: *R. LIN 1 , Y. LIANG 2 , R. RAY 2 , C.-G. HAHN 2 , S. J. SIEGEL 2 ;<br />
1 Pharmacol., 2 Psychiatry, Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Background: Nonadherence is a major problem in schizophrenia. Numerous studies<br />
have shown benefits in patient outcome <strong>for</strong> those that maintain proper medication. Improvements<br />
in adherence have been made with <strong>the</strong> advent of depot injections. However, we believe that <strong>the</strong><br />
benefits seen in depots are not only due to improved adherence, but to differences in receptor<br />
regulation following continuous infusion as compared to intermittent oral dosing.<br />
Hypo<strong>the</strong>sis: We hypo<strong>the</strong>size that continuous <strong>an</strong>tipsychotic treatment with risperidone PLGA<br />
impl<strong>an</strong>ts will have similar added benefits over intermittent <strong>an</strong>tipsychotic treatment with oral<br />
risperidone. We provide preliminary evidence to support our claim using behavioral <strong>an</strong>imal<br />
models <strong>an</strong>d molecular measures of receptor expression.<br />
Results: In mice treated with risperidone impl<strong>an</strong>ts, we saw reversal of amphetamine-induced<br />
locomotor activity back to control (saline) conditions. In contrast, mice treated with oral<br />
risperidone initially had reduced locomotor activity followed 4 hrs later by higher locomotor<br />
activity compared to control, saline-treated mice. Additionally, we examined mRNA levels <strong>for</strong><br />
D1 <strong>an</strong>d D2 receptor tr<strong>an</strong>scripts <strong>an</strong>d found fluctuations in mRNA levels <strong>for</strong> both receptors in <strong>the</strong><br />
oral group but not in <strong>the</strong> impl<strong>an</strong>t group.<br />
Conclusions: This data supports our hypo<strong>the</strong>sis that continuous risperidone treatment has distinct<br />
behavioral <strong>an</strong>d molecular effects in comparison to intermittent oral treatment. These differences<br />
may contribute to <strong>the</strong> observed benefits of depot injections relative to comparable oral dosing.<br />
Disclosures: R. Lin, None; Y. Li<strong>an</strong>g, None; R. Ray, None; C. Hahn, None; S.J. Siegel, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.17/W7<br />
Topic: C.15.d. Animal models<br />
Support: NIH R01-MH57683 (PO'D)
NIH R01-DA015718 (GS)<br />
NIH K01-DA021609 (MRR)<br />
HMMI training gr<strong>an</strong>t 52003756<br />
LY379268 kindly provided by Eli Lilly & Co<br />
Title: Systemically administered mGluR 2/3 agonist attenuates impairments of value-based<br />
decision making in <strong>the</strong> neonatal hippocampal lesion model of schizophrenia<br />
Authors: *A. J. GRUBER 1 , M. R. ROESCH 1 , I. SHUSTERMAN 3 , G. SCHOENBAUM 2 , P.<br />
O'DONNELL 2 ;<br />
1 Dept Anat & Neurobiol, 2 Dept Anat & Neurobiol <strong>an</strong>d Psychiatry, Univ. Maryl<strong>an</strong>d, Baltimore,<br />
MD; 3 Univ. Maryl<strong>an</strong>d Baltimore Co, Catonsville, MD<br />
Abstract: Adult rats with neonatal ventral hippocampal lesions (NVHL) exhibit several<br />
behavioral, neurochemical <strong>an</strong>d electrophysiological <strong>an</strong>omalies resembling phenomena observed<br />
in schizophrenia. We have previously shown that NVHL rats demonstrate poor per<strong>for</strong>m<strong>an</strong>ce on a<br />
binary choice task <strong>for</strong> unequal amounts of reward. Compared to control rats, NVHL rats made<br />
more response errors when required to respond <strong>for</strong> small amounts of reward, but also made more<br />
responses <strong>for</strong> small rewards when permitted free choice of ei<strong>the</strong>r response <strong>for</strong> rein<strong>for</strong>cement.<br />
Electrophysiological recordings <strong>from</strong> medial prefrontal cortex (mPFC) revealed excessive<br />
activity in NVHL <strong>an</strong>imals, as more units were found to be task modulated <strong>an</strong>d <strong>the</strong>se had higher<br />
firing rates th<strong>an</strong> in controls. We hypo<strong>the</strong>size that such increased mPFC activity may result <strong>from</strong> a<br />
state of insufficient inhibition, probably as a consequence of impaired interneuron modulation in<br />
NVHL rats <strong>an</strong>d likely to model prefrontal dysfunction produced by loss of interneuron function<br />
in schizophrenia. Thus, we sought to test whe<strong>the</strong>r reducing cortical pyramidal cell firing would<br />
improve task per<strong>for</strong>m<strong>an</strong>ce in NVHL rats. We tested task per<strong>for</strong>m<strong>an</strong>ce of NVHL rats 30 minutes<br />
after systemic administration of a group II metabotropic glutamate receptor agonist (LY379268;<br />
1 mg/kg, ip), which reduces glutamate release by acting on presynaptic receptors. Compared to<br />
vehicle alone, drug administration had no signific<strong>an</strong>t effect on reaction times, but did reduce<br />
errors when rats were required to respond <strong>for</strong> small rewards (17.6±8.9% vs. 11.6±9.2%; p=0.02;<br />
n=4) <strong>an</strong>d also reduced responding <strong>for</strong> small rewards on free choice trials (49.9±6.6% vs.<br />
35.2±6.3%; p=0.01; n=4). Thus, attenuating <strong>the</strong> consequences of excessive mPFC firing with a<br />
mGluR2/3 agonist improved decision making in NVHL rats without affecting motivation as<br />
assessed by reaction times, adding fur<strong>the</strong>r support <strong>for</strong> a <strong>the</strong>rapeutic benefit <strong>for</strong> this class of<br />
compound in <strong>the</strong> treatment of schizophrenia <strong>an</strong>d o<strong>the</strong>r disorders associated with <strong>an</strong> imbal<strong>an</strong>ce of<br />
cortical excitation <strong>an</strong>d inhibition.<br />
Disclosures: A.J. Gruber, None; M.R. Roesch, None; I. Shusterm<strong>an</strong>, None; G. Schoenbaum,<br />
None; P. O'Donnell, None.<br />
Poster
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.18/W8<br />
Topic: C.15.d. Animal models<br />
Title: Behavioral <strong>an</strong>d neurochemical characterization of GPR88 knock-out mice: A novel target<br />
<strong>for</strong> schizophrenia<br />
Authors: *A. RASSOULPOUR, J. DANAO, O. MURPHY, M. CUEVA, J. ABLE, M.<br />
GIORGETTI, J. TREANOR;<br />
Amgen, South S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Schizophrenia is a complex, chronic debilitating disease that is characterized by<br />
positive <strong>an</strong>d negative symptoms, as well as deficits in cognition. Currently marketed medications<br />
have limitations in <strong>the</strong> domains of <strong>the</strong> disease that <strong>the</strong>y treat, <strong>the</strong>ir side effects profile, <strong>an</strong>d a large<br />
number of non-responsive patients. Thus, <strong>the</strong>re is a large unmet medical need <strong>for</strong> <strong>the</strong> treatment<br />
of schizophrenia <strong>an</strong>d to date very few targets have proven successful as novel <strong>the</strong>rapeutic targets.<br />
The orph<strong>an</strong> GPCR GPR88 has a high level of expression in <strong>the</strong> striatum, a region believed to be<br />
import<strong>an</strong>t in schizophrenia pathophysiology. In <strong>an</strong> attempt to evaluate GPR88 as a novel<br />
schizophrenia target we set out to characterize GPR88 KO <strong>an</strong>imals in <strong>an</strong>imal behavior <strong>an</strong>d<br />
neurochemistry studies. As previously reported (Pausch et al, 2006) we found GPR88 KO<br />
<strong>an</strong>imals to have a disruption in pre-pulse inhibition, a preclinical correlate of sensorimotor gating<br />
deficits. In addition super-sensitivity to psychostimul<strong>an</strong>ts was observed in <strong>the</strong> GPR88 KO<br />
<strong>an</strong>imals, such that locomotor activity was greater in KO <strong>an</strong>imals compared to wild-type<br />
littermate controls when <strong>an</strong>imals were administered ei<strong>the</strong>r amphetamine (2.5 mg/kg) or ketamine<br />
(20 mg/kg). The effects of amphetamine were attenuated in KO <strong>an</strong>imals pre-treated with <strong>the</strong><br />
typical <strong>an</strong>tipsychotic haloperidol (0.25 mg/kg) <strong>an</strong>d <strong>the</strong> atypical <strong>an</strong>tipsychotic risperidone (0.1<br />
mg/kg), <strong>an</strong>d a modest reduction was observed in separate <strong>an</strong>imals pre-treated with lithium (1<br />
mg/kg). In addition, microdialysis experiments demonstrated similar levels of striatal DA release<br />
in WT <strong>an</strong>d KO <strong>an</strong>imals following administration of amphetamine or ketamine. GPR88 KO<br />
<strong>an</strong>imals also displayed a deficit in motor coordination, as revealed by a reduced latency to fall in<br />
<strong>the</strong> rotorod test. Finally we examined <strong>the</strong> levels of dopamine (DA), serotonin (5HT), glutamate<br />
(Glu) <strong>an</strong>d glycine (Gly) in <strong>the</strong> pre-frontal cortex, striatum, <strong>an</strong>d hippocampus of WT <strong>an</strong>d KO<br />
<strong>an</strong>imals. No differences in DA, 5HT, or Glu were observed between tissues <strong>from</strong> WT <strong>an</strong>d KO<br />
<strong>an</strong>imals in <strong>an</strong>y of <strong>the</strong> regions tested. However, KO <strong>an</strong>imals displayed a signific<strong>an</strong>t increase in <strong>the</strong><br />
levels of hippocampal Gly. We are currently exploring <strong>the</strong> possibility that this increased Gly may<br />
play a role in <strong>the</strong> cognitive per<strong>for</strong>m<strong>an</strong>ce of GPR88 KO <strong>an</strong>imals. Toge<strong>the</strong>r, our data provide<br />
fur<strong>the</strong>r support <strong>for</strong> <strong>the</strong> role of GPR88 as a novel target in <strong>the</strong> treatment of schizophrenia, <strong>an</strong>d<br />
demonstrate <strong>the</strong> utility of <strong>the</strong>se <strong>an</strong>imals in testing <strong>an</strong>tipsychotic efficacy.
Disclosures: A. Rassoulpour, None; J. D<strong>an</strong>ao, None; O. Murphy, None; M. Cueva, None; J.<br />
Able, None; M. Giorgetti, None; J. Tre<strong>an</strong>or, None.<br />
Poster<br />
546. Schizophrenia <strong>an</strong>d Bi-polar Disorder: Animal Models II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 546.19/W9<br />
Topic: C.15.d. Animal models<br />
Support: DFG, Schm 1710<br />
NSERC<br />
Title: Nicotine receptor subtypes differentially effect prepulse inhibition of startle<br />
Authors: T. K. BROWN 1 , F. S. PINNOCK 1 , L. WALKER 1 , D. BOSCH 2,3 , J. S. YEOMANS 2 ,<br />
*S. SCHMID 1,2,3 ;<br />
1 Univ. Western Ontario, London, ON, C<strong>an</strong>ada; 2 Psychology, Univ. of Toronto, Toronto, ON,<br />
C<strong>an</strong>ada; 3 Univ. of Tuebingen, Tuebingen, Germ<strong>an</strong>y<br />
Abstract: There is evidence <strong>from</strong> patients suffering <strong>from</strong> mental disorders associated with a<br />
disruption of prepulse inhibition (PPI) that nicotine consumption partly restores this deficit.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> α7-nicotinic receptor has been identified as a susceptibility gene <strong>for</strong><br />
schizophrenia. There<strong>for</strong>e, we hypo<strong>the</strong>size that nicotine receptors have <strong>an</strong> import<strong>an</strong>t function in<br />
mediating or modulating PPI.<br />
We tested <strong>the</strong> role of different nicotine receptor subtypes in mediating PPI in rats by three<br />
approaches: we injected <strong>the</strong> α7 specific <strong>an</strong>tagonist MLA or <strong>the</strong> non α7 <strong>an</strong>tagonist TMPH<br />
systemically (i.p., 5 mg/kg), <strong>an</strong>d stereotaxically into <strong>the</strong> caudal pontine reticular <strong>for</strong>mation (PnC)<br />
through chronically impl<strong>an</strong>ted c<strong>an</strong>nulae 30 min. prior to testing <strong>for</strong> PPI. After habituation, rats<br />
were exposed to startle stimuli alone (baseline startle), or with 2 ms white noise prepulses (85<br />
db) at different ISI (6-250 ms) in a pseudo-r<strong>an</strong>domized order. We also per<strong>for</strong>med patch-clamp<br />
recordings <strong>from</strong> startle mediating neurons in brainstem slices <strong>an</strong>d perfused nicotine as well as<br />
MLA while monitoring synaptic tr<strong>an</strong>smission, membr<strong>an</strong>e resist<strong>an</strong>ce <strong>an</strong>d resting membr<strong>an</strong>e<br />
potential.<br />
Systemic injections of 5 mg/kg MLA or TMPH both enh<strong>an</strong>ced PPI at <strong>an</strong> ISI of 100 ms (<strong>an</strong>d at<br />
250 ms by MLA) <strong>from</strong> 58% <strong>an</strong>d 61% of baseline startle after saline injection to73% after both,<br />
MLA <strong>an</strong>d TMPH injections (p
<strong>an</strong>tagonist MLA (0.1 mM; 0.5µL), signific<strong>an</strong>tly inhibited PPI by 13.0% (±4.3%) compared to<br />
saline injections (p=0.014), <strong>an</strong>d this effect was exclusively seen at <strong>the</strong> interstimulus interval of<br />
12 ms. There were no effects on PPI with longer ISI. Local injections of TMPH (0.1mM; 0.5 µL)<br />
had no signific<strong>an</strong>t effect on PPI at <strong>an</strong>y ISI. Electrophysiological recordings revealed that nicotine<br />
reduces synaptic currents <strong>an</strong>d membr<strong>an</strong>e resist<strong>an</strong>ce in PnC neurons while <strong>the</strong> resting membr<strong>an</strong>e<br />
potential becomes more positive. All effects were reversed by MLA <strong>an</strong>d persisted when<br />
cadmium (2mM) was perfused in order to block synaptic tr<strong>an</strong>smission.<br />
The results of <strong>the</strong> electrophysiology <strong>an</strong>d local injections indicate that α7-nicotinic receptors are<br />
expressed in <strong>the</strong> PnC <strong>an</strong>d <strong>the</strong>ir activation is import<strong>an</strong>t <strong>for</strong> PPI at short interstimulus intervals.<br />
This is in accord<strong>an</strong>ce with <strong>the</strong>ir fast activation <strong>an</strong>d inactivation kinetics. In contrast, systemic<br />
administration of nicotine <strong>an</strong>tagonists seems to target both α7 <strong>an</strong>d non-α7-nicotinic receptors.<br />
They are presumably located in higher brain regions known to modulate PPI, such as <strong>the</strong><br />
prefrontal cortex, hippocampus, or striatum. This is in accord<strong>an</strong>ce to <strong>the</strong> effects at longer ISI.<br />
Disclosures: T.K. Brown, None; F.S. Pinnock, None; L. Walker, None; D. Bosch, None; J.S.<br />
Yeom<strong>an</strong>s, None; S. Schmid, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.1/W10<br />
Topic: C.12. Neuro-Oncology<br />
Title: Common mech<strong>an</strong>ism of action shown by metallic toxic compounds <strong>an</strong>d metallic <strong>an</strong>tic<strong>an</strong>cer<br />
drugs: Calcium signaling<br />
Authors: *D. BUSSELBERG 1 , A.-M. FLOREA 2 ;<br />
1 2<br />
Paul L. Foster SOM, Texas Tech. Univ, HSC, EL Paso, TX; Neuropathology, Univ. Clin.,<br />
Dusseldorf, Germ<strong>an</strong>y<br />
Abstract: Exposure to metals or metal compounds is inevitable in our daily life. While specific<br />
metals are essential <strong>for</strong> cellular function (e.g. Se, Zn, Fe), o<strong>the</strong>r metals are (neuro-)toxic <strong>an</strong>d/or<br />
carcinogens (e.g. Sb, Hg, As). Metals c<strong>an</strong> trigger cell death by necrosis or apoptosis. Regulation<br />
of <strong>the</strong> apoptotic process is critical <strong>for</strong> toxicity, c<strong>an</strong>cer development, as well as, <strong>for</strong> c<strong>an</strong>cer<br />
treatment. Inhibition of apoptosis could lead to uncontrolled cell growth <strong>an</strong>d tumor <strong>for</strong>mation<br />
while <strong>the</strong> induction of apoptosis by drugs might be crucial <strong>for</strong> c<strong>an</strong>cer treatment. Ch<strong>an</strong>ges in<br />
[Ca 2+ ]i are import<strong>an</strong>t <strong>for</strong> induction of cell death. Metal compounds alter [Ca 2+ ]i signals <strong>the</strong>reby<br />
inducing cell death. In this study clinical <strong>an</strong>d environmental relev<strong>an</strong>t metallic compounds
influence calcium homeostasis. The metal compounds cisplatin (CDDP), arsenic trioxide (As2O3)<br />
<strong>an</strong>d trimethyltin chloride (TMT) were applied to tumor (SY5Y, HelaS3) <strong>an</strong>d non tumor cells<br />
(HEK293). We show that <strong>the</strong>se metallic compounds trigger a concentration dependent [Ca 2+ ]i<br />
increase to steady state elevated level. The source of <strong>the</strong> [Ca 2+ ]i responsible <strong>for</strong> <strong>the</strong> elevation is<br />
however different depending on <strong>the</strong> metallic compounds involved. As2O3 <strong>an</strong>d TMT released<br />
calcium <strong>from</strong> <strong>the</strong> intracellular calcium stores, while CDDP triggered a calcium influx <strong>from</strong> <strong>the</strong><br />
extracellular matrix. The rise of [Ca 2+ ]i was also directly correlated with induction of apoptosis.<br />
Interestingly, combination calcium release <strong>from</strong> <strong>the</strong> stores <strong>an</strong>d influx of calcium to <strong>the</strong> cell<br />
triggered by different metals resulted in synergistic effects, which were also resulted in a<br />
stronger induction of apoptosis. Results vary depending on <strong>the</strong> order of application of <strong>the</strong>se<br />
metal compounds. These findings could represent a possible strategy that to improve <strong>an</strong>tic<strong>an</strong>cer<br />
<strong>the</strong>rapy.<br />
Disclosures: D. Busselberg, None; A. Florea, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.2/W11<br />
Topic: C.12. Neuro-Oncology<br />
Support: NCI 1-UO1-CA84314<br />
Title: Neurofibromatosis-1 heterozygosity results in increased cAMP-dependent retinal g<strong>an</strong>glion<br />
cell apoptosis in vitro <strong>an</strong>d in vivo <strong>an</strong>d reduced neuronal survival<br />
Authors: *J. A. BROWN 1 , S. M. GIANINO 2 , D. H. GUTMANN 2 ;<br />
1 St Louis, MO; 2 Neurol., Washington Univ. Sch. of Med., St Louis, MO<br />
Abstract: Neurofibromatosis type 1 (NF1) is one of <strong>the</strong> most common autosomal domin<strong>an</strong>t<br />
inherited c<strong>an</strong>cer syndromes affecting <strong>the</strong> nervous system: 15-20% of children with NF1 develop<br />
low-grade glial cell neoplasms affecting optic nerve/chiasm (optic glioma). Almost 50% of<br />
children with NF1-associated optic glioma exhibit visual loss at initial presentation, <strong>an</strong>d few<br />
regain vision following treatment. To determine whe<strong>the</strong>r <strong>the</strong>se early <strong>an</strong>d seemingly irreversible<br />
visual deficits reflect <strong>an</strong> underlying vulnerability of NF1+/- retinal g<strong>an</strong>glion cells (RGCs), as<br />
seen in patients with NF1, we examined several aspects of neuronal function in Nf1+/- mouse<br />
RGCs. Surprisingly, we found that Nf1 heterozygosity in RGCs is associated with signific<strong>an</strong>tly<br />
reduced growth cone areas <strong>an</strong>d neurite lengths as well as increased cell death. We fur<strong>the</strong>r show
that <strong>the</strong>se Nf1+/- RGC phenotypes reflect reduced cAMP levels <strong>an</strong>d are completely reversed by<br />
treatment with agents (<strong>for</strong>skolin or rolipram) that restore normal cAMP levels in vitro. Finally,<br />
we demonstrate that rolipram treatment nearly completely eliminates <strong>the</strong> RGC apoptosis<br />
associated with tumor <strong>for</strong>mation in Nf1 genetically-engineered mice (GEM) in vivo.<br />
Disclosures: J.A. Brown , None; S.M. Gi<strong>an</strong>ino, None; D.H. Gutm<strong>an</strong>n, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.3/W12<br />
Topic: C.12. Neuro-Oncology<br />
Support: Cali<strong>for</strong>nia Institute <strong>for</strong> Regenerative Medicine (CIRM) Gr<strong>an</strong>t 445169-18315<br />
Title: Cellular consequences of radiation-induced oxidative stress in hum<strong>an</strong> neural stem cells<br />
Authors: *M. M. ACHARYA 1 , E. GIEDZINSKI 1 , M. L. LAN 2 , C. L. LIMOLI 1 ;<br />
1 2<br />
Dept of Radiation Oncology, Sue <strong>an</strong>d Bill Gross Stem Cell Res. Ctr., Univ. of Cali<strong>for</strong>nia<br />
Irvine, Irvine, CA<br />
Abstract: Cr<strong>an</strong>ial irradiation remains a front-line treatment <strong>for</strong> brain c<strong>an</strong>cer, but leads to normal<br />
tissue side effects that are often dose limiting. However, even lower doses c<strong>an</strong> elicit cognitive<br />
dysfunction that is thought to be caused by <strong>the</strong> depletion of radiosensitive neural stem <strong>an</strong>d<br />
precursor cells in <strong>the</strong> brain. In order to decipher <strong>the</strong> mech<strong>an</strong>isms underlying radiation-induced<br />
stem cell dysfunction, neural precursor cells (hNSCs) derived <strong>from</strong> H9 hum<strong>an</strong> embryonic stem<br />
cells were <strong>an</strong>alyzed <strong>for</strong> acute (0-48 h) ch<strong>an</strong>ges in apoptosis, DNA double str<strong>an</strong>d break (γ-H2AX)<br />
<strong>an</strong>d reactive oxygen/nitrogen species (ROS/RNS) following exposure to γ-rays (0.5-5 Gy).<br />
Irradiated hNSCs exhibited <strong>an</strong> acute, dose-dependent increase in apoptosis accomp<strong>an</strong>ied by<br />
elevated ROS <strong>an</strong>d RNS that persisted over a 48h period. Acute irradiation also led to <strong>the</strong><br />
phosphorylation of histone H2AX <strong>for</strong>ming γ-H2AX, widely used as a surrogate marker <strong>for</strong> DNA<br />
double-str<strong>an</strong>d breakage. Moreover, irradiated hNSCs undergone activation of cell cycle<br />
checkpoints, leading to altered cell cycle progression. These data suggest that <strong>the</strong> radiation<br />
response of hNSC involves <strong>the</strong> production of secondary reactive species that trigger a stress<br />
response not atypical of o<strong>the</strong>r primary cells. These stress response pathways dictate <strong>the</strong><br />
functional <strong>an</strong>d regenerative capacities of tissue-specific stem cells. These effects must be<br />
understood in ef<strong>for</strong>ts to coerce endogenous stem cell pools to undergo productive tissue<br />
remodeling following injury. We propose that <strong>the</strong> persistent oxidative stress found after
irradiation inhibits neurogenesis <strong>an</strong>d provides a biochemical mech<strong>an</strong>ism underlying <strong>the</strong><br />
development of radiation-induced cognitive impairment.<br />
This work is supported by Cali<strong>for</strong>nia Institute <strong>for</strong> Regenerative Medicine (CIRM) gr<strong>an</strong>t 445169-<br />
18315 to CLL.<br />
Disclosures: M.M. Acharya, Cali<strong>for</strong>nia Institute <strong>for</strong> Regenerative Medicine, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received); E. Giedzinski, None; M.L. L<strong>an</strong>, None; C.L. Limoli, Cali<strong>for</strong>nia Institute <strong>for</strong><br />
Regenerative Medicine (CIRM), B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.4/W13<br />
Topic: C.12. Neuro-Oncology<br />
Support: KOSEF <strong>2009</strong>-0058805<br />
BK21<br />
Title: Interplay between G1 phase cell cycle arrest <strong>an</strong>d apoptosis in hum<strong>an</strong> brain metastatic cell<br />
line by rare sugar D-allose<br />
Authors: *N. NAHA 1,2 , M.-O. KIM 1 ;<br />
1 2<br />
Neurobio., Gyeongs<strong>an</strong>g Natl. Univ., Jinju, Republic of Korea; Indi<strong>an</strong> Council of Med.<br />
Research, ROHC, Kolkata, India<br />
Abstract: Brain metastatic cell line derived <strong>from</strong> late stage hormone-refractory prostate c<strong>an</strong>cer<br />
exposed to rare sugar D-allose, enter mitochondria mediated caspase-dependent apoptotic<br />
pathway without <strong>an</strong>y side effects on normal brain cells. In <strong>the</strong> present study we systematically<br />
demonstrate <strong>the</strong> involvement of cyclin-cdk machinery during cell cycle employing synchronized<br />
brain metastatic cell line, DU145, as dysregulated cell cycle is <strong>the</strong> hallmark of c<strong>an</strong>cer where<br />
cyclin-cdk plays a crucial role. The up regulation of WAF1/p21, KIP1/p27 <strong>an</strong>d INK4a/p16<br />
proteins expression with concomit<strong>an</strong>t down regulation of cyclin D1-cdk4 <strong>an</strong>d cyclin E-cdk2<br />
complexes resulted in a signific<strong>an</strong>t G1 phase arrest of cell cycle as a consequence of D-allose<br />
treatment. The high sensitivity of G1 phase-specific DU145 cell line towards apoptosis<br />
characterized by differential expression of <strong>an</strong>ti-<strong>an</strong>d pro-apoptotic proteins, cell shrinkage, loss of
cell-cell integrity, DNA fragmentation <strong>an</strong>d membr<strong>an</strong>e blebbing. We also prove <strong>the</strong> dosedependent<br />
up regulations of WAF1/p21, Apaf-1 <strong>an</strong>d Bax in G1 phase-specific DU145 cell line by<br />
D-allose are independent of p53 expression. Taken toge<strong>the</strong>r, our study <strong>for</strong> <strong>the</strong> first time suggest<br />
that D-allose modulates G1 phase-specific ckis in late stage hum<strong>an</strong> brain metastasis in vitro,<br />
<strong>the</strong>reby inhibits corresponding cyclin-cdk complexes, which causes <strong>an</strong> irreversible arrest of cells<br />
at G0-G1 phase ultimately leading to apoptosis. Fur<strong>the</strong>r pharmacokinetic study of (D)-allose<br />
using in vivo brain metastatic model is warr<strong>an</strong>ted. (This work was supported by KOSEF <strong>2009</strong>-<br />
0058805 <strong>an</strong>d BK21).<br />
Disclosures: N. Naha, None; M. Kim, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.5/W14<br />
Topic: C.12. Neuro-Oncology<br />
Support: BK21<br />
KOSEF<br />
Title: Bcl-2/bcl-xL bispecific <strong>an</strong>tisense oligonucleotide in combination with pl<strong>an</strong>t flavonoid<br />
baicalin attenuate brain metastatic cell growth: A novel <strong>the</strong>rapeutic approach<br />
Authors: *A. DAS 1,2 , N. NAHA 2 , M.-O. KIM 2 , K.-C. CHANG 2 ;<br />
1 B<strong>an</strong>galore Univ., B<strong>an</strong>galore, India; 2 Gyeongs<strong>an</strong>g Natl. Univ., Jinju, Republic of Korea<br />
Abstract: Pl<strong>an</strong>t secondary metabolite baicalin has been referred to as ‘Nature’s biological<br />
response modifier” because of its strong inherit<strong>an</strong>ce to modify body’s reaction to carcinogen.<br />
Bcl-2/Bcl-xL bi-specific <strong>an</strong>tisense oligonucleotide induces apoptosis in a variety of c<strong>an</strong>cer cell<br />
lines. In <strong>the</strong> present study, we investigated <strong>the</strong> possible existence of <strong>an</strong> apoptotic cross-link<br />
between pl<strong>an</strong>t flavonoid baicalin <strong>an</strong>d Bcl-2 family member(s) in brain metastatic cell line,<br />
DU145 that derived <strong>from</strong> prostate c<strong>an</strong>cer. Our studies showed down regulation of <strong>an</strong>ti-apoptotic<br />
proteins with concomit<strong>an</strong>t up regulation of apoptotic inducers, loss of mitochondrial membr<strong>an</strong>e<br />
potential <strong>an</strong>d subsequent release of cytochrome C <strong>an</strong>d smac <strong>from</strong> mitochondria, leads to <strong>the</strong><br />
<strong>for</strong>mation of apoptosome <strong>an</strong>d finally <strong>the</strong> cleavage of caspase 3 <strong>an</strong>d poly (ADP-ribose)<br />
polymerase. These findings suggest that down-regulation of Bcl-2 <strong>an</strong>d Bcl-xL in favor of<br />
apoptosis by Bcl-2/Bcl-xL bi-specific <strong>an</strong>tisense oligonucleotide become more pronounced when
applied in combination with baicalin, while normal brain cell lines, HCN1A <strong>an</strong>d HCN2, showed<br />
no remarkable effect. Fur<strong>the</strong>rmore, cell death induced by baicalin in combination with Bcl-2/BclxL<br />
<strong>an</strong>tisense was markedly associated with G1 phase arrest of <strong>the</strong> cell cycle mediated via G1<br />
phase cyclins-cdks <strong>an</strong>d corresponding cyclin kinase inhibitors. In conclusion, <strong>the</strong> combined<br />
targeting of Bcl-2 <strong>an</strong>d Bcl-xL with baicalin may have a rational to be a novel treatment regime<br />
towards brain metastasis originated <strong>from</strong> adv<strong>an</strong>ce stage prostate c<strong>an</strong>cer. (Gr<strong>an</strong>t support: BK21<br />
<strong>an</strong>d KOSEF)<br />
Disclosures: A. Das, None; N. Naha, None; M. Kim, None; K. Ch<strong>an</strong>g, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.6/W15<br />
Topic: C.12. Neuro-Oncology<br />
Support: DODW8IXWH-07-1-0601 (Fisher, PI)<br />
Title: Cisplatin negatively affects progenitor cells in <strong>the</strong> central nervous system<br />
Authors: *A. T. NUNES, M. NOBLE;<br />
Univ. of Rochester, Rochester, NY<br />
Abstract: Patients that are treated with chemo<strong>the</strong>rapy have reported ch<strong>an</strong>ges in olfactory<br />
function decreasing quality of life. Patients have reported a reduced ability to smell <strong>an</strong>d detect<br />
odors that continues after chemo<strong>the</strong>rapy treatment has ended (Steinbach et al <strong>2009</strong>, Wickham et<br />
al 1999). Our lab has previously shown that chemo<strong>the</strong>rapy, including cisplatin, decreases neural<br />
progenitor cell division <strong>an</strong>d increases cell death in <strong>the</strong> mouse brain (Dietrich et al 2006). The<br />
olfactory system is a site of continued cell division throughout life <strong>an</strong>d has not yet been<br />
examined on <strong>the</strong> cellular level as a target of chemo<strong>the</strong>rapeutic agents. We have found that<br />
cisplatin negatively affects progenitor cells in three areas of <strong>the</strong> central nervous system (CNS):<br />
progenitor cells migrating <strong>from</strong> <strong>the</strong> subventricular zone to <strong>the</strong> olfactory bulb, <strong>the</strong> olfactory<br />
epi<strong>the</strong>lium, <strong>an</strong>d <strong>the</strong> corpus callosum. Stem cells found in <strong>the</strong> subventricular zone (SVZ) <strong>for</strong>m<br />
neuroblasts that migrate through <strong>the</strong> rostral migratory stream (RMS) to <strong>the</strong> olfactory bulb (OB)<br />
where <strong>the</strong>y differentiate into olfactory bulb interneurons. In <strong>the</strong> SVZ, RMS <strong>an</strong>d OB we found <strong>an</strong><br />
increase in cell death <strong>an</strong>d a signific<strong>an</strong>t decrease in <strong>the</strong> neuroblast population by examining <strong>the</strong><br />
decrease in proliferating neuroblasts. This ch<strong>an</strong>ge in progenitor cell numbers remained<br />
signific<strong>an</strong>t compared to vehicle control <strong>for</strong> as long as six months. In <strong>the</strong> olfactory epi<strong>the</strong>lium, we
found a signific<strong>an</strong>t decrease in proliferation of olfactory receptor neuron (ORN) progenitor cells.<br />
The ORNs are necessary <strong>for</strong> identifying both novel <strong>an</strong>d familiar odors <strong>an</strong>d relaying <strong>the</strong><br />
in<strong>for</strong>mation to <strong>the</strong> olfactory bulb. There was also a decrease in proliferation <strong>an</strong>d <strong>an</strong> increase in<br />
cell death in <strong>the</strong> oligodendrocyte precursor cell (OPC) population of <strong>the</strong> corpus callosum.<br />
Oligodendrocyte precursor cells (OPCs) in <strong>the</strong> corpus callosum differentiate into<br />
oligodendrocytes, allowing <strong>for</strong> a const<strong>an</strong>t supply of myelin throughout adulthood. Additionally,<br />
we were able to isolate OPCs <strong>an</strong>d when <strong>the</strong>se cells were treated with cisplatin in vitro, we found<br />
<strong>an</strong> increase in differentiated oligodendrocytes in <strong>the</strong> treated group <strong>an</strong>d no ch<strong>an</strong>ge in OPC cell<br />
death. These findings help us underst<strong>an</strong>d <strong>the</strong> effect cisplatin has on progenitor cells in <strong>the</strong> CNS<br />
<strong>an</strong>d may help to develop treatments to prevent <strong>the</strong> neurotoxic side effects of cisplatin, improving<br />
quality of life.<br />
Disclosures: A.T. Nunes, None; M. Noble, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.7/W16<br />
Topic: C.12. Neuro-Oncology<br />
Support: NIH Gr<strong>an</strong>t 1P20R021937-01A2<br />
Title: Bone-marrow-derived cells based n<strong>an</strong>oparticle plat<strong>for</strong>m <strong>for</strong> delivery in glioma<br />
Authors: *H. DOU, C. B. GROTEPAS;<br />
Univ. Nebraska Med. Ctr., Omaha, NE<br />
Abstract: Clinical limitations of glioma treatment are complicated due to poor penetr<strong>an</strong>ce of <strong>the</strong><br />
blood-brain barrier (BBB), infiltrative tumor growth, migrating dist<strong>an</strong>tly <strong>from</strong> <strong>the</strong> solid tumor<br />
mass <strong>an</strong>d infiltrating normal brain tissue. In particular, <strong>the</strong> deep filtration <strong>an</strong>d dissemination of<br />
tumor cells make surgery <strong>an</strong>d radiation <strong>the</strong>rapy difficult. To address <strong>the</strong>se difficulties, we<br />
developed bone-marrow-derived cells (BMDCs) n<strong>an</strong>oparticles (NP) delivery system <strong>for</strong><br />
combination of <strong>an</strong>titumor gene immune <strong>the</strong>rapy <strong>an</strong>d chemo<strong>the</strong>rapy. BMDCs have showed <strong>an</strong><br />
extraordinary ability to cross BBB, migration toward tumor, <strong>an</strong>d home to a site of tumor growth.<br />
We utilized BMDCs as a vehicle loaded with combo NP Interleukin-12 (IL-12) <strong>an</strong>d paclitaxel<br />
(PTX) <strong>an</strong>d <strong>for</strong> glioma. With surfact<strong>an</strong>t coating by PLGA syn<strong>the</strong>ses to fluoresceinamine, PLGA-<br />
NP-IL-12 size was tested by ZetaPlus measurement. NP-PTX was made by high-pressure<br />
homogenization of crystalline drug with surfact<strong>an</strong>t coating <strong>an</strong>d surface modifications to optimize
cell uptake. The NP-IL-12 size was arr<strong>an</strong>ged 239-278 nm <strong>an</strong>d <strong>the</strong> average size of NP-PTX was<br />
140 nm. NP-PTX <strong>an</strong>d NP-IL-12 uptake were assessed by static light scattering, laser doppler<br />
electrophoresis, <strong>an</strong>d confocal microscopy. The results showed uptake into cytoplasmic lysosomal<br />
vesicles. Mitochondrial function determined no signific<strong>an</strong>t ch<strong>an</strong>ge in BMDCs with or without<br />
loaded NP-IL-12. Cell uptake of NP was linked to size <strong>an</strong>d coating <strong>an</strong>d occurred in 10 minutes<br />
<strong>an</strong>d maxim uptake obtained in 8-12 hours. IL-12 released <strong>from</strong> BMDCs showed dose-dependent<br />
m<strong>an</strong>ner <strong>an</strong>d produce signific<strong>an</strong>t IFNγ in co-culture with macrophages. PTX were released into<br />
culture media over 7 days with subst<strong>an</strong>tive <strong>an</strong>ti-tumor efficacy to U87 culture. These results<br />
support <strong>the</strong> concept <strong>for</strong> BMDCs-mediated delivery using NP <strong>for</strong> combo of immuno<strong>the</strong>rapy <strong>an</strong>d<br />
chemo<strong>the</strong>rapy <strong>for</strong> glioma.<br />
Disclosures: H. Dou, None; C.B. Grotepas, None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.8/W17<br />
Topic: C.12. Neuro-Oncology<br />
Support: PENED 03ED778/GGET<br />
Title: Nucleocytoplasmic shuttling of neurofibromin in gliomas is regulated by phosphorylation<br />
Authors: X. KOLIOU 1 , G. LEONDARITIS 2 , N. SAKELLARIDIS 2 , *D. A. MANGOURA 1 ;<br />
1 Neurosci. Div., Fndn. Biomed Rese, A<strong>the</strong>ns, Greece; 2 Sch. of Med., Univ. of Thessaly, Larissa,<br />
Greece<br />
Abstract: Recent genome wide profiling studies have shown that neurofibromin is a<br />
glioblastoma tumor suppressor gene, as a signific<strong>an</strong>t percentage of sporadic glioblastomas have<br />
neurofibromin somatic mutations, while patients with germline mutations in <strong>the</strong> Nf1 gene often<br />
develop gliomas. A fundamental mech<strong>an</strong>ism <strong>for</strong> <strong>the</strong> function of tumor suppressor proteins is<br />
proving to be nuclear localization. M<strong>an</strong>y tumor suppressor proteins such as p53, APC/betacatenin,<br />
<strong>an</strong>d PTEN must be present in <strong>the</strong> cell nucleus to per<strong>for</strong>m <strong>the</strong>ir policing activities, <strong>an</strong>d<br />
<strong>the</strong>ir shuttling is, in most cases, phosphorylation-dependent. We have previously reported after<br />
confocal <strong>an</strong>alysis of immunofluorescent neurofibromin its developmentally-regulated subcellular<br />
shuttling in neural cells. More recently we showed that phosphorylation of neurofibromin at its<br />
C-tail is required <strong>for</strong> differentiation of neuroblastoma SH-SY5Y cells. To begin addressing <strong>the</strong><br />
specific function of neurofibromin’s nucleocytoplasmic shuttling we examined its expression <strong>an</strong>d
subcellular localization during a cell cycle of SF268 glioblastoma cells. Using cells synchronized<br />
at G1/S, S, G2, or G2/M after a double thymidine <strong>an</strong>d nocodazole block, we found that <strong>the</strong> levels<br />
of neurofibromin tr<strong>an</strong>scripts increased during <strong>the</strong> S phase, peaked at late S <strong>an</strong>d declined by early<br />
G2. We verified by immunofluorescence <strong>an</strong>alysis, subcellular <strong>an</strong>d subnuclear fractionation<br />
studies, <strong>an</strong>d co-immunoprecipitations with lamin A/C that nuclear neurofibromin was dynamic<br />
regulated throughout <strong>the</strong> cell cycle, as neurofibromin progressively accumulates in <strong>the</strong> nucleus<br />
throughout <strong>the</strong> duration of <strong>the</strong> S phase, a process that it is reversed at <strong>the</strong> beginning of <strong>the</strong> G2<br />
phase, with <strong>the</strong> protein slowly becoming primarily cytosolic. More import<strong>an</strong>tly, we found that<br />
neurofibromin’s association with <strong>the</strong> nuclear matrix started diminishing as neurofibromin<br />
became hyperphosphorylated on C-terminal, PKC-specific sites. Our results indicate that<br />
neurofibromin actively shuttles between <strong>the</strong> nucleus <strong>an</strong>d <strong>the</strong> cytoplasm in a phosphorylation<br />
specific way <strong>an</strong>d induction of intermolecular interactions <strong>for</strong> functions possibly related to neural<br />
cell mitosis (PENED gr<strong>an</strong>t 03ED778).<br />
Disclosures: X. Koliou, None; G. Leondaritis, None; N. Sakellaridis, None; D.A. M<strong>an</strong>goura,<br />
None.<br />
Poster<br />
547. Neurooncology II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 547.9/W18<br />
Topic: C.12. Neuro-Oncology<br />
Support: NIH NCRR P20RR016456<br />
Title: Delay of staurosporine-induced apoptosis by glutamate in brain tumor glia <strong>an</strong>d normal<br />
astrocytes<br />
Authors: *M. A. DECOSTER, J. MCNAMARA, K. COTTON, R. MASVEKAR;<br />
Louisi<strong>an</strong>a Tech. Univ., Ruston, LA<br />
Abstract: The protein kinase inhibitor staurosporine (STS) has been widely used to induce<br />
apoptosis in m<strong>an</strong>y cell models. As we previously reported, <strong>the</strong> STS-induced apoptotic cascade<br />
exhibits a time delay when astrocytes are pretreated with glutamate (GLU). Vital staining with<br />
calcein, metabolic tetrazole-based assays such as MTT, <strong>an</strong>d immunostaining <strong>for</strong> cytochrome c<br />
release all indicated that we were observing apoptotic ra<strong>the</strong>r th<strong>an</strong> necrotic ch<strong>an</strong>ges in our<br />
experiments. We now show this effect in brain tumor cells that express GLU receptors, such as<br />
<strong>the</strong> CRL 2199 gliosarcoma cell line (obtained <strong>from</strong> ATCC). <strong>When</strong> treated with a single addition
of 4 mM GLU 5 minutes prior to treatment with 2 µM STS, activated caspase-3 (casp3) levels<br />
were signific<strong>an</strong>tly suppressed at 3 hours post-STS treatment compared to STS treatment alone.<br />
Both normal astrocytes <strong>an</strong>d CRL 2199 brain tumor cells showed casp3 suppression by GLU at<br />
<strong>the</strong> 3 hour time point post-STS. We have seen as much as 53% suppression of casp3 activity in<br />
c<strong>an</strong>cer cells <strong>an</strong>d 38% in normal astrocytes. By 6 hours post-STS treatment, casp3 levels in both<br />
normal astrocytes <strong>an</strong>d brain tumor cells pretreated with GLU had returned close to levels<br />
measured in STS-treatments alone (7% <strong>an</strong>d 12% suppression, respectively). As expected, Jurkat<br />
T-cells, a leukemia cell line used as a control, showed no inhibition of STS-induced casp3 due to<br />
GLU pretreatment, using identical treatment concentrations <strong>an</strong>d observation timepoints, as <strong>the</strong>y<br />
lack GLU receptors <strong>an</strong>d/or tr<strong>an</strong>sporter complexes. The ability <strong>for</strong> both normal brain astrocytes<br />
<strong>an</strong>d brain tumor glia to suppress apoptosis due to <strong>the</strong> endogenous excitatory neurotr<strong>an</strong>smitter<br />
GLU may indicate <strong>an</strong> import<strong>an</strong>t focal point <strong>for</strong> communication between normal <strong>an</strong>d c<strong>an</strong>cerous<br />
glia. One potential mech<strong>an</strong>ism <strong>for</strong> this <strong>an</strong>ti-apoptotic action includes calcium influx. We found<br />
that both normal astrocytes <strong>an</strong>d brain tumor glia in our model demonstrated modest calcium<br />
influx or intracellular calcium oscillations due to 4 mM GLU stimulus. Since STS inhibits<br />
protein kinase C (PKC), among o<strong>the</strong>r kinases, m<strong>an</strong>y of which require calcium <strong>for</strong> activation,<br />
GLU pretreatment in our experiments may provide a pre-stimulus of PKC be<strong>for</strong>e <strong>the</strong> proapoptotic<br />
STS, thus delaying onset of apoptosis as reflected in our results measuring activated<br />
caspase-3 levels. Since brain tumor glia have been shown to release GLU, our results reported<br />
here support <strong>the</strong> idea of GLU as <strong>an</strong> <strong>an</strong>ti-apoptotic signal in <strong>the</strong> brain <strong>for</strong> both normal brain glia<br />
<strong>an</strong>d brain tumor cells.<br />
Disclosures: M.A. DeCoster, None; J. McNamara, None; K. Cotton, None; R. Masvekar,<br />
None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.1/W19<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: NIH/NIMH Gr<strong>an</strong>t RO1 #MH 9R01MH84282-04A1<br />
C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research CNE #85117<br />
Title: Neuroimaging <strong>an</strong>d genetic testing: Perceptions of benefit <strong>an</strong>d risk by people with mood<br />
disorders
Authors: *D. BUCHMAN, J. ILLES;<br />
Med., Natl. Core For Neuroethics, Univ. of British Columbia, V<strong>an</strong>covuer, BC, C<strong>an</strong>ada<br />
Abstract: The aim of <strong>the</strong> current study is to explore stakeholder attitudes <strong>an</strong>d beliefs about <strong>the</strong><br />
potential clinical tr<strong>an</strong>slation of neuroimaging <strong>an</strong>d genetic testing <strong>for</strong> predicting, diagnosing <strong>an</strong>d<br />
treating mood disorders. We conducted one-on-one semi-structured interviews with adults<br />
(N=17) diagnosed with ei<strong>the</strong>r unipolar depression or bipolar disorder to explore <strong>the</strong> prospects of<br />
neuroimaging or genetic testing in <strong>the</strong> context of: a) prediction, diagnosis, <strong>an</strong>d treatment of<br />
mental illness; b) <strong>an</strong>ticipated long-term risks <strong>an</strong>d benefits; c) projected impact on burden of<br />
disease <strong>an</strong>d privacy; <strong>an</strong>d d) expected interactions with identity <strong>an</strong>d stigma. Each interview was<br />
tr<strong>an</strong>scribed <strong>an</strong>d org<strong>an</strong>ized into categories <strong>an</strong>d <strong>the</strong>mes by multiple reviewers using a grounded<br />
<strong>the</strong>ory approach. The tri<strong>an</strong>gulation process continued until no new <strong>the</strong>oretical or conceptual<br />
domains were identified. We identified three primary <strong>the</strong>mes common to stakeholders views on<br />
neuroimaging <strong>an</strong>d genetic technologies: (1) in<strong>for</strong>mation <strong>from</strong> neuroimaging or a genetic test<br />
would be interpreted as conclusive evidence <strong>for</strong> <strong>the</strong> biological basis of mental illness, akin to<br />
o<strong>the</strong>r medical conditions; (2) a biological expl<strong>an</strong>ation of mental illness derived <strong>from</strong><br />
neuroimaging or a genetic test would decrease stigma <strong>an</strong>d <strong>the</strong> sense of self-blame by validating<br />
mental disorders as a legitimate medical illness; <strong>an</strong>d (3) predictive use of imaging or genetic<br />
testing would likely reduce <strong>the</strong> number of psychiatric visits by creating opportunities <strong>for</strong> earlier<br />
diagnosis, <strong>an</strong>d would lessen <strong>the</strong> time required <strong>an</strong>d potential side-effects associated with<br />
medication tailoring. We conclude that adults diagnosed with mood disorders have a tendency to<br />
be optimistic about <strong>the</strong> ability of both neuroimaging <strong>an</strong>d genetic testing to ease <strong>the</strong> burden of<br />
disease. The data support <strong>the</strong> need <strong>for</strong> upstream incorporation of patient views into <strong>the</strong><br />
development <strong>an</strong>d design of imaging <strong>an</strong>d genetic capabilities to maximize <strong>the</strong> efficacy of clinical<br />
tr<strong>an</strong>slation, <strong>an</strong>d ensure that fundamental ethics principles are incorporated <strong>an</strong>d addressed<br />
proactively. Outreach, education initiatives, <strong>an</strong>d accessible science communication are essential<br />
<strong>for</strong> elucidating <strong>the</strong> promises <strong>an</strong>d limits of <strong>the</strong>se technologies.<br />
Disclosures: D. Buchm<strong>an</strong>, None; J. Illes, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.2/W20<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)
Title: Functional connectivity in <strong>the</strong> Default Mode Network at rest is related to its<br />
neurotr<strong>an</strong>smitter patterns - A combined fMRI/MRS approach in healthy hum<strong>an</strong>s <strong>an</strong>d depressive<br />
patients<br />
Authors: *D. I. HORN 1 , J. BUCHMANN 1 , J. KAUFMANN 2 , A. OSOBA 1 , U. ECKERT 1 , T.<br />
M. MALONE 1 , J. STEINER 1 , B. BISWAL 3 , B. BOGERTS 1 , M. WALTER 1 ;<br />
1 Dept. of Psychiatry, Otto-von-Guericke Univ., Magdeburg, Germ<strong>an</strong>y; 2 Dept. of Neurology,<br />
Otto-von-Guericke Univ., Magdeburg, Germ<strong>an</strong>y; 3 Radiology, New Jersey Med. Sch., Newark,<br />
NJ<br />
Abstract: Objective: Consistent Resting State Networks (RSN) across healthy subjects <strong>an</strong>d<br />
differences in BOLD signal ch<strong>an</strong>ges of several regions in Major Depressive Disorder (MDD)<br />
have been described (Greicius et al 2007). Recent studies support <strong>the</strong> involvement of<br />
glutamatergic mech<strong>an</strong>isms in MDD. Our goal was thus to test <strong>the</strong> interdependence of altered<br />
neurotr<strong>an</strong>smitter concentrations in patients <strong>an</strong>d corresponding functional connectivity between<br />
regions of <strong>the</strong> Default Mode Network (DMN) which was recently reported to be of specific<br />
interest in depression (Walter et al <strong>2009</strong>).<br />
Methods: Functional Magnetic Reson<strong>an</strong>ce Imaging (fMRI) <strong>an</strong>d MR-Spectroscopy (MRS) on a<br />
3T MRI System was applied on 30 healthy <strong>an</strong>d 15 depressed hum<strong>an</strong>s. We used a single voxel<br />
(PRESS) sequence (TE 80 ms) <strong>for</strong> Glutamate(Glu)/Glutamine(Gln) separation. The MRS voxels<br />
of task positive regions such as dorsolateral prefrontal (dlPFC), <strong>an</strong>terior Insula/frontal<br />
Operculum (aIfO) <strong>an</strong>d dorsal <strong>an</strong>terior cingulate cortex(dACC) <strong>an</strong>d of task negative, DMN<br />
regions such as pregenual <strong>an</strong>terior cingulate cortex (pgACC) <strong>an</strong>d posterior cingulate cortex<br />
(pCC) defined <strong>the</strong> regions of interest (ROI) used <strong>for</strong> <strong>the</strong> fMRI data <strong>an</strong>alysis with AFNI.<br />
Connectivity is defined as <strong>the</strong> correlation of <strong>the</strong> BOLD activity timecourses measured over 488<br />
timepoints (TR 1.25 s) in different regions.<br />
Results: During resting state we found signific<strong>an</strong>tly different patterns of functional connectivity<br />
in <strong>the</strong> DMN of depressed patients.<br />
We found that connectivity between pgACC <strong>an</strong>d pCC correlates signific<strong>an</strong>tly with <strong>the</strong><br />
concentration of neurotr<strong>an</strong>smitters in <strong>the</strong>se regions. Lower Glu concentrations in PCC correlate<br />
with higher connectivity between pCC <strong>an</strong>d pgACC. Depressed patients show stronger<br />
connectivities between <strong>the</strong>se regions. The opposite pattern was observed <strong>for</strong> correlations between<br />
task positive <strong>an</strong>d task negative regions. Here, higher Glu levels predict a stronger segregation of<br />
<strong>the</strong> two networks in terms of functional connectivity.<br />
Conclusion: The observed interactions of functional connectivity with metabolic state support<br />
<strong>the</strong> specific involvement of previously described DMN regions in MDD. Our results emphasize<br />
<strong>the</strong> import<strong>an</strong>t role of combined metabolic <strong>an</strong>d functional studies in specific RSN’s. Fur<strong>the</strong>rmore,<br />
we could show that functional abnormalities observed in a number of conventional fMRI studies<br />
c<strong>an</strong> be traced back to a basic functional deviation, characterized both in functional <strong>an</strong>d metabolic<br />
terms.<br />
The clinical impact of <strong>the</strong> resting state DMN as a biomarker is especially high because of <strong>the</strong><br />
easy use concerning limited ability of depressed patients to concentrate on specific tasks.<br />
Disclosures: D.I. Horn, None; J. Buchm<strong>an</strong>n, None; J. Kaufm<strong>an</strong>n, None; A. Osoba, None; U.<br />
Eckert, None; T.M. Malone, None; J. Steiner, None; B. Biswal, None; B. Bogerts, None; M.<br />
Walter, None.
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.3/W21<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: Fonds National de la Recherche Scientifique (FNRS, Belgium; gr<strong>an</strong>t number<br />
3.4516.05 to M.D.)<br />
S.S.)<br />
University of Liege (Belgium)<br />
Queen Elisabeth Medical Foundation (Belgium)<br />
Swiss National Science Foundation (gr<strong>an</strong>ts #310000-114008, #3200B0-104100 to<br />
Title: Neurobiological bases of suicidality in major depression<br />
Authors: *M. DESSEILLES 1 , S. SCHWARTZ 2 , T. DANG-VU 3 , V. STERPENICH 3 , G.<br />
ALBOUY 3 , C. SCHMIDT 3 , E. SALMON 3 , A. DARSAUD 3 , G. VANDEWALLE 3 , M.<br />
ANSSEAU 4 , P. MAQUET 3 ;<br />
1 Univ. of Liege, Cyclotron Res. Ctr., Liege, Belgium; 2 Dept. of Neurosciences <strong>an</strong>d Geneva<br />
Neurosci. Ctr., Univ. of Geneva, Geneva, Switzerl<strong>an</strong>d; 3 Cyclotron Res. Ctr., 4 Dept. of<br />
psychiatry, Univ. of Liège, Liège, Belgium<br />
Abstract: Objectives<br />
Suicide is a major public health problem that typically occurs in <strong>the</strong> context of a depression. We<br />
used functional magnetic reson<strong>an</strong>ce imaging to reveal <strong>the</strong> neurobiological correlates of<br />
suicidality in depressed patients.<br />
Methods<br />
The present 3 Tesla fMRI study was conducted in 25 non-medicated patients with a first episode<br />
of unipolar MDD <strong>an</strong>d 25 matched controls. During sc<strong>an</strong>ning, subjects per<strong>for</strong>med two tasks<br />
imposing two different levels of attentional load at fixation (easy or difficult, i.e. low or high<br />
attentional load), while irrelev<strong>an</strong>t stimuli (i.e. faces) were presented in <strong>the</strong> periphery. To obtain<br />
<strong>an</strong> unbiased measure of suicidality <strong>from</strong> <strong>the</strong> Hamilton Rating Scale <strong>for</strong> Depression (HRSD)<br />
scores, we computed <strong>the</strong>ir singular value decomposition, a ma<strong>the</strong>matical procedure related to<br />
principal component <strong>an</strong>alysis.<br />
Functional MRI data were <strong>an</strong>alyzed using a two-step procedure taking into account <strong>the</strong> intra-
individual <strong>an</strong>d inter-individual vari<strong>an</strong>ce. The summary statistics images of <strong>the</strong> individual level<br />
were entered into a second-level one-way ANOVA implemented in SPM5<br />
(http://www.fil.ion.ucl.ac.uk) to assess r<strong>an</strong>dom-effects group comparisons.<br />
We per<strong>for</strong>med additional whole-brain second-level independent correlation <strong>an</strong>alyses <strong>for</strong> <strong>the</strong> main<br />
contrasts of interest using a component of <strong>the</strong> HDRS as covariates with singular value<br />
decomposition of <strong>the</strong> scale.<br />
Results<br />
<strong>When</strong> asked to engage attention in a cognitive task (high > low attentional load) depressed<br />
patients activated <strong>the</strong> mesopontine reticular <strong>for</strong>mation (noradrenergic locus coeruleus,<br />
serotonergic raphé nuclei) in proportion of <strong>the</strong>ir suicidality. No such correlation was observed in<br />
healthy particip<strong>an</strong>ts, whose suicidality scores were within normal r<strong>an</strong>ges.<br />
Conclusions<br />
In <strong>an</strong>imals, activity in <strong>the</strong> mesopontine reticular <strong>for</strong>mation c<strong>an</strong> promote rapid behavioral shifts in<br />
response to cognitive challenges by facilitating <strong>the</strong> functional reorg<strong>an</strong>ization of cortical<br />
networks. Our results suggest that, in <strong>the</strong> context of high attentional dem<strong>an</strong>ds, depressed patients<br />
may be prone to impulsive shifts in behavioral states. These findings provide import<strong>an</strong>t clues<br />
about <strong>the</strong> mech<strong>an</strong>isms causing impulsive behavior at <strong>the</strong> beginning of <strong>an</strong>tidepress<strong>an</strong>t treatments.<br />
Disclosures: M. Desseilles, None; S. Schwartz, None; T. D<strong>an</strong>g-Vu, None; V. Sterpenich,<br />
None; G. Albouy, None; C. Schmidt, None; E. Salmon, None; A. Darsaud, None; G.<br />
V<strong>an</strong>dewalle, None; M. Ansseau, None; P. Maquet, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.4/W22<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: NIH Gr<strong>an</strong>t F31MH078346<br />
NIMH Gr<strong>an</strong>t R01MH68376<br />
Sackler Scholar in Psychobiology Research Gr<strong>an</strong>t<br />
Title: Associations between serotonin tr<strong>an</strong>sporter genotype <strong>an</strong>d action monitoring: A possible<br />
endophenotype explaining increased vulnerability to depression
Authors: *A. J. HOLMES 1 , R. BOGDAN 2 , D. PIZZAGALLI 2 ;<br />
1 Clin. Psyc, 2 Harvard Univ., Cambridge, MA<br />
Abstract: Objective: Functional polymorphisms of <strong>the</strong> serotonin tr<strong>an</strong>sporter gene (5-HTTLPR)<br />
have been associated with <strong>an</strong> increased risk to develop Major Depressive Disorder (MDD).<br />
Individuals with MDD are characterized by executive dysfunction <strong>an</strong>d abnormal activation<br />
within <strong>the</strong> <strong>an</strong>terior cingulate cortex (ACC), particularly in situations requiring adaptive<br />
behavioral adjustments following errors <strong>an</strong>d response-conflict (action monitoring). Impaired<br />
action monitoring may increase vulnerability to depression, but <strong>the</strong> neural mech<strong>an</strong>isms<br />
underlying <strong>the</strong>se dysfunctions remain largely unknown.<br />
Method: The goal of this functional magnetic reson<strong>an</strong>ce imaging study was to test whe<strong>the</strong>r<br />
psychiatrically healthy S carriers would demonstrate abnormalities mirroring those previously<br />
observed in MDD subjects. To this end, 19 S carriers <strong>an</strong>d 14 L/L homozygotes per<strong>for</strong>med a<br />
modified Fl<strong>an</strong>ker task known to induce errors, response-conflict, <strong>an</strong>d activations in various ACC<br />
subdivisions<br />
Results: Relative to L/L homozygotes, S allele carriers showed (1) impaired post-error <strong>an</strong>d postconflict<br />
behavioral adjustments; (2) larger error-related rostral ACC activation; <strong>an</strong>d (3) lower<br />
conflict-related dorsal ACC activity.<br />
Conclusions: These data indicate that <strong>the</strong> presence of <strong>the</strong> S allele is associated with a<br />
dysregulated action monitoring system. Because similar behavioral <strong>an</strong>d neural dysfunctions have<br />
been recently described in MDD samples, <strong>the</strong> current results indicate that impaired action<br />
monitoring <strong>an</strong>d associated ACC dysregulation may represent risk factors associated with<br />
increased vulnerability to depression.<br />
Disclosures: A.J. Holmes, None; R. Bogd<strong>an</strong>, None; D. Pizzagalli, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.5/W23<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: NIH Gr<strong>an</strong>t R01MH64769<br />
Title: Limbic <strong>an</strong>d prefrontal brain activation in children with preschool onset depression<br />
Authors: A. BELDEN, J. LUBY, *D. M. BARCH;<br />
Dept Psychol & Psychiatry, Washington Univ., St. Louis, MO
Abstract: A growing body of work points to <strong>the</strong> clinical reality of preschool onset depression, a<br />
early onset psychiatric disorder that shares m<strong>an</strong>y of <strong>the</strong> typical features of adolescent <strong>an</strong>d adult<br />
depression, though in a developmentally appropriate <strong>for</strong>m. However, little is know about <strong>the</strong><br />
neurobiological ch<strong>an</strong>ges associated with such preschool onset <strong>for</strong>ms of depression. Adult<br />
depression is associated with both structural <strong>an</strong>d functional ch<strong>an</strong>ges in a circuit of limbicprefrontal<br />
regions known to be critical <strong>for</strong> emotion processing <strong>an</strong>d regulation, including <strong>the</strong><br />
ventral medial prefrontal cortex (VMPFC), insular cortex, cingulate cortex, amygdala <strong>an</strong>d<br />
hippocampus. We hypo<strong>the</strong>sized that preschool onset depression would also be associated with<br />
functional <strong>an</strong>d structural ch<strong>an</strong>ges in at least a subset of <strong>the</strong>se regions, with more severe <strong>an</strong>d<br />
chronic MDD in children being associated with greater impairment in this circuit. To test this<br />
hypo<strong>the</strong>sis, we are examining both <strong>the</strong> structural <strong>an</strong>d functional integrity of <strong>the</strong>se regions in a<br />
large sample of prospectively followed children (currently ages 8-10) who were identified<br />
between <strong>the</strong> ages of 3-5 as having ei<strong>the</strong>r Major Depression (MDD), Anxiety disorders, disruptive<br />
disorders or no psychiatric illness (healthy controls). We used a well-validated facial emotionprocessing<br />
task in which children are shown faces that vary in emotional content (happy, neutral,<br />
sad, <strong>an</strong>gry <strong>an</strong>d fearful) <strong>an</strong>d emotional intensity. We collected BOLD contrast images during<br />
per<strong>for</strong>m<strong>an</strong>ce of this task, using a Siemen’s 3T Tim TRIO <strong>an</strong>d a 12 ch<strong>an</strong>nel head coil. The<br />
children with preschool onset MDD showed greater activation th<strong>an</strong> controls <strong>for</strong> <strong>the</strong> comparison<br />
of sad to happy faces in pregenual cingulate, VMPFC <strong>an</strong>d left Amygdala. In addition we<br />
correlated dimensional characterizations of depression (assessed over multiple ages <strong>for</strong> each<br />
child) <strong>for</strong> depression, <strong>an</strong>xiety, <strong>an</strong>d disruptive behavior with brain activity in response to<br />
emotional faces, using partial correlations that controlled <strong>for</strong> vari<strong>an</strong>ce in <strong>the</strong> o<strong>the</strong>r dimensions.<br />
Greater severity in <strong>the</strong> depression dimension - even when controlling <strong>for</strong> <strong>an</strong>xiety <strong>an</strong>d disruptive<br />
symptom severity - correlated with greater activity to sad faces in <strong>the</strong> VMPFC <strong>an</strong>d <strong>the</strong><br />
amygdala/hippocampal complex. The disruptive dimension showed little correlation with brain<br />
activity to sad faces, <strong>an</strong>d <strong>the</strong> <strong>an</strong>xiety dimension primarily showed correlations with lateral<br />
temporal regions <strong>for</strong> sad faces. These initial data provide <strong>the</strong> first evidence that preschool onset<br />
depression is associated with functional brain activation alterations in at least some of <strong>the</strong> same<br />
regions found to show abnormalities in adolescent <strong>an</strong>d adult depression.<br />
Disclosures: A. Belden, None; J. Luby, None; D.M. Barch, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.6/W24<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)
Support: KAKENHI (20790841)<br />
Title: Brain activation during a verbal fluency task in recovered depressive patients with silent<br />
cerebral infarction<br />
Authors: *G. OKADA 1 , Y. OKAMOTO 1 , H. TAKAMI 2 , H. YAMASHITA 1 , S.<br />
YAMAWAKI 1 ;<br />
1 Hiroshima Univ. Sch. Med., Hiroshima, Jap<strong>an</strong>; 2 Futaba Hosp., Hiroshima, Jap<strong>an</strong><br />
Abstract: Depression in <strong>the</strong> elderly is complicated by cerebrovascular disease which is believed<br />
to contribute to <strong>the</strong> limited efficacy of <strong>an</strong>tidepress<strong>an</strong>t treatment. However, <strong>the</strong> neurobiologic<br />
basis of <strong>the</strong> depressive syndrome <strong>an</strong>d treatment response in vascular depression has not been<br />
established. The aim of this study was to investigate cerebral brain function in recovered<br />
depressed elderly <strong>an</strong>d investigate <strong>the</strong> relationship between this brain function <strong>an</strong>d silent cerebral<br />
infarction (SCI), as detected by magnetic reson<strong>an</strong>ce imaging (Yamashita et al., 2001). Sixteen<br />
elderly depressive patients in recovery were included in this study. The depressive patients were<br />
classified as SCI(+) (n = 7) or SCI(-) (n = 9). Functional magnetic reson<strong>an</strong>ce imaging was<br />
per<strong>for</strong>med in both groups during a verbal fluency task. The data were <strong>an</strong>alyzed using statistical<br />
parametric mapping software. The examinations were conducted under a protocol that was<br />
approved by <strong>the</strong> Ethics Committee of Hiroshima University School of Medicine. After complete<br />
description of <strong>the</strong> study to <strong>the</strong> subjects, written in<strong>for</strong>med consent was obtained. For both groups,<br />
verbal fluency task resulted in <strong>the</strong> signific<strong>an</strong>t activation of <strong>the</strong> bilateral <strong>an</strong>d medial prefrontal<br />
cortex. Although <strong>the</strong>re were no signific<strong>an</strong>t differences in areas of activation between <strong>the</strong> SCI(+)<br />
group <strong>an</strong>d <strong>the</strong> SCI(-) group in <strong>the</strong> direct comparison, <strong>the</strong> SCI(-) group showed signific<strong>an</strong>t<br />
activation in <strong>the</strong> cingulate cortex, while <strong>the</strong> SCI(+) group did not. Fur<strong>the</strong>r study is needed to<br />
assess <strong>the</strong> functional brain abnormality related to vascular depression.<br />
Disclosures: G. Okada, None; Y. Okamoto, None; H. Takami, None; H. Yamashita,<br />
None; S. Yamawaki, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.7/W25<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: NIH 5R21MH080258
NIH P01-AG024831<br />
NIH AG13308<br />
NIH P50 AG 16570<br />
NIH MH/AG58156<br />
NIH MH52453; AG10123; M01-RR00865<br />
DOE DE-FC03-87-ER60615<br />
Title: Protein binding in patients with late-life depression detected using [18f]FDDNP positron<br />
emission tomography<br />
Authors: *O. A. AJILORE 1 , A. KUMAR 2 , V. KEPE 3 , J. R. BARRIO 3 , P. SIDDARTH 4 , V.<br />
MANOUKIAN 4 , V. ELDERKIN-THOMPSON 4 , G. SMALL 4 ;<br />
2 1 3 4<br />
Psychiatry, Univ. of Illinois-Chicago, Chicago, IL; Mol. <strong>an</strong>d Med. Pharmacol., Psychiatry,<br />
UCLA, Los Angeles, CA<br />
Abstract: The objective of our study was to examine protein (amyloid <strong>an</strong>d tau) binding in<br />
critical brain regions in patients diagnosed with late-life major depression (MDD) <strong>an</strong>d healthy<br />
controls using [18F]FDDNP (fluorine-18 FDDNP (2-(1-{6-[(2-[18F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile)<br />
positron emission tomography (PET). Our samples<br />
comprised 20 patients diagnosed with MDD <strong>an</strong>d 19 healthy subjects of comparable age, gender<br />
<strong>an</strong>d education. Relative distribution volume in regions of interest was used as <strong>the</strong> measure of<br />
FDDNP binding in all subjects. FDDNP binding was signific<strong>an</strong>tly higher in <strong>the</strong> posterior<br />
cingulate <strong>an</strong>d lateral temporal regions in <strong>the</strong> MDD group when compared with controls, while<br />
<strong>the</strong> <strong>an</strong>terior cingulate <strong>an</strong>d mesial temporal regions showed higher binding levels that approached<br />
statistical signific<strong>an</strong>ce. These findings suggest that neuronal injury secondary to higher protein<br />
load in key brain regions might provide <strong>an</strong> import<strong>an</strong>t mech<strong>an</strong>ism in <strong>the</strong> pathophysiology of<br />
MDD in late-life, <strong>an</strong>d have implications <strong>for</strong> <strong>the</strong> <strong>the</strong>rapeutics of depression in <strong>the</strong> elderly.<br />
Disclosures: O.A. Ajilore, None; A. Kumar, None; V. Kepe, None; J.R. Barrio, None; P.<br />
Siddarth, None; V. M<strong>an</strong>ouki<strong>an</strong>, None; V. Elderkin-Thompson, None; G. Small, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 548.8/W26<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: NIH gr<strong>an</strong>t P50-MH60451<br />
NIH gr<strong>an</strong>t K23-AG028982<br />
Title: Aging specific alteration in neural substrates associated with emotion <strong>an</strong>d cognition in<br />
geriatric depression<br />
Authors: *L. WANG, Dr. 1 , R. KRISHNAN 2,3 , D. C. STEFFENS 2 , G. MCCARTHY 4 ;<br />
1 BRAIN IMAGING & ANALYSIS Center/Duke Univ. Med. Ctr., Durham, NC; 2 Dept. of<br />
Psychiatry, Duke Univ. Med. Ctr., Durham, NC; 3 Duke-NUS Grad. Med. Sch. Singapore,<br />
Singapore, Singapore; 4 Yale Univ., Department of Psychology, CT<br />
Abstract: Studies on young <strong>an</strong>d middle-aged patients with major depression disorder (MDD)<br />
using functional magnetic reson<strong>an</strong>ce imaging (fMRI) have consistently found activation ch<strong>an</strong>ges<br />
related to emotional <strong>an</strong>d cognitive processing in <strong>the</strong> limbic-thalamic-cortical network. Patients<br />
with geriatric depression may have different deficits in <strong>the</strong> limbic-thalamic-cortical network th<strong>an</strong><br />
young depressed adults due to age-related pathology. However, experiments systematically<br />
examining <strong>the</strong> interplay of aging <strong>an</strong>d MDD on <strong>the</strong> alteration of neural substrates are lacking. To<br />
address this question, we compared brain activations evoked during <strong>an</strong> emotional oddball task in<br />
middle-aged <strong>an</strong>d older patients with MDD. The data were collected <strong>from</strong> 19 middle-aged <strong>an</strong>d 17<br />
older adults with acute MDD, as well as 20 middle-aged <strong>an</strong>d 20 older healthy controls. The<br />
emotional oddball task included infrequently presented attentional targets <strong>an</strong>d sad <strong>an</strong>d neutral<br />
pictures as novel distracters. ANOVA <strong>an</strong>alysis on brain activations in response to sad pictures<br />
<strong>an</strong>d attentional targets were conducted using age (middle-aged, older) <strong>an</strong>d study group (acute<br />
MDD, control) as covariates. Age was associated with decreased activation in <strong>the</strong> bilateral<br />
fusi<strong>for</strong>m gyrus <strong>an</strong>d <strong>the</strong> left inferior frontal cortex in response to negative emotional stimuli. In<br />
addition, age was associated with decreased activation in <strong>the</strong> dorsal <strong>an</strong>terior cingulate <strong>an</strong>d<br />
increased activation in <strong>the</strong> premotor cortex <strong>an</strong>d inferior parietal cortex in response to target<br />
stimuli. Relative to both control groups, middle-aged <strong>an</strong>d older depressed patients demonstrated<br />
decreased activation in <strong>the</strong> right dorsolateral prefrontal cortex in response to attentional targets.<br />
Decreased activation in <strong>the</strong> <strong>an</strong>terior portion of posterior cingulate <strong>an</strong>d middle temporal cortex<br />
seems to be geriatric depression unique. Mapping age-specific ch<strong>an</strong>ges in emotional <strong>an</strong>d<br />
executive systems in MDD would help to better underst<strong>an</strong>d <strong>the</strong> pathology of geriatric depression.<br />
Disclosures: L. W<strong>an</strong>g, None; R. Krishn<strong>an</strong>, P50-MH60451(PD: R<strong>an</strong>ga Krishn<strong>an</strong>l);, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); D.C. Steffens, None; G. McCarthy, None.<br />
Poster
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.9/W27<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Title: Identifying multivari<strong>an</strong>t biomarkers in primary neuronal cultures treated with mood<br />
stabilizers - A potential strategy <strong>for</strong> screening novel <strong>the</strong>rapeutic agents<br />
Authors: *J. K. KETTERMAN 1 , S. HAGGARTY 4,1 , T. NIELAND 2 , D. LOGAN 3 , J. PAN 1 ;<br />
1 Psychiatric Dis. Initiative, 2 RNAi Plat<strong>for</strong>m, 3 Imaging Plat<strong>for</strong>m, Broad Inst. of Harvard <strong>an</strong>d MIT,<br />
Cambridge, MA; 4 Dept. of Neurol., Massachusetts Gen. Hosp., Boston, MA<br />
Abstract: Lithium (Li + ), valproate, <strong>an</strong>d <strong>an</strong>tipsychotic drugs such as Quetiapin are common<br />
treatments <strong>for</strong> m<strong>an</strong>ic depressive (bipolar) disorder. Previous experiments treating cultured<br />
neurons with <strong>the</strong>se drug compounds have proven inconclusive. Using primary neuronal cultures<br />
as a cellular model, we visualized <strong>the</strong> effects of <strong>the</strong>se drug compounds using axonal <strong>an</strong>d<br />
dendritic markers <strong>an</strong>d <strong>the</strong> image <strong>an</strong>alysis tools CellProfiler <strong>an</strong>d CellProfiler Analyst to identify<br />
<strong>an</strong>d qu<strong>an</strong>tify morphological biomarkers. Treatment of primary neurons with Li + reduced average<br />
neurite length <strong>an</strong>d <strong>the</strong> average number of neurites per neuron in a dose-dependent m<strong>an</strong>ner. In<br />
addition, ch<strong>an</strong>ges in axonal thickness were shown upon Li + treatment. Fur<strong>the</strong>r <strong>an</strong>alysis of<br />
primary neuronal cultures by western blot showed a dose-dependent increase in two downstream<br />
targets of GSK-3β inhibition, <strong>the</strong> microtubule associated protein Tau <strong>an</strong>d <strong>the</strong> tr<strong>an</strong>scriptional<br />
activator, β-Catenin. These results were consistent with reports that Li + modulated GSK-3β in<br />
neurons. Fur<strong>the</strong>r tests of known benchmark inhibitors of GSK-3β indicated that GSK-3β<br />
inhibition c<strong>an</strong> account <strong>for</strong> <strong>the</strong> majority of <strong>the</strong> identified biomarkers of lithium effects in cultured<br />
neurons. We are applying this same principle to cultured neurons treated with valproate <strong>an</strong>d<br />
Quietiapine. Our goal is to identify morphological markers common to different classes of drug<br />
compounds <strong>for</strong> bipolar disorder <strong>an</strong>d to develop a high-throughput, imaged-based screening<br />
method <strong>for</strong> use in <strong>the</strong> discovery of novel <strong>the</strong>rapeutic agents.<br />
Disclosures: J.K. Ketterm<strong>an</strong>, Broad Institute of Harvard <strong>an</strong>d MIT, A. Employment (full or<br />
part-time); S. Haggarty, None; T. Niel<strong>an</strong>d, None; D. Log<strong>an</strong>, Broad Institute of Harvard <strong>an</strong>d<br />
MIT, A. Employment (full or part-time); J. P<strong>an</strong>, Broad Institute of Harvard <strong>an</strong>d MIT, A.<br />
Employment (full or part-time).<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.10/W28<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research (C) (16591170)<br />
Title: Regulation of <strong>the</strong> hum<strong>an</strong> tryptoph<strong>an</strong> hydroxylase-2(TPH2) gene promoter activity by<br />
CCAAT enh<strong>an</strong>cer-binding protein alpha in RN46A cells<br />
Authors: Y. INOUE 1 , H. TOMOKO 1 , Y. OKADA 1 , R. KUWABARA 1 , H. KOUYAMA 1 , H.<br />
HASEGAWA 2 , S. MARUTA 2 , A. ADACHI 2 , K. OSADA 2 , M. ASAKURA 2 , N.<br />
YAMAGUCHI 2 , *H. MATSUI 1 ;<br />
1 St. Mari<strong>an</strong>na Univ. Grad Sch. Med., Kawasaki, K<strong>an</strong>agawa, Jap<strong>an</strong>; 2 Dept. Neuropsychiatry,<br />
St.Mari<strong>an</strong>na Univ. Sch. Med., Kawasaki, Jap<strong>an</strong><br />
Abstract: Promoter polymorphism of <strong>the</strong> hum<strong>an</strong> tryptoph<strong>an</strong> hydroxylase-2 (hTPH2) gene<br />
coding <strong>for</strong> <strong>the</strong> rate-limiting enzyme of serotonin (5-HT) syn<strong>the</strong>sis in <strong>the</strong> brain is thought to be<br />
related to neuropsychiatric diseases as well as personality traits. Elucidation of mech<strong>an</strong>isms<br />
involved in <strong>the</strong> tr<strong>an</strong>scriptional regulation of <strong>the</strong> hTPH2 gene will assist our underst<strong>an</strong>ding of how<br />
<strong>the</strong> expression of <strong>the</strong> hTPH2 gene could be altered by various pathologic states. To address <strong>the</strong>se<br />
questions more directly, we tested <strong>the</strong> hTPH2 gene promoter activity <strong>an</strong>d found a critical element<br />
necessary <strong>for</strong> <strong>the</strong> hTPH2 gene expression. A 2-kb fragment upstream to <strong>the</strong> tr<strong>an</strong>slation start site<br />
(-1991 to -1; adenine of ATG was assigned to +1) was cloned into pGL4-Basic (TPH2-55) <strong>an</strong>d a<br />
series of deletion <strong>an</strong>d substitution mut<strong>an</strong>ts were constructed. Promoter activities were assessed<br />
by tr<strong>an</strong>sient tr<strong>an</strong>sfections into rat serotonergic RN46A cells (a generous gift <strong>from</strong> Dr.<br />
Whittemore SR., Univ Louisville, KY). A NRSE-like sequence was found at <strong>the</strong> vicinity (-133 to<br />
-106) of <strong>the</strong> tr<strong>an</strong>scription start site of <strong>the</strong> hTPH2 gene (Patel PD et al., J BC 282 (2007): 26717-<br />
26724). RT-PCR <strong>an</strong>alysis confirmed <strong>the</strong> expression of NRSF in RN46A cells. Thus, <strong>the</strong><br />
expression of <strong>the</strong> hTPH2 gene seems to be strictly controlled through <strong>the</strong> NRSF-NRSE system in<br />
RN46A cells. Accordingly, <strong>the</strong> promoter activity of <strong>the</strong> TPH2-55 construct was very low in<br />
RN46A cells. To explore <strong>the</strong> tr<strong>an</strong>scription factors that could surpass <strong>the</strong> NRSE-mediated<br />
repression of <strong>the</strong> hTPH2 gene expression, <strong>the</strong> sequence (-1991 to -1) was searched <strong>for</strong> <strong>the</strong>ir<br />
binding sites. A multiple potential binding sites <strong>for</strong> CCAAT enh<strong>an</strong>cer-binding proteins (C/EBPs)<br />
were found. Overexpression of C/EBP alpha, not C/EBP beta, increased <strong>the</strong> promoter activity of<br />
<strong>the</strong> TPH2-55 construct. By tr<strong>an</strong>sfections of mut<strong>an</strong>t constructs, a critical sequence (-273 to -185)<br />
involved in <strong>the</strong> C/EBP alpha-mediated increase in <strong>the</strong> hTPH2 gene promoter activity was<br />
identified. Supershift <strong>an</strong>d ChIP assays fur<strong>the</strong>r confirmed <strong>the</strong> C/EBP alpha binding to this<br />
particular sequence. These results indicate that <strong>the</strong> C/EBP alpha could surpass <strong>the</strong> NRSFmediated<br />
repression of <strong>the</strong> hTPH2 gene tr<strong>an</strong>scription in RN46A cells. C/EBP alpha may<br />
contribute to <strong>the</strong> complex regulation of <strong>the</strong> TPH2 gene tr<strong>an</strong>scription <strong>an</strong>d consequently lead to <strong>an</strong><br />
increase in TPH2 protein <strong>an</strong>d 5-HT syn<strong>the</strong>sis in <strong>the</strong> brain. This work was supported by Gr<strong>an</strong>t-in-<br />
Aid <strong>for</strong> Scientific Research (C) (16591170).
Disclosures: Y. Inoue, None; H. Tomoko, None; Y. Okada, None; R. Kuwabara, None; H.<br />
Kouyama, None; H. Hasegawa, None; S. Maruta, None; A. Adachi, None; K. Osada,<br />
None; M. Asakura, None; N. Yamaguchi, None; H. Matsui, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.11/W29<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: International Research Scholars gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Howard Hughes Medical Institute<br />
Gr<strong>an</strong>t <strong>from</strong> Agencia Nacional de Promocion Cientifica y Tecnologica, Argentina<br />
Title: Cysteine residues in <strong>the</strong> large extracellular loop (EC2) are essential <strong>for</strong> <strong>the</strong> function of <strong>the</strong><br />
stress-regulated glycoprotein M6a<br />
Authors: *B. FUCHSOVA 1 , M. E. FERNÁNDEZ 2 , J. ALFONSO 3 , A. C. FRASCH 4 ;<br />
1 Inst. De Investigaciones Biotecnológicas, S<strong>an</strong> Martín, Buenos Aires, Argentina; 2 Biozentrum,<br />
Univ. of Basel, Basel, Switzerl<strong>an</strong>d; 3 Heidelberg Univ., Heidelberg, Germ<strong>an</strong>y; 4 Inst. de<br />
Investigaciones Biotecnológicas, S<strong>an</strong> Martín, Buenos Aires, Argentina<br />
Abstract: Glycoprotein M6a was identified as a stress-responsive gene in <strong>the</strong> hippocampal<br />
<strong>for</strong>mation. This gene is down-regulated in <strong>the</strong> hippocampus of both socially <strong>an</strong>d physically<br />
stressed <strong>an</strong>imals, <strong>an</strong>d this effect c<strong>an</strong> be reversed by <strong>an</strong>tidepress<strong>an</strong>t treatment. Previously we<br />
showed that <strong>the</strong> stress-regulated protein M6a is a key modulator <strong>for</strong> neurite outgrowth <strong>an</strong>d<br />
filopodium/spine <strong>for</strong>mation. In <strong>the</strong> present work, mutational <strong>an</strong>alysis was used to characterize <strong>the</strong><br />
action of M6a at <strong>the</strong> molecular level. Using site-directed mutagenesis we constructed a p<strong>an</strong>el of<br />
M6a mut<strong>an</strong>ts with <strong>the</strong> series of point mutations at <strong>the</strong> putative biologically signific<strong>an</strong>t amino acid<br />
residues. Four cysteines C162, C174, C192 <strong>an</strong>d C202 within EC2 are shown to be critical. The<br />
presence of cysteines C162 <strong>an</strong>d C202 is essential <strong>for</strong> <strong>the</strong> efficient cell surface expression of M6a<br />
protein. In contrast, cysteines C174 <strong>an</strong>d C192 are not required <strong>for</strong> <strong>the</strong> efficient surface expression<br />
of M6a. Their mutation to al<strong>an</strong>ine does not interfere with <strong>the</strong> localization of M6a to filopodial<br />
protrusions in primary hippocampal neurons. Never<strong>the</strong>less, tr<strong>an</strong>sfection experiments revealed<br />
that neurons expressing C174A <strong>an</strong>d C192A mut<strong>an</strong>ts display decreased filopodia number. In nonpermeabilized<br />
cells <strong>the</strong>se mut<strong>an</strong>t proteins are not recognized by a function-blocking monoclonal<br />
<strong>an</strong>tibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A<br />
mut<strong>an</strong>t display signific<strong>an</strong>tly lower density of presynaptic clusters over <strong>the</strong>ir dendrites. Taken
toge<strong>the</strong>r, this study demonstrates that EC2 domain is critical <strong>for</strong> <strong>the</strong> role of M6a in filopodium<br />
outgrowth <strong>an</strong>d synaptogenesis.<br />
Disclosures: B. Fuchsova, None; M.E. Fernández, None; J. Alfonso, None; A.C. Frasch,<br />
None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.12/W30<br />
Topic: C.16.b. Mood disorders: Hum<strong>an</strong> biomarkers (imaging, genetics, biochemistry)<br />
Support: Rackham Research Gr<strong>an</strong>t<br />
Title: Repression of tryptoph<strong>an</strong> hydroxylase 2 by <strong>the</strong> microRNA miR-370<br />
Authors: *L. OWN 1,2 , P. D. PATEL 3 ;<br />
1 Ann Arbor, MI; 2 Neurosci., 3 MBNI, Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: The neuronal specific iso<strong>for</strong>m, tryptoph<strong>an</strong> hydroxylase 2 (TPH2), is <strong>the</strong> rate-limiting<br />
enzyme <strong>for</strong> brain serotonin syn<strong>the</strong>sis. Serotonin modulates a spectrum of behaviors, including<br />
emotion, motivation, aggression, <strong>an</strong>d cognition. Mental illnesses, including OCD, ADHD,<br />
affective disorders, <strong>an</strong>d schizophrenia, are linked to disturb<strong>an</strong>ces in serotonergic levels <strong>an</strong>d<br />
tr<strong>an</strong>smission. Linkage <strong>an</strong>d association studies implicate TPH2 polymorphisms in reduced<br />
serotonin levels in mice, suggesting variability in TPH2 function or expression may underlie<br />
vulnerability. However, despite <strong>the</strong> growing number of genomic studies, little is known<br />
regarding <strong>the</strong> regulation of TPH2. Here, we sought to assess whe<strong>the</strong>r TPH2 expression is<br />
regulated by microRNAs. Using a novel algorithm accounting <strong>for</strong> gapped sequence matches, we<br />
identified <strong>an</strong> evolutionarily conserved target <strong>for</strong> miR-370 in <strong>the</strong> TPH2 5’ untr<strong>an</strong>slated region<br />
(UTR). In vitro, co-tr<strong>an</strong>sfection of miR-370 dsRNA with a TPH2 5’-UTR regulated construct<br />
demonstrates that <strong>the</strong> TPH2 5’ UTR is a functional miR-370 target. Interestingly, miR-370<br />
targets a sequence which overlaps <strong>an</strong> neural restrictive silencing element (NRSE), a sequence<br />
which recruits <strong>the</strong> epigenetic repressor, Re-1 Silencing Tr<strong>an</strong>scription Factor (REST). However,<br />
no interactions were observed between miR-370 <strong>an</strong>d REST repression. Finally, we per<strong>for</strong>med a<br />
developmental profile of miR-370 in embryonic, postnatal, <strong>an</strong>d adult mice. In situ hybridization<br />
suggests low levels of expression across development <strong>an</strong>d tissue, with minimal levels of neuronal<br />
expression in adult. These results suggest that miR-370 likely functions as a global basal
epressor of TPH2 expression. In summary, our results demonstrate that miR-370 regulates<br />
TPH2 <strong>an</strong>d provides support <strong>for</strong> miRNA targeting outside of <strong>the</strong> 3’ UTR.<br />
Disclosures: L. Own, None; P.D. Patel, None.<br />
Poster<br />
548. Mood Disorders: Hum<strong>an</strong> Biomarkers (Imaging, Cellular, <strong>an</strong>d Molecular)<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 548.13/W31<br />
Topic: C.16.d. Mood disorders: Animal models<br />
Support: Martin & Sharleen Cohen Foundation <strong>for</strong> Biomedical Research<br />
Title: Whole, <strong>an</strong>terior <strong>an</strong>d posterior hippocampal volumes in behaviorally depressed<br />
cynomolgus macaques (Macaca fascicularis): A magnetic reson<strong>an</strong>ce imaging study<br />
Authors: *S. L. WILLARD 1 , R. A. KRAFT 2 , J. B. DAUNAIS 3 , C. A. SHIVELY 4 ;<br />
1 Neurosci. Program, 2 Biomed. Engin., 3 Physiol. & Pharmacol., 4 Pathology (Comparative<br />
Medicine), Wake Forest Univ. Sch. of Med., Winston-Salem, NC<br />
Abstract: Alterations in hippocampal (HC) volume have been implicated in <strong>the</strong> pathophysiology<br />
of depression. Meta-<strong>an</strong>alyses suggest that HC volume measured with MRI is reduced bilaterally<br />
in depressed patients. Hum<strong>an</strong> <strong>an</strong>d rodent studies indicate functional differentiation along <strong>the</strong><br />
<strong>an</strong>terior-posterior extent of <strong>the</strong> HC such that <strong>the</strong> <strong>an</strong>terior HC is associated with emotional <strong>an</strong>d<br />
<strong>an</strong>xiety-related behavioral functioning. Hum<strong>an</strong> studies are complicated by heterogeneity in<br />
subject characteristics known to differentially affect HC volume, <strong>an</strong>d few report separate<br />
measurement of <strong>an</strong>terior <strong>an</strong>d posterior HC. We recently showed <strong>an</strong> average 16% reduction in<br />
<strong>an</strong>terior HC volume in post mortem assessments of behaviorally depressed monkeys. To confirm<br />
<strong>the</strong>se observations in vivo, we used MRI to measure whole, <strong>an</strong>terior, <strong>an</strong>d posterior HC volumes<br />
in a matched sample of adult female cynomolgus macaques characterized <strong>for</strong> behavioral<br />
depression (n=6 depressed, 6 nondepressed). High resolution structural MRIs were acquired <strong>for</strong><br />
each monkey using a 3 Tesla GE sc<strong>an</strong>ner, <strong>an</strong>d T1-weighted images were converted to<br />
ANALYZE <strong>for</strong>mat. Using MRIcro, HC regions of interest (ROIs) were m<strong>an</strong>ually segmented on<br />
each 2D slice of all pl<strong>an</strong>ar acquisitions within. Volumes were computed by adding voxels within<br />
<strong>the</strong> ROI <strong>from</strong> each slice <strong>an</strong>d multiplying by <strong>the</strong> voxel volume. HC structures included in<br />
volumetric <strong>an</strong>alyses were <strong>the</strong> cornu ammonis (CA1-CA3), dentate gyrus, subiculum, <strong>an</strong>d alveus.<br />
Anterior <strong>an</strong>d posterior HC were delineated by <strong>the</strong> presence of <strong>the</strong> uncus at <strong>the</strong> point at which <strong>the</strong><br />
HC head is separate <strong>from</strong> <strong>the</strong> body. Preliminary results indicate global HC volume reductions in
depressed compared to nondepressed monkeys, with <strong>an</strong> average 22% reduction in whole<br />
(p
impulsivity using <strong>the</strong> Barratt Impulsiveness Scale (BIS) <strong>an</strong>d mental <strong>an</strong>d personality disorders<br />
following DSM-IV using SCID-I <strong>an</strong>d SCID-II (Structured Clinical Interview <strong>for</strong> DSM-IV).<br />
The 25% of <strong>the</strong>se subjects showed a mental disease according to SCID-I of DSM-IV. In<br />
particular, 29% of <strong>the</strong> subjects presented major depressive disorders in <strong>the</strong> past <strong>an</strong>d 4% at <strong>the</strong><br />
moment of <strong>the</strong> study, 15% depressive disorder not o<strong>the</strong>rwise specified; 32% has suffered of<br />
alcohol dependence <strong>an</strong>d 31% of alcohol abuse, 11% of post-traumatic stress disorder, 7% of<br />
social phobia. According to Axis-II 75% of <strong>the</strong>se subjects had personality disorders <strong>an</strong>d among<br />
<strong>the</strong>m 50% presented <strong>an</strong>tisocial behavior.<br />
A signific<strong>an</strong>t decrease in 5-HTP levels was observed in subjects with a self-reported general<br />
aggressive behaviour of MacCVI (0.059 ± 0.003 µg/ml vs 0.079 ± 0.007 µg/ml; me<strong>an</strong> ± S.E.).<br />
5-HTP levels had also a negative <strong>an</strong>d signific<strong>an</strong>t correlation (Spearm<strong>an</strong>’s rho= -0.238; p=0.022)<br />
with <strong>the</strong> total number of aggressive acts measured using <strong>the</strong> MacCVI.<br />
5-HTP serum levels showed a signific<strong>an</strong>t <strong>an</strong>d negative correlation with <strong>the</strong> attentional<br />
impulsiveness of BIS (Spearm<strong>an</strong>’s rho= -0.226; p=0.03) as well as 5-HTP/Trp ratio (Spearm<strong>an</strong>’s<br />
rho= -0.252; p=0.015), <strong>an</strong> index of <strong>the</strong> tryptoph<strong>an</strong> hydroxylase activity.<br />
5-HTP/Trp ratio is also signific<strong>an</strong>tly decreased (5.53 ± 0.33 vs 7.75 ± 0.91) in subjects with a<br />
personality disorders according to SCID-II. Our preliminary data confirm that in people with<br />
violent behavior <strong>the</strong>re are ch<strong>an</strong>ges in <strong>the</strong> serum concentrations of tryptoph<strong>an</strong> metabolites via<br />
serotonin thus influencing <strong>the</strong>ir brain availability <strong>for</strong> <strong>the</strong> syn<strong>the</strong>sis of <strong>the</strong> neurotr<strong>an</strong>smitter<br />
serotonin. Fur<strong>the</strong>rmore, <strong>the</strong> decreases of 5-HTP serum levels <strong>an</strong>d of 5-HTP/Trp ratio fur<strong>the</strong>r<br />
confirm <strong>the</strong> hypo<strong>the</strong>sis that serotonin deficiency is secondary to a decrease in tryptoph<strong>an</strong><br />
hydroxylase activity.<br />
Disclosures: S. Comai, None; A. Bertazzo, None; J. Vachon, None; G. Cote, None; C.V.L.<br />
Costa, None; G. Gobbi, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.1/W33<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: A neuropeptide y y5 receptor <strong>an</strong>tagonist exerts <strong>an</strong>xiolytic-like <strong>an</strong>d <strong>an</strong>tidepress<strong>an</strong>t-like<br />
effects<br />
Authors: A. W. LEE 1 , T. D. WOLINSKY 1 , V. JUBIAN 1 , G. CHANDRASENA 1 , H. ZHONG 1 ,<br />
X. HUANG 1 , S. MILLER 1 , L. G. HEGDE 1 , D. A. MARSTELLER 1 , M. R. MARZABADI 1 , M.<br />
PAPP 2 , D. H. OVERSTREET 3 , C. P. G. GERALD 1 , D. A. CRAIG 1 , *M. W. WALKER 1 ;
1 Dept of Biol. Res., Lundbeck Res. USA, Paramus, NJ; 2 Inst. of Pharmacology, Polish Acad. of<br />
Sci., Krakow, Pol<strong>an</strong>d; 3 Univ. of North Carolina at Chapel Hill, Chapel Hill, NC<br />
Abstract: Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y1,<br />
Y2, Y4, Y5, y6). The Y5 receptor is well-known <strong>for</strong> its role in appetite. Based on expression in <strong>the</strong><br />
limbic system, we hypo<strong>the</strong>sized that <strong>the</strong> Y5 receptor might also modulate stress-sensitivity. We<br />
identified a novel Y5 receptor-selective <strong>an</strong>tagonist, Lu AA33810, that bound to cloned rat Y5<br />
receptors (Ki = 1.5 nM) <strong>an</strong>d <strong>an</strong>tagonized NPY-evoked cAMP <strong>an</strong>d calcium mobilization in vitro.<br />
Lu AA33810 (3-30 mg/kg, p.o.) blocked feeding elicited by i.c.v. injection of Y5 receptorselective<br />
agonist cPP(1-7), NPY(19-23),Ala 31 ,Aib 32 , Gln 34 ] in Sprague-Dawley rats. In vivo<br />
effects of Lu AA33810 were correlated with brain exposure > 50 ng/g <strong>an</strong>d ex vivo Y5 receptor<br />
occup<strong>an</strong>cy of 22-95%. Lu AA33810 was subsequently evaluated in models of stress-sensitivity.<br />
In Fischer 344 rats, Lu AA33810 (30 mg/kg, p.o.) attenuated increases in plasma ACTH <strong>an</strong>d<br />
cortisol elicited by i.c.v. injection of cPP(1-7),NPY(19-23),Ala 31 ,Aib 32 , Gln 34 ]. In Sprague<br />
Dawley rats subjected to <strong>the</strong> social interaction test, Lu AA33810 (3-30 mg/kg, p.o.) produced<br />
<strong>an</strong>xiolytic-like effects after acute or chronic treatment. In Flinders Sensitive Line rats, chronic<br />
dosing of Lu AA33810 (10 mg/kg/day, i.p.) produced <strong>an</strong>xiolytic-like effects in <strong>the</strong> social<br />
interaction test, plus <strong>an</strong>tidepress<strong>an</strong>t-like effects in <strong>the</strong> <strong>for</strong>ced swim test. In Wistar rats exposed to<br />
chronic mild stress, chronic dosing of Lu AA33810 (3, 10 mg/kg/day, i.p.) produced<br />
<strong>an</strong>tidepress<strong>an</strong>t-like activity, i.e. normalization of stress-induced decrease in sucrose<br />
consumption. We propose that Y5 receptors may function as part of <strong>an</strong> endogenous stress-sensing<br />
system to mediate social <strong>an</strong>xiety <strong>an</strong>d reward- or motivational-deficits in selected rodent models.<br />
We are currently investigating <strong>the</strong> underlying neuronal circuits using c-fos<br />
immunohistochemistry as a detection method.<br />
Disclosures: A.W. Lee, Lundbeck Research USA, A. Employment (full or part-time); T.D.<br />
Wolinsky, Lundbeck Research USA, A. Employment (full or part-time); V. Jubi<strong>an</strong>, Lundbeck<br />
Research USA, A. Employment (full or part-time); G. Ch<strong>an</strong>drasena, Lundbeck Research USA,<br />
A. Employment (full or part-time); H. Zhong, Lundbeck Research USA, A. Employment (full or<br />
part-time); X. Hu<strong>an</strong>g, Lundbeck Research USA, A. Employment (full or part-time); S. Miller,<br />
Lundbeck Research USA, A. Employment (full or part-time); L.G. Hegde, Lundbeck Research<br />
USA, A. Employment (full or part-time); D.A. Marsteller, Lundbeck Research USA, A.<br />
Employment (full or part-time); M.R. Marzabadi, Lundbeck Research USA, A. Employment<br />
(full or part-time); M. Papp, Lundbeck Research USA, C. O<strong>the</strong>r Research Support (receipt of<br />
drugs, supplies, equipment or o<strong>the</strong>r in-kind support); D.H. Overstreet, Lundbeck Research<br />
USA, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind<br />
support); C.P.G. Gerald, Lundbeck Research USA, A. Employment (full or part-time); D.A.<br />
Craig, Lundbeck Research USA, A. Employment (full or part-time); M.W. Walker, Lundbeck<br />
Research USA, A. Employment (full or part-time).<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.2/W34<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: NIMH MH72894<br />
Title: Modulation of behavior by non-erythropoietic EPO-like compounds<br />
Authors: *C. H. DUMAN 1 , M. BRINES 2 , S. S. NEWTON 1 ;<br />
1 Dept. of Psychiatry, Yale Univ., New Haven, CT; 2 Warren Pharmaceuticals, Ossining, NY<br />
Abstract: Erythropoietin (EPO) has been widely studied <strong>for</strong> its role in elevating erythropoiesis<br />
<strong>an</strong>d is used successfully <strong>for</strong> <strong>the</strong> treatment <strong>for</strong> <strong>an</strong>emia. The expression of EPO receptors in<br />
neuronal cell lines <strong>an</strong>d brain suggests that actions of EPO are not restricted to hematopoiesis. In<br />
fact, subst<strong>an</strong>tial evidence indicates that EPO c<strong>an</strong> exert robust neurotrophic <strong>an</strong>d <strong>an</strong>giogenic<br />
effects in <strong>the</strong> central nervous system (Ribatti et al., 2003; Brines <strong>an</strong>d Cerami, 2005). The trophic<br />
hypo<strong>the</strong>sis of depression, which states that deficits in trophic support <strong>an</strong>d signaling could<br />
contribute to neuronal atrophy, dysfunction <strong>an</strong>d behavioral depression while <strong>an</strong>tidepress<strong>an</strong>ts are<br />
able to elevate trophic signaling <strong>an</strong>d alleviate or reverse <strong>the</strong>se abnormalities, suggested that EPO<br />
might have <strong>an</strong>tidepress<strong>an</strong>t properties. Our recent study confirmed that EPO is induced in brain<br />
by <strong>an</strong> <strong>an</strong>tidepress<strong>an</strong>t treatment <strong>an</strong>d demonstrated that EPO c<strong>an</strong> produce <strong>an</strong>tidepress<strong>an</strong>t-like<br />
behavioral responses in rodent models (Girgenti et al., <strong>2009</strong>). These results are consistent with<br />
hum<strong>an</strong> studies showing that EPO improves mood (Miskowiak et al., 2008).<br />
The investigation of potential <strong>the</strong>rapeutic utility of EPO in depression is currently impeded by<br />
<strong>the</strong> erythropoietic effect that is seen with long term administration . In order to address this<br />
limitation we are testing non-erythropoietic EPO derivatives/compounds in rodent behavioral<br />
models. WP206 is <strong>an</strong> EPO-derivative that differs <strong>from</strong> native EPO in structure <strong>an</strong>d in EPO<br />
receptor binding affinity. We administered WP206 to mice <strong>for</strong> 10 days via continuous<br />
subcut<strong>an</strong>eous infusion using osmotic minipumps (Alzet, Durect Corp., Cupertino, CA). We<br />
measured freezing behavior following fear conditioning to a tone cue as a well-characterized<br />
behavioral measure that is sensitive to alterations in neuronal plasticity. This administration was<br />
well tolerated <strong>an</strong>d produced no overt behavioral abnormalities in mice. Freezing in response to<br />
<strong>the</strong> tone, measured 24 hr after conditioning was signific<strong>an</strong>tly increased in WP206-treated mice.<br />
Freezing behavior was not signific<strong>an</strong>tly altered in response to <strong>the</strong> conditioning context. The<br />
ability of WP206 administration to alter fear-conditioned behavior indicates that a nonerythropoietic<br />
EPO derivative which does not elevate hematopoiesis even when continuously,<br />
administered at high doses, c<strong>an</strong> effectively influence this measure of neural plasticity. Fur<strong>the</strong>r<br />
behavioral characterization <strong>an</strong>d investigation of correlative gene expression ch<strong>an</strong>ges will extend<br />
our underst<strong>an</strong>ding of mech<strong>an</strong>isms that may underlie <strong>the</strong> neurotrophic actions of EPO <strong>an</strong>d EPOlike<br />
compounds.
Disclosures: C.H. Dum<strong>an</strong>, None; M. Brines, Warren Pharmaceuticals, A. Employment (full or<br />
part-time); Warren Pharmaceuticals, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); S.S. Newton, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.3/W35<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Servier support<br />
Title: The novel <strong>an</strong>tidepress<strong>an</strong>t agomelatine normalizes hippocampal neuronal activity <strong>an</strong>d<br />
stimulates neurogenesis in chronically stressed rats<br />
Authors: *G. DAGYTE 1,2 , E. A. VAN DER ZEE 1 , P. MEERLO 1 , F. POSTEMA 1 , A.<br />
TRENTANI 2 , C. GABRIEL 3 , E. MOCAER 3 , P. G. M. LUITEN 1 , J. A. DEN BOER 2 ;<br />
1 Dept. of Mol. Neurobio., Univ. of Groningen, Haren, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Dept. of Psychiatry, Univ.<br />
of Groningen, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 Inst. de Recherches Internationales Servier, Courbevoie,<br />
Fr<strong>an</strong>ce<br />
Abstract: Agomelatine is a novel <strong>an</strong>tidepress<strong>an</strong>t which acts as a melatonergic (MT1/MT2)<br />
receptor agonist <strong>an</strong>d 5-HT2C receptor <strong>an</strong>tagonist. The <strong>an</strong>tidepress<strong>an</strong>t properties of agomelatine<br />
have been demonstrated in <strong>an</strong>imal models as well as in clinical studies. Several preclinical<br />
studies report agomelatine-induced effects on brain plasticity, mainly under basal conditions<br />
(1,2). Yet, it is import<strong>an</strong>t to unravel agomelatine-mediated ch<strong>an</strong>ges in <strong>the</strong> brain affected by<br />
psychopathology. Since stress is involved in <strong>the</strong> etiology of depression, chronic stress in<br />
laboratory <strong>an</strong>imals is used to create a vulnerable brain, a model suitable to investigate<br />
<strong>an</strong>tidepress<strong>an</strong>t-induced effects. Here, we sought to determine ch<strong>an</strong>ges in <strong>the</strong> brain after<br />
agomelatine treatment in chronically stressed rats. Adult male rats were treated with BrdU (300<br />
mg/kg i.p.) <strong>an</strong>d 4 days later subjected to daily footshock stress (3) <strong>an</strong>d agomelatine treatment (40<br />
mg/kg i.p.), <strong>for</strong> 21 consecutive days. Control rats only received daily injections with vehicle (1%<br />
HEC). Blood samples were collected in order to assess plasma levels of ACTH <strong>an</strong>d<br />
corticosterone. Samples were taken be<strong>for</strong>e <strong>an</strong>d after <strong>the</strong> footshock session on day 1, 11 <strong>an</strong>d 21 of<br />
<strong>the</strong> experiment. One day after <strong>the</strong> last stress exposure <strong>an</strong>d agomelatine treatment, brains were<br />
collected <strong>an</strong>d processed <strong>for</strong> immunohistochemistry. Rats subjected to a chronic footshock<br />
procedure showed robust increases in ACTH <strong>an</strong>d corticosterone. Simult<strong>an</strong>eous treatment with<br />
agomelatine led to a blunting of <strong>the</strong> stress-induced ACTH response at <strong>the</strong> end of <strong>the</strong> 3-week
procedure but did not ch<strong>an</strong>ge <strong>the</strong> corticosterone response. Moreover, whereas chronic exposure<br />
to daily footshock stress reduced c-Fos expression in <strong>the</strong> dentate gyrus of <strong>the</strong> hippocampus,<br />
agomelatine treatment reversed this effect <strong>an</strong>d normalized neuronal activity to basal levels. Also,<br />
chronic agomelatine led to increased hippocampal cell proliferation (Ki-67 labeling) <strong>an</strong>d survival<br />
(BrdU labeling) in stressed but not in control rats. Fur<strong>the</strong>rmore, agomelatine treatment reversed<br />
<strong>the</strong> stress-induced decrease in <strong>the</strong> number of doublecortin-positive neurons. Taken toge<strong>the</strong>r,<br />
<strong>the</strong>se data show that agomelatine counteracts <strong>the</strong> deleterious effects of stress on <strong>the</strong> brain by<br />
restoring stress-affected neuronal activation <strong>an</strong>d promoting hippocampal neurogenesis, which is<br />
in line with <strong>the</strong> previous findings in <strong>an</strong>imal models of depression (4,5).<br />
(1) B<strong>an</strong>asr et al., Biol Psychiatry 2006, 59:1087-1096.<br />
(2) Conboy et al., Int J Neuropsychopharmacol <strong>2009</strong>, 12:329-341.<br />
(3) Trent<strong>an</strong>i et al., Neurobiol Dis 2003, 14:602-618.<br />
(4) Maccari et al., Am Soc Neurosci, 2005, 566.8.<br />
(5) Païz<strong>an</strong>is et al., Eur Neuropsychopharmacol 2008, 18 (1), S316.<br />
Disclosures: G. Dagyte, None; E.A. V<strong>an</strong> der Zee, None; P. Meerlo, None; F. Postema,<br />
None; A. Trent<strong>an</strong>i, None; C. Gabriel, None; E. Mocaer, None; P.G.M. Luiten, None; J.A.<br />
Den Boer, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.4/W36<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: Exendin-4 stimulates hippocampal neurogenesis <strong>an</strong>d exerts affective <strong>an</strong>d cognition<br />
associated effects in behavioral models<br />
Authors: O. ZACHRISSON, R. ISACSON, E. NIELSEN, G. BERTILSSON, K. DANNAEUS,<br />
*L. B. WIKSTROM;<br />
NeuroNova AB, Stockholm, Sweden<br />
Abstract: Neurogenesis in <strong>the</strong> adult hippocampus plays a central role in key aspects of<br />
hippocampal functioning. The glucagon-like peptide 1 receptor agonist exendin-4 (Ex-4) has<br />
been shown to stimulate neurogenesis in <strong>the</strong> SVZ/medial striatum as previously shown<br />
(Bertilsson et al, J Neurosci Res, 2008). The aim of this study was to assess <strong>the</strong> effect of Ex-4<br />
treatment in rodents on hippocampal neurogenesis, <strong>an</strong>d on mood <strong>an</strong>d cognition associated<br />
behavior. Ex-4 is used clinically (Byetta) to treat diabetes type II <strong>an</strong>d displays gluco-
egulatory effects including glucose dependent enh<strong>an</strong>cement of insulin secretion <strong>an</strong>d glucose<br />
dependent suppression of high glucagon secretion. Following one week bidaily treatment with<br />
Ex-4, BrdU incorporation, <strong>an</strong> indicator of cellular proliferation, was increased in <strong>the</strong><br />
hippocampal dentate gyrus of mice in a dose dependent m<strong>an</strong>ner. Moreover, <strong>the</strong> mRNA<br />
expression of Ki67, a mitotic marker, Mash-1, a marker <strong>for</strong> neuronal differentiation, <strong>an</strong>d<br />
doublecortin, a marker <strong>for</strong> neuronal progenitors, were all upregulated in <strong>the</strong> dentate gyrus of<br />
hippocampus following treatment with Ex-4 as seen using qu<strong>an</strong>titative PCR. In addition, <strong>the</strong><br />
number of doublecortin positive cells was increased in <strong>the</strong> rat hippocampal dentate gyrus as<br />
shown by histochemical staining. To test <strong>the</strong> effect on behavior, radial maze <strong>an</strong>d <strong>for</strong>ced swim<br />
test were employed. Rodents were treated with Ex-4, 0.1µg/kg, bi-daily <strong>for</strong> 1-2 weeks. The time<br />
necessary <strong>for</strong> rats to solve a radial maze task <strong>an</strong>d <strong>the</strong> duration of immobility <strong>for</strong> mice in <strong>the</strong><br />
<strong>for</strong>ced swim test were signific<strong>an</strong>tly reduced compared to respective vehicle groups. In contrast to<br />
<strong>the</strong> positive control imipramine, a single injection of Ex-4 1 h prior to <strong>the</strong> challenge in <strong>the</strong> <strong>for</strong>ced<br />
swim test had no effect. It is worth noting that only chronic treatment precipitated <strong>the</strong> behavioral<br />
effects, suggesting that a (neuro)plastic event is involved in <strong>the</strong> key aspects of its mech<strong>an</strong>ism.<br />
Taken toge<strong>the</strong>r, <strong>the</strong> ch<strong>an</strong>ge in behavior <strong>an</strong>d <strong>the</strong> increase of mRNA <strong>an</strong>d protein expression seen<br />
following chronic treatment with Ex-4 are conson<strong>an</strong>t to <strong>an</strong> increase in hippocampal<br />
neurogenesis. Thus, Ex-4 is proposed as a c<strong>an</strong>didate <strong>for</strong> <strong>the</strong> alleviation of mood <strong>an</strong>d cognitive<br />
disorders, through its action on hippocampal neurogenesis in <strong>the</strong> adult brain.<br />
Disclosures: O. Zachrisson, NeuroNova AB, A. Employment (full or part-time); R. Isacson,<br />
NeuroNova AB, A. Employment (full or part-time); E. Nielsen, NeuroNova AB, A. Employment<br />
(full or part-time); G. Bertilsson, NeuroNova AB, A. Employment (full or part-time); K.<br />
D<strong>an</strong>naeus, NeuroNova AB, A. Employment (full or part-time); L.B. Wikstrom, NeuroNova<br />
AB, A. Employment (full or part-time).<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.5/W37<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: NIH MH077681<br />
NIH DA00436.<br />
Title: Effect of acetylcholine esterase activity on depression-like behaviors in mice
Authors: *Y. S. MINEUR, A. OBAYEMI, Jr., M. R. PICCIOTTO;<br />
Psychiatry, Yale Univ. Sch. Med., New Haven, CT<br />
Abstract: Recent studies suggest that decreasing cholinergic tone in <strong>the</strong> brain through blockade<br />
of high affinity nicotinic acetylcholine receptors (nAChRs) results in <strong>an</strong>tidepress<strong>an</strong>t-like effects<br />
in both hum<strong>an</strong>s <strong>an</strong>d <strong>an</strong>imal models. This is consistent with early work demonstrating that<br />
treatment with physostigmine (<strong>an</strong> acetylcholinesterase (AChE) inhibitor that increases<br />
cholinergic tone by decreasing activity of <strong>the</strong> principal ACh degrading enzyme) increases <strong>the</strong><br />
symptoms of unipolar depression. These data suggest that increasing cholinergic activity could<br />
result in pro-depress<strong>an</strong>t-like effects <strong>an</strong>d that decreasing cholinergic signaling could be<br />
<strong>an</strong>tidepress<strong>an</strong>t. We tested this hypo<strong>the</strong>sis by altering AChE activity in mouse brain <strong>an</strong>d<br />
investigating its relationship with depression-like behaviors in mice.<br />
Acute treatment with various doses of physostigmine induced a pro-depress<strong>an</strong>t-like effect in <strong>the</strong><br />
tail suspension test, a model of <strong>an</strong>tidepress<strong>an</strong>t efficacy. Concomit<strong>an</strong>tly, <strong>an</strong> overall dosedependent<br />
decrease of acetylcholinesterase activity was noted in several brain regions.<br />
Interestingly, hippocampal levels of AChE activity correlated well with <strong>the</strong> behavioral results.<br />
<strong>When</strong> infused into <strong>the</strong> hippocampus, physotigmine induced a pro-depress<strong>an</strong>t-like effect, fur<strong>the</strong>r<br />
suggesting that AChE activity in this brain area may be involved in mediating depression-like<br />
behaviors.<br />
Unexpectedly, chronic treatment (14 days) with several AChE inhibitors induced <strong>an</strong><br />
<strong>an</strong>tidepress<strong>an</strong>t-like effect in <strong>the</strong> tail suspension test. The effect size was similar to that induced<br />
by fluoxetine, a classical <strong>an</strong>tidepress<strong>an</strong>t. Fur<strong>the</strong>r <strong>an</strong>alyses revealed that both AChE<br />
concentrations <strong>an</strong>d activity levels were increased following chronic treatment with AChE<br />
inhibitors, suggesting a functional compensation induced by chronic inhibition. Interestingly,<br />
chronic treatment with fluoxetine dramatically increased AChE activity in several brain regions<br />
including <strong>the</strong> hippocampus.<br />
Taken toge<strong>the</strong>r, <strong>the</strong>se data strongly suggest that cholinergic activity is critical in regulating<br />
depression-like symptoms. Fur<strong>the</strong>rmore, <strong>the</strong>se data suggest that treatment of Alzheimer’s <strong>an</strong>d<br />
Parkinson’s patients with AChE inhibitors could contribute to mood ch<strong>an</strong>ges in <strong>the</strong>se subjects.<br />
Disclosures: Y.S. Mineur, None; A. Obayemi, None; M.R. Picciotto, NIH MH077681 <strong>an</strong>d<br />
DA00436, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.6/W38
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: KB099520, a highly selective ER-beta agonist, exerts <strong>an</strong>tidepress<strong>an</strong>t effects in rodent<br />
behavioral models of depression<br />
Authors: *P. FAGERGREN 1 , A. A. WALF 2 , M. EL YACOUBI 3 , P. RHÖNNSTAD 1 , T.<br />
APELQVIST 1 , M. NILSSON 1 , S. NILSSON 1 , J.-M. VAUGEOIS 3 , C. A. FRYE 2 , M. K.<br />
ÖSTERLUND 1 ;<br />
1 Karobio AB, Huddinge, Sweden; 2 Psychology, Biology, Neuro- & Life Sci., Univ. of Alb<strong>an</strong>y,<br />
Alb<strong>an</strong>y, NY; 3 Lab. de Neuropharmacologie, Faculté de Pharmacie - Univ. de Lyon 1, Lyon,<br />
Fr<strong>an</strong>ce<br />
Abstract: Estrogens c<strong>an</strong> have <strong>an</strong>tidepress<strong>an</strong>t effects in part through actions at estrogen receptor<br />
beta (ER-beta). We have developed a highly selective ER-beta agonist, KB099520,<br />
demonstrating 600 fold ER-beta over ER-alpha potency in a cellular tr<strong>an</strong>s-activation gene<br />
reporter assay. The effects of KB099520 in several models of affective behavior are presented. In<br />
c57Bl/6 mice, both acute high dosing (30 mg/kg), <strong>an</strong>d sub-chronic moderate dosing (10 mg/kg),<br />
of KB099520 decreased time spent immobile in <strong>the</strong> <strong>for</strong>ced swim test (FST). Similarly, reduced<br />
immobility in <strong>the</strong> FST was demonstrated in ovariectomized Long Ev<strong>an</strong>s rats. Rats were<br />
administered KB099520 (10 <strong>an</strong>d 30 mg/kg) <strong>an</strong>d 17-beta estradiol (0.1 mg/kg) once weekly <strong>an</strong>d<br />
were tested 44-48 hours later in <strong>the</strong> elevated plus maze (EPM), light dark tr<strong>an</strong>sition (LTD), <strong>an</strong>d<br />
FST, each week respectively. Both KB099520 <strong>an</strong>d 17-beta estradiol signific<strong>an</strong>tly reduced time<br />
spent immobile in <strong>the</strong> FST, whereas no signific<strong>an</strong>t <strong>an</strong>xiolytic effects were seen in <strong>the</strong> EPM or<br />
LDT. To fur<strong>the</strong>r investigate <strong>an</strong>ti-depress<strong>an</strong>t activity of KB099520, <strong>an</strong> <strong>an</strong>imal model of<br />
endogenous depression, helpless H/Rouen mice, was studied. Similar to fluoxetine (10 mg/kg),<br />
KB099520 decreased time spent immobile in <strong>the</strong> tail suspension test (TST), at 1 but not 10<br />
mg/kg, already 30 minutes after <strong>the</strong> first s.c. injection. The drug administrations were continued<br />
daily <strong>for</strong> 21 days <strong>an</strong>d <strong>the</strong> <strong>an</strong>tidepress<strong>an</strong>t effect was sustained at every testing occasion (20 h after<br />
last administration at days 2, 8, 15 <strong>an</strong>d 22). No effects of KB099520 or fluoxetine were present<br />
in <strong>the</strong> control non-helpless NH/Rouen mice, ruling out unw<strong>an</strong>ted "depress<strong>an</strong>t" activity. Taken<br />
toge<strong>the</strong>r, we have demonstrated in several behavioral models of depression, <strong>an</strong>tidepress<strong>an</strong>t<br />
effects of a highly selective ER-beta agonist, KB099520.<br />
Disclosures: P. Fagergren, None; A.A. Walf, None; M. El Yacoubi, None; P. Rhönnstad,<br />
None; T. Apelqvist, None; M. Nilsson, None; S. Nilsson, None; J. Vaugeois, None; C.A. Frye,<br />
None; M.K. Österlund, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 549.7/X1<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: Pharmacological characteristics of SEP230864: Application towards <strong>the</strong> treatment of<br />
neuropsychiatric diseases<br />
Authors: *R. LEW, R. SCHREIBER, C. BOWEN, S. ENGEL, L. HARDY, J. MA, M.<br />
QUINTON, L. SHAO, N. SPICER, U. CAMPBELL;<br />
Discovery & Early Clin. Res., Sepracor Inc, Marlborough, MA<br />
Abstract: Monoamine uptake inhibitors are compounds that c<strong>an</strong> increase neurotr<strong>an</strong>smitter levels<br />
by blocking neuronal re-uptake <strong>an</strong>d are effective in <strong>the</strong> treatment of neuropsychiatric diseases<br />
<strong>an</strong>d some <strong>for</strong>ms of pain. However, elevation of 5-HT, NE & DA levels must be carefully titrated<br />
to maximize <strong>the</strong> efficacy to adverse effect ratio. In <strong>the</strong> present study, we describe <strong>the</strong> in vitro <strong>an</strong>d<br />
in vivo pharmacological characteristics of SEP230864, a monoamine uptake inhibitor of 5-HT,<br />
NE & DA <strong>an</strong>d consider possible disease indications based upon its pharmacological profile. In<br />
vitro functional uptake studies using CHO cells expressing ei<strong>the</strong>r hum<strong>an</strong> recombin<strong>an</strong>t SERT,<br />
NET or DAT, showed SEP230864 to block uptake at SERT, NET & DAT with potencies (IC50<br />
+ S.D) of 1 + 0.1 nM, 3 + 0.3 nM & 11 + 2 nM respectively. A similar pharmacological profile<br />
was observed with rat recombin<strong>an</strong>t SERT, NET & DAT although at lower potency (SERT, NET<br />
& DAT IC50 = 12 nM, 62 nM & 85 nM). Fur<strong>the</strong>rmore, a p<strong>an</strong>el screen including 50 receptor<br />
targets & ion ch<strong>an</strong>nels showed no activity at 10 uM SEP230864. In vivo studies showed <strong>the</strong><br />
MED <strong>for</strong> SEP230864 in <strong>the</strong> rat <strong>for</strong>ced swim test <strong>an</strong>d <strong>the</strong> mouse tail suspension test to be 30<br />
mg/kg po & 10 mg/kg po respectively. Analgesic studies (<strong>for</strong>malin flinch test & chronic<br />
constriction injury model) showed <strong>the</strong> MED of SEP230864 to be 30 mg/kg po. Rat microdialysis<br />
was used to examine <strong>the</strong> neurochemical mech<strong>an</strong>ism of SEP230864. In rat prefrontal cortex,<br />
SEP230864 at 3, 10 & 30 mg/kg po signific<strong>an</strong>tly increased 5-HT & NE levels up to ~ 500% over<br />
baseline respectively in a dose dependent m<strong>an</strong>ner. In striatum, SEP230864 at 30 mg/kg po<br />
signific<strong>an</strong>tly increased DA levels to ~ 200% above baseline. Ex vivo receptor occup<strong>an</strong>cy studies<br />
in mouse brain were also per<strong>for</strong>med. Using 3 H-Citalopram (SERT), 3 H-Nisoxetine (NET) & 3 H-<br />
WIN35428 (DAT), <strong>the</strong> RO50 <strong>for</strong> SEP230864 at SERT, NET & DAT was 3 mg/kg po, 1 mg/kg po<br />
<strong>an</strong>d >30 mg/kg po respectively. Finally, SPECT imaging studies in baboon were per<strong>for</strong>med to<br />
determine <strong>the</strong> efficacious dose r<strong>an</strong>ge of SEP230864. Using 123 I-mZINCT (SERT) <strong>an</strong>d 123 I-PE2I<br />
(DAT), <strong>the</strong> RO50 of SEP230864 was estimated at 3 ng/ml & 53 ng/ml total plasma concentration.<br />
The present studies indicate that SEP230864 is efficacious at SERT & NET <strong>an</strong>d may be effective<br />
in treating neuropsychiatric diseases such as depression <strong>an</strong>d some <strong>for</strong>ms of pain.<br />
Disclosures: R. Lew, None; R. Schreiber, None; C. Bowen, None; S. Engel, None; L. Hardy,<br />
None; J. Ma, None; M. Quinton, None; L. Shao, None; N. Spicer, None; U. Campbell, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.8/X2<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Sepracor Inc.<br />
Title: Pharmacological characterization of <strong>the</strong> triple monoamine tr<strong>an</strong>sporter uptake inhibitor SEP<br />
225289<br />
Authors: *R. SCHREIBER 1 , R. LEW 1 , L. HARDY 1 , T. CREMERS 2 , K. FANG 1 , U.<br />
CAMPBELL 1 ;<br />
1 Sepracor, Inc., Marlborough, MA; 2 Brains-On-Line, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Single <strong>an</strong>d dual inhibitors of tr<strong>an</strong>sporters <strong>for</strong> serotonin (SERT), norepinephrine<br />
(NET), <strong>an</strong>d dopamine (DAT) are effective <strong>for</strong> <strong>the</strong> treatment of various CNS disorders, including<br />
major depression. Clinical studies with <strong>the</strong> NET/DAT inhibitor bupropion as add-on treatment to<br />
o<strong>the</strong>r monoamine tr<strong>an</strong>sporter inhibitors reported improvements in efficacy <strong>an</strong>d tolerability,<br />
suggesting that compounds that combine inhibitory activity against all three tr<strong>an</strong>sporters might<br />
possess a superior <strong>the</strong>rapeutic profile. Herein, we report <strong>the</strong> pharmacological profiles of two such<br />
compounds: SEP-225289 (SEP-289) <strong>an</strong>d DOV-216303 (DOV). In in vitro functional uptake<br />
studies using a CHO cell line expressing hum<strong>an</strong> recombin<strong>an</strong>t SERT, NET or DAT, SEP-289 <strong>an</strong>d<br />
DOV blocked <strong>the</strong>se tr<strong>an</strong>sporters (IC50’s, SEP-289: 15, 4 <strong>an</strong>d 3 nM; DOV (Skolnick 2006): 14,<br />
20, <strong>an</strong>d 78 nM, respectively). A similar profile was obtained <strong>for</strong> inhibition of rat recombin<strong>an</strong>t<br />
tr<strong>an</strong>sporters (IC50’s, SEP-289: 14, 4 <strong>an</strong>d 2 nM; DOV (Skolnick 2003): 28, 35, <strong>an</strong>d 50 nM,<br />
respectively). In separate mouse ex vivo tr<strong>an</strong>sporter occup<strong>an</strong>cy studies using 3 H-citalopram<br />
(SERT), 3 H-nisoxetine (NET) <strong>an</strong>d 3 H-WIN35428 (DAT), <strong>the</strong> 50% tr<strong>an</strong>sporter occup<strong>an</strong>cy <strong>for</strong><br />
SEP-289 were 10, 5, <strong>an</strong>d 1 mg/kg po <strong>an</strong>d <strong>for</strong> DOV was 7, 2 <strong>an</strong>d >30 mg/kg, po, respectively.<br />
SPECT imaging studies in baboons using 123 I-mZINT (SERT) <strong>an</strong>d 123 I-PE2I (DAT) found that<br />
SEP-289 at 0.2 mg/kg iv inhibited binding at SERT by 22% (midbrain) <strong>an</strong>d at DAT by 87%<br />
(striatum) respectively. Functional effects were demonstrated in rat microdialysis studies; each<br />
compound increased monoamine levels in <strong>the</strong> prefrontal cortex <strong>an</strong>d striatum, however, SEP-289<br />
induced a greater increase in prefrontal DA levels th<strong>an</strong> DOV. In summary, in <strong>the</strong>se preclinical<br />
studies SEP-289 was a potent DAT inhibitor <strong>an</strong>d this profile may be of particular interest <strong>for</strong><br />
disorders where <strong>an</strong> increase in dopaminergic activity is desirable.<br />
Disclosures: R. Schreiber, Sepracor Inc., A. Employment (full or part-time); R. Lew, Sepracor<br />
Inc., A. Employment (full or part-time); L. Hardy, Sepracor Inc., A. Employment (full or parttime);<br />
T. Cremers, Brains-on-Line, A. Employment (full or part-time); University of Groningen,<br />
A. Employment (full or part-time); Brains-on-Line, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); K. F<strong>an</strong>g, Sepracor Inc., A. Employment (full or part-time);<br />
U. Campbell, Sepracor Inc., A. Employment (full or part-time).
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.9/X3<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: The novel triple reuptake inhibitor SEP-0228425 exhibits <strong>an</strong>tidepress<strong>an</strong>t, <strong>an</strong>xiolytic <strong>an</strong>d<br />
<strong>an</strong>algesic efficacy in rodent behavioral tests<br />
Authors: *U. C. CAMPBELL 1 , S. ENGEL 1 , L. HARDY 1 , M. HEWITT 1 , B. OLIVIER 2 , L.<br />
SHAO 1 , N. SPICER 1 , R. SCHREIBER 1 ;<br />
1 Sepracor, Marlboro, MA; 2 Univ. of Utrecht, Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: In <strong>the</strong> present study, <strong>the</strong> in vitro, ex vivo <strong>an</strong>d behavioral profile of <strong>the</strong> novel triple<br />
reuptake inhibitor, SEP-0228425 was characterized. In vitro, SEP-0228425 inhibited serotonin<br />
(5-HT), norepinephrine (NE) <strong>an</strong>d dopamine (DA) uptake by recombin<strong>an</strong>t hum<strong>an</strong> tr<strong>an</strong>sporters<br />
with IC50 values of 0.8 ± 0.4, 10 ± 5.3, <strong>an</strong>d 28 ± 0.4 nM, respectively. SEP-0228425 also<br />
inhibited <strong>the</strong> uptake of 5-HT, NE <strong>an</strong>d DA into rat brain synaptosomes with IC50 values of 5.0 ±<br />
1.4, 3.2 ± 0.5, <strong>an</strong>d 25 ± 2.6 nM, respectively. Mouse ex vivo binding studies showed that SEP-<br />
0228425 had similar affinity <strong>for</strong> cortical 5-HT <strong>an</strong>d NE tr<strong>an</strong>sporters (OCC50 = 2.8 <strong>an</strong>d 2.8 mg/kg<br />
PO, respectively), with approximately 8-fold lower affinity <strong>for</strong> striatal DA tr<strong>an</strong>sporters (0CC50 =<br />
20.6 mg/kg PO). The <strong>an</strong>tidepress<strong>an</strong>t-like profile of SEP-0228425 was evaluated using <strong>the</strong> mouse<br />
tail suspension (TS) <strong>an</strong>d rat <strong>for</strong>ced swim (FS) tests. In both tests, SEP-0228425 dose-<br />
dependently (3-30 mg/kg, PO) reduced time spent immobile <strong>an</strong>d efficacy was comparable to that<br />
of desipramine (20 mg/kg, IP). SEP-0228425 did not alter baseline locomotor activity indicating<br />
no general locomotor activating or inhibitory effect. In a rat model of sexual dysfunction, unlike<br />
paroxetine, no inhibition of sexual behavior was observed following chronic SEP-0228425 (30<br />
mg/kg, PO) administration. Anxiolytic-like effects of SEP-0228425 were demonstrated using a<br />
guinea pig pup distress vocalization model. In this model, SEP-0228425 dose-dependently (3-30<br />
mg/kg, IP) reduced total number <strong>an</strong>d duration of vocalizations <strong>an</strong>d efficacy was similar to <strong>the</strong><br />
positive control fluoxetine (10 mg/kg, IP). The effects of SEP-0228425 (3-30 mg/kg, PO) were<br />
also evaluated in rat models of acute (hot plate, tail flick) <strong>an</strong>d neuropathic (spinal nerve ligation,<br />
SNL) pain. SEP-0228425 displayed no <strong>an</strong>algesic effects in rat models of acute <strong>the</strong>rmal pain <strong>an</strong>d<br />
robust <strong>an</strong>tihyperalgesic effects were observed in <strong>the</strong> SNL model of neuropathic pain.<br />
Antihyperalgesic efficacy of SEP-0228425 was of a magnitude comparable to or greater th<strong>an</strong><br />
that observed with gabapentin (100 mg/kg, IP). Taken toge<strong>the</strong>r, <strong>the</strong> results indicate that SEP-<br />
0228425 is a novel triple reuptake inhibitor which may have efficacy in <strong>the</strong> treatment of<br />
depression, <strong>an</strong>xiety <strong>an</strong>d neuropathic pain or o<strong>the</strong>r persistent pain states in hum<strong>an</strong>s.
Disclosures: U.C. Campbell, None; S. Engel, None; L. Hardy, None; M. Hewitt, None; B.<br />
Olivier, None; L. Shao, None; N. Spicer, None; R. Schreiber, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.10/X4<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: H. Lundbeck A/S<br />
Title: Pharmacological profile of Lu AA21004, a novel multitarget drug <strong>for</strong> <strong>the</strong> treatment of<br />
mood disorders<br />
Authors: *A. MORK 1 , L. T. BRENNUM 1 , S. M. FALLON 2 , S. BISULCO 2 , K.<br />
FREDERIKSEN 1 , B. BANG-ANDERSEN 1 , A. B. LASSEN 1 , H. ZHONG 2 , J. G. PATEL 2 , P.<br />
POON 2 , D. G. SMITH 2 , C. SÁNCHEZ 2 , T. B. STENSBØL 1 , S. HOGG 1 ;<br />
1 H. Lundbeck A/S, Copenhagen-Valby, Denmark; 2 Lundbeck Res. USA, Paramus, NJ<br />
Abstract: Signific<strong>an</strong>t unmet needs in <strong>the</strong> treatment of depression <strong>an</strong>d <strong>an</strong>xiety remain, despite <strong>the</strong><br />
existence of several current treatment options. Lu AA21004 is a novel compound with a unique<br />
pharmacological profile. Lu AA21004 is a 5-HT enh<strong>an</strong>cer with <strong>an</strong>tagonist effect at h5-HT3<br />
receptors (Ki = 4.5 nM), agonist effect at h5-HT1A receptors (Ki = 15 nM) <strong>an</strong>d inhibitory effect on<br />
<strong>the</strong> serotonin tr<strong>an</strong>sporter (IC50 = 5.4 nM). The aim of <strong>the</strong> present research was to study <strong>the</strong><br />
effects of Lu AA21004 on neurotr<strong>an</strong>smitter levels in <strong>the</strong> brain <strong>an</strong>d to test <strong>the</strong> compound in<br />
preclinical models predictive of <strong>an</strong>xiolytic- <strong>an</strong>d <strong>an</strong>tidepress<strong>an</strong>t-like effects. The acute effects of<br />
Lu AA21004 (2.5 - 10 mg/kg, sc) on extracellular neurotr<strong>an</strong>smitter [acetylcholine (ACh),<br />
norepinephrine (NE), dopamine (DA), serotonin (5-HT)] levels were measured by microdialysis<br />
in <strong>the</strong> prefrontal cortex <strong>an</strong>d ventral hippocampus of freely moving rats. Subchronic effects of Lu<br />
AA21004 on extracellular 5-HT levels in <strong>the</strong> hippocampus <strong>an</strong>d 5-HT tr<strong>an</strong>sporter (5-HTT)<br />
occup<strong>an</strong>cy were measured following a 3-day administration (5 mg/kg/day, sc, by osmotic<br />
minipumps). In vivo 5-HTT occup<strong>an</strong>cy of Lu AA21004 following <strong>the</strong> 3-day administration was<br />
determined using 3 H-MADAM as radiolig<strong>an</strong>d. Effects of Lu AA21004 on <strong>the</strong> 5-HT3 receptor<br />
were studied by measuring <strong>the</strong> Bezold-Jarisch-like reflex in rats. Antidepress<strong>an</strong>t potential was<br />
assessed in <strong>the</strong> mouse <strong>for</strong>ced swim <strong>an</strong>d tail suspension tests, <strong>an</strong>d <strong>an</strong>xiolytic potential was<br />
assessed using <strong>the</strong> rat conditioned fear <strong>an</strong>d rat social interaction tests. Lu AA21004 signific<strong>an</strong>tly<br />
increased extracellular ACh, NE, DA <strong>an</strong>d 5-HT levels in a dose-dependent m<strong>an</strong>ner. After a 3-day<br />
administration, Lu AA21004 signific<strong>an</strong>tly increased extracellular 5-HT levels at low (41%) 5-
HTT occup<strong>an</strong>cy. Lu AA21004 dose-dependently inhibited <strong>the</strong> Bezold-Jarisch-like reflex,<br />
demonstrating a 5-HT3 receptor <strong>an</strong>tagonistic action in vivo. Lu AA21004 produced dosedependent<br />
<strong>an</strong>tidepress<strong>an</strong>t-like effects in <strong>the</strong> mouse <strong>for</strong>ced swim - <strong>an</strong>d tail suspension tests.<br />
Moreover, Lu AA21004 showed a signific<strong>an</strong>t <strong>an</strong>d dose-dependent <strong>an</strong>xiolytic-like profile in <strong>the</strong><br />
conditioned fear <strong>an</strong>d social interaction tests. Lu AA21004 affects brain levels of multiple<br />
neurotr<strong>an</strong>smitters assumed to be involved in <strong>the</strong> pathophysiology of mood disorders <strong>an</strong>d shows<br />
robust <strong>an</strong>tidepress<strong>an</strong>t- <strong>an</strong>d <strong>an</strong>xiolytic-like actions in preclinical models. This profile is different<br />
<strong>from</strong> that of o<strong>the</strong>r <strong>an</strong>tidepress<strong>an</strong>ts <strong>an</strong>d may tr<strong>an</strong>slate into a unique <strong>the</strong>rapeutic profile.<br />
Disclosures: A. Mork, None; L.T. Brennum, None; S.M. Fallon, None; S. Bisulco, None; K.<br />
Frederiksen, None; B. B<strong>an</strong>g-Andersen, None; A.B. Lassen, None; H. Zhong, None; J.G.<br />
Patel, None; P. Poon, None; D.G. Smith, None; C. Sánchez, None; T.B. Stensbøl, None; S.<br />
Hogg, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.11/X5<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: The triple reuptake inhibitor DOV216,303, a putative new <strong>an</strong>tidepress<strong>an</strong>t, decreases ICSS<br />
thresholds without producing withdrawal effects<br />
Authors: *J. PRINS 1,2,3 , P. J. KENNY 4 , I. DOOMERNIK 2,3 , B. OLIVIER 2,3 , S. M. KORTE 2,3 ;<br />
1 Psychopharmacology, Utrecht Univ., Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Utrecht Inst. <strong>for</strong> Pharmaceut. Sci.,<br />
Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 Rudolf Magnus Inst. of Neurosci., Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 Mol.<br />
Therapeut., The Scripps Res. Inst., Jupiter, FL<br />
Abstract: Selective serotonin reuptake inhibitors (SSRIs) are <strong>the</strong> most frequently prescribed<br />
<strong>an</strong>tidepress<strong>an</strong>t agents, <strong>an</strong>d are relatively safe to use. Never<strong>the</strong>less, SSRIs c<strong>an</strong> induce adverse side<br />
effects <strong>an</strong>d have a long <strong>the</strong>rapeutic lag-time. New promising <strong>an</strong>tidepress<strong>an</strong>ts are triple reuptake<br />
inhibitors (TRIs), which not only enh<strong>an</strong>ce serotonin <strong>an</strong>d norepinephrine neurotr<strong>an</strong>smission, but<br />
signific<strong>an</strong>tly, also increase brain dopamine levels. By impacting dopamine tr<strong>an</strong>smission, TRIs<br />
are believed to have faster <strong>the</strong>rapeutic onset th<strong>an</strong> SSRIs, <strong>an</strong>d may be particularly useful <strong>for</strong> <strong>the</strong><br />
treatment of <strong>an</strong>hedonia (loss of pleasure <strong>an</strong>d reward sensitivity), one of <strong>the</strong> core symptoms of<br />
depression.<br />
Import<strong>an</strong>tly, <strong>the</strong> increased dopamine tr<strong>an</strong>smission induced by TRIs raises concerns related to<br />
<strong>the</strong>ir abuse potential <strong>an</strong>d possible rein<strong>for</strong>cing effects. In this study, we directly assessed <strong>the</strong>
eward-related effects of <strong>the</strong> TRI, DOV216,303. Specifically, we compared <strong>the</strong> effects of<br />
DOV216,303 <strong>an</strong>d amphetamine, <strong>an</strong>d <strong>the</strong>ir withdrawal, on intracr<strong>an</strong>ial self stimulation (ICSS)<br />
thresholds in rats. ICSS is considered a direct measure of brain reward function. Bipolar<br />
stimulating electrodes were impl<strong>an</strong>ted into <strong>the</strong> lateral hypothalamus of rats, <strong>an</strong>d <strong>an</strong>imals were<br />
trained in a discrete-trial current-threshold ICSS procedure. After stable ICSS reward thresholds<br />
were established, <strong>an</strong>imals received one injection per day of DOV216,303 (20 mg/kg) or<br />
amphetamine (5 mg/kg) <strong>for</strong> four consecutive days. ICSS thresholds were assessed 3, 6, <strong>an</strong>d 23<br />
hours after injection, <strong>an</strong>d daily <strong>for</strong> ten days after <strong>the</strong> last injection.<br />
Both DOV216,303 <strong>an</strong>d amphetamine decreased ICSS thresholds 3 hours after injection,<br />
suggesting a drug-induced potentiation of brain reward function. In contrast, whereas withdrawal<br />
<strong>from</strong> amphetamine induced signific<strong>an</strong>t elevations of ICSS thresholds 23 hours after each<br />
injection, threshold elevations were not observed at <strong>an</strong>y time-point after DOV216,303<br />
administration. These data suggest that DOV216,303 c<strong>an</strong> tr<strong>an</strong>siently activate brain reward<br />
systems, but under present conditions does not induced long-term compensatory adaptations in<br />
reward circuitries similar to those induced by drugs of abuse like amphetamine.<br />
Disclosures: J. Prins, None; P.J. Kenny, None; I. Doomernik, None; B. Olivier, None; S.M.<br />
Korte, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.12/X6<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: The estrogen receptor beta agonist KB099520 rapidly increases serotonergic activity in <strong>the</strong><br />
raphe nucleus <strong>an</strong>d cell proliferation in dentate gyrus; A potential new <strong>an</strong>tidepress<strong>an</strong>t<br />
Authors: *K. OSTERLUND 1 , J. DAHLLUND 1 , E. ANDERSSON 1 , T. APELQVIST 1 , P.<br />
RHÖNNSTAD 1 , R. JEGGO 2 , S. GUSTAFSSON 1 , P. SVENNINGSSON 3 , M. EGELAND 3 , P.<br />
FAGERGREN 1 , S. NILSSON 1 ;<br />
1 Karo Bio AB, S-141 57 Huddinge, Sweden; 2 Neurosolutions Ltd., M<strong>an</strong>chester, United<br />
Kingdom; 3 Dept. of physiology <strong>an</strong>d pharmacology, Karolinska Inst., Stockholm, Sweden<br />
Abstract: Selective estrogen receptor (ER) β agonists, avoiding <strong>the</strong> ERα mediated breast <strong>an</strong>d<br />
uterine side effects, represent a novel possibility <strong>for</strong> treatment of depression. KB099520 is a<br />
highly selective ERβ agonist, both in vitro <strong>an</strong>d in vivo, that exerts <strong>an</strong>tidepress<strong>an</strong>t-like behavior in<br />
<strong>an</strong>imal models of depression; possible underlying mech<strong>an</strong>isms <strong>for</strong> <strong>the</strong> <strong>an</strong>tidepress<strong>an</strong>t properties
are presented. Treatment effects on spont<strong>an</strong>eous firing of serotonergic neurons in <strong>the</strong> dorsal<br />
raphe nucleus were evaluated by in vivo electrophysiology recording under <strong>an</strong>es<strong>the</strong>sia. Male rats<br />
were treated s.c. with vehicle, KB099520 (30 µmol/kg), or Citalopram (20 mg/kg) <strong>for</strong> 3 days <strong>an</strong>d<br />
recordings were per<strong>for</strong>med 24h after <strong>the</strong> last dose. Compared to vehicle, KB099520 signific<strong>an</strong>tly<br />
increased spont<strong>an</strong>eous firing of serotonergic neurons whereas no effect was observed in <strong>the</strong><br />
Citalopram group. At <strong>the</strong> end of <strong>the</strong> experiment, auto-inhibition of firing rate was investigated by<br />
challenges of acute i.v. injections of Citalopram in accumulated doses. There was no ch<strong>an</strong>ge in<br />
sensitivity to acute Citalopram-induced reduction of firing rate after 3 days of KB099520<br />
treatment compared to vehicle treated controls. Thus, <strong>the</strong> increased firing did not appear to be<br />
due to desensitization of <strong>the</strong> 5HT1A receptors. Moreover, acute KB099520 treatment in c57Bl/6<br />
mice (males <strong>an</strong>d ovariectomized females) induces <strong>the</strong> expression of tryptoph<strong>an</strong> hydroxylase 1<br />
(TPH1) mRNA, <strong>the</strong> rate limiting enzyme in <strong>the</strong> serotonin syn<strong>the</strong>sis, in <strong>the</strong> raphe nucleus. The<br />
induction of TPH1 mRNA was dose-dependent <strong>an</strong>d no increase in uterine weight was observed<br />
in <strong>the</strong> female mice at doses up to 800 p.o. or 180 s.c. µmol/kg. A slight effect on estradiol<br />
regulated genes in <strong>the</strong> uterus was observed at <strong>the</strong> highest doses. An increased number of Ki-67<br />
immunoreactive cells were observed in <strong>the</strong> dentate gyrus of male c57Bl/6 mice 24h after a single<br />
injection of KB099520, compared to vehicle treatment, thus indicating enh<strong>an</strong>ced cell<br />
proliferation. In conclusion, KB099520 rapidly increased serotonergic activity in <strong>the</strong> raphe<br />
nucleus <strong>an</strong>d increased <strong>the</strong> number of proliferating cells in <strong>the</strong> dentate gyrus, with a large safety<br />
margin to undesired effects in <strong>the</strong> uterus.<br />
Disclosures: K. Osterlund, Karo Bio AB, A. Employment (full or part-time); J. Dahllund,<br />
Karo Bio AB, A. Employment (full or part-time); E. Andersson, Karo Bio AB, A. Employment<br />
(full or part-time); T. Apelqvist, Karo Bio AB, A. Employment (full or part-time); P.<br />
Rhönnstad, Karo Bio AB, A. Employment (full or part-time); R. Jeggo, Neurosolution Ltd, A.<br />
Employment (full or part-time); S. Gustafsson, Karo Bio AB, A. Employment (full or parttime);<br />
P. Svenningsson, None; M. Egel<strong>an</strong>d, None; P. Fagergren, Karo Bio AB, A.<br />
Employment (full or part-time); S. Nilsson, Karo Bio AB, A. Employment (full or part-time).<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.13/X7<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Azalea (1972) Endowment Fund<br />
Title: Elucidating beneficial effects of Wolfberry (Lycium barbarum) in experimnetal depression
Authors: E. ZHANG 1 , R. C. C. CHANG 1,2 , *K.-F. SO 1,2 ;<br />
1 2<br />
Anat., Li Ka Shing Fac. of Med., Hong Kong, China; Res. Ctr. of Heart, Brain, Hormone <strong>an</strong>d<br />
Healthy aging, Hong Kong, China<br />
Abstract: The polysaccharides of Wolfberry (LBP), has been shown to exhibit protective effect<br />
on retinal neurons <strong>from</strong> a chronic ocular hypertension insult or cortical neurons <strong>from</strong> A-beta<br />
toxicity. Apart <strong>from</strong> <strong>the</strong>se two experimental models, increasing line of evidence have proved a<br />
sharp decline of new neurons generation in dendate gyrus in depression. There<strong>for</strong>e, we<br />
investigated whe<strong>the</strong>r LBP could also play a neuroprotective role on <strong>the</strong> neurogenesis in <strong>the</strong><br />
dentate gyrus. To test this hypo<strong>the</strong>sis, we studied <strong>the</strong> effects of 21-day LBP (1mg/kg) oral<br />
treatment on <strong>the</strong> neurogenesis in a rat depression model involving daily injection corticosterone<br />
(40 mg/Kg) <strong>for</strong> 14 days. Our results showed that 21-day LBP treatment reversed <strong>the</strong> inhibitory<br />
effect of corticosterone on <strong>the</strong> neurogenesis in <strong>the</strong> dentate gyrus, while 21-day LBP treatment<br />
alone did not affect neurogenesis in rats without <strong>the</strong> injection of corticosterone. Fur<strong>the</strong>rmore, our<br />
data showed that LBP treatment decreased depression-like behavior since <strong>the</strong> period of<br />
immobility in <strong>for</strong>ced swimming test in corticosterone-treated rats was signific<strong>an</strong>tly decreased. In<br />
conclusion, our data demonstrate that LBP is able to block <strong>the</strong> reduction of neurogenesis induced<br />
by <strong>the</strong> corticosterone injection <strong>an</strong>d ameliorates <strong>the</strong> behavioral per<strong>for</strong>m<strong>an</strong>ce in depressed rats. The<br />
underlying mech<strong>an</strong>isms of this neuroprotective effects of LBP need to be fur<strong>the</strong>r explored.<br />
Disclosures: E. Zh<strong>an</strong>g, None; R.C.C. Ch<strong>an</strong>g, None; K. So, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.14/X8<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Title: RS 67333 induces desensitization of serotonergic autoreceptor function: Implication <strong>for</strong><br />
<strong>an</strong>tidepress<strong>an</strong>t-like effects by 5-HT4 agonism<br />
Authors: M. M. VAN GAALEN, F. POHLKI, *G. GROSS, K. WICKE;<br />
Abbott, Ludwigshafen, Germ<strong>an</strong>y<br />
Abstract: There is abund<strong>an</strong>t evidence that 5-HT4 receptor agonists have pro-cognitive effects.<br />
Recently, it has been demonstrated that <strong>the</strong>se compounds exhibit <strong>an</strong>tidepress<strong>an</strong>t-like properties<br />
as well. In <strong>the</strong> present study, we aimed to confirm <strong>the</strong> effects of <strong>the</strong> 5-HT4 partial agonist RS<br />
67333 in <strong>the</strong> <strong>for</strong>ced swim test, a model with predictive validity <strong>for</strong> <strong>an</strong>tidepress<strong>an</strong>ts in mice <strong>an</strong>d
ats. Fur<strong>the</strong>rmore, to gain underst<strong>an</strong>ding of <strong>the</strong> mech<strong>an</strong>ism by which 5-HT4 agonism leads to<br />
<strong>an</strong>tidepress<strong>an</strong>t-like effects, we assessed if subchronic treatment with RS 67333 desensitizes 5-<br />
HT1A somatodendritic autoreceptor function, as has been described <strong>for</strong> clinically used<br />
<strong>an</strong>tidepress<strong>an</strong>ts.<br />
In rats, we confirmed that RS 67333 has <strong>an</strong>tidepress<strong>an</strong>t-like effect in <strong>the</strong> <strong>for</strong>ced swim test at 1<br />
mg/kg. In a subsequent study, rats were treated with RS 67333 <strong>for</strong> seven days via minipumps<br />
(1.5 mg/kg/day). After subchronic treatment, extracellular recordings <strong>from</strong> spont<strong>an</strong>eously active<br />
dorsal raphe (DR) serotonergic neurons were per<strong>for</strong>med with glass microelectrodes. After<br />
baseline recording, <strong>the</strong> 5-HT1A agonist 8-OH-DPAT was administered iv by cumulative dosing.<br />
RS 67333 shifted <strong>the</strong> dose response of 8-OH-DPAT to approximately 3-fold higher values.<br />
Similar effects have been described <strong>for</strong> subchronic treatment with established <strong>an</strong>tidepress<strong>an</strong>ts. In<br />
a separate experiment, we found that acutely iv applied RS 67333 does not affect firing rate of<br />
spont<strong>an</strong>eously active DR serotonergic neurons. In mice, however, no effect of RS 67333 (0.3 -<br />
3.0 mg/kg) was found in <strong>the</strong> <strong>for</strong>ced swim test. 8-OH-DPAT-induced hypo<strong>the</strong>rmia in mice has<br />
been attributed to activation of somatodendritic 5-HT1A autoreceptors <strong>an</strong>d is attenuated by<br />
chronic treatment with various <strong>an</strong>tidepress<strong>an</strong>ts. Three or seven days of treatment with RS 67333<br />
via minipump (1.5 or 3.0 mg/kg/day) did not affect 8-OH DPAT-induced hyper<strong>the</strong>rmia.<br />
We confirmed that RS 67333 has <strong>an</strong>tidepress<strong>an</strong>t-like effects in rats but not in mice. The<br />
electrophysiological effects of subchronic treatment with RS 67333 in rats may indicate a<br />
desensitization of <strong>the</strong> 5-HT1A autoreceptors controlling <strong>the</strong> firing of DR serotonergic neurons.<br />
The reason why <strong>the</strong> <strong>an</strong>tidepress<strong>an</strong>t-like effect <strong>an</strong>d somatodendritic receptor desensitization of RS<br />
67333 is species-dependent needs fur<strong>the</strong>r investigation.<br />
Disclosures: M.M. v<strong>an</strong> Gaalen, Abbott, A. Employment (full or part-time); F. Pohlki, Abbott,<br />
A. Employment (full or part-time); G. Gross, Abbott, A. Employment (full or part-time); K.<br />
Wicke, Abbott, A. Employment (full or part-time).<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.15/X9<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Ministerio de Educación y Ciencia (SAF-07/61862)<br />
Fundación Alicia Koplowitz<br />
Fundación de Investigacion Médica Mutua Madrileña
Title: Neuroplastic ch<strong>an</strong>ges associated to <strong>an</strong>tidepress<strong>an</strong>t efficacy after a short treatment with a 5-<br />
HT4 receptor agonist<br />
Authors: J. PASCUAL-BRAZO 1,2 , *F. PILAR-CUELLAR 1,2 , I. RUIZ 1,2 , A. MARTIN 1,2 , A.<br />
PAZOS 1,2 , E. VALDIZAN 1,2 , R. VIDAL 1,2 , E. CASTRO 1,2 ;<br />
1 IBBTEC (UC-CSIC-IDICAN), CIBERSAM, S<strong>an</strong>t<strong>an</strong>der, Spain; 2 Dpto. Fisiología y<br />
Farmacología, Univ. de C<strong>an</strong>tabria, S<strong>an</strong>t<strong>an</strong>der, Spain<br />
Abstract: The 5-HT4 receptor agonist RS67333 has been recently proposed to possess<br />
<strong>an</strong>tidepress<strong>an</strong>t-like properties in rats after a treatment of only 3 days of duration. This result<br />
suggest a new strategic <strong>the</strong>rapy <strong>for</strong> depressive disorders with a short onset of action. It has been<br />
described that certain hippocampal neuroplastic ch<strong>an</strong>ges induced by classical <strong>an</strong>tidepress<strong>an</strong>ts, as<br />
<strong>the</strong> increase in hippocampal BDNF levels or in <strong>the</strong> number of new neurons generated in <strong>the</strong><br />
dentate gyrus, occur only after several weeks of treatment. Here, immunohistochemical <strong>an</strong>d in<br />
situ hybridization studies have carried out in order to assess if a 3-days treatment with this 5-HT4<br />
receptor agonist induces neuroplastic ch<strong>an</strong>ges comparable to those observed after several weeks<br />
of treatment with typical <strong>an</strong>tidepress<strong>an</strong>ts. RS67333 (1,5 mg/kg/day) was delivered s.c. through 3days<br />
osmotic minipumps to male Sprague-Dawley rats. RS67333 administration <strong>for</strong> 3 days<br />
increases <strong>the</strong> expression of <strong>the</strong> neurotrophic factor BDNF mRNA in hippocampus (CA3 field<br />
28,2% Student-t test unpaired data, p
Topic: C.16.g. Mood disorders: Experimental <strong>the</strong>rapeutics, behavioral models<br />
Support: China postdoctoral science foundation Gr<strong>an</strong>t 023205002<br />
Title: Enh<strong>an</strong>cement of cognitive control of emotions by elevated brain magnesium leads to <strong>an</strong>tidepress<strong>an</strong>ts<br />
like effect<br />
Authors: *N. ABUMARIA 1 , B. YIN 1 , L. ZHANG 1 , L. ZHAO 1 , G. LIU 1,2 ;<br />
1 2<br />
Ctr. <strong>for</strong> learning <strong>an</strong>d Memory, Tsinghua Univ., Beijing, China; Ctr. <strong>for</strong> learning <strong>an</strong>d memory,<br />
Univ. of Texas at Austin, Austin, TX<br />
Abstract: Facilitating learning <strong>an</strong>d memory abilities has been long a major aim of m<strong>an</strong>y<br />
researchers. Though associative fear-memories are import<strong>an</strong>t <strong>for</strong> survival, <strong>the</strong> lack of control<br />
over such memories increases <strong>the</strong> risk <strong>for</strong> affective <strong>an</strong>d <strong>an</strong>xiety disorders. There<strong>for</strong>e while<br />
tempting to enh<strong>an</strong>ce <strong>the</strong> cognitive ability of individuals; one should consider <strong>the</strong> impact on<br />
associative fear-memories <strong>an</strong>d subsequent potential impact on psychiatric disorders.<br />
A previous study demonstrated that elevated extracellular magnesium level leads to increase in<br />
synaptic density/plasticity in vitro (1). Recently, using a novel magnesium compound MgT, we<br />
found that elevated brain magnesium leads to increase in synaptic density/plasticity in <strong>the</strong><br />
hippocampus <strong>an</strong>d enh<strong>an</strong>cement of working, spatial <strong>an</strong>d recognition memories in rats (2). Here,<br />
we investigated whe<strong>the</strong>r elevated brain magnesium has effect on different <strong>for</strong>ms of fearmemories;<br />
<strong>an</strong>d more import<strong>an</strong>tly on <strong>the</strong> extinction <strong>an</strong>d attenuation of fear memories. Using fear<br />
conditioning paradigm, we found that chronic MgT treatment enh<strong>an</strong>ces hippocampus-dependent<br />
but not amygdala-dependent fear-memory in rats. Interestingly, prefrontal cortex-dependent<br />
retention of extinction was facilitated, <strong>an</strong>d recovery of previously extinguished fear-memories<br />
was attenuated.<br />
These data suggest that MgT might enh<strong>an</strong>ce <strong>the</strong> coping ability with aversive events <strong>an</strong>d control<br />
of emotional responses, prerequisites <strong>for</strong> treatment of affective <strong>an</strong>d <strong>an</strong>xiety disorders such as<br />
depression <strong>an</strong>d PTSD. This prompted us to fur<strong>the</strong>r test whe<strong>the</strong>r MgT treatment has<br />
<strong>an</strong>tidepress<strong>an</strong>t-like effect in <strong>an</strong>imal models of depression. In <strong>the</strong> <strong>for</strong>ced swimming test, MgT<br />
treated rats exhibited less behavioral despair compared with control. Remarkably, in <strong>the</strong> learned<br />
helplessness paradigm, MgT treatment signific<strong>an</strong>tly reversed <strong>the</strong> escape deficits induced by<br />
inescapable shock during both FR-1 <strong>an</strong>d FR-2 tests. Finally, at <strong>the</strong> structural <strong>an</strong>d molecular<br />
levels, synaptic density, BDNF expression <strong>an</strong>d CREB phosphorylation were increased in both<br />
hippocampus <strong>an</strong>d prefrontal cortex but not in <strong>the</strong> amygdala. This is inline with our behavioral<br />
data above.<br />
Our data show that elevating brain magnesium might be a novel strategy to dampen traumatic<br />
memories <strong>an</strong>d treat affective <strong>an</strong>d <strong>an</strong>xiety disorders in clinics. The enh<strong>an</strong>cement of neural<br />
plasticity in hippocampus <strong>an</strong>d PFC, brain regions responsible <strong>for</strong> highly processed emotional<br />
responses, could be <strong>the</strong> underlying mech<strong>an</strong>isms.<br />
1.Slutsky,I., Sadeghpour,S., Li,B. & Liu,G. Neuron 44, 835-849 (2004).<br />
2.Slutsky I*, Abumaria N*, Wu L J, Hu<strong>an</strong>g C, Li B, Govindaraj<strong>an</strong> A, ZhaoMG, Zhuo M,<br />
Tonegawa S, Liu G. Nat medicine, revised m<strong>an</strong>uscript submitted. (<strong>2009</strong>).<br />
Disclosures: N. Abumaria, None; B. Yin, None; L. Zh<strong>an</strong>g, None; L. Zhao, None; G. Liu,<br />
None.
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.17/X11<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Neurogen Corporation<br />
Title: Effects of CRF1 receptor <strong>an</strong>tagonists on <strong>the</strong> enh<strong>an</strong>cement of acoustic startle <strong>an</strong>d heart rate<br />
activation induced by stimulation of central CRF or NE pathways<br />
Authors: *D. C. HOFFMAN 1 , J. H. KEHNE 2 ;<br />
1 Neurogen Corp, Br<strong>an</strong><strong>for</strong>d, CT; 2 Tr<strong>an</strong>slational Neuropharm. Consulting, LLC, Rockville, MD<br />
Abstract: The 41 amino acid peptide corticotropin releasing factor (CRF) is a key mediator of<br />
<strong>the</strong> behavioral, autonomic, <strong>an</strong>d endocrine responses to stressors, <strong>an</strong>d dysfunctional<br />
hyperactivation of CRF neurons acting on CRF1 receptor subtypes (“CRF1 pathways”) has been<br />
implicated in <strong>the</strong> etiology of numerous stress-related psychiatric disorders. Given clinical data<br />
<strong>for</strong> concomit<strong>an</strong>t hyperactivation of brain norepinephrine (NE) pathways, <strong>an</strong>d considerable<br />
evidence <strong>for</strong> brain CRF-NE interactions, evaluating <strong>the</strong> degree to which CRF1 receptor<br />
<strong>an</strong>tagonists c<strong>an</strong> reverse states of both CRF <strong>an</strong>d NE hyperactivation is of <strong>the</strong>rapeutic interest. The<br />
present study evaluated <strong>the</strong> ability of orally-active, small molecule CRF1 receptor <strong>an</strong>tagonists to<br />
reverse <strong>the</strong> autonomic (heart rate) <strong>an</strong>d behavioral (acoustic startle) activation produced by<br />
pharmacologically-induced stimulation of central CRF or NE pathways. Heart rate was measured<br />
wirelessly in freely-moving male Sprague-Dawley rats impl<strong>an</strong>ted with telemetry tr<strong>an</strong>smitter<br />
probes (Data Sciences International). Acoustic startle was measured in rect<strong>an</strong>gular Plexiglas<br />
cages (Med Associates) located in sound-attenuating chambers. Startle-induced displacement of<br />
<strong>the</strong> cage was qu<strong>an</strong>tified by a load cell plat<strong>for</strong>m placed beneath <strong>the</strong> cage. In <strong>the</strong> first study,<br />
pharmacological activation of ei<strong>the</strong>r CRF (i.c.v. infusion of <strong>the</strong> peptide r/h CRF, 0.1-1.0 µg) or<br />
NE (i.p. injection of <strong>the</strong> α2-adrenergic receptor <strong>an</strong>tagonist, yohimbine, 0.3-10 mg/kg) pathways<br />
produced dose-related increases in heart rate <strong>an</strong>d acoustic startle. In <strong>the</strong> second study, rats were<br />
pretreated with ei<strong>the</strong>r <strong>the</strong> CRF1 <strong>an</strong>tagonist, R121919 (10, 30 mg/kg p.o.) or NGD 98-2 (30 mg/kg<br />
p.o.), <strong>an</strong>d following <strong>the</strong> appropriate pretreatment time, injected with yohimbine or CRF at doses<br />
which were shown in <strong>the</strong> first study to cause signific<strong>an</strong>t heart rate or acoustic startle elevations.<br />
NGD 98-2 <strong>an</strong>d R121919 reduced both CRF-induced elevations in heart rate <strong>an</strong>d startle. In<br />
contrast, CRF1 receptor <strong>an</strong>tagonism under <strong>the</strong>se conditions did not attenuate <strong>the</strong> increases in<br />
heart rate or startle produced by yohimbine. These results indicate that blockade of CRF1<br />
receptors c<strong>an</strong> attenuate <strong>the</strong> autonomic <strong>an</strong>d behavioral activation resulting <strong>from</strong> hyperactivation of
CRF, but not NE, pathways. These data fur<strong>the</strong>rmore suggest <strong>the</strong> possibility that, in clinical<br />
populations with concomit<strong>an</strong>t hyperactivation of CRF <strong>an</strong>d NE pathways, optimal <strong>the</strong>rapeutic<br />
efficacy of CRF1 receptor <strong>an</strong>tagonists may require augmentation by agents with o<strong>the</strong>r<br />
pharmacological mech<strong>an</strong>isms (e.g. α2-NE agonists or α1-NE <strong>an</strong>tagonists) which c<strong>an</strong> effectively<br />
normalize <strong>the</strong> dysfunctional NE component. Supported by Neurogen Corporation.<br />
Disclosures: D.C. Hoffm<strong>an</strong>, Neurogen Corporation, A. Employment (full or part-time); J.H.<br />
Kehne, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.18/X12<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: Ministerio de Educación y Ciencia (SAF07-61862)<br />
Pl<strong>an</strong> Nacional de Drogas (2006)<br />
Title: Effect of chronic delta 9 -THC in combination with fluoxetine upon <strong>the</strong> functionality of<br />
endoc<strong>an</strong>nabionid system<br />
Authors: *E. CASTRO-FERNÁNDEZ 1,2 , A. DIAZ 1,2 , E. M. VALDIZAN 1,2 , A. MARTIN 1,2 ,<br />
R. VIDAL 1,2 , A. PAZOS 1,2 ;<br />
1 IBBTEC (UC-CSIC-IDICAN). Univ. De C<strong>an</strong>tabria, S<strong>an</strong>t<strong>an</strong>der, Spain; 2 CIBERSAM, S<strong>an</strong>t<strong>an</strong>der,<br />
Spain<br />
Abstract:<br />
Several reports indicate that brain endoc<strong>an</strong>nabinoid (EC) system may be involved in<br />
depression <strong>an</strong>d in <strong>an</strong>tidepress<strong>an</strong>t-like activity as demonstrated in some experimental models.<br />
In addition, recent data indicate that EC activity might be modulated by different<br />
<strong>an</strong>tidepress<strong>an</strong>t paradigms. To extend our knowledge on <strong>the</strong> crosstalk mech<strong>an</strong>isms between<br />
brain EC <strong>an</strong>d serotonergic systems <strong>an</strong>d <strong>the</strong>ir implication in depression, we have evaluated <strong>the</strong><br />
effects of chronic in vivo exposure to <strong>the</strong> SSRI drug fluoxetine alone or in combination with<br />
∆ 9 - tetrahydroc<strong>an</strong>nabinol (∆ 9 -THC) on <strong>the</strong> density <strong>an</strong>d functionality of brain c<strong>an</strong>nabinoid CB1<br />
receptors as well as on <strong>the</strong> behavioural responses in depression/<strong>an</strong>xiety tests (open-field <strong>an</strong>d<br />
elevated-plus maze). Animals (male Sprague-Dawley rats) were treated <strong>for</strong> 21 days with<br />
vehicle (saline), ∆ 9 -THC (10 mg/kg/day, i.p.), fluoxetine (10 mg/kg/day, p.o.) <strong>an</strong>d ∆ 9 -THC plus
fluoxetine. No signific<strong>an</strong>t ch<strong>an</strong>ges were observed in <strong>the</strong> open-field <strong>an</strong>d elevated-plus maze<br />
paradigms with <strong>an</strong>y of <strong>the</strong> drug regimes. CB1 receptor density ([ 3 H]CP55,940 saturation<br />
binding) was signific<strong>an</strong>tly decreased by chronic ∆ 9 -THC (% reduction= 52.4 ± 7.3, p< 0.05;<br />
one-way ANOVA) <strong>an</strong>d fluoxetine (% reduction= 42.4 ± 5.8, p< 0.05; one-way ANOVA) in <strong>the</strong><br />
rat prefrontal cortex. However, <strong>the</strong>se ch<strong>an</strong>ges in <strong>the</strong> receptor density were similar to those<br />
observed by <strong>the</strong> concomit<strong>an</strong>t administration of ∆ 9 -THC plus fluoxetine. In addition, <strong>the</strong><br />
maximal effect of <strong>the</strong> c<strong>an</strong>nabinoid agonist WIN55,212-2 to stimulate [ 35 S]GTPγS binding was<br />
attenuated by chronic ∆ 9 -THC alone or in combination with fluoxetine; however chronic<br />
fluoxetine did not alter CB1 agonist-stimulated [ 35 S]GTPγS binding. Finally, <strong>the</strong> maximal<br />
ability of WIN55,212-2 to inhibit AC (<strong>for</strong>skolin-stimulated-cAMP accumulation) was<br />
signific<strong>an</strong>tly enh<strong>an</strong>ced (Emaxvehicle= 64.5 ± 8.3 vs Emaxfluoxetine= 48.5 ± 13.4; p < 0.01; oneway<br />
ANOVA) <strong>an</strong>d decreased (Imax∆9-THC = 84.5 ± 7.4 %; p < 0.01 vs vehicle; one-way<br />
ANOVA) in <strong>the</strong> prefrontal cortex of rats treated with fluoxetine <strong>an</strong>d ∆ 9 -THC respectively. By<br />
contrast, in <strong>the</strong> ∆ 9 -THC plus fluoxetine treated group <strong>the</strong> inhibition of AC induced by<br />
WIN55,212-2 was not signific<strong>an</strong>tly different <strong>from</strong> <strong>the</strong> vehicle group. Our data indicate that<br />
chronic stimulation of EC tr<strong>an</strong>smission desensitizes <strong>the</strong> receptorial <strong>an</strong>d postreceptorial<br />
mech<strong>an</strong>isms coupled to CB receptors. They also suggest that <strong>the</strong> association of a CB1 agonist<br />
with SSRIs does not potentiate <strong>the</strong> ch<strong>an</strong>ges in <strong>the</strong> functionality of CB receptors induced by<br />
<strong>an</strong>tidepress<strong>an</strong>t drugs.<br />
Disclosures: E. Castro-Fernández, None; A. Diaz, None; E.M. Valdiz<strong>an</strong>, None; A. Martin,<br />
None; R. Vidal, None; A. Pazos, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.19/X13<br />
Topic: C.16.f. Mood disorders: Experimental <strong>the</strong>rapeutics, preclinical studies<br />
Support: fully sponsored by BuddhaBiopharm, Ltd.<br />
Title: Effects of imipramine <strong>an</strong>d insulin receptor sensitizer dicholine succinate on stress-induced<br />
ahedonia <strong>an</strong>d <strong>an</strong>xiety in mice<br />
Authors: *T. STREKALOVA 1 , V. VAN MIEGEM 2 , R. CESPUGLIO 3 , G. LARDE 2 , I.<br />
POMYTKIN 2 , H. W. STEINBUSCH 1 ;<br />
1 Sch. <strong>for</strong> Mental Hlth. <strong>an</strong>d Neurosci., Maastricht Univ., Maastricht, Ne<strong>the</strong>rl<strong>an</strong>ds;
2 3<br />
BuddhaBiopharm, Ltd, Helsinki, Finl<strong>an</strong>d; Lab. of Sleep <strong>an</strong>d Wakefulness, Univ. of C. Bernard,<br />
CNRS, Lyon, Fr<strong>an</strong>ce<br />
Abstract: The neuronal insulin receptor, which is close to <strong>the</strong> TrkB receptor in structure <strong>an</strong>d<br />
brain distribution, is suggested to be implicated in <strong>the</strong> mech<strong>an</strong>isms of affective disorders. Here,<br />
we addressed <strong>the</strong> question whe<strong>the</strong>r a chronic administration of Dicholine Succinate (DS), a<br />
sensitizer of <strong>the</strong> neuronal insulin receptor, which was shown to potentiate neuronal insulin<br />
receptor signaling <strong>an</strong>d enh<strong>an</strong>ce insulin action via <strong>the</strong> activation of insulin receptor kinase,<br />
interferes with a development of depressive-like <strong>an</strong>d <strong>an</strong>xiety behavior in a mouse model of<br />
stress-induced <strong>an</strong>hedonia, a core symptom of clinical depression. We used a chronic stress<br />
depression model, in which rat exposure <strong>an</strong>d social defeat stress evoke hedonic deficit, in a<br />
subset of CD1 mice. Chronic delivery of <strong>an</strong>tidepress<strong>an</strong>t imipramine (8 mg/kg/day) delivered 1<br />
week be<strong>for</strong>e <strong>an</strong>d during a 4-week stress procedure with drinking water, counteracts induction of<br />
<strong>an</strong>hedonia, defined by a decrease in sucrose intake <strong>an</strong>d preference <strong>an</strong>d reduces a percentage of<br />
<strong>an</strong>hedonic subjects. Similarly, <strong>the</strong> i.p. administration of <strong>the</strong> DS (25 mg/kg/day) during 2 weeks<br />
be<strong>for</strong>e <strong>the</strong> onset of stress diminished <strong>the</strong> percentage of <strong>an</strong>hedonic individuals counteracted a<br />
decrease in sucrose intake <strong>an</strong>d preference. In addition, DS-treated stressed mice showed<br />
decreased scores of <strong>an</strong>xiety-like behavior in <strong>the</strong> dark/light box in comparison to non-treated<br />
mice. Toge<strong>the</strong>r, <strong>the</strong>se data let to suggest <strong>an</strong> import<strong>an</strong>ce of neuronal insulin receptors function in<br />
<strong>the</strong> pathogenesis of depressive- <strong>an</strong>d <strong>an</strong>xiety-like behavioral ch<strong>an</strong>ges induced by chronic stress.<br />
Disclosures: T. Strekalova, BuddhaBiopharm, Ltd., B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
BuddhaBiopharm, Ltd., C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or<br />
o<strong>the</strong>r in-kind support); BuddhaBiopharm, Ltd., E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); V. v<strong>an</strong> Miegem, None; R. Cespuglio, None; G. Larde,<br />
None; I. Pomytkin, BuddhaBiopharm, Ltd., A. Employment (full or part-time);<br />
BuddhaBiopharm, Ltd., B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); BuddhaBiopharm, Ltd., C. O<strong>the</strong>r Research<br />
Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); BuddhaBiopharm, Ltd.,<br />
E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); H.W.<br />
Steinbusch, None.<br />
Poster<br />
549. Mood Disorders: Experimental Therapeutics <strong>an</strong>d Novel Compounds<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 549.20/X14<br />
Topic: C.16.d. Mood disorders: Animal models
Title: Peripheral infusion of <strong>the</strong> peptide hormone amylin <strong>an</strong>d its clinical c<strong>an</strong>didate <strong>an</strong>alog,<br />
PSN0041, produces <strong>an</strong>tidepress<strong>an</strong>t effects without sexual side effects<br />
Authors: *B. J. CALDARONE 1 , C. MACK 2 , T. HANANIA 1 , D. BRUNNER 1 , M. SAMANT 2 ,<br />
D. PARKES 2 , V. SRIVASTAVA 3 , M. HANLEY 2 , P. MCGONIGLE 1 , W. ROTE 2 , B.<br />
OLIVIER 1 , A. GHAVAMI 1 ;<br />
1 2 3<br />
PsychoGenics Inc, Tarrytown, NY; Amylin Pharmaceuticals Inc., S<strong>an</strong> Diego, CA; New<br />
Engl<strong>an</strong>d Peptide, Boston, MA<br />
Abstract: Peptide hormones possess appealing drug profiles based on <strong>the</strong>ir role as natural<br />
integrators of physiological systems. We have recently discovered <strong>an</strong>tidepress<strong>an</strong>t-like effects of<br />
<strong>the</strong> peptide hormone amylin in both mouse <strong>an</strong>d rat preclinical models. Infusion of amylin (1<br />
mg/kg/d) via SC osmotic minipumps <strong>for</strong> 7-21 days in mice decreased immobility in <strong>the</strong> <strong>for</strong>ced<br />
swim test (FST). In contrast to known <strong>an</strong>tidepress<strong>an</strong>ts, acute administration of amylin (IP or SC)<br />
had no effect in <strong>the</strong> mouse FST. In olfactory bulbectomized (OBX) rats a suppression of <strong>the</strong><br />
OBX-induced hyperactivity was obtained as early as one day after amylin (0.3mg/kg/d) <strong>an</strong>d<br />
imipramine (10mg/kg/d) infusion. One week after cessation of 14 days of amylin treatment,<br />
OBX-induced hyperactivity remained comparable to sham-operated <strong>an</strong>imals, indicating long<br />
lasting effects of amylin. Chronic amylin effects on prefrontal cortex (PFC) monoaminergic<br />
tr<strong>an</strong>smission were <strong>the</strong>n examined by in vivo microldialysis in mice. Unlike DOV-216,303 (a<br />
dopamine (DA), serotonin (5-HT), norepinephrine (NE) reuptake inhibitor), nei<strong>the</strong>r acute<br />
(10mg/kg) nor chronic amylin infusion (0.3, 1, <strong>an</strong>d 3mg/kg/d) <strong>for</strong> 21 days increased DA, 5-HT,<br />
<strong>an</strong>d NE level in <strong>the</strong> PFC indicating that <strong>an</strong>tidepress<strong>an</strong>t effects of amylin might be independent of<br />
monoamine release. Based on <strong>the</strong>se results, PSN0041, a novel amylin mimetic petide, was<br />
identified after screening more th<strong>an</strong> 200 amylin <strong>an</strong>alogs through PsychoGenics proprietary in<br />
vivo drug discovery technologies. In mice, PSN0041 was more potent th<strong>an</strong> amylin in reducing<br />
immobility in <strong>the</strong> FST (MED=0.03 vs 1 mg/kg/d, 7d infusion), <strong>an</strong>d in decreasing <strong>the</strong> number of<br />
marbles in <strong>the</strong> marble burying (MRB) test (MED=0.3mg/kg vs 3mg/kg IP). PSN0041 was also<br />
optimized through SAR studies to increase duration of action. The effect of PSN0041 (3 mg/kg,<br />
IP) in <strong>the</strong> MRB test was sustained up to 4 hr compared to 30 min with amylin treatment (3<br />
mg/kg, IP). Finally, in rats PSN0041 showed a clear superiority compared to known<br />
<strong>an</strong>tidepress<strong>an</strong>ts; PSN0041 enh<strong>an</strong>ced cognitive per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> novel object recognition test,<br />
induced weight loss, <strong>an</strong>d did not produce sexual dysfunction (0.1, 0.3, <strong>an</strong>d 1mg/kg/d, 21d<br />
infusion). In conclusion, PSN0041 is a unique, first-in-class compound with enh<strong>an</strong>ced<br />
pharmacological properties over native amylin <strong>an</strong>d is <strong>an</strong>ticipated to differentiate <strong>from</strong> current<br />
marketed <strong>an</strong>tidepress<strong>an</strong>ts.<br />
Disclosures: B.J. Caldarone, PsychoGenics, A. Employment (full or part-time); Psylin, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); PsychoGenics, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); C. Mack, Amylin<br />
Pharmaceuticals Inc., A. Employment (full or part-time); Amylin Pharmaceuticals Inc., E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); T. H<strong>an</strong><strong>an</strong>ia,<br />
PsychoGenics, A. Employment (full or part-time); Psylin, E. Ownership Interest (stock, stock<br />
options, patent or o<strong>the</strong>r intellectual property); PsychoGenics, E. Ownership Interest (stock, stock<br />
options, patent or o<strong>the</strong>r intellectual property); D. Brunner, PsychoGenics, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); PsychoGenics, A. Employment (full
or part-time); Psylin, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); M. Sam<strong>an</strong>t, Amylin Pharmaceuticals Inc., A. Employment (full or part-time); Amylin<br />
Pharmaceuticals Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); D. Parkes, Amylin Pharmaceuticals Inc, A. Employment (full or part-time); Amylin<br />
Pharmaceuticals Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); V. Srivastava, None; M. H<strong>an</strong>ley, Amylin Pharmaceuticals Inc., A. Employment (full<br />
or part-time); Amylin Pharmaceuticals Inc., E. Ownership Interest (stock, stock options, patent<br />
or o<strong>the</strong>r intellectual property); P. McGonigle, PsychoGenics, A. Employment (full or part-time);<br />
Psylin, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
PsychoGenics, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
W. Rote, Amylin Pharmaceuticals Inc., A. Employment (full or part-time); Amylin<br />
Pharmaceuticals Inc., E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); B. Olivier, PsychoGenics, A. Employment (full or part-time); Psylin, E. Ownership<br />
Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); PsychoGenics, E. Ownership<br />
Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); A. Ghavami, PsychoGenics,<br />
A. Employment (full or part-time); PsychoGenics, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.1/X15<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH NIDA Gr<strong>an</strong>t DA020686<br />
NIH NIDA Gr<strong>an</strong>t DA023915<br />
The L<strong>an</strong>denberger Foundation Gr<strong>an</strong>t<br />
B<strong>an</strong>k of America Fellowship<br />
Title: Motivational drives in obesity: Evidence <strong>for</strong> addiction-like compulsive responding <strong>for</strong><br />
palatable food<br />
Authors: *P. M. JOHNSON 1 , P. J. KENNY 2 ;<br />
2 Mol. Therapeut., 1 The Scripps Res. Inst., Jupiter, FL
Abstract: In recent years it has been speculated that <strong>the</strong> powerful hedonic aspects of palatable<br />
food are mediated through similar neurobiological substrates as drugs of abuse <strong>an</strong>d may<br />
<strong>the</strong>re<strong>for</strong>e confer addictive properties. However, empirical evidence supporting this notion has<br />
been lacking. In rats, extended access to addictive drugs c<strong>an</strong> result in <strong>the</strong> tr<strong>an</strong>sition <strong>from</strong> casual<br />
to compulsive use, <strong>an</strong> effect that time-locked with reduced activity in brain reward circuits,<br />
reflected in elevated intracr<strong>an</strong>ial self-stimulation (ICSS) thresholds. This state of negative reward<br />
is hypo<strong>the</strong>sized to increase <strong>the</strong> motivation to consume palatable food. Thus, brain reward deficits<br />
are thought to be a hallmark of addiction. Here we show that <strong>an</strong>imals permitted extended (>18<br />
h), but not restricted (1 h), daily access to a “cafeteria-style” diet consisting of high-fat palatable<br />
food readily available <strong>for</strong> hum<strong>an</strong> consumption exhibited rapid weight gain <strong>an</strong>d a shift in dietary<br />
preference <strong>for</strong> <strong>the</strong> palatable food. Import<strong>an</strong>tly, <strong>the</strong> development of obesity was time-locked to<br />
profound reductions in brain reward function, as measured by elevated ICSS thresholds. Dietinduced<br />
reward dysfunction lasted <strong>for</strong> at least two weeks during abstinence <strong>from</strong> <strong>the</strong> palatable<br />
diet. Fur<strong>the</strong>rmore, extended access to <strong>the</strong> cafeteria diet resulted in enh<strong>an</strong>ced motivation to<br />
consume <strong>the</strong> diet, reflected in persistent consumption of palatable food in obese <strong>an</strong>imals even<br />
under conditions of punishment (foot-shock). These findings suggest that extended access to a<br />
cafeteria diet m<strong>an</strong>ifest addiction-like deficits in brain reward function that may enh<strong>an</strong>ce <strong>the</strong><br />
motivation to consume palatable foods, thus promoting <strong>the</strong> development <strong>an</strong>d mainten<strong>an</strong>ce of<br />
obesity. These findings parallel those previously observed with extensive access to drugs of<br />
abuse, supporting <strong>the</strong> notion that obesity <strong>an</strong>d addiction may result <strong>from</strong> common<br />
neuroadaptations.<br />
Disclosures: P.M. Johnson, None; P.J. Kenny, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.2/X16<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: Smith Family Award <strong>for</strong> Excellence in Biomedical Research<br />
NIH Gr<strong>an</strong>t DK065872<br />
NIH Gr<strong>an</strong>t DA023760<br />
Title: Effects of physical exercise on mesolimbic dopamine neurotr<strong>an</strong>smission
Authors: *B. M. GEIGER 1,2 , M. KAROUANI 1,2 , J. MUNRO 2 , R. KANAREK 3 , E. N.<br />
POTHOS 1,2 ;<br />
1 Pharmacol. <strong>an</strong>d Exptl. Therapeut., 2 Program in Neurosci., Tufts Univ., Boston, MA;<br />
3 Psychology, Tufts Univ., Med<strong>for</strong>d, MA<br />
Abstract: Physical activity has been connected to a broad r<strong>an</strong>ge of beneficial health outcomes<br />
including cardio-respiratory, weight loss, <strong>an</strong>d c<strong>an</strong>cer prevention. While this suggests <strong>the</strong><br />
potential of exercise <strong>for</strong> preventing disease, very little is known about its effects on addictive<br />
behaviors <strong>an</strong>d hyperphagia. Even less is known about its effects on <strong>the</strong> central dopamine systems<br />
linked to <strong>the</strong>se behaviors. Here we investigate <strong>the</strong> effects of voluntary physical exercise in <strong>the</strong><br />
<strong>for</strong>m of wheel running on central dopaminergic neurotr<strong>an</strong>smission in a rat model. We<br />
hypo<strong>the</strong>size that chronic voluntary physical activity would be linked to a signific<strong>an</strong>t upregulation<br />
of dopamine release in <strong>the</strong> dopaminergic pathways of <strong>the</strong> midbrain through presynaptic<br />
mech<strong>an</strong>isms. We determine real time release of dopamine <strong>from</strong> <strong>the</strong> nucleus accumbens shell <strong>an</strong>d<br />
dorsal striatum using carbon fiber amperometry <strong>an</strong>d measure ch<strong>an</strong>ges in mRNA expression in<br />
<strong>the</strong> cell bodies <strong>from</strong> <strong>the</strong> VTA of dopamine syn<strong>the</strong>sis <strong>an</strong>d signaling genes with qu<strong>an</strong>titative PCR.<br />
We found that male Long-Ev<strong>an</strong>s rats that had ad lib access to running wheels <strong>for</strong> at least 2<br />
months have higher average evoked dopamine levels th<strong>an</strong> sedentary control rats. In <strong>the</strong> nucleus<br />
accumbens shell, evoked dopamine release was 159.8x10 6 + 13.9x10 6 molecules <strong>for</strong> runners vs.<br />
73.8x10 6 + 10.6x10 6 molecules <strong>for</strong> controls (n=10 pulses <strong>from</strong> 2 slices in <strong>the</strong> control <strong>an</strong>imals <strong>an</strong>d<br />
n=15 pulses <strong>from</strong> 3 slices in <strong>the</strong> exercised <strong>an</strong>imals). In <strong>the</strong> dorsal striatum, evoked dopamine<br />
release was 219.3x10 6 + 29.6x10 6 molecules <strong>for</strong> runners vs. 78.9x10 6 + 5.9x10 6 molecules <strong>for</strong><br />
controls (n=15 pulses <strong>from</strong> 3 slices per group). Additionally, tyrosine hydroxylase (TH) <strong>an</strong>d<br />
dopamine reuptake tr<strong>an</strong>sporter (DAT) mRNA expression levels are more th<strong>an</strong> 35 <strong>an</strong>d 350 times<br />
higher in exercised rats over <strong>the</strong>ir sedentary counterparts. These results are in contrast to<br />
hypoactive <strong>an</strong>d hyperphagic inbred obesity-prone rats. Evoked dopamine release <strong>from</strong> <strong>the</strong><br />
nucleus accumbens shell in <strong>the</strong>se rats was 36.8x10 6 + 4.8x10 6 molecules (n=45 pulses in 9 slices)<br />
compared to 78.6x10 6 + 11.5x10 6 molecules (n=53 pulses <strong>from</strong> 11 slices) released <strong>from</strong> <strong>the</strong><br />
obesity-resist<strong>an</strong>t controls. These results suggest that physical exercise may be protective against<br />
drug addiction <strong>an</strong>d hyperphagia through <strong>an</strong> upregulation of central dopamine neurotr<strong>an</strong>smission.<br />
Disclosures: B.M. Geiger, None; M. Karou<strong>an</strong>i, None; J. Munro, None; R. K<strong>an</strong>arek,<br />
None; E.N. Pothos, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.3/X17
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: CIHR gr<strong>an</strong>t RN014705<br />
Title: Opioid-induced neuroplasticity of dopaminergic neurons in <strong>the</strong> ventral tegmental area<br />
mediates rein<strong>for</strong>cement of natural reward<br />
Authors: *K. K. PITCHERS, C. M. COPPENS, J. FULLER, S. VAN, L. M. COOLEN;<br />
Anat. <strong>an</strong>d Cell Biol., Univ. Western Ontario, London, ON, C<strong>an</strong>ada<br />
Abstract: There is a large body of evidence suggesting that natural rewards <strong>an</strong>d drugs of abuse<br />
converge on <strong>the</strong> mesolimbic circuit. Chronic exposure to morphine, <strong>an</strong> exogenous opiate, has<br />
been found to induce biochemical <strong>an</strong>d morphological alterations in dopaminergic neurons of <strong>the</strong><br />
ventral tegmental area (VTA). However, it is unknown if endogenous opioids affect <strong>the</strong> VTA<br />
dopamine neurons in a similar m<strong>an</strong>ner. We previously demonstrated that endogenous opioids act<br />
on mu-opioid receptors in <strong>the</strong> VTA during male sexual behavior, a natural rewarding behavior. It<br />
has been proposed that stimulation of mu-opioid receptors on GABAergic interneurons of <strong>the</strong><br />
VTA results in this inhibition, which in turn leads to <strong>the</strong> disinhibition of VTA dopaminergic<br />
neurons <strong>an</strong>d subsequent release of dopamine into <strong>the</strong> nucleus accumbens as observed during<br />
male sexual behavior. The current study tested <strong>the</strong> hypo<strong>the</strong>ses that mating-induced release of<br />
endogenous opioids in <strong>the</strong> VTA causes morphological ch<strong>an</strong>ges of VTA dopamine cells that in<br />
turn are critical <strong>for</strong> <strong>the</strong> long-term rein<strong>for</strong>cement of sexual behavior. First, <strong>an</strong>imals mated during 5<br />
consecutive days <strong>an</strong>d were examined <strong>for</strong> VTA dopamine cell morphology. We showed using<br />
immunocytochemistry <strong>for</strong> tyrosine hydroxylase (dopamine marker) that sexually experienced<br />
male rats had decreased VTA dopamine soma size 1 <strong>an</strong>d 7, but not 30 days following last mating<br />
session compared to sexually naïve males. Next, it was demonstrated that this neuroplasticity<br />
was dependent on <strong>the</strong> action of endogenous opioids. Blocking opioid receptors with systemic<br />
naloxone (10 mg/kg; s.c.) prior to introduction of a female blocked <strong>the</strong> sexual experienceinduced<br />
decrease in DA soma size. Subsequently, <strong>the</strong> relev<strong>an</strong>ce of <strong>the</strong> neuroplasticity <strong>for</strong><br />
experience-induced ch<strong>an</strong>ges in sexual behavior was tested. Male rats received naloxone or saline,<br />
ei<strong>the</strong>r systemically (10 mg/kg) or intra-VTA (bilateral infusions; 10 µg/hemisphere) prior to each<br />
mating session. All males showed facilitation of sexual behavior during <strong>the</strong> 5 consecutive mating<br />
tests, evidenced by shorter latencies on <strong>the</strong> fifth <strong>an</strong>d final day compared to <strong>the</strong> first day of mating<br />
<strong>an</strong>d naloxone had no effect on facilitation of behavior. However, when <strong>an</strong>imals were tested 7<br />
days following <strong>the</strong> final mating session, facilitation of sexual behavior was not maintained in<br />
naloxone treated <strong>an</strong>imals. Hence, endogenous opioids during mating induce alterations in VTA<br />
dopamine neurons that in turn are critical <strong>for</strong> <strong>the</strong> long-term rein<strong>for</strong>cement of sex behavior.<br />
Toge<strong>the</strong>r, <strong>the</strong>se data provide novel insight into <strong>the</strong> functional relev<strong>an</strong>ce of neuroplasticity in <strong>the</strong><br />
mesolimbic dopamine system induced by endogenous opioids during naturally rewarding<br />
behavior.<br />
Disclosures: K.K. Pitchers, None; C.M. Coppens, None; J. Fuller, None; S. V<strong>an</strong>, None; L.M.<br />
Coolen, None.
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.4/X18<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: CIHR RN014705<br />
Title: Natural reward induces long-term neural plasticity in <strong>the</strong> nucleus accumbens<br />
Authors: *A. R. DI SEBASTIANO, S. SCHMID, K. K. PITCHERS, M. N. LEHMAN, L. M.<br />
COOLEN;<br />
Univ. Western Ontario, London, ON, C<strong>an</strong>ada<br />
Abstract: Drugs of abuse act on <strong>the</strong> mesolimbic system which mediates motivation <strong>an</strong>d reward<br />
of natural behaviors. Repeated exposure to drugs results in neural adaptations in this system,<br />
consisting of tr<strong>an</strong>scriptional <strong>an</strong>d morphological alterations, as well as ch<strong>an</strong>ges in synaptic<br />
strength. This neuroplasticity contributes to <strong>the</strong> development <strong>an</strong>d expression of drug-related<br />
memories <strong>an</strong>d addiction. However, it is not clear if similar ch<strong>an</strong>ges are also induced by naturally<br />
rewarding <strong>an</strong>d rein<strong>for</strong>cing behaviors. Recently, our laboratory has shown that repeated male<br />
sexual behavior (sexual experience) induces long lasting locomotor <strong>an</strong>d reward sensitization to<br />
psychostimul<strong>an</strong>ts, as well as increased dendritic arbors <strong>an</strong>d spines in <strong>the</strong> nucleus accumbens. The<br />
current study investigated if sexual experience also alters <strong>the</strong> synaptic strength of nucleus<br />
accumbens neurons. Male Sprague Dawley rats mated to one ejaculation during 5 consecutive<br />
days. One day, 7 days, or 30 days after last mating session, electrophysiological recordings were<br />
per<strong>for</strong>med in order to determine <strong>the</strong> AMPA/NMDA ratio of synaptic currents in <strong>the</strong> nucleus<br />
accumbens shell. Control <strong>an</strong>imals were h<strong>an</strong>dled <strong>an</strong>d exposed to female odors, but did not mate.<br />
Whole-cell patch clamp recordings of medium spiny neurons in <strong>the</strong> nucleus accumbens shell<br />
revealed a reduced AMPA/NMDA ratio at all time intervals following last mating session<br />
compared to synaptic currents in slices <strong>from</strong> sexually naïve controls. Separate experiments<br />
confirmed that <strong>the</strong> reduced AMPA/NMDA ratio was correlated with sensitization of sexual<br />
behavior <strong>an</strong>d psychostimul<strong>an</strong>t responses. Specifically, sexually experienced males displayed<br />
facilitation of sexual behavior 1, 7, or 30 after final mating session. Similarly, males displayed <strong>an</strong><br />
enh<strong>an</strong>ced locomotor response to amphetamine after 1, 7, <strong>an</strong>d 30 days after final mating session<br />
compared to sexually naïve control males. Toge<strong>the</strong>r, <strong>the</strong>se data show that repeated sexual<br />
experience causes neural alterations in <strong>the</strong> nucleus accumbens at a synaptic level that c<strong>an</strong> be<br />
detected immediately after mating <strong>an</strong>d which are long-lasting. These alterations may contribute<br />
to <strong>the</strong> long lasting rein<strong>for</strong>cement of sexual behavior, suggesting common mech<strong>an</strong>isms <strong>for</strong><br />
rein<strong>for</strong>cement of natural <strong>an</strong>d drug reward. However, <strong>the</strong> sexual experience-induced alteration is<br />
different <strong>from</strong> drug-induced plasticity as <strong>the</strong> latter appears in a bidirectional m<strong>an</strong>ner with
increased AMPA/NMDA ratio with prolonged periods of drug withdrawal. Thus, toge<strong>the</strong>r, <strong>the</strong>se<br />
studies suggest that <strong>the</strong> bidirectional ch<strong>an</strong>ge plays <strong>an</strong> essential role in <strong>the</strong> development of<br />
addictive behavior.<br />
Disclosures: A.R. Di Sebasti<strong>an</strong>o, None; S. Schmid, None; K.K. Pitchers, None; M.N.<br />
Lehm<strong>an</strong>, None; L.M. Coolen, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.5/X19<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: T32 DA07255<br />
RO1 DA09397<br />
Title: Uncertainty of saccharin rein<strong>for</strong>cement leads to enh<strong>an</strong>ced responding to amphetamine <strong>an</strong>d<br />
alterations in dendritic spine density in <strong>the</strong> nucleus accumbens<br />
Authors: *B. F. SINGER 1 , J. SCOTT-RAILTON 2 , V. BINDOKAS 3 , P. VEZINA 1,2 ;<br />
1 2 3<br />
Committee on Neurobiol, Dept. of Psychiatry, Dept. of Neurobiology, Pharmacol. <strong>an</strong>d<br />
Physiol., Univ. Chicago, Chicago, IL<br />
Abstract: Drug naïve, non-deprived male rats in separate groups were trained to lever press <strong>for</strong><br />
non-caloric saccharin under escalating fixed-ratio (FR) or variable-ratio (VR) schedules of<br />
rein<strong>for</strong>cement. As <strong>the</strong> schedules escalated over days, <strong>the</strong> relation between lever pressing <strong>an</strong>d<br />
saccharin remained fixed <strong>for</strong> rats under <strong>the</strong> FR schedules but became increasingly uncertain <strong>for</strong><br />
rats under <strong>the</strong> VR schedules, even though <strong>the</strong> average number of rein<strong>for</strong>cers remained <strong>the</strong> same<br />
<strong>for</strong> both groups throughout training. After 55 days of training, rats worked reliably under ei<strong>the</strong>r<br />
FR-19 or VR-19 schedules of rein<strong>for</strong>cement, respectively. Starting two weeks after <strong>the</strong> last<br />
saccharin training session, all rats were tested <strong>for</strong> <strong>the</strong>ir locomotor response to amphetamine. A<br />
threshold amphetamine challenge (0.5 mg/kg IP) produced a signific<strong>an</strong>tly greater locomotor<br />
response in VR- compared to FR-trained rats. In addition, three subsequent injections of<br />
amphetamine (1 mg/kg IP, one injection every third day) produced more locomotor activity in<br />
VR-trained <strong>an</strong>imals. Finally, brains were harvested 2-3 days later <strong>an</strong>d dendritic spine density was<br />
assessed in <strong>the</strong> nucleus accumbens. Rats previously trained on <strong>the</strong> VR rein<strong>for</strong>cement schedules<br />
showed reduced dendritic spine density relative to rats trained on <strong>the</strong> FR schedules. Toge<strong>the</strong>r,
<strong>the</strong>se findings suggest that exposure to uncertainty c<strong>an</strong> produce long-lasting ch<strong>an</strong>ges in<br />
behavioral responding to amphetamine that are accomp<strong>an</strong>ied by persistent ch<strong>an</strong>ges in dendritic<br />
morphology. These latter ch<strong>an</strong>ges produced by uncertain rein<strong>for</strong>cement may reflect common<br />
neuroadaptations underlying different addictions, such as gambling <strong>an</strong>d stimul<strong>an</strong>t abuse, <strong>an</strong>d thus<br />
support <strong>an</strong> integrative model of addiction.<br />
Disclosures: B.F. Singer, None; J. Scott-Railton, None; V. Bindokas, None; P. Vezina, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.6/X20<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH Gr<strong>an</strong>t RO1- DA022202<br />
NIH Gr<strong>an</strong>t F32-DA025447<br />
NIH Gr<strong>an</strong>t T32 NS051156-03<br />
Title: MeCP2 mediates neuronal adaptation in response to psychostimul<strong>an</strong>t treatment<br />
Authors: *J. DENG 1 , R. M. RODRIGUIZ 2 , A. N. HUTCHINSON 3 , W. C. WETSEL 2 , A. E.<br />
WEST 1 ;<br />
1 2 3<br />
Duke Univ., Durham, NC; Dept. of Psychiatry <strong>an</strong>d Behavioral Sci., Dept. of Neurobio., Duke<br />
Univ. Med. Ctr., Durham, NC<br />
Abstract: Drugs of abuse alter behavior <strong>an</strong>d c<strong>an</strong> lead to addiction by driving long-lasting<br />
ch<strong>an</strong>ges in mesolimbic neural circuits. These adaptations are mediated, in part, by drug-regulated<br />
expression of gene tr<strong>an</strong>scription. Epigenetic mech<strong>an</strong>isms of chromatin regulation have emerged<br />
as strong c<strong>an</strong>didates to mediate persistent behavioral adaptations to drugs of abuse. Here we<br />
show that mice bearing a hypomorphic mutation in <strong>the</strong> methyl-DNA binding protein MeCP2<br />
show enh<strong>an</strong>ced sensitivity <strong>an</strong>d altered locomotor sensitization to amphetamine. Mecp2 mut<strong>an</strong>t<br />
(308 truncation) mice have increased numbers of striatal GABAergic synapses <strong>an</strong>d show<br />
dysregulation of amphetamine-induced immediate-early gene expression, which may contribute<br />
to behavioral abnormalities in <strong>the</strong>se mice. Intriguingly, psychostimul<strong>an</strong>ts drive phosphorylation<br />
of MeCP2 at Ser421, a site shown to regulate its function as a tr<strong>an</strong>scriptional repressor.<br />
Phosphorylation is selectively induced in parvalbumin-positive GABAergic interneurons of <strong>the</strong>
nucleus accumbens (NAc) <strong>an</strong>d <strong>the</strong> extent of phosphorylation strongly predicts <strong>the</strong> degree of<br />
behavioral sensitization in individual <strong>an</strong>imals. Taken toge<strong>the</strong>r, <strong>the</strong>se data reveal a novel role <strong>for</strong><br />
MeCP2 in psychostimul<strong>an</strong>t-induced behavioral sensitization, <strong>an</strong>d suggest that this response may<br />
be mediated by a select population of NAc GABAergic interneurons.<br />
Disclosures: J. Deng, None; R.M. Rodriguiz, None; A.N. Hutchinson, None; W.C. Wetsel,<br />
None; A.E. West, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.7/X21<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH DA19666<br />
NIH DA07234<br />
NIH DA23750<br />
Title: Stressful experience modifies cellular function in <strong>the</strong> nucleus accumbens<br />
Authors: *P. E. ROTHWELL, S. KOURRICH, B. L. LACROIX, J. R. KLUG, M. J.<br />
THOMAS;<br />
Neurosci, Univ. Minnesota, Minneapolis, MN<br />
Abstract: Stressful life experience is <strong>an</strong> import<strong>an</strong>t predisposing factor <strong>for</strong> <strong>the</strong> development of<br />
psychiatric disease, including mood disorders <strong>an</strong>d addiction. We have examined <strong>the</strong> impact of<br />
stressful experience on cellular function in <strong>the</strong> nucleus accumbens, a critical component of <strong>the</strong><br />
brain reward system. After exposing mice to a novel environment <strong>for</strong> 60 minutes (a stressful<br />
event), we prepared acute brain slices <strong>an</strong>d per<strong>for</strong>med whole-cell recordings <strong>from</strong> medium spiny<br />
neurons in <strong>the</strong> core <strong>an</strong>d shell subregions of nucleus accumbens. A single stressful event<br />
enh<strong>an</strong>ced <strong>the</strong> AMPA/NMDA ratio, a measure of excitatory synaptic strength. This ch<strong>an</strong>ge was<br />
selectively observed in <strong>the</strong> shell (t46 = 2.92, p = .005) but not <strong>the</strong> core (t14
literature on cellular modifications caused by stressful experience that may increase vulnerability<br />
to drug addiction <strong>an</strong>d o<strong>the</strong>r psychiatric diseases.<br />
Disclosures: P.E. Rothwell, None; S. Kourrich, None; B.L. LaCroix, None; J.R. Klug,<br />
None; M.J. Thomas, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.8/X22<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIDA 5F32 DA023741<br />
NIDA RO1 DA09264<br />
Title: Pathway specific cellular <strong>an</strong>d synaptic properties of medium spiny neurons in <strong>the</strong> nucleus<br />
accumbens core<br />
Authors: *B. A. GRUETER, R. C. MALENKA;<br />
Psychiatry & Behavioral Sci., St<strong>an</strong><strong>for</strong>d Univ., Palo Alto, CA<br />
Abstract: The nucleus accumbens (NAc) is a component of <strong>the</strong> mesolimbic dopamine system<br />
thought to contribute to behavioral ch<strong>an</strong>ges associated with drugs of abuse. Similar to <strong>the</strong> dorsal<br />
striatum, neural circuits in <strong>the</strong> NAc are thought to flow through <strong>the</strong> basal g<strong>an</strong>glia in distinct<br />
parallel circuits, termed <strong>the</strong> direct <strong>an</strong>d indirect pathways. Despite <strong>the</strong> import<strong>an</strong>ce of <strong>the</strong>se<br />
pathways, little is known about differences in <strong>the</strong> cellular <strong>an</strong>d synaptic properties of <strong>the</strong>se two<br />
populations of NAc medium spiny neurons (MSNs) <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>y express different <strong>for</strong>ms of<br />
synaptic plasticity. Utilizing bacterial artificial chromosome tr<strong>an</strong>sgenic mice that express GFP in<br />
indirect pathway, D2 dopamine receptor-expressing MSNs, we have begun to examine <strong>the</strong><br />
cellular <strong>an</strong>d synaptic properties of <strong>the</strong>se different cell populations in <strong>the</strong> NAc core using targeted<br />
whole-cell patch clamp electrophysiological techniques. We find that <strong>the</strong> NAc D2-expressing<br />
MSNs have a higher firing rate in response to depolarizing current injection as well as greater<br />
mEPSC frequency <strong>an</strong>d NMDA/AMPA ratios. Fur<strong>the</strong>rmore, LTD elicited by 10 Hz, 5 minute<br />
stimulation is robustly expressed in D2-expressing but not D2-lacking MSNs. Pharmacological<br />
<strong>an</strong>d genetic m<strong>an</strong>ipulations have allowed us to tease apart novel signaling mech<strong>an</strong>isms underlying<br />
LTD of excitatory synapses onto D2-expressing MSNs. These results demonstrate that <strong>the</strong>se two<br />
populations of NAc MSNs express different cellular <strong>an</strong>d synaptic properties.
Disclosures: B.A. Grueter, None; R.C. Malenka, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.9/X23<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: Hope Foundation <strong>for</strong> Depression Research<br />
Cocaine was supplied in part by <strong>the</strong> Drug Supply Program of NIH NIDA<br />
Title: Differential effects of passive <strong>an</strong>d active cocaine exposure on membr<strong>an</strong>e excitability of<br />
nucleus accumbens neurons<br />
Authors: P. MU, *M. ISHIKAWA, Y. ZHANG, Y. DONG;<br />
Dept of VCAPP, Washington State Univ., Pullm<strong>an</strong>, WA<br />
Abstract: The membr<strong>an</strong>e excitability of nucleus accumbens (NAc) neurons is one of <strong>the</strong> primary<br />
targets <strong>for</strong> drugs of abuse to produce addiction-related behaviors. It has long been known that<br />
different patterns of drug exposure may result in m<strong>an</strong>y different behavioral consequences. In <strong>an</strong><br />
attempt to determine <strong>the</strong> cellular basis underlying <strong>the</strong> pattern-dependent behavioral alterations by<br />
drugs of abuse, we compared <strong>the</strong> effects of intraperitoneal (i.p.) cocaine injection <strong>an</strong>d<br />
intravenous (i.v.) cocaine self-administration on <strong>the</strong> membr<strong>an</strong>e excitability of NAc neurons.<br />
More specifically, whole-cell current-clamp recordings were per<strong>for</strong>med to measure <strong>the</strong><br />
membr<strong>an</strong>e excitability of NAc medium spiny neurons <strong>from</strong> rats that previously received i.p. (15<br />
mg/kg/day) or i.v. (0.75 mg/kg per 0.1ml/6 sec) cocaine exposure. We first examined rats treated<br />
with 5-day cocaine exposure, followed by a 2-day withdrawal. Compared to saline control<br />
groups, <strong>the</strong> basal membr<strong>an</strong>e excitability of NAc neurons in both passive (i.p.) <strong>an</strong>d active (i.v.)<br />
group was signific<strong>an</strong>tly decreased, accomp<strong>an</strong>ied by a concurrent increase in <strong>the</strong> medium<br />
component of <strong>the</strong> afterhyperpolarization potential. However, <strong>the</strong> resting membr<strong>an</strong>e potential of<br />
NAc neurons, which was not altered by i.p. cocaine injections, was signific<strong>an</strong>tly depolarized by<br />
cocaine self-administration. At a longer withdrawal time point (3 weeks), we observed that <strong>the</strong><br />
membr<strong>an</strong>e excitability of NAc neurons remained low in rats received i.p. cocaine injections. In<br />
contrast, <strong>the</strong> membr<strong>an</strong>e excitability of NAc neurons returned to <strong>the</strong> baseline in self-administrated<br />
rats at this long withdrawal time point. Fur<strong>the</strong>rmore, upon re-exposure to cocaine after 3 weeks<br />
of withdrawal, <strong>the</strong> membr<strong>an</strong>e excitability of NAc neurons <strong>from</strong> both i.p. <strong>an</strong>d self-administration<br />
groups became higher th<strong>an</strong> that of <strong>the</strong> saline control groups. Thus, <strong>the</strong> membr<strong>an</strong>e excitability of
NAc neurons is differentially regulated by different patterns of cocaine exposure as well as by<br />
different withdrawal time. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that dynamic regulation of<br />
membr<strong>an</strong>e excitability of NAc neurons may differentially contribute to different aspects of<br />
addiction-related behavioral alterations.<br />
Disclosures: P. Mu, None; M. Ishikawa, None; Y. Zh<strong>an</strong>g, None; Y. Dong, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.10/X24<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: DA00962<br />
DA015835<br />
DA000453<br />
Title: Mech<strong>an</strong>isms underlying AMPA receptor plasticity associated with <strong>the</strong> incubation of<br />
cocaine craving<br />
Authors: *C. R. FERRARIO, M. MILOVANOVIC, K. A. FORD, X. WANG, M. E. WOLF;<br />
Rosalind Fr<strong>an</strong>klin Univ. Med. Sci., North Chicago, IL<br />
Abstract: Our lab has previously demonstrated that GluR2-lacking AMPA receptors are added<br />
to nucleus accumbens (NAc) synapses after prolonged (45 days) but not short (1 day) withdrawal<br />
<strong>from</strong> cocaine self-administration <strong>an</strong>d that activation of <strong>the</strong>se receptors mediates <strong>the</strong> expression of<br />
enh<strong>an</strong>ced (incubated) cocaine craving after prolonged withdrawal. In order to examine synaptic<br />
alterations in this model, we prepared a post-synaptic density (PSD) fraction of <strong>the</strong> NAc <strong>from</strong><br />
rats given 1 or 45 days of withdrawal <strong>from</strong> cocaine or saline self-administration. Whole-cell<br />
patch clamp recordings confirmed <strong>the</strong> presence of GluR2-lacking AMPA receptors at withdrawal<br />
day 45 in <strong>the</strong> NAc of a subset of rats in this cohort. In <strong>the</strong> cocaine-withdrawal day 45 group, we<br />
found increased CaMKII <strong>an</strong>d ERK2 activation compared to saline controls. We also observed <strong>an</strong><br />
increase in total protein levels of SAP-97, <strong>the</strong> only MAGUK protein known to directly interact<br />
with <strong>the</strong> GluR1 subunit. These data are consistent with <strong>an</strong> increase in synaptic incorporation of<br />
GluR1 <strong>an</strong>d may be indicative of synaptic remodeling. O<strong>the</strong>r signaling pathways that may link<br />
glutamate tr<strong>an</strong>smission to synaptic remodeling are currently being studied. Our previous
electrophysiological experiments showed that blockade of GluR2-lacking AMPA receptors with<br />
Naspm reduced evoked EPSC amplitude by ~30% in NAc slices <strong>from</strong> cocaine-withdrawal day<br />
45 rats, indicating that at least a portion of new GluR2-lacking AMPA receptors are added to<br />
synapses after prolonged withdrawal <strong>from</strong> cocaine self-administration. However, biochemical<br />
measures have indicated a variable but sometimes extremely robust increase in total GluR1<br />
protein at <strong>the</strong> same withdrawal time (150-250% of saline control levels, depending on <strong>the</strong> cohort<br />
of rats). To test <strong>the</strong> hypo<strong>the</strong>sis that some GluR2-lacking AMPA receptors are added<br />
extrasynaptically, we are using subcellular fractionation procedures to assess synaptic <strong>an</strong>d<br />
extrasynaptic levels of AMPA receptors, <strong>an</strong>d NMDA receptors, after withdrawal <strong>from</strong> cocaine<br />
self-administration.<br />
Disclosures: C.R. Ferrario, None; M. Milov<strong>an</strong>ovic, None; K.A. Ford, None; X. W<strong>an</strong>g,<br />
None; M.E. Wolf, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.11/X25<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: DA009621<br />
DA015835<br />
DA000453<br />
Title: Mech<strong>an</strong>isms underlying increased AMPA receptor surface expression in cocainesensitized<br />
rats<br />
Authors: *X. WANG 1 , C. R. FERRARIO 1 , M. MILOVANOVIC 1 , Z. XIE 2 , P. PENZES 2 , M. E.<br />
WOLF 1 ;<br />
1 2<br />
Neurosci., Rosalind Fr<strong>an</strong>klin Univ. of Med. <strong>an</strong>d Sci., North Chicago, IL; Northwestern Univ.,<br />
Chicago, IL<br />
Abstract: Repeated cocaine administration produces enduring molecular, cellular <strong>an</strong>d behavioral<br />
plasticity. Although <strong>the</strong> underlying mech<strong>an</strong>isms are still not clear, considerable evidence<br />
suggests that increases in glutamate neurotr<strong>an</strong>smission <strong>an</strong>d postsynaptic signaling in nucleus<br />
accumbens (NAc) play import<strong>an</strong>t roles in behavioral sensitization to cocaine. Prior work <strong>from</strong>
our lab demonstrated increased surface expression of GluR1/2-containing AMPA receptors in<br />
<strong>the</strong> NAc of cocaine-sensitized rats. AMPA receptor upregulation developed during <strong>the</strong> first week<br />
of withdrawal <strong>an</strong>d was sustained at least through withdrawal day 21 (Boudreau & Wolf, J<br />
Neurosci 2005; Boudreau et al., J Neurochem <strong>2009</strong>). In <strong>the</strong>se prior studies, AMPA receptor<br />
surface expression was assessed using a BS 3 protein crosslinking assay. The goal of <strong>the</strong> present<br />
study was to fur<strong>the</strong>r explore <strong>the</strong> molecular mech<strong>an</strong>isms underlying increased AMPA receptor<br />
surface expression, <strong>an</strong>d to investigate <strong>the</strong> relationship between AMPA receptor upregulation <strong>an</strong>d<br />
signaling pathways that might be responsible <strong>for</strong> <strong>the</strong> morphological plasticity of spines <strong>an</strong>d<br />
dendrites observed after cocaine administration. To this end, we used a cocaine regimen (15<br />
mg/kg <strong>for</strong> 8 days) that increases spine density in <strong>the</strong> NAc core on withdrawal day 14 (Li et al.,<br />
Eur J Neurosci 2004). We employed a subcellular fractionation procedure to yield a<br />
synaptosomal membr<strong>an</strong>e fraction (LP1), which is enriched in postsynaptic density proteins. The<br />
subcellular fractionation procedure was validated by <strong>the</strong> use of protein markers <strong>for</strong> subcellular<br />
compartments. GluR1 expression was signific<strong>an</strong>tly increased in <strong>the</strong> LP1 fraction prepared <strong>from</strong><br />
cocaine-sensitized rats on withdrawal day 14, in agreement with our prior findings using <strong>the</strong> BS 3<br />
crosslinking assay. Experiments are underway to <strong>an</strong>alyze signaling molecules that may link<br />
postsynaptic glutamate signaling to <strong>the</strong> regulation of cytoskeletal dynamics.<br />
Disclosures: X. W<strong>an</strong>g, None; C.R. Ferrario, None; M. Milov<strong>an</strong>ovic, None; Z. Xie, None; P.<br />
Penzes, None; M.E. Wolf, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.12/X26<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: DA009621<br />
DA000453<br />
Title: Brain-derived neurotrophic factor (BDNF) <strong>an</strong>d AMPA receptor trafficking in <strong>the</strong> nucleus<br />
accumbens core<br />
Authors: *X. LI 1 , A. BAHI 3 , M. R. DEJOSEPH 2 , J. H. URBAN 2 , J.-L. DREYER 4 , M. E.<br />
WOLF 1 ;<br />
1 Neurosci., 2 Physiol. <strong>an</strong>d Biophysics, Rosalind Fr<strong>an</strong>klin Univ. of Med. <strong>an</strong>d Sci., North Chicago,
IL; 3 Psychiatry, Yale Univ. Sch. of Med., New Haven, CT; 4 Biochem., Univ. of Fribourg,<br />
Fribourg, Switzerl<strong>an</strong>d<br />
Abstract: BDNF has been suggested to play <strong>an</strong> import<strong>an</strong>t role in drug addiction. Some results<br />
indicate that elevating BDNF levels in <strong>the</strong> nucleus accumbens (NAc) c<strong>an</strong> enh<strong>an</strong>ce cocainerelated<br />
behavioral responses, including cocaine seeking after withdrawal (Bahi et al.,<br />
Psychopharmacol. 2007; Graham et al., Nat. Neurosci. 2007). The time-dependent intensification<br />
(incubation) of cocaine seeking after prolonged withdrawal <strong>from</strong> long-access cocaine selfadministration<br />
is accomp<strong>an</strong>ied by increased BDNF levels in <strong>the</strong> NAc (Grimm et al., J. Neurosci.<br />
2003). Previously, we have shown that incubation of cocaine craving after <strong>the</strong> same regimen is<br />
mediated by increased levels of GluR2-lacking AMPA receptors in <strong>the</strong> NAc core (Conrad et al.,<br />
Nature 2008). The goal of <strong>the</strong> present study is to test <strong>the</strong> hypo<strong>the</strong>sis that BDNF mediates <strong>the</strong><br />
increase in GluR2-lacking AMPA receptors. This is based on results in o<strong>the</strong>r systems showing<br />
that BDNF c<strong>an</strong> promote plasticity at glutamate synapses through mech<strong>an</strong>isms that include<br />
increased synaptic delivery of homomeric GluR1 receptors (Caldeira et al., J. Biol. Chem. 2007;<br />
Li <strong>an</strong>d Keifer, Neurosci. 2008). However, whe<strong>the</strong>r BDNF c<strong>an</strong> have a similar effect in <strong>the</strong> NAc is<br />
not known. In order to study <strong>the</strong> in vivo effect of BDNF on AMPA receptor distribution in <strong>the</strong><br />
NAc, we infused BDNF (0.75ug/0.5ul/side) into <strong>the</strong> NAc core through guide c<strong>an</strong>nulae. Using a<br />
BS 3 protein crosslinking assay <strong>an</strong>d Western blotting, we are able to distinguish surface-expressed<br />
<strong>an</strong>d intracellular AMPA receptor subunits. In a time-course study, we measured GluR1<br />
distribution 30 min, 3 hrs <strong>an</strong>d 24 hrs after BDNF infusion <strong>an</strong>d found no signific<strong>an</strong>t differences in<br />
GluR1 surface expression in NAc core between BDNF-treated <strong>an</strong>d vehicle-treated <strong>an</strong>imals.<br />
These results suggest that BDNF may not play <strong>the</strong> same role in <strong>the</strong> NAc as it does in cultured<br />
hippocampal slices, where GluR1 synaptic delivery was observed over <strong>the</strong> same time period<br />
studied in our experiments (Caldeira et al., 2007). Alternatively, <strong>the</strong> delivery system we chose<br />
may not be suited to detecting biochemical ch<strong>an</strong>ges because of possible neuronal damage <strong>from</strong><br />
<strong>the</strong> infusion. Experiments are underway to use intracr<strong>an</strong>ial infusion of lentiviral vectors to ei<strong>the</strong>r<br />
over-express BDNF or knockdown <strong>the</strong> TrkB receptor in NAc core, in order to test BDNF’s role<br />
in mediating <strong>the</strong> increase in GluR2-lacking AMPA receptors that underlies <strong>the</strong> incubation of<br />
cocaine craving.<br />
Disclosures: X. Li, None; A. Bahi, None; M.R. DeJoseph, None; J.H. Urb<strong>an</strong>, None; J.<br />
Dreyer, None; M.E. Wolf, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.13/X27
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: DA015835<br />
DA000453<br />
Title: Effect of brain-derived neurotrophic factor (BDNF) on AMPA receptor trafficking in<br />
primary cultures of nucleus accumbens neurons<br />
Authors: *J. M. REIMERS, M. E. WOLF;<br />
Rosalind Fr<strong>an</strong>klin Univ. Med. Sci., North Chicago, IL<br />
Abstract: BDNF is a peptide that binds to <strong>the</strong> TrkB receptor <strong>an</strong>d activates intracellular signaling<br />
cascades. It has been shown to play a role in synaptic plasticity <strong>an</strong>d in <strong>the</strong> survival <strong>an</strong>d function<br />
of midbrain dopamine neurons. Increases in BDNF protein levels have been observed in nucleus<br />
accumbens (NAc), ventral tegmental area, <strong>an</strong>d amygdala in association with <strong>the</strong> time-dependent<br />
intensification (incubation) of cocaine craving after withdrawal <strong>from</strong> cocaine self-administration<br />
(Grimm et al., 2003). Using <strong>the</strong> same incubation of cocaine craving model, our lab has shown <strong>an</strong><br />
increase in GluR2-lacking AMPA receptors in <strong>the</strong> NAc (Conrad et al., 2008). As BDNF has been<br />
shown to increase AMPA receptor synaptic delivery in o<strong>the</strong>r systems, we hypo<strong>the</strong>sized that<br />
increased BDNF levels might underlie synaptic addition of GluR2-lacking AMPA receptors in<br />
<strong>the</strong> incubation model. As a first step towards testing this hypo<strong>the</strong>sis, we are studying <strong>the</strong> effects<br />
of BDNF on AMPA receptor expression <strong>an</strong>d distribution in medium spiny neurons of <strong>the</strong> NAc in<br />
vitro. We per<strong>for</strong>med two types of studies. First, we treated high-density primary NAc cultures<br />
with 100ng/mL BDNF <strong>for</strong> 30 min, 3 hr, or 24 hr. After BDNF treatment, a BS3 protein<br />
crosslinking assay was used to distinguish surface <strong>from</strong> intracellular AMPA receptor subunits.<br />
Preliminary data have shown no signific<strong>an</strong>t effect of BDNF on surface-expressed or intracellular<br />
GluR1. Studies are underway to examine o<strong>the</strong>r AMPA receptor subunits <strong>an</strong>d to assess ERK<br />
activation. The NAc does not contain intrinsic glutamate neurons, so pure NAc cultures do not<br />
contain glutamate synapses. There<strong>for</strong>e, to study <strong>the</strong> effect of BDNF on synaptic AMPA receptor<br />
expression, primary NAc neurons <strong>from</strong> P1 rats were co-cultured with prefrontal cortex neurons<br />
<strong>from</strong> P1 enh<strong>an</strong>ced cy<strong>an</strong> fluorescent protein (ECFP) expressing mice. Prefrontal cortical neurons<br />
restore excitatory inputs onto <strong>the</strong> NAc medium spiny neurons, but cell types c<strong>an</strong> be distinguished<br />
based on fluorescence. Co-cultures (14-21 DIV) were treated with 100ng/mL BDNF <strong>for</strong> 30 min,<br />
3 hr, or 24 hr. Live-cell staining was used to visualize surface GluR1 <strong>an</strong>d GluR2. Preliminary<br />
results <strong>from</strong> <strong>the</strong>se studies show a trend towards <strong>an</strong> increase in <strong>the</strong> number of GluR1 puncta in <strong>the</strong><br />
30 min group <strong>an</strong>d a signific<strong>an</strong>t decrease in <strong>the</strong> 3 hr <strong>an</strong>d 24 hr groups compared to <strong>the</strong> 30 min<br />
condition. Similar effects were found when measuring GluR1 staining area. Experiments are<br />
underway to examine <strong>the</strong> effect of both shorter <strong>an</strong>d longer BDNF treatments, <strong>an</strong>d to investigate a<br />
possible role <strong>for</strong> BDNF in synaptic scaling in <strong>the</strong> NAc.<br />
Disclosures: J.M. Reimers, None; M.E. Wolf, None.<br />
Poster
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.14/X28<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: P50 DA15369<br />
T32 DA07288<br />
Title: Alterations in brain-derived neurotrophic factor levels in brain regions involved in drug<br />
addiction induced by intracr<strong>an</strong>ial electrical stimulation of <strong>the</strong> rodent prefrontal cortex<br />
Authors: *S. A. EISENSTEIN 1 , Y. JIANG 2 , Z. NAHAS 2 , J. F. MCGINTY 1 ;<br />
1 Neurosci., 2 Mood Disorders Program, Med. Univ. of South Carolina, Charleston, SC<br />
Abstract: Intracr<strong>an</strong>ial electrical stimulation (ICES) is a procedure in which electrodes that are<br />
impl<strong>an</strong>ted in <strong>the</strong> brain are electrically stimulated, inducing ch<strong>an</strong>ges in neuronal activity at <strong>the</strong> site<br />
of stimulation as well as in neuronal networks encompassing <strong>the</strong> stimulation site. Recent studies<br />
have shown that ICES of <strong>the</strong> nucleus accumbens (NAc) <strong>an</strong>d <strong>the</strong> medial prefrontal cortex (mPFC)<br />
suppresses cocaine-seeking in cocaine-addicted rats, indicating that ICES exerts beneficial<br />
effects on <strong>the</strong> neural circuitry underlying cocaine addiction. Our laboratory has previously found<br />
that cocaine-seeking is suppressed <strong>an</strong>d glutamatergic tr<strong>an</strong>smission in <strong>the</strong> NAc is normalized by<br />
mPFC infusion of exogenous brain-derived neurotrophic factor (BDNF), a peptide<br />
neurotr<strong>an</strong>smitter that is released upon increased synaptic activity <strong>an</strong>d induces ch<strong>an</strong>ges in <strong>the</strong><br />
expression of immediate-early genes that are import<strong>an</strong>t in synaptic plasticity. There<strong>for</strong>e, we<br />
hypo<strong>the</strong>sized that one mech<strong>an</strong>ism by which ICES of <strong>the</strong> mPFC suppresses cocaine-seeking is by<br />
enh<strong>an</strong>cing BDNF levels <strong>an</strong>d altering immediate-early gene expression in <strong>the</strong> mPFC <strong>an</strong>d <strong>the</strong> NAc,<br />
<strong>the</strong>reby restoring normal neural activity. As a first step towards investigating this hypo<strong>the</strong>sis, we<br />
examined <strong>the</strong> effect of two hours of high-frequency ICES of <strong>the</strong> mPFC on BDNF levels in <strong>the</strong><br />
NAc. Rats with electrodes impl<strong>an</strong>ted in <strong>the</strong> mPFC were ei<strong>the</strong>r stimulated (130 Hz, 100 µA) or<br />
sham-stimulated (no stimulation) <strong>for</strong> two hours <strong>an</strong>d decapitated immediately. NAc tissue was<br />
punched <strong>an</strong>d assayed <strong>for</strong> BDNF protein levels using enzyme-linked immunosorbent assay<br />
(ELISA). Preliminary results indicate a trend toward increased BDNF protein in <strong>the</strong> NAc of<br />
stimulated rats relative to sham stimulated rats. The effects of duration of mPFC stimulation <strong>an</strong>d<br />
stimulation parameters (frequency <strong>an</strong>d amplitude) on cocaine-seeking, BDNF levels <strong>an</strong>d<br />
immediate-early gene expression in brain regions involved in drug addiction are currently under<br />
investigation. These studies will aid in identifying a neurochemical mech<strong>an</strong>ism <strong>for</strong> <strong>the</strong> beneficial<br />
effects of ICES <strong>an</strong>d will fur<strong>the</strong>r investigate <strong>the</strong> ability of ICES of <strong>the</strong> mPFC to suppress cocaineseeking<br />
in <strong>an</strong> <strong>an</strong>imal model of cocaine addiction.<br />
Disclosures: S.A. Eisenstein, None; Y. Ji<strong>an</strong>g, None; Z. Nahas, None; J.F. McGinty, None.
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.15/X29<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: DA017905<br />
DA00354<br />
Title: Altered binding of MeCP2 to <strong>the</strong> promoter region of BDNF in <strong>the</strong> medial prefrontal cortex<br />
of prenatally cocaine-exposed adult mice<br />
Authors: *Z. R. DARUWALLA 1,2 , S. RA 2 , A. RAJADHYAKSHA 1,2 , B. KOSOFSKY 1,2 ;<br />
1 Dept. of Neuroscience, Weill Cornell Grad. Sch. of Biomed. Sci., New York, NY; 2 Dept. of<br />
Pediatric Neurology, Weill Med. Col. of Cornell Univ., New York, NY<br />
Abstract: Prenatal cocaine exposure results in persistent ch<strong>an</strong>ges in <strong>the</strong> structure <strong>an</strong>d function of<br />
<strong>the</strong> brain possibly due to epigenetic modifications of <strong>the</strong> genome. Because brain derived<br />
neurotrophic factor (BDNF) tr<strong>an</strong>scribed <strong>from</strong> exon IV is <strong>an</strong> import<strong>an</strong>t regulator of cocaineinduced<br />
plasticity we focused on its role in mediating such ch<strong>an</strong>ges. Recently, <strong>the</strong> lab identified<br />
increased BDNF mRNA expression <strong>from</strong> <strong>the</strong> exon IV promoter in <strong>the</strong> medial prefrontal cortex<br />
(mPFC) of prenatally cocaine-exposed (PCE) adult mice. Epigenetic ch<strong>an</strong>ges in <strong>the</strong> methylation<br />
status of CpG isl<strong>an</strong>ds, in <strong>the</strong> promoter region of BDNF exon IV influences its expression by<br />
altering <strong>the</strong> binding of <strong>the</strong> repressor, methyl cytosine binding protein 2 (MeCP2). We were<br />
interested in determining whe<strong>the</strong>r <strong>the</strong> ch<strong>an</strong>ge in BDNF expression in <strong>the</strong> mPFC of PCE adult<br />
mice was a result of epigenetic modifications of <strong>the</strong> BDNF exon IV promoter region affecting<br />
binding of MeCP2. We found that in PCE adult mice <strong>the</strong>re was a signific<strong>an</strong>t decrease in <strong>the</strong><br />
binding of MeCP2 to <strong>the</strong> BDNF exon IV promoter in <strong>the</strong> mPFC but not in <strong>the</strong> hippocampus. To<br />
explore behavioral ch<strong>an</strong>ges attributable to <strong>the</strong>se molecular phenomena we studied <strong>the</strong> freezing<br />
behavior in PCE adult mice immediately after acute footshock, a behavior that has been shown to<br />
be dependent on <strong>the</strong> mPFC. We saw a signific<strong>an</strong>t increase in <strong>the</strong> freezing behavior in <strong>the</strong> PCE<br />
adult mice over a five-minute observation period following footshock. We are now working to<br />
identify <strong>the</strong> exact CpG isl<strong>an</strong>d altering MeCP2 binding to <strong>the</strong> BDNF exon IV promoter <strong>an</strong>d if<br />
<strong>the</strong>re exists a persistent ch<strong>an</strong>ge in <strong>the</strong> phosphorylation status of MeCP2 after PCE, molecular<br />
adaptations which may additionally underlie o<strong>the</strong>r aspects of <strong>the</strong> phenotype in mice <strong>an</strong>d hum<strong>an</strong>s.
Disclosures: Z.R. Daruwalla, None; S. Ra, None; A. Rajadhyaksha, None; B. Kosofsky,<br />
None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.16/X30<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: Hartwell Foundation (AR)<br />
NIDA Gr<strong>an</strong>t R21DA023686-01A1 (AR)<br />
Austri<strong>an</strong> Science Fund P20670 (JS)<br />
Title: Differential regulation of MeCP2-mediated BDNF splice vari<strong>an</strong>t expression by L-type<br />
Cav1.2 <strong>an</strong>d Cav1.3 Ca2+ ch<strong>an</strong>nels in <strong>the</strong> mouse hippocampus <strong>an</strong>d cortex<br />
Authors: *A. M. RAJADHYAKSHA 1 , S. C. R. HAUCK 1 , Z. DARUWALLA 1 , J.<br />
STRIESSNIG 2 , B. E. KOSOFSKY 1 ;<br />
1 Weill Med. Coll, Cornell Univ., NY, NY; 2 Dept. of Pharmacol. <strong>an</strong>d Toxicology, Inst. of<br />
Pharmacy, Univ. of Innsbruck, Innsbruck, Austria<br />
Abstract: Dysregulation of BDNF gene expression by <strong>the</strong> tr<strong>an</strong>scriptional regulator MeCP2 has<br />
been implicated in several developmental brain disorders. Ca2+ plays a critical role in<br />
tr<strong>an</strong>scriptional regulation of BDNF exon IV splice vari<strong>an</strong>t by MeCP2, with voltage-gated L-type<br />
Ca2+ ch<strong>an</strong>nels (LTCCs) serving as <strong>the</strong> primary route of activity-dependent tr<strong>an</strong>scriptional<br />
control in neurons. The precise brain-specific LTCC subunit regulating BDNF gene expression<br />
remains unknown. In this study we have explored <strong>the</strong> roles of <strong>the</strong> two brain specific LTCC<br />
subunits, Cav1.2 <strong>an</strong>d Cav1.3 in <strong>the</strong> mouse hippocampus <strong>an</strong>d cortex using Cav1.2<br />
dihydropyridine (DHP) insensitive (Cav1.2DHP-/-) mice. In homozygous mut<strong>an</strong>t mice Cav1.2<br />
subunits are insensitive to dihydropyridines allowing us to directly test <strong>the</strong> role of Cav1.3. Using<br />
<strong>the</strong> LTCC DHP activator, BayK (4 mg/kg, i.p.) <strong>an</strong>d splice-vari<strong>an</strong>t specific primers in real time<br />
PCR experiments we find that in <strong>the</strong> hippocampus, Cav1.3 LTCCs activate BDNF exon IV<br />
tr<strong>an</strong>scription with no ch<strong>an</strong>ge in exon I, II or VI, <strong>the</strong> o<strong>the</strong>r brain-specific splice vari<strong>an</strong>ts. Using<br />
chromatin immunoprecipitation (ChIP) assays to examine regulation of MeCP2 binding to exon<br />
IV promoter reveals that <strong>the</strong> increase in Cav1.3-mediated BDNF ex IV in <strong>the</strong> hippocampus is<br />
associated with a Cav1.3-mediated decrease in MeCP2 binding to exon IV promoter suggesting
that MeCP2 acts as a repressor at this site. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d in <strong>the</strong> cortex, BDNF exon IV<br />
tr<strong>an</strong>scription is activated by Cav1.2 LTCCs whereas Cav1.3 LTCCs activate tr<strong>an</strong>scription of<br />
BDNF exon I splice vari<strong>an</strong>t. ChIP experiments reveal that <strong>the</strong> increase in Cav1.3 LTCCmediated<br />
BDNF exon I splice vari<strong>an</strong>t is associated with <strong>an</strong> increase in Cav1.3-mediated MeCP2<br />
binding to this promoter site suggesting that MeCP2 could possibly act as <strong>an</strong> activator at <strong>the</strong><br />
exon I promoter. Regulation of MeCP2 binding by Cav1.2 LTCCs on exon IV promoter in <strong>the</strong><br />
cortex is currently being examined. We are also pursuing MECP2 phosphorylation studies to<br />
fur<strong>the</strong>r examine LTCC Ca2+ mech<strong>an</strong>isms that regulate MeCP2 function. In summary, we find<br />
that Cav1. 2 <strong>an</strong>d Cav1.3 differentially regulate BDNF splice vari<strong>an</strong>t gene tr<strong>an</strong>scription <strong>an</strong>d<br />
MeCP2 may act as ei<strong>the</strong>r a tr<strong>an</strong>scriptional activator or repressor in a promoter <strong>an</strong>d regionspecific<br />
m<strong>an</strong>ner.<br />
Disclosures: A.M. Rajadhyaksha, None; S.C.R. Hauck, None; Z. Daruwalla, None; J.<br />
Striessnig, None; B.E. Kosofsky, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.17/X31<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIDA Gr<strong>an</strong>t R21DA023686-01A1 (AR)<br />
Title: Ventral tegmental area Cav1.3 L-type Ca2+ ch<strong>an</strong>nels mediate amphetamine-induced<br />
molecular <strong>an</strong>d behavioral plasticity<br />
Authors: *J. HAO 1 , A. TAL 1 , X.-W. SUN 1 , S. S. SATPUTE 4 , G. WANG 2 , C. E.<br />
INTURRISI 2,3 , A. M. RAJADHYAKSHA 1,2 ;<br />
1 Pediatric Neurology, Pediatrics, 2 Neurol. <strong>an</strong>d Neurosci., 3 Pharmacol., Weill Cornell Med. Col.,<br />
New York, NY; 4 Neurol., Univ. of Maryl<strong>an</strong>d Med. Ctr., Baltimore, MD<br />
Abstract: The ventral tegmental area (VTA) is <strong>the</strong> primary <strong>an</strong>atomical site responsible <strong>for</strong> <strong>the</strong><br />
initiation of persistent behavioral adaptations following psychostimul<strong>an</strong>t exposure. Calcium<br />
signaling via VTA L-type Ca 2+ ch<strong>an</strong>nels (LTCCs) plays <strong>an</strong> import<strong>an</strong>t role in mediating<br />
psychostimul<strong>an</strong>t-induced ch<strong>an</strong>ges. However, distinguishing between <strong>the</strong> roles of Cav1.2 <strong>an</strong>d<br />
Cav1.3, <strong>the</strong> two LTCC subtypes expressed in <strong>the</strong> brain, has been hindered due to <strong>the</strong> lack of<br />
subunit-specific pharmacological agents. Recent evidence suggests <strong>the</strong> Cav1.3 subunit is critical<br />
to psychostimul<strong>an</strong>t-induced neuroplasticity, because Cav1.3 knockout mice do not exhibit
development or expression of behavioral sensitization after exposure to amphetamine (Giord<strong>an</strong>o<br />
III et al, J. Neurochem. 2006). In <strong>the</strong> present study, we explored molecular ch<strong>an</strong>ges in <strong>the</strong> VTA<br />
of Cav1.3 knockout mice following amphetamine exposure. Using Western blot <strong>an</strong>alysis, we find<br />
that Cav1.3 LTCCs mediate acute <strong>an</strong>d chronic amphetamine-induced Ser 133 CREB<br />
phosphorylation in <strong>the</strong> VTA. In addition, examination of phospho-ERK revealed that Cav1.3<br />
LTCCs mediate chronic but not acute amphetamine induced P-ERK. Finally, to fur<strong>the</strong>r explore<br />
<strong>the</strong> role of VTA-specific Cav1.3 LTCCs in amphetamine-induced behavioral ch<strong>an</strong>ges, we<br />
generated recombin<strong>an</strong>t adenoassociated viral (rAAV) vectors to deliver Cav1.3-specific short<br />
interfering RNA (siRNA) directly into <strong>the</strong> VTA. Mice were stereotaxically microinjected with<br />
rAAV-Cav1.3siRNA <strong>an</strong>d behaviorally tested 3-4 weeks post-surgery. Our preliminary results<br />
indicate that knockdown of Cav1.3 LTCCs in <strong>the</strong> VTA blunts <strong>the</strong> development of behavioral<br />
sensitization. Immunohistological studies are currently underway to identify ch<strong>an</strong>ges in levels of<br />
CREB <strong>an</strong>d ERK phosphorylation following Cav1.3 knockdown. In summary, converging<br />
evidence <strong>from</strong> our lab suggests that Cav1.3 LTCCs in <strong>the</strong> VTA are critically involved in<br />
mediating <strong>the</strong> molecular <strong>an</strong>d behavioral adaptations induced by exposure to amphetamine.<br />
Disclosures: J. Hao, None; A. Tal, None; X. Sun, None; S.S. Satpute, None; G. W<strong>an</strong>g,<br />
None; C.E. Inturrisi, None; A.M. Rajadhyaksha, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.18/X32<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH Gr<strong>an</strong>t DA11289<br />
NIH Gr<strong>an</strong>t DA024527<br />
Title: Stress <strong>an</strong>d drugs of abuse impair plasticity of inhibitory synapses onto VTA dopamine<br />
neurons<br />
Authors: *J. L. NIEHAUS, J. A. KAUER;<br />
Brown Univ., Providence, RI<br />
Abstract: Addiction to drugs of abuse is a pathological state hypo<strong>the</strong>sized to result, in part, <strong>from</strong><br />
<strong>the</strong> pharmacological effects of addictive drugs on brain circuits that process naturally rewarding<br />
stimuli. Synaptic ch<strong>an</strong>ges in components of reward processing in <strong>the</strong> brain are induced by
multiple drugs of abuse, not only at excitatory synapses, but also inhibitory synapses. A number<br />
of diverse addictive drugs potentiate excitatory synapses onto dopamine neurons in <strong>the</strong> ventral<br />
tegmental area (VTA) with a single exposure. Acute stress also potentiates excitatory synapses to<br />
a comparable degree, suggesting that drugs of abuse <strong>an</strong>d stress similarly affect synaptic plasticity<br />
of VTA dopamine neurons. In contrast to <strong>the</strong> effect on excitatory synapses, a similar in vivo<br />
injection of morphine prevented long-term potentiation of inhibitory synapses onto VTA<br />
dopamine neurons (LTPGABA).<br />
To determine whe<strong>the</strong>r drugs of abuse <strong>an</strong>d stress might also reduce inhibitory synaptic<br />
tr<strong>an</strong>smission onto VTA dopamine neurons, we measured LTPGABA in brain slices <strong>from</strong> rats<br />
administered a drug of abuse or exposed to acute stress. Nitric oxide (NO) is required to induce<br />
LTPGABA, but becomes ineffective after a single in vivo dose of morphine (Nugent et al., Nature<br />
446: 1086, 2007). In this study, we used bath application of <strong>the</strong> NO donor, SNAP, to observe<br />
potentiation of GABAA IPSCs. In brain slices prepared <strong>from</strong> <strong>an</strong>imals injected 24 h prior with<br />
cocaine (15 mg/kg), we found that IPSCs were potentiated to a lesser extent (131 ± 2.0 % of<br />
control values, n = 10) th<strong>an</strong> in brain slices <strong>from</strong> saline-injected <strong>an</strong>imals (166 ± 1.4 % of control<br />
values, n = 10). These results suggest that like morphine, cocaine also weakens plasticity of<br />
inhibitory synapses on dopamine neurons <strong>for</strong> at least 24 h after initial exposure. A second group<br />
of <strong>an</strong>imals was exposed to a brief, acute stress 24 h prior to brain slice preparation. Acute stress<br />
strongly impaired LTPGABA (102 ± 2.5 % of control values, n = 8) in slices <strong>from</strong> <strong>an</strong>imals that<br />
received vehicle injection 30 min prior to <strong>the</strong> acute stress, but not in slices <strong>from</strong> <strong>an</strong>imals that<br />
received RU486, a glucocorticoid receptor <strong>an</strong>tagonist, prior to <strong>the</strong> acute stress (163 ± 4.5 % of<br />
control values, n = 8). Thus, two different drugs of abuse <strong>an</strong>d acute stress impair LTPGABA at<br />
synapses on VTA dopamine neurons. These findings demonstrate a shared neuroadaptation<br />
produced by stress or addictive drugs. The reduction in inhibitory plasticity occurring<br />
simult<strong>an</strong>eously with drug- or stress-induced LTP of excitatory synapses is expected to increase<br />
dopamine cell excitability, <strong>an</strong>d may contribute to <strong>the</strong> early development of addiction.<br />
Disclosures: J.L. Niehaus , None; J.A. Kauer, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.19/X33<br />
Topic: C.17.i. Cocaine: Neural mech<strong>an</strong>isms of addiction<br />
Support: CNRS<br />
University of Strasbourg
Fondation pour la Recherche Medicale<br />
Fédération pour la recherche sur le cerveau<br />
ENINET (LHSM-CT-2005-019063)<br />
Title: The GABAergic tail of <strong>the</strong> ventral tegmental area: A new control center <strong>for</strong> <strong>the</strong><br />
dopaminergic system<br />
Authors: J. KAUFLING, P. VEINANTE, S. PAWLOWSKI, *R. SCHLICHTER, M.-J.<br />
FREUND-MERCIER, M. BARROT;<br />
CNRS UPR 3212, Univ. Strasbourg, Strasbourg 67084, Fr<strong>an</strong>ce<br />
Abstract: The tr<strong>an</strong>scription factor ∆FosB is implicated in <strong>the</strong> plasticity induced by drugs of<br />
abuse. We showed that psychostimul<strong>an</strong>ts induce ∆FosB in GABA cells of a caudal subregion of<br />
<strong>the</strong> ventral tegmental area (VTA) which was named tail of <strong>the</strong> VTA (tVTA). While tVTA mostly<br />
shares VTA inputs, its outputs remained to be characterized. tVTA efferents were studied by<br />
iontophoretic injections of <strong>the</strong> <strong>an</strong>terograde tracer biotinylated dextr<strong>an</strong> amine (BDA). To fur<strong>the</strong>r<br />
study VTA inputs arising <strong>from</strong> tVTA, injections of <strong>the</strong> retrograde tracer Fluoro-Gold® were<br />
combined with multiple labeling by immunohistochemistry in rats treated with cocaine. The<br />
indirect projection <strong>from</strong> <strong>the</strong> tVTA to <strong>the</strong> nucleus accumbens was assessed by using a doubletracing<br />
approach, with cholera toxin B subunit (CTB) delivered in <strong>the</strong> nucleus accumbens <strong>an</strong>d<br />
BDA in <strong>the</strong> tVTA. Tract-tracing studies showed that tVTA heavily projects to <strong>the</strong> midbrain<br />
dopaminergic system <strong>an</strong>d revealed terminal appositions with dopamine cells in <strong>the</strong> VTA.<br />
Double-labeling studies demonstrated that this tVTA output is mostly GABAergic, that it<br />
includes cells in which cocaine exposure induces ∆FosB, <strong>an</strong>d that it contacts dopamine cells<br />
projecting to <strong>the</strong> nucleus accumbens. GABA neurons expressing ∆FosB in <strong>the</strong> tVTA after<br />
cocaine exposure thus project to <strong>the</strong> dopamine mesolimbic neurons.<br />
This work was supported by <strong>the</strong> Centre national de la Recherche Scientifique (contracts<br />
UMR7168 <strong>an</strong>d UPR3212), <strong>the</strong> University of Strasbourg (contract UMR7168), <strong>the</strong> Fondation<br />
pour la Recherche Médicale (M.B.), <strong>the</strong> Fédération pour la Recherche sur le Cerveau (P.V.) <strong>an</strong>d<br />
<strong>the</strong> ENINET (Europe<strong>an</strong> Commission contract LHSM-CT-2005-019063).<br />
Disclosures: J. Kaufling, None; P. Vein<strong>an</strong>te, None; S. Pawlowski, None; R. Schlichter ,<br />
None; M. Freund-Mercier, None; M. Barrot, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 550.20/X34<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH Gr<strong>an</strong>t AA09125<br />
NIH Gr<strong>an</strong>t AA05846<br />
Title: Low concentrations of eth<strong>an</strong>ol <strong>an</strong>tagonize time-dependent reversal of dopamine inhibition<br />
in <strong>the</strong> VTA<br />
Authors: *M. S. BRODIE, S. NIMITVILAI;<br />
Dept Physiol & Biophys M/C901, Univ. Illinois-Chicago, Chicago, IL<br />
Abstract: Alteration of mech<strong>an</strong>isms of plasticity of dopaminergic (DAergic) ventral tegmental<br />
area (VTA) neurons may play import<strong>an</strong>t roles in ch<strong>an</strong>ges in <strong>the</strong> mesocorticolimbic system that<br />
lead to <strong>the</strong> development of addictive behaviors. For example, specific mech<strong>an</strong>isms that underlie<br />
plasticity of DAergic VTA neurons may be altered by eth<strong>an</strong>ol which could interfere with<br />
in<strong>for</strong>mation processing related to reward/rein<strong>for</strong>cement. We previously reported that long-term<br />
in vitro application of moderate concentrations of dopamine (DA, 1-10 µM) induced ch<strong>an</strong>ges in<br />
DAergic VTA neurons that resulted in time-dependent, long-lasting decreases in <strong>the</strong> inhibitory<br />
effectiveness of dopamine, which we termed “dopamine inhibition reversal”, through a<br />
mech<strong>an</strong>ism that is, at least in part, glutamatergic. In <strong>the</strong> present study, we used extracellular<br />
recordings to examine <strong>the</strong> effect of eth<strong>an</strong>ol on dopamine inhibition reversal of DAergic VTA<br />
neurons in rat coronal brain slices. In control cells, when DA (r<strong>an</strong>ge <strong>from</strong> 1-10 µM) was applied<br />
<strong>for</strong> 40 minutes, we observed <strong>an</strong> initial DA-induced inhibition of about 70% in <strong>the</strong> first 10<br />
minutes, which gradually decreased with time; at 40 min, <strong>the</strong> inhibition was about 20% of<br />
baseline, which is typical of inhibition reversal seen in most cells. In o<strong>the</strong>r cells, low<br />
concentrations of eth<strong>an</strong>ol were administered prior to <strong>an</strong>d during <strong>the</strong> DA administration. Eth<strong>an</strong>ol<br />
(1-10 mM) alone did not increase <strong>the</strong> firing rate signific<strong>an</strong>tly (n=29); this is consistent with our<br />
previous work indicating that <strong>the</strong>se concentrations are below those needed to increase <strong>the</strong><br />
spont<strong>an</strong>eous firing rate. In <strong>the</strong> presence of 10 mM eth<strong>an</strong>ol, dopamine inhibition reversal was<br />
blocked reliably (6 cells of 6 tested), such that <strong>the</strong> level of inhibition at 10 minutes <strong>an</strong>d 40<br />
minutes was not signific<strong>an</strong>tly different. At lower concentrations (1-5 mM), eth<strong>an</strong>ol blocked <strong>the</strong><br />
dopamine inhibition reversal in some cells (14 of 23 neurons tested). These results suggest that<br />
prolonged DA inhibition of DAergic VTA neurons c<strong>an</strong> result in reduced sensitivity of <strong>the</strong><br />
neurons to DA, a type of plasticity that might be import<strong>an</strong>t in in<strong>for</strong>mation processing related to<br />
reward, <strong>an</strong>d our data indicate that very low concentrations of eth<strong>an</strong>ol c<strong>an</strong> interfere with this<br />
plasticity, possibly through a glutamatergic mech<strong>an</strong>ism.<br />
Disclosures: M.S. Brodie, None; S. Nimitvilai, None.<br />
Poster
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.21/X35<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH Gr<strong>an</strong>t AA015521<br />
NIH Gr<strong>an</strong>t 1F31AA017553<br />
Title: In vivo eth<strong>an</strong>ol exposure enh<strong>an</strong>ces inositol triphosphate-mediated calcium signaling in<br />
ventral tegmental area dopamine neurons<br />
Authors: *B. E. BERNIER 1 , H. MORIKAWA 2 ;<br />
1 Univ. Texas at Austin, austin, TX; 2 Neurobio., Univ. Texas at Austin, Austin, TX<br />
Abstract: Dopamine neurons of <strong>the</strong> ventral tegmental area (VTA) are critically involved in<br />
reward-based learning <strong>an</strong>d <strong>the</strong> development of addiction to drugs of abuse, including eth<strong>an</strong>ol. In<br />
behaving <strong>an</strong>imals, dopamine neurons “learn” to respond to reward-predicting cues with a burst of<br />
action potentials after repeated cue-reward pairing. NMDA-type glutamate receptors are known<br />
to play a predomin<strong>an</strong>t role in <strong>the</strong> generation of <strong>the</strong>se bursts. We have recently reported long-term<br />
potentiation (LTP) of NMDA receptor-mediated tr<strong>an</strong>smission onto dopamine neurons that may<br />
contribute to <strong>the</strong> acquisition of <strong>the</strong> conditioned burst response. This <strong>for</strong>m of plasticity is induced<br />
in a m<strong>an</strong>ner dependent on inositol triphosphate (IP3)-mediated facilitation of burst-evoked Ca 2+<br />
signals. In this study, we per<strong>for</strong>med patch-clamp recordings <strong>an</strong>d flash photolysis of caged IP3 in<br />
acutely prepared mouse midbrain slices to examine <strong>the</strong> effects of repeated in vivo eth<strong>an</strong>ol<br />
exposure on IP3-mediated Ca 2+ signaling in VTA dopamine neurons. C57BL/6 mice were<br />
injected with eth<strong>an</strong>ol (2 g/kg, i.p.) or saline 3 times per day <strong>for</strong> 7 days, <strong>an</strong>d midbrain slices<br />
containing <strong>the</strong> VTA were prepared 1 day after <strong>the</strong> final injection. Flash photolysis of caged IP3<br />
activates Ca 2+ -sensitive K + currents (IIP3) via release of Ca 2+ <strong>from</strong> intracellular stores in<br />
dopamine neurons. Repeated eth<strong>an</strong>ol exposure signific<strong>an</strong>tly reduced <strong>the</strong> flash intensity required<br />
to elicit half-maximal IIP3 without altering <strong>the</strong> maximal IIP3 amplitude, suggesting increased IP3<br />
receptor sensitivity. Additionally, IP3-induced facilitation of action potential-evoked Ca 2+ signals<br />
was enh<strong>an</strong>ced in eth<strong>an</strong>ol-treated mice. Stimulation of <strong>the</strong> cyclic AMP pathway with <strong>for</strong>skolin (30<br />
µM) also increased IP3 receptor sensitivity, suggesting that protein kinase A-mediated<br />
phosphorylation of IP3 receptors may underlie <strong>the</strong> effect of in vivo eth<strong>an</strong>ol treatment. Finally, we<br />
found that our eth<strong>an</strong>ol treatment protocol resulted in increased eth<strong>an</strong>ol consumption assessed<br />
using a two-bottle choice paradigm. These results demonstrate that repeated eth<strong>an</strong>ol exposure<br />
enh<strong>an</strong>ces <strong>the</strong> Ca 2+ signal critical <strong>for</strong> <strong>the</strong> induction of NMDA receptor plasticity in VTA<br />
dopamine neurons. This may promote <strong>the</strong> learning of cues associated with eth<strong>an</strong>ol consumption,<br />
such as <strong>the</strong> taste <strong>an</strong>d smell of eth<strong>an</strong>ol, <strong>the</strong>reby contributing to <strong>the</strong> development of eth<strong>an</strong>ol<br />
drinking behavior <strong>an</strong>d alcoholism.
Disclosures: B.E. Bernier, None; H. Morikawa, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.22/X36<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIH Gr<strong>an</strong>t AA106559<br />
Title: Eth<strong>an</strong>ol blocks long-term potentiation of GABAergic synapses in <strong>the</strong> ventral tegmental<br />
area involving µ-opioid receptors<br />
Authors: Y.-Z. GUAN 1 , *J. YE 2 ;<br />
1 Anes<strong>the</strong>siol., 2 UMDNJ New Jersey Med. Sch., Newark, NJ<br />
Abstract: Addiction is caused, in part, by powerful <strong>an</strong>d long-lasting memories of <strong>the</strong> drug<br />
experience, <strong>an</strong>d <strong>the</strong> long-term ch<strong>an</strong>ges in chemical synaptic action in response to drug<br />
administration are believed to be <strong>the</strong> c<strong>an</strong>didate mech<strong>an</strong>ism. It has been well documented that<br />
exposure to abused drugs c<strong>an</strong> induced long term ch<strong>an</strong>ges in <strong>the</strong> excitatory synapses; emerging<br />
evidence indicates that abused drugs c<strong>an</strong> also induced long term ch<strong>an</strong>ges in <strong>the</strong> inhibitory<br />
synapses. In <strong>the</strong> current study, using whole-cell electrophysiological recording in rat brain slices,<br />
we assessed <strong>the</strong> ch<strong>an</strong>ges induced by eth<strong>an</strong>ol on <strong>the</strong> inhibitory synapses. Consistent with previous<br />
reports, high-frequency stimulation (100 Hz <strong>for</strong> 1 second, repeated twice 20s apart) induced<br />
long-term potentiation in <strong>the</strong> fast inhibitory GABA(A) receptor synapses (LTPGABA) onto<br />
dopamine neurons in <strong>the</strong> ventral tegmental area, a brain region essential <strong>for</strong> reward-seeking<br />
behavior. Exposure to eth<strong>an</strong>ol both in vivo (2 g/kg body weight, by intraperitoneal injection, 24<br />
hours be<strong>for</strong>e electrophysiological recordings) <strong>an</strong>d in vitro (40 mM eth<strong>an</strong>ol, acutely applied to <strong>the</strong><br />
brain slices) prevented LTPGABA. Fur<strong>the</strong>rmore, eth<strong>an</strong>ol blockade of LTPGABA was reversed by a µ<br />
opioid receptor <strong>an</strong>tagonist naloxone (in vivo 10 mg/kg by intraperitoneal injection, 24 hours<br />
be<strong>for</strong>e electrophysiological recordings) <strong>an</strong>d in vitro (5 µM, acutely applied to brain slices). These<br />
data suggest that eth<strong>an</strong>ol c<strong>an</strong> induce long term ch<strong>an</strong>ges in <strong>the</strong> inhibitory synapses in <strong>the</strong><br />
mesolimbic circuitry by a mech<strong>an</strong>ism involving <strong>the</strong> activation of µ opioid receptors. These<br />
neuroadaptations to eth<strong>an</strong>ol might contribute to early stage of addiction.<br />
Disclosures: Y. Gu<strong>an</strong>, None; J. Ye, None.
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.23/X37<br />
Topic: C.17.i. Cocaine: Neural mech<strong>an</strong>isms of addiction<br />
Support: BID 1728/OC-AR PICT # 1728<br />
SECYT<br />
Title: Cocaine sensitization is correlated to increases in synaptic plasticity <strong>an</strong>d to a<br />
“sensitization” of nitric oxide synthase activity in hippocampus<br />
Authors: *M. F. PEREZ 1 , L. GABACH 2 , V. CARLINI 2 , L. MAGLIO 2 , S. RUBIALES 2 , O.<br />
RAMÍREZ 2 ;<br />
1 IFEC F, 2 IFEC, Farmacología, Fac Ciencias Químicas, Cordoba, Argentina<br />
Abstract: Hippocampus (HP) is a limbic structure that participates in learning <strong>an</strong>d memory<br />
<strong>for</strong>mation. Ch<strong>an</strong>ges in HP synaptic tr<strong>an</strong>smission constitute some of <strong>the</strong> neuroadaptations<br />
occurred after exposure of drugs of abuse. Repeated cocaine (COC) administration induces<br />
sensitization to its effects on locomotion <strong>an</strong>d nitric oxide (NO) could be involved in <strong>the</strong><br />
acquisition <strong>an</strong>d mainten<strong>an</strong>ce of <strong>the</strong> behavioral effects of COC, because inhibition of <strong>the</strong> enzyme<br />
NO synthase (NOS) attenuates <strong>the</strong> development of sensitization. Moreover, <strong>the</strong> role of NO in <strong>the</strong><br />
mech<strong>an</strong>isms involved in COC sensitization has not been described. In <strong>the</strong> present work we<br />
combined behavioral experiments toge<strong>the</strong>r with electrophysilogical <strong>an</strong>d neurochemical<br />
experiments in order to: 1- determine if COC sensitization is associated to increases in synaptic<br />
plasticity, 2- establish a correlation between sensitization <strong>an</strong>d NO production, measured by <strong>the</strong><br />
conversion of citruline H 3 to arginine H 3 , 3- to determine if <strong>the</strong> observed ch<strong>an</strong>ges were<br />
consequence of sensitization or due to withdrawal <strong>from</strong> COC <strong>an</strong>d 4- examine <strong>the</strong> dependence on<br />
NO of <strong>the</strong>se events. Male Wistar rats were administered with COC (15 mg/kg/day, i.p.), NOS<br />
inhibitor (7-NI 50 mg/kg/day. i.p.) + COC or saline (SAL) <strong>for</strong> 5 days <strong>an</strong>d locomotor activity was<br />
evaluated on days 1 <strong>an</strong>d 5. Sensitized (S) rats were separated <strong>from</strong> non-sensitized rats (NS). The<br />
electrophysiologycal <strong>an</strong>d neurochemical studies were per<strong>for</strong>med on day 5 (non-W) or after 3<br />
days of withdrawal (W). Rats <strong>from</strong> <strong>the</strong> S non-W group showed a reduction in threshold to<br />
generate LTP (SAL 102 ± 8 vs. S non-W 27 ± 4 Hz). The same results were observed in <strong>the</strong> S W<br />
group. The increase in synaptic plasticity was correlated to <strong>the</strong> increase on <strong>the</strong> NOS activity in<br />
<strong>the</strong> S non-W group (SAL 0.4 ± 0.1 vs. S non-W 2.83 ± 0.6 pmol/mg protein). The NOS activity<br />
showed a “sensitized” pattern of activity in <strong>an</strong>imals <strong>from</strong> S non-W group compared to <strong>an</strong>imals<br />
that received acute COC (S non-W 775 ± 158 vs. acute COC 308 ± 51 %). Administration of 7-<br />
NI be<strong>for</strong>e COC not only prevented sensitization, but also increased <strong>the</strong> threshold to generate LTP
<strong>an</strong>d prevented <strong>the</strong> increased NOS activity. These findings indicate that NO <strong>an</strong>d <strong>the</strong> pathways<br />
activated by this neuromodulator may have <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> ch<strong>an</strong>ges induced by COC in<br />
hippocampal synaptic tr<strong>an</strong>smission, contributing to <strong>the</strong> development of sensitization <strong>an</strong>d<br />
withdrawal.<br />
Disclosures: M.F. Perez, None; L. Gabach, None; V. Carlini, None; L. Maglio, None; S.<br />
Rubiales, None; O. Ramírez, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.24/X38<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIDA Gr<strong>an</strong>t R01DA02503601<br />
T32 DA007287-12<br />
Title: Morphine-induced ch<strong>an</strong>ges in AMPA receptor expression <strong>an</strong>d composition that may be<br />
persistent at hippocampal synapses<br />
Authors: *N. L. BJORKLUND, S. K. BILLA, B. KRISHNAN, J. LIU, J. A. MORON;<br />
Ctr. <strong>for</strong> Addiction Research, Pharmacol. <strong>an</strong>d Toxicology, Univ. of Texas Med. Br., Galveston,<br />
TX<br />
Abstract: Repeated administration of morphine c<strong>an</strong> cause long-lasting effects, both at <strong>the</strong><br />
behavioral <strong>an</strong>d at <strong>the</strong> biological level. These neuroadaptations include ch<strong>an</strong>ges in α-amino-3hydroxy-5-methyl-4-isoxazole<br />
propionic acid receptor (AMPAR) function which affects<br />
glutamate neurotr<strong>an</strong>smission. Previous studies in our laboratory have found that 12 hours after a<br />
behaviorally-sensitizing morphine administration paradigm, <strong>the</strong>re is <strong>an</strong> increased expression of<br />
AMPARs lacking glutamate receptor 2 (GluR2-lacking) in <strong>the</strong> hippocampus, <strong>an</strong> area import<strong>an</strong>t<br />
<strong>for</strong> learning <strong>an</strong>d memory. This increase in GluR2-lacking receptors suggests that morphine<br />
treatment leads to ch<strong>an</strong>ges in AMPARs composition which affects <strong>the</strong> functionality of <strong>the</strong>se<br />
receptors <strong>an</strong>d consequently glutamatergic neurotr<strong>an</strong>smission. The current work focuses on <strong>the</strong><br />
study of ch<strong>an</strong>ges in localization of <strong>the</strong> different AMPAR subunits (GluR1-3) at <strong>the</strong> synapse<br />
(intracellular or membr<strong>an</strong>e bound) <strong>an</strong>d in <strong>the</strong> composition of <strong>the</strong> expressed receptors in <strong>the</strong><br />
hippocampus at both 12 hours <strong>an</strong>d one week after morphine treatment. These time points allow<br />
<strong>an</strong>alysis of <strong>the</strong> ch<strong>an</strong>ges occurring during development of sensitization <strong>an</strong>d also determine <strong>the</strong>
persistence of <strong>the</strong>se neuroadaptations. Receptor crosslinking studies in hippocampal<br />
homogenates show that <strong>the</strong>re is no ch<strong>an</strong>ge in intracellular AMPAR subunits 12 hours <strong>an</strong>d one<br />
week after morphine treatment. However, total expression of GluR1 <strong>an</strong>d GluR1 phosphorylated<br />
at serine 845 are increased 12 hours after morphine treatment suggesting <strong>an</strong> increase in cell<br />
surface expression. Ongoing studies will examine <strong>the</strong> total expression levels after one week<br />
withdrawal to determine cell surface expression at this time point. In addition, future studies will<br />
examine receptor membr<strong>an</strong>e expression in synaptic fractions after morphine treatment.<br />
Qu<strong>an</strong>titative co-immunoprecipitations have been developed in mouse hippocampus to selectively<br />
pull down <strong>the</strong> different AMPA receptor subunits. Preliminary studies show that 12 hours after<br />
morphine treatment <strong>the</strong>re may be <strong>an</strong> increased association of GluR1 with GluR3. Fur<strong>the</strong>r studies<br />
will look at <strong>the</strong> persistence of <strong>the</strong>se AMPA receptor composition ch<strong>an</strong>ges. In summary, we find<br />
that repeated morphine administration alters <strong>the</strong> expression <strong>an</strong>d composition of AMPA glutamate<br />
receptors in <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong>se effects may persist over time <strong>an</strong>d be responsible <strong>for</strong><br />
long-term behavioral sensitization induced by repeated morphine administration.<br />
This work was supported by gr<strong>an</strong>ts T32 DA007287-12 (NLB) <strong>an</strong>d R01 DA02503601(JAM).<br />
Disclosures: N.L. Bjorklund, None; S.K. Billa, None; B. Krishn<strong>an</strong>, None; J. Liu, None; J.A.<br />
Moron, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 550.25/Y1<br />
Topic: C.17.bb. Neural plasticity, dependence <strong>an</strong>d addiction<br />
Support: NIAAA, INIA-Stress Gr<strong>an</strong>t 1U01AA016670-01<br />
Title: Effects of voluntary eth<strong>an</strong>ol consumption on GABA-A receptor plasticity in socially<br />
isolated C57BL/6J mice<br />
Authors: *E. SANNA 1 , M. C. MOSTALLINO 2 , P. P. SECCI 1 , N. OBILI 1 , G. TALANI 1 , L.<br />
MURRU 1 , C. UTZERI 1 , P. OLLA 1 , F. BIGGIO 1 , G. GORINI 3 , P. FOLLESA 1 , G. BIGGIO 1,2 ;<br />
1 Dept Exp Biol, Univ. Cagliari, Monserrato 09042, Italy; 2 Inst. of Neurosci., C.N.R., Cagliari,<br />
Italy; 3 Waggoner Ctr. <strong>for</strong> Alcohol <strong>an</strong>d Addiction Research, Col. of Natural Sci., The Univ. of<br />
Texas at Austin, Austin, TX<br />
Abstract: Social isolation is a model of prolonged mild stress that has been shown to be<br />
associated to marked behavioral alterations, a decrease in <strong>the</strong> brain <strong>an</strong>d plasma concentrations of
neuroactive steroids, <strong>an</strong>d by <strong>an</strong> abnormal response to acute stressful stimuli as well as by <strong>an</strong><br />
increased neurosteroidogenic effect induced by <strong>the</strong> acute administration of eth<strong>an</strong>ol. We used<br />
social isolation in <strong>the</strong> C57BL/6J mouse strain as a model of prolonged mild stress in which to<br />
investigate <strong>the</strong> effect eth<strong>an</strong>ol in <strong>the</strong> free-choice drinking paradigm on gene expression <strong>an</strong>d<br />
function of GABA-A receptors in <strong>the</strong> hippocampus. Socially isolated <strong>an</strong>d group-housed mice<br />
were exposed <strong>for</strong> 6 weeks to <strong>the</strong> two-bottle choice (eth<strong>an</strong>ol versus water). Both groups of<br />
<strong>an</strong>imals were given, during <strong>the</strong> whole period of isolation free access to eth<strong>an</strong>ol <strong>for</strong> 2 h in <strong>the</strong>ir<br />
home cage, beginning at 0.5 h prior <strong>the</strong> start of <strong>the</strong> dark cycle. Mice <strong>from</strong> both experimental<br />
groups were individually housed <strong>for</strong> <strong>the</strong> 2-h procedure. Specific GABA-A receptor subunit<br />
expression was measured by RNase protection assay <strong>an</strong>d immunohistochemistry. GABA-A<br />
receptor function was evaluated by conventional whole-cell patch clamp recording in brain<br />
slices. We found a signific<strong>an</strong>t increase in <strong>the</strong> abund<strong>an</strong>ce of both α4 <strong>an</strong>d δ subunits of <strong>the</strong> GABA-<br />
A receptor in <strong>the</strong> hippocampus of social isolated mice (20 <strong>an</strong>d 26% respectively p
Title: Toluene intoxication: Structural reorg<strong>an</strong>ization <strong>an</strong>d alteration on behavior in adolescent<br />
<strong>an</strong>d adult rats<br />
Authors: *L. CHILACHAVA 1 , L. GELAZONIA 2 , N. JAPARIDZE 2 , R. MIKAVA 3 , M.<br />
ZHVANIA 2 ;<br />
1 I.Chavchavadze State Univ., Tbilisi, Georgia; 2 I.Beritashvili Inst. of Physiol., Tbilisi, Georgia;<br />
3 Independent Univ. of Zugdidi, Zugdidi, Georgia<br />
Abstract: In <strong>the</strong> last period <strong>the</strong> problem of adolescent snuffers-addicts became relev<strong>an</strong>t,<br />
especially in developing countries. To get <strong>the</strong> narcotic high adolescents frequently use<br />
inexpensive industrial products, <strong>the</strong> main ingredient of which is Toluene. Recent data denote<br />
addictive nature of toluene. In particular, inhalation of toluene by rats causes activation of<br />
dopaminergic neurons of mesolimbic reward pathway <strong>an</strong>d by me<strong>an</strong>s of this c<strong>an</strong> provide abuse<br />
potential of toluene containing subst<strong>an</strong>ces.<br />
It is known that periadolescence is a time, during which signific<strong>an</strong>t ch<strong>an</strong>ges in some receptors,<br />
neuroendocrine control, neuro<strong>an</strong>atomy of brain occur. Thus, subst<strong>an</strong>ce abuse in adolescence may<br />
have effects that are different <strong>an</strong>d perhaps more subst<strong>an</strong>tial th<strong>an</strong> in adults. The adolescent CNS<br />
remains plastic in response to different m<strong>an</strong>ipulations. At <strong>the</strong> last time <strong>the</strong> special interest<br />
provokes structural reorg<strong>an</strong>ization of neurons as a result of drug abuse (Morphine, Cocaine,<br />
Nicotine etc.): <strong>the</strong> length, diameter <strong>an</strong>d qu<strong>an</strong>tity of dendritic br<strong>an</strong>ches, number <strong>an</strong>d <strong>for</strong>m spines<br />
signific<strong>an</strong>tly differs. From this point of view, toluene is not studied.<br />
Our aims are to clarify whe<strong>the</strong>r toluene intoxication cause persistent structural reorg<strong>an</strong>ization of<br />
neurons in adolescent <strong>an</strong>d adult rat limbic structures <strong>an</strong>d to determine corresponding persistent<br />
ch<strong>an</strong>ges in behavior.<br />
Inhalation applied at 30 <strong>an</strong>d 120-day old rats, <strong>for</strong> 40 days, six days per week <strong>for</strong> approximately<br />
3-4 min until sidewise position. We investigate 1) spiny medium size neurons of n. accumbens<br />
core, which are involved in corticomesolimbic circle <strong>an</strong>d are especially sensitive to various<br />
addictive drugs; 2) <strong>the</strong> hippocampus, where intoxication provokes cell death. Material is stained<br />
by Nissl, Golgi <strong>an</strong>d Gallia methods.The special computer program is used <strong>for</strong> investigation of:<br />
dendrites number, diameter, direction, spines number <strong>an</strong>d shape. Besides to reveal whe<strong>the</strong>r<br />
toluene intoxication produce long lasting alterations in <strong>the</strong> behaviour, <strong>the</strong> emotional <strong>an</strong>d<br />
explorative sphere of <strong>the</strong>se <strong>an</strong>imals is studied by open field test.<br />
Disclosures: L. Chilachava, None; L. Gelazonia, None; N. Japaridze, None; R. Mikava,<br />
None; M. Zhv<strong>an</strong>ia, None.<br />
Poster<br />
550. Neural Plasticity, Dependence, <strong>an</strong>d Addiction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 550.27/Y3<br />
Topic: C.06.k. Animal models <strong>an</strong>d mech<strong>an</strong>isms<br />
Support: USPHS Gr<strong>an</strong>t DA020654<br />
Title: Age matters<br />
Authors: J. E. MCCUTCHEON, *M. MARINELLI;<br />
Cell & Molec Pharmacol, Rosalind Fr<strong>an</strong>klin Univ/Chicago Med., North Chicago, IL<br />
Abstract: The age of <strong>an</strong> experimental <strong>an</strong>imal c<strong>an</strong> be a critical variable yet age matters are often<br />
overlooked within neuroscience. M<strong>an</strong>y studies make use of young <strong>an</strong>imals without considering<br />
possible differences between immature <strong>an</strong>d mature subjects. This c<strong>an</strong> be especially problematic<br />
when attempting to model traits or diseases that do not emerge until adulthood. Here, we<br />
illustrate this trend with a systematic review of published articles on long-term potentiation<br />
(LTP). We examined all articles published on LTP in rats or mice between 1997 <strong>an</strong>d 2007 <strong>an</strong>d<br />
restricted our search to <strong>the</strong> journals Nature, Science, Journal of Neuroscience, <strong>an</strong>d The Europe<strong>an</strong><br />
Journal of Neuroscience. For each publication we tabulated <strong>the</strong> age of <strong>an</strong>imals used, <strong>the</strong> methods<br />
used, <strong>an</strong>d <strong>the</strong> age that <strong>the</strong> authors reported. We found that of studies using ex vivo techniques,<br />
only a minority (34%) were conducted in adult <strong>an</strong>imals (>P63), <strong>the</strong> majority (59%) were<br />
conducted in <strong>an</strong>imals that had not yet reached adulthood <strong>an</strong>d in 7% of studies age was not<br />
specified. <strong>When</strong> we confined our <strong>an</strong>alysis of ex-vivo studies to experiments using voltage- or<br />
current-clamp slice electrophysiology (patch clamp or sharp electrodes) we found that 75% of<br />
studies used young <strong>an</strong>imals <strong>an</strong>d only 20% examined adult <strong>an</strong>imals; <strong>the</strong> rest (5%) did not specify<br />
<strong>the</strong> age of <strong>the</strong> experimental subjects. These figures contrast with experiments conducted in vivo<br />
in which <strong>the</strong> majority of work was conducted in adult <strong>an</strong>imals (65%), a minority in young<br />
<strong>an</strong>imals (26%) <strong>an</strong>d in 9% of studies age was not specified. The most worrying finding was that in<br />
60% of studies young <strong>an</strong>imals were incorrectly defined as adults. Ano<strong>the</strong>r concerning trend was<br />
that of pooling <strong>an</strong>imals of different ages. In m<strong>an</strong>y cases (30%) <strong>an</strong>imals were pooled whose ages<br />
varied by 3-4 weeks, often sp<strong>an</strong>ning critical age periods such as puberty. In conclusion, <strong>the</strong>re is<br />
much variation in <strong>the</strong> age of <strong>an</strong>imals used in neuroscience studies <strong>an</strong>d this is often misreported.<br />
These findings should encourage <strong>the</strong> community, whe<strong>the</strong>r reading, reviewing or practicing<br />
science to pay closer attention to age.<br />
Disclosures: J.E. McCutcheon, None; M. Marinelli, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 551.1/Y4<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: Norwegi<strong>an</strong> Research Council Gr<strong>an</strong>t 170534/v40<br />
Title: The acute behavioural response to heroin in mice is mediated by <strong>the</strong> initial metabolite 6acetylmorphine<br />
<strong>an</strong>d not morphine<br />
Authors: J. ANDERSEN, Å. RIPEL, F. BOIX, P. NORMANN, *J. MORLAND;<br />
Norwegi<strong>an</strong> Inst. Publ. Hlth., Oslo, Norway<br />
Abstract: Heroin, <strong>the</strong> most widely abused opioid in <strong>the</strong> world, has a higher intoxicating potency<br />
th<strong>an</strong> o<strong>the</strong>r effective opioids, like morphine, even though it is rapidly metabolized <strong>an</strong>d has low<br />
affinity <strong>for</strong> opiate receptors. Thus, heroin may act as a pro-drug, <strong>the</strong> effects being mediated by its<br />
metabolites. Morphine has been considered to be <strong>the</strong> major active metabolite after heroin<br />
exposure <strong>an</strong>d is used as a heroin surrogate in m<strong>an</strong>y studies. However, <strong>the</strong> pharmacological active<br />
6-acetylmorphine (6-AM) is present earlier th<strong>an</strong> morphine <strong>an</strong>d could mediate <strong>the</strong> effects of<br />
heroin. To evaluate <strong>the</strong> contribution of heroin itself <strong>an</strong>d its metabolites, we related <strong>the</strong><br />
stimulation of locomotor activity induced by heroin <strong>an</strong>d <strong>the</strong> metabolites to <strong>the</strong>ir pharmacokinetic<br />
profiles.<br />
Two equimolar doses (5 <strong>an</strong>d 15 µmol/kg) of heroin, 6-AM or morphine were administered sc to<br />
male C57BL/6J-Bom mice. Thereafter, <strong>the</strong> mice were ei<strong>the</strong>r placed in a cage where <strong>the</strong>ir<br />
locomotor activity was followed <strong>for</strong> five hours, or sacrificed at different time points after<br />
injection. Brain samples were <strong>an</strong>alysed <strong>for</strong> heroin, 6-AM <strong>an</strong>d morphine by LC-MSMS.<br />
Heroin <strong>an</strong>d 6-AM induced a signific<strong>an</strong>t much higher increase in both maximal <strong>an</strong>d total<br />
locomotor activity compared to equimolar doses of morphine. There were no differences<br />
between heroin <strong>an</strong>d 6-AM in <strong>the</strong> total response.<br />
Heroin was detected in brain tissue after its injection, but <strong>the</strong> levels were too low <strong>an</strong>d its presence<br />
too short-lived to be responsible <strong>for</strong> <strong>the</strong> behavioural response observed.<br />
The brain concentration of 6-AM increased shortly after administration of both heroin <strong>an</strong>d 6-<br />
AM, <strong>an</strong>d <strong>the</strong> concentration profile of this metabolite during <strong>the</strong> first hour reflected <strong>the</strong> ch<strong>an</strong>ges in<br />
locomotor activity observed after administration of <strong>the</strong> two drugs, indicating that 6-AM was <strong>the</strong><br />
drug causing <strong>the</strong> behavioural effect.<br />
The morphine concentration in brain increased slowly after injection of each subst<strong>an</strong>ce. The<br />
morphine levels achieved after heroin or 6-AM administration could not explain <strong>the</strong>ir immediate<br />
behavioural effect when compared with <strong>the</strong> response obtained after morphine alone.<br />
In summary, <strong>the</strong> locomotor activity response observed after injection of heroin follows <strong>the</strong><br />
pharmacokinetic profile of 6-AM in <strong>the</strong> brain. The concentration of morphine was not high<br />
enough to account <strong>for</strong> <strong>the</strong> immediate increase in activity, although a possible effect on <strong>the</strong> later<br />
portion of <strong>the</strong> heroin-induced behavioural response curve c<strong>an</strong> not be rejected. These findings,<br />
toge<strong>the</strong>r with observations <strong>from</strong> o<strong>the</strong>r studies indicating that 6-AM might have a receptor<br />
binding profile somewhat different <strong>from</strong> morphine, may question <strong>the</strong> use of morphine as a model<br />
subst<strong>an</strong>ce <strong>for</strong> heroin in <strong>an</strong>imal experiments.
Disclosures: J. Andersen, None; Å. Ripel, None; F. Boix, None; P. Norm<strong>an</strong>n, None; J.<br />
Morl<strong>an</strong>d, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.2/Y5<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIH K99 ES015428<br />
NIH T32 ES007326<br />
NIH R01 ES015687<br />
Title: Discriminative stimulus effects of cocaine in rats following developmental exposure to<br />
polychlorinated biphenyls (PCBs)<br />
Authors: *H. J. SABLE 1 , S. MONAIKUL 2 , E. POON 2 , P. A. EUBIG 3 , S. L. SCHANTZ 4 ;<br />
1 Psychology, Univ. Memphis, Memphis, TN; 2 Neurosci. Program, 3 Vet. Biosci., 4 Neurosci.<br />
Program <strong>an</strong>d Vet. Biosci., Univ. Illinois at Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a, IL<br />
Abstract: Exposure to <strong>the</strong> ubiquitous environmental contamin<strong>an</strong>t polychlorinated biphenyls<br />
(PCBs) c<strong>an</strong> result in a number of hum<strong>an</strong> health concerns. Developmental exposure to PCBs in<br />
rats has been shown to produce alterations in brain dopamine (DA) concentrations that persist<br />
into adulthood. These alterations suggest <strong>the</strong> rein<strong>for</strong>cing properties of drugs of abuse that act on<br />
<strong>the</strong> DA system may be affected by developmental PCB exposure. The current study was<br />
conducted to determine if <strong>the</strong> interoceptive effects of cocaine <strong>an</strong>d d-amphetamine would be<br />
altered by developmental exposure to <strong>an</strong> environmentally relev<strong>an</strong>t PCB mixture. Female Long-<br />
Ev<strong>an</strong>s rats were orally exposed to 0, 3 or 6 mg/kg/day PCBs beginning 4 weeks prior to breeding<br />
<strong>an</strong>d continuing until litters were we<strong>an</strong>ed on postnatal day 21. One adult male <strong>an</strong>d female per<br />
litter were trained to discriminate cocaine (10.0 mg/kg, i.p.) <strong>from</strong> saline by repeatedly pairing<br />
cocaine injections with rein<strong>for</strong>cement on one response lever, <strong>an</strong>d saline injections with<br />
rein<strong>for</strong>cement on <strong>the</strong> o<strong>the</strong>r lever. Once <strong>the</strong> training criterion was met, each rat was given a series<br />
of generalization tests (three replicates/dose) to four lower doses of cocaine (1.25, 2.5, 5.0, <strong>an</strong>d<br />
7.5 mg/kg) <strong>an</strong>d four doses of amphetamine (0.125, 0.25, 0.50, <strong>an</strong>d 1.0 mg/kg), as well as crossgeneralization<br />
tests following 5.0 mg/kg of <strong>the</strong> non-DA mediated drug pentobarbital (all<br />
administered i.p.). Overall, PCB exposure signific<strong>an</strong>tly altered <strong>the</strong> interoceptive properties of <strong>the</strong>
highest dose of cocaine <strong>an</strong>d amphetamine, with <strong>the</strong> alteration being in opposite directions <strong>for</strong> <strong>the</strong><br />
two drugs. Signific<strong>an</strong>t sex differences were not found, but visual inspection of <strong>the</strong> data revealed<br />
<strong>the</strong> effect was more profound in <strong>the</strong> PCB-exposed males who showed increased responding on<br />
<strong>the</strong> cocaine lever <strong>for</strong> <strong>the</strong> two highest cocaine doses relative to non-exposed controls, <strong>an</strong>d<br />
decreased responding <strong>for</strong> <strong>the</strong> two highest amphetamine doses. A similar pattern was seen at <strong>the</strong><br />
highest cocaine <strong>an</strong>d amphetamine dose in <strong>the</strong> females, but responding at <strong>the</strong> lower doses was<br />
more variable. Responding on <strong>the</strong> cocaine-paired lever following pentobarbital was low <strong>for</strong> both<br />
sexes. Overall, <strong>the</strong>se results suggest that developmental PCB exposure c<strong>an</strong> alter <strong>the</strong><br />
discriminative stimulus properties of cocaine <strong>an</strong>d amphetamine. These findings also suggest that<br />
<strong>the</strong> alterations are due to ch<strong>an</strong>ges in interoceptive DA cues ra<strong>the</strong>r th<strong>an</strong> ch<strong>an</strong>ges in overall<br />
rein<strong>for</strong>cement, as responding on <strong>the</strong> drug lever was not altered by pentobarbital, a drug whose<br />
rein<strong>for</strong>cing properties are not DA mediated.<br />
Disclosures: H.J. Sable, None; S. Monaikul, None; E. Poon, None; P.A. Eubig, None; S.L.<br />
Sch<strong>an</strong>tz, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.3/Y6<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: DA011054<br />
RR00168<br />
Title: Attenuation of <strong>the</strong> discriminative stimulus effects of cocaine by <strong>the</strong> mGluR1 <strong>an</strong>tagonist<br />
JNJ16259685 in squirrel monkeys<br />
Authors: C. ACHAT-MENDES, D. M. PLATT, *R. D. SPEALMAN;<br />
NEPRC, Div. of Neurosci., Harvard Med. Sch., Southborough, MA<br />
Abstract: Group I, but not Group II, metabotropic glutamate receptors (mGluRs) have been<br />
implicated in <strong>the</strong> tr<strong>an</strong>sduction of cocaine’s discriminative stimulus (DS) effects in monkeys.<br />
Previous work has shown that <strong>an</strong>tagonism at <strong>the</strong> mGluR5 subtype of Group I receptors attenuates<br />
<strong>the</strong> discriminative stimulus (DS) effects of cocaine, but <strong>the</strong> role of <strong>the</strong> mGluR1 subtype has been<br />
less fully explored. In <strong>the</strong> present study, <strong>the</strong> effects of cocaine after pretreatment with <strong>the</strong><br />
selective mGluR1 <strong>an</strong>tagonist JNJ16259685 <strong>an</strong>d its vehicle were determined in squirrel monkeys
(n=6) trained to discriminate cocaine (0.3 or 0.42 mg/kg) <strong>from</strong> saline using a two-lever drug<br />
discrimination procedure in which lever pressing was maintained under a fixed-ratio schedule of<br />
food rein<strong>for</strong>cement. Following pretreatment with vehicle, cocaine engendered dose-related<br />
increases in <strong>the</strong> percentage of responses on <strong>the</strong> cocaine-associated lever, reaching a maximum of<br />
>90% cocaine-lever responses after 0.3mg/kg or greater. Pretreatment with JNJ16259685 (0.18<br />
<strong>an</strong>d 0.56 mg/kg) signific<strong>an</strong>tly attenuated <strong>the</strong> DS effects of cocaine, producing parallel rightward<br />
shifts in <strong>the</strong> cocaine dose-response function. ED50 (dose resulting in 50% cocaine-lever<br />
responses) <strong>an</strong>d EDmin (lowest dose producing a signific<strong>an</strong>t increase in % cocaine-lever<br />
responses) were increased ≥1.5-fold after pretreatment with JNJ16259685 compared to values<br />
obtained after pretreatment with vehicle. Under both training <strong>an</strong>d testing conditions, cocaine<br />
(0.18-0.42 mg/kg) signific<strong>an</strong>tly increased <strong>the</strong> rate of lever-pressing compared to <strong>the</strong> response rate<br />
after saline administration. Pretreatment with JNJ16259685 blocked <strong>the</strong> response-rate increasing<br />
effects of cocaine without producing signific<strong>an</strong>t decreases in response rate compared to <strong>the</strong> rate<br />
after saline administration. These findings, which are similar to those reported previously <strong>for</strong> <strong>the</strong><br />
selective mGluR5 <strong>an</strong>tagonist MPEP, suggest that both subtypes of <strong>the</strong> group I mGluR family<br />
play a role in <strong>the</strong> tr<strong>an</strong>sduction of cocaine’s subjective effects.<br />
Disclosures: C. Achat-Mendes, None; D.M. Platt, None; R.D. Spealm<strong>an</strong>, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.4/Y7<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: CIHR Gr<strong>an</strong>t Ref # 79919<br />
Title: Oreo cookies = cocaine?<br />
Authors: *A. LEVY, F. LERI;<br />
Psychology, Univ. of Guelph, Guelph, ON, C<strong>an</strong>ada<br />
Abstract: We have been exploring <strong>the</strong> hypo<strong>the</strong>sis that highly palatable foods c<strong>an</strong> be as addictive<br />
as drugs of abuse. More specifically, we tested whe<strong>the</strong>r differences in sensitivity to <strong>the</strong><br />
motivational properties of Oreo cookies are associated with differences in intravenous (IV) selfadministration<br />
(SA) of cocaine, reinstatement of cocaine-seeking (by Oreos <strong>an</strong>d by 20 mg/kg<br />
cocaine prime) <strong>an</strong>d locomotor reactivity to cocaine. A total of 72 non-food deprived, male<br />
Sprague-Dawley rats received 8 conditioning sessions (one 30 min session/day) during which
<strong>the</strong>y had access to ei<strong>the</strong>r a single Oreo (12g) in one compartment or no food in <strong>the</strong> o<strong>the</strong>r. Eleven<br />
days following <strong>the</strong> CPP test, 48 of <strong>the</strong>se rats self-administered cocaine (0.25 mg/kg/inf) on a<br />
continuous schedule of rein<strong>for</strong>cement <strong>for</strong> one 3h session/day <strong>for</strong> 8 days. Following acquisition,<br />
rats underwent extinction training, one 3h session/day <strong>for</strong> 11 days <strong>an</strong>d <strong>the</strong>n reinstatement testing<br />
precipitated first, by <strong>the</strong> consumption of Oreos <strong>an</strong>d secondly, by cocaine prime. The o<strong>the</strong>r 24 rats<br />
were injected with cocaine (0, 3, 15, <strong>an</strong>d 30 mg/kg, IP) <strong>an</strong>d confined to activity chambers <strong>for</strong> one<br />
2h locomotion test/day <strong>for</strong> 4 days. It was found that rats displayed ei<strong>the</strong>r a signific<strong>an</strong>t preference,<br />
or avoid<strong>an</strong>ce <strong>for</strong> <strong>the</strong> Oreo-paired compartment, <strong>an</strong>d this was not related to <strong>the</strong> qu<strong>an</strong>tity of Oreos<br />
consumed in <strong>the</strong> conditioning phase of CPP. During SA, preference rats displayed greater<br />
responding in acquisition <strong>an</strong>d extinction training compared to avoid<strong>an</strong>ce rats. At test,<br />
consumption of Oreos precipitated cocaine-seeking in preference but not avoid<strong>an</strong>ce rats, <strong>an</strong>d <strong>the</strong><br />
cocaine prime was effective in both groups; however, <strong>the</strong> magnitude of reinstatement was greater<br />
in preference rats. Finally, <strong>the</strong>re was a cocaine dose dependent increase in locomotion in both<br />
groups; however, locomotion was signific<strong>an</strong>tly lower in <strong>the</strong> preference rats in response to 15<br />
mg/kg cocaine. There<strong>for</strong>e, similar to drugs of abuse, Oreos exerted powerful motivational<br />
control over behaviour during CPP <strong>an</strong>d reinstatement testing. Sensitivity to <strong>the</strong> motivational<br />
properties of Oreos <strong>an</strong>d cocaine were related, whereby preference rats maintained higher<br />
responding during acquisition, <strong>an</strong>d displayed less locomotor reactivity to cocaine. These findings<br />
indicate that greater sensitivity to <strong>the</strong> motivational properties of palatable foods may reflect<br />
individual differences in vulnerability to <strong>the</strong> rein<strong>for</strong>cing effects of drugs of abuse, suggesting that<br />
<strong>for</strong> some individuals, junk food c<strong>an</strong> be considered <strong>an</strong> addictive drug.<br />
Disclosures: A. Levy, None; F. Leri, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.5/Y8<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Title: Conditioned appetitive properties of concurrent MDMA <strong>an</strong>d cocaine administration in rats<br />
Authors: *J. J. PANOS, A. C. MILSTEIN, M. R. JONES, L. A. BARNES, L. E. BAKER;<br />
Psychol Dept, Western Michig<strong>an</strong> Univ., Kalamazoo, MI<br />
Abstract: Polysubst<strong>an</strong>ce abuse is a fairly common practice among recreational users of<br />
methylenedioxymethamphetamine (MDMA or “Ecstasy”). In particular, cocaine use is more<br />
frequently reported by MDMA users th<strong>an</strong> non-drug users. Although <strong>the</strong>re is a large body of
experimental research regarding <strong>the</strong> neurobehavioral effects of MDMA <strong>an</strong>d cocaine administered<br />
separately, few controlled experimental studies have investigated <strong>the</strong> effects of concurrent<br />
administration with <strong>the</strong>se drugs. The aim of <strong>the</strong> current study was to determine <strong>the</strong> acute effects<br />
of concurrent MDMA/cocaine administration in <strong>an</strong> <strong>an</strong>imal model designed to assess conditioned<br />
reward <strong>an</strong>d aversion. Male Sprague-Dawley rats were administered cocaine (0, 10, 20 mg/kg),<br />
MDMA (0, 1.5, 3.0 mg/kg) or one of four MDMA/cocaine combinations in a conditioned place<br />
preference (CPP) procedure. After two days of habituation to <strong>the</strong> CPP apparatus, six 30 minute<br />
conditioning trials were conducted over a series of six consecutive days in which rats were<br />
individually placed in one side of <strong>the</strong> place-conditioning apparatus following drug injections <strong>an</strong>d<br />
in <strong>the</strong> o<strong>the</strong>r side following vehicle injections. Locomotor activity was also recorded during all<br />
conditioning trials. During <strong>the</strong> test trial, <strong>an</strong>imals were allowed access to both compartments <strong>for</strong><br />
15 minutes <strong>an</strong>d <strong>the</strong> amount of time spent in each compartment was recorded. Results showed that<br />
<strong>the</strong> MDMA 3.0/cocaine 20 mg/kg combination produced a greater preference <strong>for</strong> <strong>the</strong> drug-paired<br />
side compared to ei<strong>the</strong>r drug alone. This preclinical study may have import<strong>an</strong>t clinical<br />
implications <strong>an</strong>d may help explain <strong>the</strong> common practice of polysubs<strong>an</strong>ce use among MDMA<br />
users.<br />
Disclosures: J.J. P<strong>an</strong>os, None; A.C. Milstein, None; M.R. Jones, None; L.A. Barnes,<br />
None; L.E. Baker, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.6/Y9<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: Fondi di Ateneo 2006 - C26A0SLHXL<br />
PRIN - 2005050334_004<br />
Title: Drug taking <strong>an</strong>d context: Different setting preferences <strong>for</strong> heroin, cocaine, <strong>an</strong>d alcohol use<br />
in hum<strong>an</strong> addicts<br />
Authors: P. A. SPAGNOLO 1 , A. DUBLA 1 , M. CELENTANO 1 , *A. BADIANI 2 ;<br />
1 Dept. of Physiol. <strong>an</strong>d Pharmacol., Sapienza Univ. of Rome, Rome, Italy; 2 Dept Hum<strong>an</strong> Physiol,<br />
Pharmacol, Univ. of Rome La Sapienza, Rome, Italy
Abstract: Background: We have previously shown that drug preference in laboratory rats may<br />
be modulated by <strong>the</strong> context of drug taking (Caprioli et al. 2007, 2008, <strong>2009</strong>; Celent<strong>an</strong>o <strong>2009</strong>).<br />
In a tr<strong>an</strong>slational study, we have recently found <strong>an</strong> un<strong>for</strong>eseen dissociation in <strong>the</strong> preferred<br />
setting <strong>for</strong> heroin vs. cocaine taking in a population of heroin <strong>an</strong>d/or cocaine addicts (Caprioli et<br />
al. <strong>2009</strong>).<br />
Rationale: In order to fur<strong>the</strong>r characterize this phenomenon, <strong>the</strong> present study was conducted to:<br />
1) confirm our results in a larger sample of heroin <strong>an</strong>d/or cocaine addicts, 2) to compare <strong>the</strong><br />
preferred setting <strong>for</strong> current drug taking with that of early drug taking; 3) to investigate <strong>the</strong><br />
preferred setting <strong>for</strong> alcohol consumption among heavy drinkers (more th<strong>an</strong> 5 alcoholic units per<br />
day).<br />
Methods: Particip<strong>an</strong>ts were heroin <strong>an</strong>d/or cocaine addicts recruited among <strong>the</strong> outpatients of<br />
Villa Maraini Therapeutic Community. A structured interview was administered to <strong>the</strong><br />
individuals who: 1) met DSM-IVR criteria <strong>for</strong> cocaine <strong>an</strong>d/or heroin dependence, 2) did not meet<br />
DSM-IVR criteria <strong>for</strong> <strong>an</strong>y o<strong>the</strong>r major psychiatric disorder, <strong>an</strong>d were in not treatment with<br />
<strong>an</strong>tipsychotic, <strong>an</strong>tidepress<strong>an</strong>t, or <strong>an</strong>xiolytic medications, 3) had a fixed address, <strong>an</strong>d 4) reported<br />
using heroin <strong>an</strong>d/or cocaine in <strong>the</strong> past 3 months. The interview was developed specifically to<br />
ascertain <strong>the</strong> context of cocaine, heroin, <strong>an</strong>d alcohol taking. In addition, in<strong>for</strong>mation regarding<br />
gender, age, socioeconomic status, years of cocaine <strong>an</strong>d/or heroin taking, use of o<strong>the</strong>r drugs was<br />
also collected.<br />
Results: In agreement with a previous report, about 72% of particip<strong>an</strong>ts reported using heroin<br />
exclusively or mostly at home (vs. 24% using heroin exclusively or mostly outside <strong>the</strong> home)<br />
whereas only 27% of cocaine users reported using cocaine exclusively or mostly at home (vs.<br />
66% using cocaine exclusively or mostly outside <strong>the</strong> home). Setting preferences <strong>for</strong> heavy<br />
drinkers were similar to those <strong>for</strong> cocaine: about 64% of <strong>the</strong>m reported drinking exclusively or<br />
mostly at home (vs. 27% drinking exclusively or mostly outside <strong>the</strong> home). Finally, <strong>the</strong><br />
comparison between setting preferences <strong>for</strong> actual versus early drug use indicated a dramatic<br />
shift <strong>for</strong> heroin (P < 0.0001) but not <strong>for</strong> cocaine or alcohol. The setting <strong>for</strong> early heroin use was<br />
in fact a non-home environment <strong>for</strong> 74% of particip<strong>an</strong>ts.<br />
Conclusions: The present findings indicate that environmental context plays <strong>an</strong> import<strong>an</strong>t role in<br />
drug taking <strong>an</strong>d that different drugs are used in different context.<br />
Disclosures: P.A. Spagnolo, None; A. Dubla, None; M. Celent<strong>an</strong>o, None; A. Badi<strong>an</strong>i, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.7/Y10<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology
Support: Fondi di Ateneo 2006 - C26A0SLHXL<br />
PRIN - 2005050334_004<br />
Title: Drug choice <strong>an</strong>d context: The role of associative learning versus interoceptive drug effects<br />
Authors: *M. CELENTANO, Mr., A. TESTA, M. T. DE LUCA, A. BADIANI;<br />
Dept Hum<strong>an</strong> Physiol. <strong>an</strong>d Pharmacol., Univ. Rome La Sapienza, Rome, Italy<br />
Abstract: Background: Our previous studies have shown that environmental context c<strong>an</strong> exert a<br />
powerful influence over drug choice. Surprisingly, it was found a dissociation in <strong>the</strong> effect of<br />
environment on heroin vs. cocaine reward. The intake of heroin was greater in rats that were<br />
housed in <strong>the</strong> self-administration (SA) chambers (Resident rats) relative to rats that were<br />
tr<strong>an</strong>sported to <strong>the</strong> SA chambers immediately be<strong>for</strong>e <strong>the</strong> start of each session (Non Resident rats).<br />
The opposite was found <strong>for</strong> cocaine. We have also found that environmental context c<strong>an</strong><br />
influences <strong>the</strong> choice between heroin <strong>an</strong>d cocaine when <strong>the</strong> two drugs are made available<br />
simult<strong>an</strong>eously: most Non Residents rats preferring cocaine <strong>an</strong>d most Residents rats preferring<br />
heroin. Rationale: in <strong>the</strong> present study we investigated <strong>the</strong> contribution of associate learning<br />
mech<strong>an</strong>isms in <strong>the</strong> acquisition of drug preference. Methods: Thity-nine rats received a doublelumen<br />
intrajugular ca<strong>the</strong>ter <strong>an</strong>d were <strong>the</strong>n assigned to one of four environmental conditions:<br />
Paired-Resident, Unpaired-Resident, Paired-Non Resident <strong>an</strong>d Unpaired-Non Resident. The rats<br />
in all <strong>the</strong> Resident condition were housed <strong>an</strong>d tested in two-lever oper<strong>an</strong>t chambers. The rats in<br />
<strong>the</strong> Non Resident condition were tr<strong>an</strong>sferred to <strong>the</strong> SA chambers only <strong>for</strong> <strong>the</strong> self-administration<br />
sessions. In <strong>the</strong> Paired groups each drug was const<strong>an</strong>tly paired with one of <strong>the</strong> two levers,<br />
whereas in <strong>the</strong> Unpaired groups each lever was assigned <strong>an</strong> equal number of times to cocaine or<br />
heroin following a pseudo-r<strong>an</strong>dom sequence. During sessions 1-14, <strong>the</strong> rats were trained to selfadminister,<br />
on alternate days, cocaine (400 mcg/kg per infusion) <strong>an</strong>d heroin (25 mcg/kg per<br />
infusion). The schedule requirement was gradually increased <strong>from</strong> fixed ratio 1 (FR1) to FR5.<br />
During sessions 15-16, <strong>the</strong> rats were left undisturbed. During sessions 17-24, <strong>the</strong> rats were<br />
repeatedly given <strong>the</strong> opportunity to choose between <strong>an</strong> infusion of 400 mcg/kg of cocaine <strong>an</strong>d of<br />
25 mcg/kg of heroin. Results: Data were <strong>an</strong>alyzed using a bootstrapping procedure to determine<br />
<strong>the</strong> existence of a preference <strong>for</strong> cocaine or heroin. As expected on <strong>the</strong> basis of previous reports,<br />
more Paired-Resident rats preferred heroin over cocaine relative to Paired-Non Resident rats <strong>an</strong>d<br />
vice versa <strong>for</strong> <strong>the</strong> preference <strong>for</strong> cocaine over heroin. In contrast, no Unpaired rat exhibited a<br />
preference <strong>for</strong> ei<strong>the</strong>r of <strong>the</strong> two drugs. Conclusions: <strong>the</strong> present results indicate a strong effect of<br />
associative learning mech<strong>an</strong>isms in <strong>the</strong> acquisition of drug preference. In contrast, drug<br />
discriminative effects appear to play a relatively minor role.<br />
Disclosures: M. Celent<strong>an</strong>o, None; A. Testa, None; M.T. De Luca, None; A. Badi<strong>an</strong>i, None.<br />
Poster<br />
551. Behavioral Pharmacology I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.8/Y11<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: Fondi di Ateneo 2006 - C26A0SLHXL<br />
PRIN - 2005050334_004<br />
Title: Drug taking <strong>an</strong>d context: Environmental modulation of ketamine self-administration in <strong>the</strong><br />
rat<br />
Authors: *M. DE LUCA, M. CELENTANO, A. TESTA, A. BADIANI;<br />
Univ. Rome La Sapienza, Rome, Italy<br />
Abstract: Rationale: Ketamine, a drug related to phencyclidine, was developed to be used as a<br />
dissociative <strong>an</strong>es<strong>the</strong>tic but <strong>the</strong>re is increasing evidence of its abuse potential. Ketamine abuse<br />
appears to be limited mainly to people who attend d<strong>an</strong>ce clubs <strong>an</strong>d raves. This suggests that <strong>the</strong><br />
setting of drug taking plays <strong>an</strong> import<strong>an</strong>t role in modulating <strong>the</strong> reward effects of ketamine. We<br />
have previously shown (Caprioli et al. 2007, 2008) that <strong>the</strong> intake of <strong>an</strong>d <strong>the</strong> motivation <strong>for</strong><br />
cocaine <strong>an</strong>d amphetamine is greater in rats that are tr<strong>an</strong>sported to <strong>the</strong> self-administration<br />
chambers immediately be<strong>for</strong>e <strong>the</strong> start of each session (Non Resident rats) th<strong>an</strong> in rats that are<br />
housed in <strong>the</strong> self-administration chambers (Resident rats).<br />
Objectives: The goal of <strong>the</strong> present study was to extend <strong>the</strong> <strong>an</strong>imal model summarized above to<br />
ketamine. We predicted that ketamine self-administration would be facilitated in Non Resident<br />
rats relative to Resident rats.<br />
Materials <strong>an</strong>d Methods: Independent groups of rats with intravenous ca<strong>the</strong>ters were given <strong>the</strong><br />
opportunity to self-administer two different doses of ketamine (25 µg/kg or 50 µg/kg per<br />
infusion) under two environmental conditions. Some <strong>an</strong>imals were housed in <strong>the</strong> selfadministration<br />
cages (Resident group), whereas o<strong>the</strong>r rats were tr<strong>an</strong>sported to <strong>the</strong> selfadministration<br />
cages only <strong>for</strong> <strong>the</strong> test sessions (Non Resident group). The sessions lasted 3 h<br />
each <strong>an</strong>d were conducted during <strong>the</strong> dark phase. The schedule requirement was gradually<br />
increased <strong>from</strong> fixed ratio 1 (FR1) to FR5.<br />
Results: During training, Non Resident rats self administered about 6 times more ketamine th<strong>an</strong><br />
Resident rats (110.7±45.1 versus 18.6±5.3 infusions; P < 0.05).<br />
Conclusions: The present findings indicate that environmental context exerts a powerful<br />
modulatory control over <strong>the</strong> rein<strong>for</strong>cing <strong>an</strong>d interoceptive effects of ketamine.<br />
Disclosures: M. De Luca, None; M. Celent<strong>an</strong>o, None; A. Testa, None; A. Badi<strong>an</strong>i, None.<br />
Poster
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.9/Y12<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Title: Drug choice is a function of setting: A heroin versus amphetamine self-administration<br />
study in rats with double-lumen ca<strong>the</strong>ters<br />
Authors: *A. TESTA, M. CELENTANO, D. CAPRIOLI, F. LUCANTONIO, M. DE LUCA,<br />
A. BADIANI;<br />
Dept. of Physiol. <strong>an</strong>d Pharmacol., Sapienza Univ. of Rome, Rome, Italy<br />
Abstract: Background: Self-administration studies conducted in <strong>the</strong> rat have shown that drug<br />
reward is a function of environmental context. In particular, it was found that rats that were<br />
trained to self-administer heroin <strong>an</strong>d cocaine on alternate days <strong>an</strong>d <strong>the</strong>n given <strong>the</strong> opportunity to<br />
choose between <strong>the</strong> two drugs express different preferences as a function of <strong>the</strong> setting. Most rats<br />
that were tr<strong>an</strong>sported to <strong>the</strong> self-administration chambers immediately be<strong>for</strong>e <strong>the</strong> start of each<br />
session (Non Resident rats) preferred cocaine over heroin. In contrast, most rats that were housed<br />
in <strong>the</strong> self-administration chambers (Resident rats) preferred heroin over cocaine (Caprioli et al.<br />
<strong>2009</strong>). The aim of <strong>the</strong> present study was to investigate whe<strong>the</strong>r environmental context c<strong>an</strong> also<br />
influences <strong>the</strong> choice between heroin <strong>an</strong>d amphetamine when <strong>the</strong> two drugs are made available<br />
simult<strong>an</strong>eously. Methods: Thirty-eight rats received a double-lumen intrajugular ca<strong>the</strong>ter <strong>an</strong>d<br />
were <strong>the</strong>n assigned to one of two environmental conditions. The rats in <strong>the</strong> Resident condition<br />
were housed <strong>an</strong>d tested in two-lever oper<strong>an</strong>t chambers. The rats in <strong>the</strong> Non Resident condition<br />
were tr<strong>an</strong>sferred to <strong>the</strong> oper<strong>an</strong>t chambers only <strong>for</strong> <strong>the</strong> self-administration sessions. During<br />
sessions 1-10, <strong>the</strong> rats were trained to self-administer, on alternate days, amphetamine (250 or<br />
500 mcg/kg per infusion) <strong>an</strong>d heroin (25 mcg/kg per infusion). The schedule requirement was<br />
gradually increased <strong>from</strong> fixed ratio 1 (FR1) to FR5. During sessions 11-12, <strong>the</strong> rats were left<br />
undisturbed. During sessions 13-19, <strong>the</strong> rats were repeatedly given <strong>the</strong> opportunity (every 10<br />
min) to choose between amphetamine <strong>an</strong>d heroin self-administration on <strong>an</strong> FR5 schedule of<br />
rein<strong>for</strong>cement. Results: Among <strong>the</strong> 24 rats trained with 25 mcg/kg heroin <strong>an</strong>d 250 mcg/kg<br />
amphetamine, 66.7% of Resident rats exhibited a preference <strong>for</strong> heroin whereas only 41% of<br />
Non Resident rats preferred heroin. Among <strong>the</strong> 14 rats trained with 25 mcg/kg heroin <strong>an</strong>d 250<br />
mcg/kg amphetamine, 100% of Resident rats exhibited a preference <strong>for</strong> heroin whereas only<br />
66.7% of Non Resident rats preferred heroin. Conclusions: The present study shows that<br />
environmental context c<strong>an</strong> influence <strong>the</strong> choice between heroin <strong>an</strong>d amphetamine in <strong>the</strong> rat.<br />
Disclosures: A. Testa, None; M. Celent<strong>an</strong>o, None; D. Caprioli, None; F. Luc<strong>an</strong>tonio,<br />
None; M. De Luca, None; A. Badi<strong>an</strong>i, None.
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.10/Y13<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIH R01 DA 027222<br />
NIDA 101 DA 077<br />
Title: Glutamate of PFC has a signific<strong>an</strong>t role in <strong>the</strong> acute <strong>an</strong>d chronic effect of Methylphenidate<br />
Authors: S. J. WANCHOO 1 , M. J. LEE 1 , *J. C. WAYMIRE 2 , A. C. SWANN 1 , N. DAFNY 1 ;<br />
1 2<br />
Dept. of Neurobio. <strong>an</strong>d Anat., Univ. of Texas - Houston, Houston, TX; Univ. Texas- Houston<br />
Med. Sch., Houston, TX<br />
Abstract: Progressive augmentation of behavioral response following repeated psychostimul<strong>an</strong>t<br />
administrations is known as behavioral sensitization, <strong>an</strong>d is <strong>an</strong> experimental indicator of a drug’s<br />
liability <strong>for</strong> abuse. It is known that Ritalin or methylphenidate (MPD), a drug used to treat<br />
Attention-Deficit Hyperactivity Disorder (ADHD), induces sensitization in <strong>an</strong>imals following<br />
repeated injections. Given that 5-15% of Americ<strong>an</strong> children suffer ADHD, <strong>an</strong>d are treated with<br />
MPD, it is essential to know <strong>the</strong> neuronal circuitry of MPD action. It was recently reported that<br />
bilateral electric (non-specific) lesion of PFC prevented behavioral sensitization after chronic<br />
MPD administration. Since <strong>the</strong> PFC sends glutamatergic afferents to both ventral tegmental area<br />
(VTA) <strong>an</strong>d nucleus accumbens (NAc), sites that are involved in induction <strong>an</strong>d expression of<br />
behavioral sensitization to psychostimul<strong>an</strong>ts <strong>an</strong>d as PFC glutamatergic afferents are known to<br />
modulate <strong>the</strong> NAc <strong>an</strong>d VTA dopaminergic neurons, <strong>the</strong> objective of this study was to study <strong>the</strong><br />
role of glutamate <strong>from</strong> PFC in behavioral sensitization to MPD. Locomotor activity of three<br />
groups of rats- control, sham operated <strong>an</strong>d group with specific chemical lesion of glutamate<br />
neurons of PFC- was recorded using <strong>an</strong> open-field assay <strong>an</strong>d <strong>an</strong>alyzed. On experimental day 1,<br />
<strong>the</strong> rats were injected 0.8mL of saline between 06:30 <strong>an</strong>d 07:00 <strong>an</strong>d <strong>the</strong>ir locomotor activity was<br />
recorded <strong>for</strong> two hours. The next day, <strong>an</strong>imals were divided into sham, 6-OHDA lesion <strong>an</strong>d<br />
control groups (n = 8 <strong>for</strong> each group), <strong>an</strong>d <strong>the</strong> respective surgeries were per<strong>for</strong>med on sham <strong>an</strong>d<br />
lesion groups. The <strong>an</strong>imals were given five days to recover (experimental days 3 to 7) <strong>from</strong> <strong>the</strong><br />
surgery. On experimental day 8, recordings were resumed after <strong>the</strong> saline injection (post surgery<br />
baseline). On experimental days 9 - 14, <strong>the</strong> rats were given 2.5mg/kg MPD doses daily. The<br />
recordings were started immediately after each injection <strong>for</strong> 2 hours. No injections were given on<br />
days 15-18 (Washout period). The <strong>an</strong>imals were rechallenged with 2.5mg/kg MPD dose on<br />
experimental day 19, followed by <strong>the</strong> 2 hour post injection recording. It was found that PFC<br />
glutamatergic afferents are essential <strong>for</strong> <strong>the</strong> acute effect of MPD, i.e., <strong>the</strong> MPD-induced
hyperactivity <strong>an</strong>d are also involved in its chronic effects such as behavioral sensitization to<br />
multiple MPD administrations.<br />
Disclosures: S.J. W<strong>an</strong>choo, None; M.J. Lee, None; J.C. Waymire , None; A.C. Sw<strong>an</strong>n,<br />
None; N. Dafny, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.11/Y14<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIDA 027222<br />
NIDA 101 DA 077<br />
Title: Dopamine of prefrontal cortex is essential <strong>for</strong> <strong>the</strong> expression of behavioral sensitization to<br />
methylphenidate<br />
Authors: *N. DAFNY 1 , S. WANCHOO 2 , M. J. LEE 2 , A. C. SWANN 2 ;<br />
2 Dept. of Neurobio. <strong>an</strong>d Anat., 1 Univ. Texas Med. Sch., Houston, TX<br />
Abstract: Psychostimul<strong>an</strong>ts like amphetamine <strong>an</strong>d methylphenidate (MPD) are used to treat<br />
Attention Deficit Hyperactivity Disorder (ADHD). The pathophysiology of ADHD is often<br />
linked to dysfunction of <strong>the</strong> prefrontal cortex (PFC). Neuropsychological <strong>an</strong>alyses indicate that<br />
impairments of ADHD patients are similar to those with prefrontal lesions, <strong>for</strong> example, both<br />
face problems with tasks of behavioral inhibition, reward reversal, <strong>an</strong>d working memory. The<br />
mesocortical dopamine (DA) system modulates <strong>the</strong>se functions in <strong>the</strong> PFC <strong>an</strong>d ADHD patients<br />
are known to have dopaminergic aberrations in PFC. Electrical lesioning of PFC prevented <strong>the</strong><br />
expression of behavioral sensitization to MPD. Behavioral sensitization is progressively<br />
augmenting activity as a result of repetitive exposure to <strong>the</strong> drug. It was reported that<br />
dopaminergic afferents of PFC play a role in behavioral sensitization to o<strong>the</strong>r psychostimul<strong>an</strong>ts<br />
like amphetamine <strong>an</strong>d cocaine, which have chemical <strong>an</strong>d pharmacological properties similar to<br />
MPD. The objective of this study was to investigate <strong>the</strong> role of DA in PFC in mediating <strong>the</strong><br />
behavioral sensitization to repeated administration of MPD in male adult Sprague Dawley rats.<br />
Three groups of <strong>an</strong>imals - control, sham-operated <strong>an</strong>d bilateral 6-OHDA lesion in PFC were used<br />
<strong>for</strong> this purpose. The baseline locomotor activity was recorded on day 1 (pre-surgery baseline).<br />
The next day, <strong>an</strong>imals were divided into sham, 6-OHDA lesion <strong>an</strong>d control groups (n = 8 <strong>for</strong>
each group), <strong>an</strong>d <strong>the</strong> respective surgeries were per<strong>for</strong>med on sham <strong>an</strong>d lesion groups. The<br />
<strong>an</strong>imals were given five days to recover (experimental days 3 to 7) <strong>from</strong> <strong>the</strong> surgery. On<br />
experimental day 8, recordings were resumed after <strong>the</strong> saline injection (post surgery baseline).<br />
On experimental days 9 - 14, <strong>the</strong> rats were given 2.5mg/kg MPD doses daily. The recordings<br />
were started immediately after each injection <strong>for</strong> 2 hours. No injections were given on days 15-<br />
18 (Washout period). The <strong>an</strong>imals were rechallenged with 2.5mg/kg MPD dose on experimental<br />
day 19, followed by <strong>the</strong> 2 hour post injection recording. It was found that bilateral 6-OHDA<br />
lesions of PFC did not alter <strong>the</strong> locomotor baseline, nor affected <strong>the</strong> acute effect of MPD on<br />
locomotion. However, it was found that bilateral destruction of dopaminergic afferents of PFC<br />
eliminated <strong>the</strong> behavioral sensitization to MPD. This suggests that suggests that DA afferents of<br />
PFC are involved in <strong>the</strong> neural circuitry underlying behavioral sensitization to MPD.<br />
Disclosures: N. Dafny , None; S. W<strong>an</strong>choo, None; M.J. Lee, None; A.C. Sw<strong>an</strong>n, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.12/Y15<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIH R01 DA 027222<br />
NIDA 101 DA 077<br />
Title: Amphetamine sensitization is prevented by prefrontal cortex lesion<br />
Authors: A. TANG 1 , S. WANCHOO 2 , *A. C. SWANN 1 , N. DAFNY 2 ;<br />
1 2<br />
Dept Psychiat & Behav Sci., Dept of Neurobiolgoy <strong>an</strong>d Anat., Univ. Texas- Houston, Hlth. Sci.<br />
Ctr., Houston, TX<br />
Abstract: The psychostimul<strong>an</strong>t amphetamine (Amph) <strong>an</strong>d methylphenidate (MPD) are widely<br />
used treatments <strong>for</strong> Attention-deficit hyperactivity disorder. Chronic intermittent exposure to<br />
psychostimulamts induces behavioral sensitization. Recent MPD studies suggest that <strong>the</strong><br />
prefrontal cortex (PFC) is involved in behavioral sensitization. The objective of this study was to<br />
investigate <strong>the</strong> acute <strong>an</strong>d chronic effect of Amph on open-field motor activity, be<strong>for</strong>e <strong>an</strong>d after<br />
electrolytic PFC lesion. Male Sprague-Dawley rats were assigned r<strong>an</strong>domly to three groups, (1)<br />
<strong>an</strong> intact control group (N=6), (2) a sham-operated group (N=6), <strong>an</strong>d (3) a lesion group (N=8).<br />
On experimental day 1, all <strong>an</strong>imals were injected with saline <strong>an</strong>d <strong>the</strong>ir motor activity was
ecorded. On experimental day 2, <strong>the</strong> lesion group received bilateral electrolytic lesions of PFC<br />
while <strong>the</strong> sham group received <strong>the</strong> same procedure without current. After 5 days o recovery, all<br />
<strong>an</strong>imals were injected daily with 0.6 mg/kg amphetamine. On experimental days 15 to 17, no<br />
injection was given but activity was recorded as on previous days. A rechallenge injection of 0.6<br />
mg/kg amphetamine was given on experimental day 18. All <strong>the</strong> three groups showed increases in<br />
locomotor activity after acute amphetamine injection (P< 0.05). Following chronic amphetamine,<br />
<strong>the</strong> control group <strong>an</strong>d sham-operated group exhibited behavioral sensitization while <strong>the</strong> PFC<br />
lesion group failed to express behavioral sensitization (P < 0.05). These results suggest that PFC<br />
lesion dose not interfere with <strong>the</strong> acute effects of amphetamine on locomotor activity but is<br />
required <strong>for</strong> development of behavior sensitization.<br />
Disclosures: A. T<strong>an</strong>g, None; S. W<strong>an</strong>choo, None; A.C. Sw<strong>an</strong>n , None; N. Dafny, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.13/Y16<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Title: Adolescent amphetamine sensitization in a rodent model of psychosis: Ch<strong>an</strong>ges in<br />
accumbal dopamine using microdialysis<br />
Authors: M. L. SMITH 1 , J. J. SMITH 1 , D. S. ROANE 2 , *R. W. BROWN 1 ;<br />
1 2<br />
East TN State Univ., Johnson City, TN; Pharmaceut. Sci., East Tennessee State Univ., Johnson<br />
City, TN<br />
Abstract: This study <strong>an</strong>alyzed amphetamine-induced behavioral sensitization <strong>an</strong>d dopamine<br />
overflow in <strong>the</strong> nucleus accumbens (NAcc) core utilizing a rodent model of psychosis. In this<br />
study, both male <strong>an</strong>d female Sprague-dawley rats were administered <strong>the</strong> dopamine D2/D3<br />
agonist quinpirole (1mg/kg) or saline <strong>from</strong> postnatal days (P)1-11 <strong>an</strong>d raised to adolescence<br />
(P30). Neonatal quinpirole treatment has been shown to increase <strong>the</strong> sensitivity of D2-like<br />
receptors throughout <strong>the</strong> <strong>an</strong>imal’s lifetime, consistent with several psychoses. After habituation<br />
to <strong>the</strong> locomotor arena, rats were administered d-amphetamine sulfate (1mg/kg) or saline every<br />
o<strong>the</strong>r day <strong>from</strong> P35-47, <strong>an</strong>d horizontal activity was recorded. The day after sensitization was<br />
complete, all <strong>an</strong>imals were surgically impl<strong>an</strong>ted with a guide c<strong>an</strong>nula aimed at <strong>the</strong> nucleus<br />
accumbens core. Six days later, a microdialysis probe was impl<strong>an</strong>ted through <strong>the</strong> c<strong>an</strong>nula <strong>an</strong>d<br />
<strong>an</strong>imals were given a cumultiave dose of amphetamine (0.1-3m/kg or saline) <strong>an</strong>d dopamine<br />
overflow <strong>an</strong>d its metabolites recorded. Results showed that D2-primed rats treated in
adolescence with amphetamine demonstrated a signific<strong>an</strong>t increase in horizontal activity<br />
compared to all o<strong>the</strong>r groups by <strong>the</strong> end of testing. Regarding microdialysis, D2-primed rats<br />
administered amphetamine demonstrated a 170% increase in accumbal dopamine release<br />
compared to non D2-primed rats administered amphetamine. Data <strong>from</strong> males are yet to be<br />
<strong>an</strong>alyzed but will be presented. Consistent with past data in schizophrenics amphetamine<br />
produces a signific<strong>an</strong>t increase in accumbal dopamine release in D2-primed versus non D2primed<br />
rats, suggesting that psychotics may demonstrate increased positive rein<strong>for</strong>cing effects of<br />
<strong>the</strong> drug, resulting in <strong>an</strong> increased propensity to use psychostimul<strong>an</strong>ts.<br />
Disclosures: M.L. Smith, None; J.J. Smith, None; D.S. Ro<strong>an</strong>e, None; R.W. Brown, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.14/Y17<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIDA 027222<br />
Title: The affect of acute <strong>an</strong>d chronic amphetamine treatment on <strong>the</strong> circadi<strong>an</strong> rhythm activity<br />
pattern of adolescent rats<br />
Authors: C. G. JONES 1 , P. B. YANG 1 , V. COX 1 , K. D. BURAU 2 , *J. L. STEINBERG 3 , A. C.<br />
SWANN 1 , N. DAFNY 1 ;<br />
1 2<br />
UTHSC- Houston Med. Sch., Houston, TX; UTHSC Sch. of Publ. Hlth., Houston, TX;<br />
3<br />
UTMSI, Houston, TX<br />
Abstract: The psychostimul<strong>an</strong>ts considered <strong>the</strong> gold st<strong>an</strong>dard in <strong>the</strong> treatment of attention deficit<br />
hyperactivity disorder (ADHD), one of <strong>the</strong> most common childhood disorders, are also finding<br />
<strong>the</strong>ir way into <strong>the</strong> h<strong>an</strong>ds of healthy young adults to improve cognitive per<strong>for</strong>m<strong>an</strong>ce. Endogenous<br />
neurotr<strong>an</strong>smitter, neuromodulator, hormone levels, <strong>an</strong>d locomotor activity are involved in <strong>the</strong><br />
biological psychostimul<strong>an</strong>t response <strong>an</strong>d demonstrate a consistent 24 hour circadi<strong>an</strong> rhythm<br />
activity pattern. The possible long term effects of psychostimul<strong>an</strong>t exposure in adolescence are<br />
considered controversial, <strong>an</strong>d thus, <strong>the</strong> objective of this study was to investigate whe<strong>the</strong>r <strong>the</strong><br />
chronic exposure to <strong>the</strong> psychostimul<strong>an</strong>t amphetamine affects <strong>the</strong> locomotor circadi<strong>an</strong> rhythm<br />
activity patterns of female adolescent Wistar-Kyoto (WKY) rat. Ch<strong>an</strong>ge in circadi<strong>an</strong> rhythm<br />
activity pattern is used as a marker <strong>for</strong> long term effect of <strong>the</strong> treatment. Twenty-four rats were<br />
divided into two groups, control (N=12) <strong>an</strong>d experimental (N=12), <strong>an</strong>d kept in a 12:12 h
light/dark cycle in <strong>an</strong> open field cage. After 5 to 7 days of acclimation, 11 days of consecutive<br />
non-stop recordings beg<strong>an</strong>. On experimental day 1, all groups were given <strong>an</strong> injection of saline.<br />
On experimental days 2 to 7, <strong>the</strong> experimental group was injected with 0.6 mg/kg amphetamine<br />
followed by a washout phase <strong>from</strong> experimental day 8 to 10, <strong>an</strong>d amphetamine re-challenge on<br />
experimental 11 similar to experimental day 2. The locomotor movements were counted by <strong>the</strong><br />
computerized <strong>an</strong>imal activity monitoring system, <strong>an</strong>d cosinor <strong>an</strong>alysis was used to fit a 24-hour<br />
curve to <strong>the</strong> activity pattern. The horizontal activity (HA), total dist<strong>an</strong>ce (TD), number of<br />
stereotypy (NOS), vertical activity (VA), <strong>an</strong>d stereotypical movements (SM) were <strong>an</strong>alyzed to<br />
find out whe<strong>the</strong>r <strong>the</strong> circadi<strong>an</strong> rhythm activity patterns were altered. Data obtained using <strong>the</strong>se<br />
locomotor indices of circadi<strong>an</strong> rhythm activity pattern suggests amphetamine treatment<br />
signific<strong>an</strong>tly modulates <strong>the</strong> locomotor circadi<strong>an</strong> rhythm activity <strong>an</strong>d produces both acute <strong>an</strong>d<br />
chronic effects on <strong>the</strong> developing brain which suggests that amphetamine elicits long term<br />
effects on adolescent rats.<br />
Disclosures: C.G. Jones, None; P.B. Y<strong>an</strong>g, None; V. Cox, None; K.D. Burau, None; J.L.<br />
Steinberg, NIDA 027222, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t<br />
<strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); A.C. Sw<strong>an</strong>n, None; N. Dafny, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.15/Y18<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Title: Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) treatment on <strong>the</strong> ch<strong>an</strong>ges of<br />
cocaine- <strong>an</strong>d amphetamine-regulated tr<strong>an</strong>script (CART) mRNA levels in adult rat brain<br />
Authors: S. CHOI, B.-H. MOON, H. LEE, S.-H. PARK, H. KIM, S. SHIN, S. KANG, H.-J.<br />
KIM, *K. H. SHIN;<br />
Dept. of Pharmacol., Korea Univ. Coll of Med., Seoul, Republic of Korea<br />
Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) decreases food intake <strong>an</strong>d body weight<br />
gain, but underlying mech<strong>an</strong>ism is not clear. Although TCDD increases cocaine- <strong>an</strong>d<br />
amphetamine-regulated tr<strong>an</strong>script (CART) in hypothalamic arcuate nucleus (ARC), <strong>the</strong> effect of<br />
TCDD treatment on CART mRNA levels in <strong>the</strong> o<strong>the</strong>r brain areas in relation to food intake has<br />
not been studied in detail. To <strong>an</strong>swer this question, we administered male Sprague-Dawley rats<br />
with TCDD (50 µg/kg, p.o.) or vehicle <strong>an</strong>d sacrificed rats 1, 2, 4 <strong>an</strong>d 7 days later to observe<br />
time-dependent ch<strong>an</strong>ges in CART mRNA levels. In addition, to observe dose-dependent ch<strong>an</strong>ges
of CART mRNA levels in relation to food intake, we administered different doses of TCDD<br />
(12.5, 25 <strong>an</strong>d 50 µg/kg) to rats <strong>an</strong>d sacrificed rats 2 weeks later. We found that TCDD (50 µg/kg)<br />
signific<strong>an</strong>tly increased CART mRNA levels in hypothalamic paraventricular nucleus (PVN) <strong>an</strong>d<br />
nucleus accumbens shell (AcbSh) at 4 <strong>an</strong>d 7 days after TCDD treatment, respectively. TCDD<br />
treatment signific<strong>an</strong>tly decreased food intake over 2 weeks in a dose dependent m<strong>an</strong>ner, resulting<br />
in a decrease of body weight gain. These effects were accomp<strong>an</strong>ied by increases in CART<br />
mRNA levels in PVN, ARC <strong>an</strong>d AcbSh. Interestingly, cumulative food intake <strong>an</strong>d weight gain<br />
were negatively correlated with CART mRNA levels in <strong>the</strong> PVN, AcbSh <strong>an</strong>d ARC. These results<br />
suggest that TCDD increases CART mRNA levels in <strong>the</strong> PVN, ARC <strong>an</strong>d AcbSh, which may be<br />
related to decreases in food intake <strong>an</strong>d body weight gain following TCDD administration.<br />
Disclosures: S. Choi, None; B. Moon, None; H. Lee, None; S. Park, None; H. Kim, None; S.<br />
Shin, None; S. K<strong>an</strong>g, None; H. Kim, None; K.H. Shin, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.16/Y19<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIH Gr<strong>an</strong>t DA19859<br />
NIH Gr<strong>an</strong>t GM81069<br />
Title: Behavioral differences between adolescent <strong>an</strong>d adult laboratory rats in response to<br />
dissociatives<br />
Authors: *B. SULLIVAN, C. Y. HELLER, K. A. TRUJILLO;<br />
Cali<strong>for</strong>nia State Univ. S<strong>an</strong> Marcos, S<strong>an</strong> Marcos, CA<br />
Abstract: Adolescence is marked by <strong>an</strong> increased likelihood <strong>for</strong> risk taking <strong>an</strong>d sensation<br />
seeking, behaviors that are epitomized by experimentation with drugs of abuse. Most<br />
psychopharmacological research has used adult <strong>an</strong>imal models. However, to better underst<strong>an</strong>d<br />
teenage drug abuse <strong>the</strong>re is growing interest in comparing drug effects in adolescents <strong>an</strong>d adults.<br />
In recent years dissociatives, such as phencyclidine (PCP) <strong>an</strong>d ketamine, have grown in<br />
popularity among young people at d<strong>an</strong>ce clubs <strong>an</strong>d raves. In laboratory <strong>an</strong>imals <strong>the</strong>se drugs<br />
produce stimulation of activity along with ataxia <strong>an</strong>d stereotypy at sub<strong>an</strong>es<strong>the</strong>tic doses. To gain a<br />
better underst<strong>an</strong>ding of dissociative use in adolescence <strong>the</strong> present experiments compared <strong>the</strong>
effects of phencyclidine in adolescent <strong>an</strong>d adult laboratory rats. Periadolescent (30 days of age)<br />
<strong>an</strong>d young adult (60 days of age) male Sprague-Dawley rats received saline or PCP (1.0, 3.16 or<br />
10.0 mg/kg). Activity <strong>an</strong>d behavioral observations were assessed in <strong>the</strong> adolescent group at<br />
postnatal (PN) day 30, PN 40, <strong>an</strong>d PN 60, <strong>an</strong>d in <strong>the</strong> adult group at PN 60, PN 70, <strong>an</strong>d PN 90.<br />
Locomotor activity was recorded using a Kinder Scientific Open Field Motor Monitor.<br />
Behavioral observations were made at five-minute intervals during which each <strong>an</strong>imal received<br />
<strong>an</strong> ataxia (0 - 4) <strong>an</strong>d stereotypy (1 - 5) rating. PCP produced a dose dependent increase in<br />
activity, ataxia, <strong>an</strong>d stereotypy. Adolescents were less responsive to <strong>the</strong> ataxic effects across<br />
doses <strong>an</strong>d showed a concomit<strong>an</strong>t increase in locomotor activity when compared to adults. These<br />
results contribute to increasing evidence demonstrating differences in response to drugs of abuse<br />
between adolescents <strong>an</strong>d adults. The different psychoactive effects of dissociatives in adolescents<br />
may contribute to use of this club drug by young people. In addition, <strong>the</strong> findings suggest that<br />
drug use during adolescence may impact on drug response in adulthood.<br />
Disclosures: B. Sulliv<strong>an</strong>, None; C.Y. Heller, None; K.A. Trujillo, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.17/Y20<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIH Gr<strong>an</strong>t DA19859<br />
NIH Gr<strong>an</strong>t GM81069<br />
Title: Low dose depress<strong>an</strong>t effects of ketamine <strong>an</strong>d interactions with o<strong>the</strong>r drugs of abuse<br />
Authors: E. MERCADO, C. GARCIA, *K. A. TRUJILLO;<br />
Cali<strong>for</strong>nia State Univ., S<strong>an</strong> Marcos, CA<br />
Abstract: Ketamine is a dissociative drug that acts as a noncompetitive <strong>an</strong>tagonist of NMDA<br />
receptors. It produces rein<strong>for</strong>cing effects <strong>an</strong>d is often abused in club <strong>an</strong>d rave settings. Dosedependent<br />
effects of ketamine in laboratory <strong>an</strong>imals include locomotor stimulation, ataxia <strong>an</strong>d<br />
stereotypy at sub<strong>an</strong>es<strong>the</strong>tic doses, <strong>an</strong>d locomotor depression accomp<strong>an</strong>ied by <strong>an</strong>es<strong>the</strong>sia at higher<br />
doses. The current studies examined ketamine (KET) in combination with various drugs of abuse<br />
-- methamphetamine (MA), morphine (MOR) or phencyclidine (PCP) -- to fur<strong>the</strong>r investigate<br />
ketamine’s behavioral effects. Since all of <strong>the</strong> drugs used in <strong>the</strong>se studies are known to produce
locomotor stimulation at low to moderate doses, it was hypo<strong>the</strong>sized that <strong>the</strong> combinations<br />
would increase locomotor behavior beyond <strong>an</strong>y of <strong>the</strong>se drug alone. Male Sprague Dawley rats<br />
were assigned to one of four treatment groups in each study: saline control, <strong>the</strong> combination<br />
group, <strong>an</strong>d each individual drug alone. The drugs were administered toge<strong>the</strong>r subcut<strong>an</strong>eously in a<br />
single injection with KET (5.0 or 10.0 mg/kg), <strong>an</strong>d included MA (0.3 or 1.0 mg/kg), MOR (5.0<br />
mg/kg) or PCP (3.0 mg/kg). Locomotor behavior was assessed with a Kinder Scientific Open<br />
Field Motor Monitor. <strong>When</strong> administered alone, MA, MOR <strong>an</strong>d PCP produced locomotor<br />
stimulation. In contrast, KET produced a more complex dose dependent effect on locomotion. At<br />
<strong>the</strong> lower dose a mild depress<strong>an</strong>t effect was observed, whereas at <strong>the</strong> higher dose a short-lived<br />
stimulation was seen, followed by depression. <strong>When</strong> KET was combined with MA or MOR, <strong>the</strong><br />
combination produced less locomotor activity th<strong>an</strong> ei<strong>the</strong>r drug alone. Thus <strong>the</strong> overall effect of<br />
ketamine was to inhibit <strong>the</strong> stimul<strong>an</strong>t effects of MA <strong>an</strong>d MOR. Conversely, when KET was<br />
combined with PCP, <strong>the</strong> combination produced greater stimulation th<strong>an</strong> ei<strong>the</strong>r drug alone. The<br />
results indicate that <strong>the</strong> behavioral effects of KET are more complicated <strong>the</strong>n previously<br />
reported. Although this drug is typically considered a stimul<strong>an</strong>t at low to moderate doses <strong>the</strong><br />
present studies found potent depress<strong>an</strong>t effects at <strong>the</strong>se doses. The depress<strong>an</strong>t effects were<br />
especially evident when KET was administered in combination with MA or MOR. Fur<strong>the</strong>r<br />
research will clarify <strong>the</strong> complex dose-dependent effects of KET <strong>an</strong>d how <strong>the</strong>se impact on abuse<br />
of this drug.<br />
Disclosures: E. Mercado, None; C. Garcia, None; K.A. Trujillo, None.<br />
Poster<br />
551. Behavioral Pharmacology I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 551.18/Y21<br />
Topic: C.17.cc. Addiction: Behavioral pharmacology<br />
Support: NIDA/IRP<br />
Title: Role of dopamine receptors, but not α2 adrenoceptors, in yohimbine-induced<br />
reinstatement of high-fat food seeking<br />
Authors: *S. G. NAIR, T. ADAMS-DEUTSCH, C. L. PICKENS, Y. SHAHAM;<br />
Behav Neurosci Br., NIDA/IRP, NIH, Baltimore, MD<br />
Abstract: Background: A major problem in obesity treatment is high rate of relapse to<br />
maladaptive eating habits; this relapse is often provoked by stress. We adapted a reinstatement
model, commonly used to study drug relapse, to study <strong>the</strong> role of stress in relapse to food<br />
seeking. We found that <strong>the</strong> prototypical α2 adrenoceptor <strong>an</strong>tagonist yohimbine, which induces<br />
stress-like states in hum<strong>an</strong>s <strong>an</strong>d laboratory <strong>an</strong>imals, reinstates food seeking. Here, we first<br />
attempted to verify whe<strong>the</strong>r yohimbine’s effect on reinstatement is mediated by α2<br />
adrenoceptors. We <strong>the</strong>n studied <strong>the</strong> role of dopamine receptors in this reinstatement.<br />
Methods: We trained food-restricted rats (~16 g/day) to lever press <strong>for</strong> 45 mg high-fat (35%)<br />
food pellets <strong>for</strong> 9-10 training days (3 h/day, every o<strong>the</strong>r day). After extinction of oper<strong>an</strong>t<br />
responding over 10-14 days, we tested <strong>the</strong> rats <strong>for</strong> reinstatement of food seeking.<br />
Results: The effect of yohimbine on reinstatement was nei<strong>the</strong>r blocked by <strong>the</strong> α2 adrenoceptor<br />
agonist clonidine (0.04, 0.08 mg/kg, i.p.) nor mimicked by <strong>the</strong> selective α2 adrenoceptor<br />
<strong>an</strong>tagonist RS 79948 (0.5, 1.0, 1.5 mg/kg, i.p.). In contrast, yohimbine-induced reinstatement<br />
was dose-dependently attenuated by <strong>the</strong> D1-family receptor <strong>an</strong>tagonist SCH 23390 (0.005, 0.010<br />
mg/kg, s.c.) or <strong>the</strong> D2-family receptor <strong>an</strong>tagonist raclopride (0.025, 0.050 mg/kg, s.c). SCH<br />
23390 or raclopride had no effect on high rates of food-rein<strong>for</strong>ced oper<strong>an</strong>t responding during<br />
training.<br />
Conclusions: Surprisingly, our data indicate a critical role of dopamine receptors, but not α2<br />
adrenoceptors, in yohimbine-induced reinstatement of high-fat food seeking.<br />
Disclosures: S.G. Nair, None; T. Adams-Deutsch, None; C.L. Pickens, None; Y. Shaham,<br />
None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.1/Y22<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: ABMRF<br />
NIDA<br />
Title: Mu opioid receptor trafficking <strong>an</strong>d alcoholism<br />
Authors: L. HE 1 , *K. H. BERGER 2 , J. L. WHISTLER 1 ;<br />
1 Gallo Res. Ctr., Emeryville, CA; 2 Dept Neurol, Ernest Gallo Clin. & Res. Ctr., Emeryville, CA
Abstract: It is well known that <strong>the</strong> mu-opioid receptor (MOR) plays a major role in <strong>the</strong><br />
activating/rein<strong>for</strong>cing effects of eth<strong>an</strong>ol consumption; however, it is less clear whe<strong>the</strong>r or how<br />
MOR contributes to <strong>the</strong> tr<strong>an</strong>sition <strong>from</strong> impulsive to compulsive eth<strong>an</strong>ol drinking. Increasing<br />
evidence indicates that <strong>the</strong> trafficking of G-protein-coupled receptors by <strong>the</strong> process of<br />
endocytosis is one fundamental cellular mech<strong>an</strong>ism regulating receptor signaling <strong>an</strong>d function<br />
<strong>an</strong>d conversely, that failure to promote MOR trafficking could signific<strong>an</strong>tly alter cellular<br />
responsiveness. Specifically while opioid peptides, such as enkephalins or <strong>the</strong> <strong>an</strong>alog D-Ala 2 , N-<br />
MePhe 4 , Gly-ol]-enkephalin (DAMGO), induce endocytosis of <strong>the</strong> MOR, morphine does not. In<br />
addition promoting endocytosis of <strong>the</strong> MOR in response to morphine prevents toler<strong>an</strong>ce <strong>an</strong>d<br />
dependence. We hypo<strong>the</strong>sized that chronic eth<strong>an</strong>ol consumption could impede <strong>the</strong> ability of<br />
MOR to endocytose in response to enkephalin <strong>the</strong>reby converting enkephalin into morphine <strong>for</strong><br />
MOR endocytosis. The present study was <strong>the</strong>n designed to test this hypo<strong>the</strong>sis. Wistar rats were<br />
given intermittent-access to 20% eth<strong>an</strong>ol (three 24-hour sessions per week: Monday, Wednesday<br />
<strong>an</strong>d Friday) <strong>an</strong>d water was always available ad libitum. The <strong>an</strong>algesic effect of intra<strong>the</strong>cal (i.t.)<br />
DAMGO was measured at 0, 4 <strong>an</strong>d 8 weeks of eth<strong>an</strong>ol drinking. The distribution of MOR on <strong>the</strong><br />
spinal cord neurons was examined using immunohistochemical staining to evaluate <strong>the</strong> effect of<br />
eth<strong>an</strong>ol drinking on <strong>the</strong> ability of DAMGO to endocytose MOR. The functional consequence of<br />
impeded MOR endocytosis was also investigated using autoradiographic opioid-activated [ 35 S]-<br />
GTPγS binding. We found that chronic eth<strong>an</strong>ol consumption profoundly reduced <strong>the</strong> <strong>an</strong>algesic<br />
effect of i.t. DAMGO after 8 weeks of eth<strong>an</strong>ol drinking compared to water drinking. More<br />
import<strong>an</strong>tly, chronic eth<strong>an</strong>ol intake signific<strong>an</strong>tly blocked <strong>the</strong> MOR endocytosis by i.t. DAMGO<br />
in <strong>the</strong> spinal cord neurons. Moreover, DAMGO- stimulated [ 35 S]-GTPγS binding was<br />
signific<strong>an</strong>tly decreased in eth<strong>an</strong>ol-drinking group. Taken toge<strong>the</strong>r, those results offered a novel<br />
insight into <strong>the</strong> mech<strong>an</strong>ism by which MOR mediates alcoholism.<br />
Disclosures: L. He, Gallo Center, UCSF, A. Employment (full or part-time); ABMRF, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); K.H. Berger, Gallo Research Center, University of Cali<strong>for</strong>nia S<strong>an</strong><br />
Fr<strong>an</strong>cisco, A. Employment (full or part-time); ABMRF, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
J.L. Whistler, Gallo Center, UCSF, A. Employment (full or part-time); NIDA, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received).<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.2/Y23
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: DOD Gr<strong>an</strong>t W81XWH-08-1-0017<br />
Cal<strong>for</strong>nia State Funds<br />
Title: Distinct mech<strong>an</strong>isms of opioid control of eth<strong>an</strong>ol consumption via mu (MOR) <strong>an</strong>d delta-1<br />
(DOR1) <strong>an</strong>d delta-2 (DOR2) opioid receptors<br />
Authors: *J. MITCHELL 1 , E. B. MARGOLIS 2 , A. R. COKER 1 , J. R. DRISCOLL 1 , H. L.<br />
FIELDS 1 ;<br />
2 Neurol., 1 UCSF: Ernest Gallo Clin. <strong>an</strong>d Res. Ctr., Emeryville, CA<br />
Abstract: The non-selective opioid <strong>an</strong>tagonist naltrexone (NTX) is <strong>an</strong> FDA approved treatment<br />
<strong>for</strong> alcohol abuse. However, NTX is aversive in rodent <strong>an</strong>d hum<strong>an</strong> models of eth<strong>an</strong>ol (EtOH)<br />
consumption (Neurobiol Dis. <strong>2009</strong> J<strong>an</strong>; 33: 72-80), thus limiting its <strong>the</strong>rapeutic efficacy. To<br />
design a more effective, less aversive treatment <strong>for</strong> alcoholism, we have examined more<br />
selective opioid lig<strong>an</strong>ds in Lewis rats using <strong>the</strong> place conditioning <strong>an</strong>d 2-bottle choice EtOH<br />
consumption paradigms. Import<strong>an</strong>tly, we have examined place conditioning behavior in actively<br />
drinking <strong>an</strong>imals, since we previously showed that delta opioid receptor (DOR) function ch<strong>an</strong>ges<br />
with prolonged EtOH consumption such that activation of DOR in <strong>the</strong> VTA reduces EtOH<br />
consumption by inhibiting GABA-A mediated inhibitory post-synaptic currents (IPSCs) in VTA<br />
neurons (J Neurosci. 2008 Nov 26; 28: 12672-81). This effect on GABA-A IPSCs is absent in<br />
EtOH naïve rats. Here we show that this ch<strong>an</strong>ge is limited to DOR1, as <strong>the</strong> effects of <strong>the</strong> DOR2<br />
agonist deltorphin are unch<strong>an</strong>ged in EtOH consuming <strong>an</strong>imals. Using in vitro electrophysiology,<br />
we found that in drinking rats deltorphin inhibits GABA-A mediated evoked IPSCs by 20+/-11%<br />
(n=15), which is not signific<strong>an</strong>tly different <strong>from</strong> EtOH naïve rats. In contrast DPDPE inhibits<br />
GABA-A IPSCs by 31+/-9% (n=11) in EtOH drinking rats <strong>an</strong>d 11+/-2% (n=3) in EtOH naïve<br />
rats. Additionally, although intra-VTA DPDPE does not induce signific<strong>an</strong>t conditioned place<br />
preference or aversion in EtOH naïve rats, it induces a signific<strong>an</strong>t preference in chronically<br />
drinking rats (n = 12; p = .03). In contrast, intra-VTA deltorphin induces signific<strong>an</strong>t place<br />
preference in naïve rats (n = 12; p = .04) <strong>an</strong>d this preference appears unaltered in chronically<br />
drinking rats. Toge<strong>the</strong>r <strong>the</strong>se data suggest that DOR1 signaling c<strong>an</strong> ch<strong>an</strong>ge independently of<br />
DOR2 <strong>an</strong>d that <strong>the</strong> aversive qualities of NTX may in part be due to blockade of both DOR1 <strong>an</strong>d<br />
DOR2 signaling during EtOH consumption. As most DOR lig<strong>an</strong>ds have yet to be tested <strong>for</strong><br />
DOR1/DOR2 selectivity, screening such compounds may identify a more specific (<strong>an</strong>d <strong>the</strong>re<strong>for</strong>e<br />
less aversive) treatment <strong>for</strong> EtOH abuse <strong>an</strong>d dependence.<br />
Disclosures: J. Mitchell, Patent, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); E.B. Margolis, Patent, E. Ownership Interest (stock, stock options, patent<br />
or o<strong>the</strong>r intellectual property); A.R. Coker, None; J.R. Driscoll, None; H.L. Fields, Patent, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.3/Y24<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t AA 013396<br />
NIH Gr<strong>an</strong>t AA 017359<br />
Title: N-methyl-D-aspartate subunit proteins are altered by repeated eth<strong>an</strong>ol exposures during rat<br />
adolescence<br />
Authors: *R. SIRCAR 1 , K. REDDY 2 , L. WU 2 , D. SIRCAR 1 , D. GETREU 2 ;<br />
1 Albert Einstein Coll Med/FIMR, M<strong>an</strong>hasset, NY; 2 Feinstein Inst. <strong>for</strong> Med. Res., M<strong>an</strong>hasset, NY<br />
Abstract: We <strong>an</strong>d o<strong>the</strong>rs have earlier reported that eth<strong>an</strong>ol exposure in adolescent rat impairs<br />
spatial learning <strong>an</strong>d memory. The N-methyl-D-aspartate receptor (NR) is considered to be <strong>an</strong><br />
import<strong>an</strong>t target <strong>for</strong> <strong>the</strong> acute <strong>an</strong>d chronic effects of eth<strong>an</strong>ol. Acute eth<strong>an</strong>ol decreases NR while<br />
chronic eth<strong>an</strong>ol treatment <strong>an</strong>d withdrawal leads to a compensatory upregulation of <strong>the</strong>se<br />
receptors. Previously, we have shown that repeated eth<strong>an</strong>ol treatments in adolescent rat increases<br />
[ 3 H]MK-801 labeled NR binding in <strong>the</strong> frontal cortex. To fur<strong>the</strong>r underst<strong>an</strong>d <strong>the</strong> mech<strong>an</strong>ism of<br />
adolescent eth<strong>an</strong>ol-induced learning deficits, we used western blot <strong>an</strong>alysis to measure NR<br />
subunit proteins (NR1, NR2A, <strong>an</strong>d NR2B) as well as <strong>the</strong> postsynaptic density protein 95 (PSD-<br />
95) in several brain areas. Adolescent male rats were administered with eth<strong>an</strong>ol on 5 consecutive<br />
days, <strong>an</strong>d surface-expressed NMDA receptor subunits <strong>an</strong>d PSD-95 levels were measured.<br />
Eth<strong>an</strong>ol signific<strong>an</strong>tly increased NR1 levels in <strong>the</strong> frontal cortex. This was accomp<strong>an</strong>ied by<br />
signific<strong>an</strong>t increases in cortical PSD-95 levels. There was a trend toward increases in <strong>the</strong> levels<br />
of NR1 <strong>an</strong>d PSD-95 in <strong>the</strong> hippocampus but <strong>the</strong> ch<strong>an</strong>ges were not statistically signific<strong>an</strong>t. NR1<br />
<strong>an</strong>d PSD-95 levels did not show <strong>an</strong>y signific<strong>an</strong>t alterations following adolescent eth<strong>an</strong>ol<br />
exposure in <strong>the</strong> o<strong>the</strong>r brain areas studied. Eth<strong>an</strong>ol did not signific<strong>an</strong>tly alter cortical NR2A <strong>an</strong>d<br />
NR2B subunit levels. Qu<strong>an</strong>tification of <strong>the</strong> western blots suggested that repeated adolescent<br />
eth<strong>an</strong>ol increased <strong>the</strong> total number of NR1 subunits <strong>an</strong>d synaptic sites in <strong>the</strong> frontal cortex. Our<br />
findings support <strong>the</strong> hypo<strong>the</strong>sis that adolescent eth<strong>an</strong>ol-induced cogntive deficits are associated<br />
with neuroadaptations in glutamatergic tr<strong>an</strong>smission particulaly in <strong>the</strong> frontal cortex. The effects<br />
of adolescent eth<strong>an</strong>ol on NR are likely to be complex <strong>an</strong>d may also involve alterations in posttr<strong>an</strong>slational<br />
adaptations (phosphorylation, internalization), <strong>an</strong>d <strong>the</strong>se may be distinct <strong>for</strong> each<br />
NR subunit <strong>an</strong>d brain region-specific.<br />
Disclosures: R. Sircar, None; K. Reddy, None; L. Wu, None; D. Sircar, None; D. Getreu,<br />
None.
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.4/Y25<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: USPHS gr<strong>an</strong>t AA-12882<br />
E.H. L<strong>an</strong>e Foundation<br />
Title: The orexigenic peptide mel<strong>an</strong>in-concentrating hormone acts in <strong>the</strong> accumbens to stimulate<br />
eth<strong>an</strong>ol intake<br />
Authors: Y.-W. CHEN 1 , S. F. LEIBOWITZ 2 , *B. G. HOEBEL, Prof. 1 ;<br />
1 Dept. Psychology, Princeton Univ., Princeton, NJ; 2 Rockfeller Univ., New York, NY<br />
Abstract: Mel<strong>an</strong>in-concentrating hormone (MCH) is <strong>an</strong> orexigenic peptide involved in<br />
regulating energy homeostasis. It is also related to behaviors that are associated with alcohol<br />
dependence, including fluid bal<strong>an</strong>ce, depression, <strong>an</strong>d <strong>an</strong>xiety. While previous research has<br />
shown that intracerebroventricular administration of MCH c<strong>an</strong> increase eth<strong>an</strong>ol intake, <strong>the</strong><br />
mech<strong>an</strong>ism <strong>an</strong>d site underlying this phenomenon remains to be elucidated. Our current study<br />
identifies <strong>the</strong> nucleus accumbens shell (NAc shell) as one critical region involved in MCHinduced<br />
eth<strong>an</strong>ol consumption. In Exp 1, male Sprague-Dawley rats were trained to drink 7%<br />
eth<strong>an</strong>ol <strong>an</strong>d impl<strong>an</strong>ted with guide shafts <strong>for</strong> c<strong>an</strong>nulas aimed at <strong>the</strong> NAc shell (AP: 1.2 mm, ML:<br />
± 0.8 mm, DV: 4.2 mm). The results show that, with eth<strong>an</strong>ol <strong>an</strong>d food available, bilateral<br />
microinjections of MCH (1µg/0.5 µl per side) signific<strong>an</strong>tly increased eth<strong>an</strong>ol intake in <strong>the</strong> first<br />
hour post-injection, <strong>from</strong> 0.19 ± 0.04 g/kg after saline to 0.34 ± 0.07g/kg after MCH (p
Disclosures: Y. Chen, None; S.F. Leibowitz, None; B.G. Hoebel, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.5/Y26<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH gr<strong>an</strong>t R01 AA11852<br />
NIH gr<strong>an</strong>t F31 AA017568<br />
Title: Dopamine in <strong>the</strong> core-shell border of <strong>the</strong> nucleus accumbens during <strong>the</strong> initiation of<br />
eth<strong>an</strong>ol self-administration<br />
Authors: J. CARRILLO 1 , *R. A. GONZALES 2 ;<br />
1 Neurosci., 2 Pharmacol., Univ. of Texas, Austin, TX<br />
Abstract: Previous studies in our lab have suggested that accumbal dopamine signaling codes<br />
<strong>for</strong> <strong>the</strong> prediction of subsequent rewarding effects of oper<strong>an</strong>t eth<strong>an</strong>ol self-administration. These<br />
<strong>for</strong>mer studies indicate that accumbal dopamine concentrations reach a peak within 5 min of<br />
drinking, but that eth<strong>an</strong>ol concentrations are maximal after dopamine levels have returned to<br />
baseline. We looked at <strong>the</strong> subregions of <strong>the</strong> NAc, <strong>an</strong>d we did not observe a dopamine response<br />
in ei<strong>the</strong>r <strong>the</strong> core or shell of <strong>the</strong> nucleus accumbens, NAc, during eth<strong>an</strong>ol self-administration.<br />
The purpose of <strong>the</strong> present study was to fur<strong>the</strong>r characterize <strong>the</strong> accumbal subregion in which <strong>the</strong><br />
dopamine signal occurs, focusing on <strong>the</strong> core-shell border, <strong>an</strong>d <strong>the</strong> time course of development<br />
of this signal. Male Long-Ev<strong>an</strong>s rats were trained to self-administer eth<strong>an</strong>ol using a protocol that<br />
gradually increases <strong>the</strong> concentration of eth<strong>an</strong>ol to 10% in <strong>the</strong> presence of 10% sucrose over a<br />
period of 7 days. Microdialysis sampling <strong>from</strong> <strong>the</strong> core-shell border was per<strong>for</strong>med on rats selfadministering<br />
10% eth<strong>an</strong>ol + 10% sucrose. There was a signific<strong>an</strong>t increase in dopamine levels<br />
within <strong>the</strong> first 5 min of eth<strong>an</strong>ol consumption (1.6 ± 0.3 g/kg), while brain eth<strong>an</strong>ol levels reached<br />
peak concentration more th<strong>an</strong> 30 min later (n=6). To test <strong>the</strong> timeline of <strong>the</strong> dopamine response<br />
in <strong>the</strong> core-shell border during eth<strong>an</strong>ol self-administration, we used a modified training protocol<br />
where we kept <strong>the</strong> eth<strong>an</strong>ol concentration (10% sucrose + 10% eth<strong>an</strong>ol) const<strong>an</strong>t during <strong>the</strong> early<br />
phase of eth<strong>an</strong>ol self-administration. Male Long-Ev<strong>an</strong>s rats were trained to bar-press <strong>for</strong> a 20<br />
min access to a 10% sucrose solution. After 4 sessions of 10% sucrose, <strong>the</strong> rats were <strong>the</strong>n<br />
exposed to two days of oper<strong>an</strong>t self-administration of 10% sucrose + 10% eth<strong>an</strong>ol. Microdialysis<br />
in <strong>the</strong> core-shell border of <strong>the</strong> NAc was per<strong>for</strong>med on <strong>the</strong> 2nd day of eth<strong>an</strong>ol self-administration.
The me<strong>an</strong> eth<strong>an</strong>ol intake of <strong>the</strong> 10% sucrose + 10% eth<strong>an</strong>ol drinking solution on day 2 (1.5 ± 0.2<br />
g/kg) when compared to day 1 was two-fold higher (n=5). We also found a signific<strong>an</strong>t increase<br />
in dopamine within <strong>the</strong> first 5 min of eth<strong>an</strong>ol consumption (p < .05). These findings suggest that<br />
dopamine in <strong>the</strong> core-shell border of <strong>the</strong> NAc may play a role in <strong>for</strong>ming associations between<br />
cues <strong>an</strong>d rein<strong>for</strong>cers during <strong>the</strong> early stages of eth<strong>an</strong>ol self-administration. It is possible that after<br />
a single exposure to eth<strong>an</strong>ol, <strong>an</strong>imals begin to associate its pharmacological effects to <strong>the</strong> cues<br />
that are present during <strong>the</strong> first 5 minutes of oper<strong>an</strong>t self-administration.<br />
Disclosures: J. Carrillo, None; R.A. Gonzales, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.6/Y27<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t AA 12262<br />
NIH Gr<strong>an</strong>t AA 10717<br />
Title: Eth<strong>an</strong>ol stimulates glutamate release in <strong>the</strong> ventral tegmental area (VTA) of Wistar rats<br />
Authors: *Z. DING, E. A. ENGLEMAN, A. D. MORAN, B. S. DEWELL, Z. GU, W. J.<br />
MCBRIDE, Z. A. RODD;<br />
IPR, Sch. Med. IU, Indi<strong>an</strong>apolis, IN<br />
Abstract: The meso-limbic system is involved in mediating <strong>the</strong> activating <strong>an</strong>d rein<strong>for</strong>cing<br />
effects of eth<strong>an</strong>ol. Glutamatergic projections to <strong>the</strong> VTA <strong>an</strong>d nucleus accumbens (NAc) are<br />
major excitatory inputs regulating <strong>the</strong> activity of <strong>the</strong> meso-limbic system. Previous findings<br />
indicated that systemic injection of eth<strong>an</strong>ol increases extracellular glutamate levels in <strong>the</strong> NAc.<br />
The objective of <strong>the</strong> present study was to examine <strong>the</strong> effect of i.p. administration of eth<strong>an</strong>ol on<br />
extracellular levels of glutamate in <strong>the</strong> VTA. Conventional microdialysis was used to collect<br />
glutamate samples <strong>from</strong> <strong>the</strong> VTA. During microdialysis, rats received i.p. injections of 0, 0.5, 1.0<br />
or 2 g/kg (n = 5-6/group) eth<strong>an</strong>ol. Glutamate was <strong>an</strong>alyzed with HPLC-EC. Basal levels of<br />
glutamate in <strong>the</strong> VTA were 4.1 ± 0.4 µM. Eth<strong>an</strong>ol produced biphasic effects with a low dose (0.5<br />
g/kg) increasing (approximately 35% above baselines) <strong>an</strong>d a high dose (2 g/kg) decreasing<br />
(approximately 20% below baselines) extracellular glutamate levels. The intermediate dose of 1<br />
g/kg did not signific<strong>an</strong>tly alter extracellular glutamate levels. Perfusion with Ca 2+ free aCSF
educed basal glutamate levels (approximately 30% below baselines) <strong>an</strong>d prevented <strong>the</strong> eth<strong>an</strong>ol<br />
(0.5 g/kg)-induced increase. Overall <strong>the</strong> data indicate that low dose eth<strong>an</strong>ol stimulates<br />
glutamatergic projections to <strong>the</strong> VTA, suggesting that VTA glutamate may be involved in <strong>the</strong><br />
activating <strong>an</strong>d rein<strong>for</strong>cing effects of eth<strong>an</strong>ol.<br />
Disclosures: Z. Ding, None; E.A. Englem<strong>an</strong>, None; A.D. Mor<strong>an</strong>, None; B.S. DeWell,<br />
None; Z. Gu, None; W.J. McBride, None; Z.A. Rodd, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.7/Y28<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: PRIN 2006 to M.D.<br />
Fondazione B<strong>an</strong>co di Sardegna to A.T.P.<br />
Title: Restraint stress prevents acetaldehyde <strong>an</strong>d eth<strong>an</strong>ol-induced increase in dopaminergic<br />
neuronal activity in <strong>the</strong> VTA of <strong>the</strong> rat<br />
Authors: M. MEREU 1 , *M. DIANA 1 , G. MUGGIRONI 1 , A. T. PEANA 1 , D. SIRCA 2 , P.<br />
ENRICO 2 , M. FODDAI 3 ;<br />
1 Univ. Sassari, Sassari, Italy; 2 Biomed. Sci., Univ. of Sassari, Sassari, Italy; 3 Psichitria,<br />
neurobiologia,Farmacologia e Biotecnologie, Univ. of Pisa, Pisa, Italy<br />
Abstract: Exposure to stress c<strong>an</strong> profoundly affect <strong>the</strong> responsiveness to drugs of abuse in both<br />
experimental <strong>an</strong>imals <strong>an</strong>d hum<strong>an</strong>s. Self-administration studies in laboratory <strong>an</strong>imals have shown<br />
that both physical <strong>an</strong>d psychological stressors, facilitate <strong>the</strong> acquisition of drug selfadministration,<br />
possibly by enh<strong>an</strong>cing <strong>the</strong> rein<strong>for</strong>cing effects of drugs of abuse (1). Eth<strong>an</strong>ol<br />
(EtOH) is after nicotine <strong>the</strong> most abused subst<strong>an</strong>ce in <strong>the</strong> Western World. Recent data confirm<br />
<strong>the</strong> relationship between stress <strong>an</strong>d EtOH abuse, <strong>an</strong>d indicate that EtOH abuse usually occurs<br />
after exposure to stress (2). EtOH's positive motivational properties have been consistently<br />
related to its ability to activate <strong>the</strong> mesolimbic dopamine (DA) system, <strong>an</strong>d new evidences show<br />
that acetaldehyde (ACD), <strong>the</strong> main metabolite of EtOH, may contribute to <strong>the</strong> rein<strong>for</strong>cing effects<br />
of EtOH trough its own rewarding properties (3,4,5,6). On <strong>the</strong>se basis, we sought to study in vivo<br />
<strong>the</strong> effect of EtOH <strong>an</strong>d ACD administration on <strong>the</strong> bioelectrical activity of mesoaccumbens DA<br />
neurons in ventral tegmental area (VTA) in control <strong>an</strong>d stressed rats. Adult male Wistar rats
(280-300 g) were exposed to 1 hour restraint stress, using a plexiglass restrainer. Thereafter<br />
subjects were <strong>an</strong>es<strong>the</strong>tized with ureth<strong>an</strong>e (1.3 g/kg, i.p.), <strong>the</strong> femoral vein was c<strong>an</strong>nulated to<br />
allow administration of EtOH (250-1000 mg/kg) <strong>an</strong>d ACD (5-20 mg/kg). Mesoaccumbens DA<br />
cells were studied using single unit extracellular recording, coupled with <strong>an</strong>tidromic<br />
identification <strong>from</strong> <strong>the</strong> nucleus accumbens shell. Both EtOH <strong>an</strong>d ACD in control <strong>an</strong>imals, dosedependently<br />
increased <strong>the</strong> firing rate of VTA DA cells. On <strong>the</strong> contrary, in stressed <strong>an</strong>imals both<br />
EtOH <strong>an</strong>d ACD effects on mesoaccumbal neuronal activity, were drastically reduced. Our result<br />
confirm that ACD by itself increases <strong>the</strong> firing of VTA DA neurons. Considering <strong>the</strong> role of DA<br />
mesolimbic system in regulating <strong>the</strong> motivational properties of abused drugs, <strong>the</strong>se data suggest<br />
that stress may alter <strong>the</strong> effects of EtOH, by influencing DA neuronal activity.<br />
1)Piazza PV, TIPS, 1998<br />
2)Zimmerm<strong>an</strong>n U, Neuropsychopharmacology, 2003<br />
3)Foddai M, Neuropsychopharmacology, 2004<br />
4)Melis M,Eur.J.Neurosci., 2007<br />
5)Enrico P,Drug Alcohol Depend., <strong>2009</strong><br />
6)Pe<strong>an</strong>a AT,Alcohol Clin.Exp.Res., 2008<br />
Disclosures: M. Mereu, None; M. Di<strong>an</strong>a, None; G. Muggironi, None; A.T. Pe<strong>an</strong>a, None; D.<br />
Sirca, None; P. Enrico, None; M. Foddai, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.8/Y29<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: F31 AA16429<br />
AA10717<br />
AA12262<br />
Title: Alcohol drinking experience increases <strong>the</strong> sensitivity of posterior ventral tegmental area<br />
(VTA) dopamine neurons to <strong>the</strong> stimulating effects of cocaine<br />
Authors: *S. M. OSTER 1 , Z.-M. DING 2 , J. TOALSTON 1 , E. ENGLEMAN 2 , J. MURPHY 1 , W.<br />
MCBRIDE 2 , Z. RODD 2 ;<br />
1 Psychology, 2 Psychiatry, IUPUI, Indi<strong>an</strong>apolis, IN
Abstract: Poly-drug abuse may be promoted by ch<strong>an</strong>ges in neuronal functioning associated with<br />
previous drug or alcohol use. The current study was conducted to determine <strong>the</strong> effects of<br />
chronic eth<strong>an</strong>ol (EtOH) drinking on <strong>the</strong> sensitivity of <strong>the</strong> posterior VTA dopamine (DA) neurons<br />
to <strong>the</strong> local stimulating effects of cocaine (Coc). Previous research <strong>from</strong> our laboratory indicated<br />
that Coc produced activating effects in <strong>the</strong> posterior, but not <strong>an</strong>terior, VTA. Adult female<br />
Alcohol-Preferring (P) rats were allowed to consume EtOH <strong>for</strong> at least 12 weeks (concurrent<br />
access to water <strong>an</strong>d 15% EtOH) or only water. Beginning 1 week prior to surgery, EtOH bottles<br />
were removed <strong>for</strong> 4 hr each day giving <strong>the</strong> <strong>an</strong>imals neglible blood EtOH levels <strong>for</strong> surgery <strong>an</strong>d<br />
microdialysis. P rats were stereotaxically impl<strong>an</strong>ted with ipsilateral guide c<strong>an</strong>nulae aimed at <strong>the</strong><br />
posterior VTA <strong>an</strong>d <strong>the</strong> nucleus accumbens (Acb). A loop-style microdialysis probe was inserted<br />
into <strong>the</strong> Acb 6 days later. The following day, <strong>an</strong>imals were attached to microdialysis lines <strong>an</strong>d a<br />
microinjector was inserted into <strong>the</strong> posterior VTA <strong>for</strong> local administration of 25 or 50 pmol per<br />
100 nl of Coc, or aCSF. EtOH intakes exceeded 6 g/kg/day <strong>for</strong> <strong>the</strong> experimental group. In EtOHnaïve<br />
rats, microinjections of 25 pmol/100nl Coc slightly increased (122% of baseline)<br />
extracellular DA levels in <strong>the</strong> Acb; 50 pmol/100 nl Coc signific<strong>an</strong>tly increased DA release<br />
(175% of baseline). In <strong>the</strong> chronic EtOH drinking group, <strong>the</strong> 25 pmol/100 nl dose increased DA<br />
release to 178% of baseline, <strong>an</strong>d <strong>the</strong> 50 pmol/100 nl dose increased DA release to 340% of<br />
baseline. The results of this study suggest that chronic EtOH drinking by P rats c<strong>an</strong> produce<br />
neuronal alterations that increase <strong>the</strong> sensitivity of <strong>the</strong> posterior VTA to <strong>the</strong> activating effects of<br />
Coc, which may enh<strong>an</strong>ce <strong>the</strong> rein<strong>for</strong>cing properties of Coc.<br />
Disclosures: S.M. Oster, None; Z. Ding, None; J. Toalston, None; E. Englem<strong>an</strong>, None; J.<br />
Murphy, None; W. McBride, None; Z. Rodd, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.9/Y30<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH PHS gr<strong>an</strong>t AA13666<br />
Kore<strong>an</strong> Government (KRF-2008-313-E00710)<br />
Title: Acupuncture suppresses eth<strong>an</strong>ol inhibition of GABA neuron activity in <strong>the</strong> ventral<br />
tegmental area <strong>an</strong>d eth<strong>an</strong>ol self-administration via mu-opioid receptors
Authors: S. YOON 1 , S. C. STEFFENSEN 2 , D. M. HANSEN 2 , B. R. BLUMELL 2 , J. J. PARK 2 ,<br />
*C. YANG 1 ;<br />
1 Daegu Ha<strong>an</strong>y Univ., Daegu, Republic of Korea; 2 Brigham Young Univ., Provo, UT<br />
Abstract: Withdrawal <strong>from</strong> chronic eth<strong>an</strong>ol enh<strong>an</strong>ces ventral tegmental area (VTA) GABA<br />
neuron excitability <strong>an</strong>d reduces mesolimbic dopamine (DA) neurotr<strong>an</strong>smission, which is<br />
suppressed by acupuncture at Shenmen (HT7) points (Yoon et al., 2004). In <strong>the</strong> first experiment,<br />
using electrophysiological methods in mature Wistar rats, we evaluated <strong>the</strong> effects of HT7<br />
electrical stimulation on VTA GABA neuron firing rate. Compared to tail stimulation at current<br />
threshold <strong>for</strong> muscle twitch, 2 Hz stimulation of HT7 had mixed effects on VTA GABA neuron<br />
activity, but signific<strong>an</strong>tly reduced eth<strong>an</strong>ol suppression of VTA GABA neuron firing rate. As<br />
VTA GABA neurons express mu-opioid receptors, we evaluated <strong>the</strong> effects of <strong>the</strong> mu-opioid<br />
<strong>an</strong>tagonist naloxone on HT7 stimulation. Intravenous administration of naloxone abolished HT7<br />
block of eth<strong>an</strong>ol suppression of VTA GABA neuron firing rate. In <strong>the</strong> second experiment, we<br />
examined <strong>the</strong> effect of acupuncture at HT7 on eth<strong>an</strong>ol-rein<strong>for</strong>ced behavior using <strong>an</strong> oral selfadministration<br />
paradigm. Acupuncture at HT7, but not tail, signific<strong>an</strong>tly decreased eth<strong>an</strong>olrein<strong>for</strong>ced<br />
responding. Additionally, intraperitoneal injection of morphine blocked <strong>the</strong> inhibition<br />
of eth<strong>an</strong>ol self-administration by acupuncture. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that<br />
acupuncture modulates mesolimbic DA release <strong>an</strong>d suppresses <strong>the</strong> rein<strong>for</strong>cing effects of eth<strong>an</strong>ol<br />
via opioid modulation of VTA GABA neurons.<br />
Disclosures: S. Yoon, None; S.C. Steffensen, None; D.M. H<strong>an</strong>sen, None; B.R. Blumell,<br />
None; J.J. Park, None; C. Y<strong>an</strong>g, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.10/Y31<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: Public Health Service Gr<strong>an</strong>ts P20 RR17701<br />
NARSAD Young Investigator Award<br />
The Intramural Research Support gr<strong>an</strong>t <strong>from</strong> The University of Mississippi Medical<br />
Center (Jackson, MS)
Title: Eth<strong>an</strong>ol increases GAPDH-TIEG2-MAO B cell death cascade in <strong>the</strong> prefrontal cortex of<br />
eth<strong>an</strong>ol-preferring rats<br />
Authors: *X. OU 1 , C. JOHNSON 1 , D. LU 1 , I. A. PAUL 1 , M. C. AUSTIN 1 , A. H. IYO 1 , J. J.<br />
MIGUEL-HIDALGO 1 , J. WANG 2 , D. B. SITTMAN 3 ;<br />
1 2 3<br />
Dept Psychiatry & Hum<strong>an</strong> Behavior, Dept Pathology, Dept Biochem., Univ. Mississippi Med.<br />
Ctr., Jackson, MS<br />
Abstract: Long-term, heavy eth<strong>an</strong>ol drinking clearly affects <strong>the</strong> brain. Although brain cell loss<br />
has been reported in subjects with alcoholism, <strong>the</strong> molecular mech<strong>an</strong>isms that account <strong>for</strong> this<br />
loss is unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) <strong>an</strong>d monoamine oxidase<br />
B (MAO B) reportedly play a role in cellular dysfunction under stressful conditions. We have<br />
recently reported that both GAPDH <strong>an</strong>d MAO B proteins are increased in <strong>the</strong> brain-derived cell<br />
lines upon eth<strong>an</strong>ol treatment (Ou et al., <strong>2009</strong>, Neurotox Res). GAPDH interacts with <strong>the</strong><br />
tr<strong>an</strong>scriptional activator, tr<strong>an</strong>s<strong>for</strong>ming growth factor-beta-inducible early gene 2 (TIEG2) to<br />
augment TIEG2-mediated MAO B activation, resulting in neuronal cell damage as a result of<br />
exposure to eth<strong>an</strong>ol. The current study investigates whe<strong>the</strong>r <strong>the</strong> GAPDH-TIEG2-MAO B<br />
cascade is also active in rats that are treated with eth<strong>an</strong>ol.<br />
Ten male eth<strong>an</strong>ol-preferring Wistar rats were fed with a liquid diet containing eth<strong>an</strong>ol, which<br />
contained increasing amounts of eth<strong>an</strong>ol until a final diet containing 36% of calories <strong>from</strong><br />
eth<strong>an</strong>ol (6.4% eth<strong>an</strong>ol) was attained <strong>for</strong> a total of 28 days. Ten control rats were pair fed <strong>the</strong><br />
liquid diet without eth<strong>an</strong>ol. The expression of GAPDH protein, TIEG2 protein <strong>an</strong>d <strong>the</strong> MAO B<br />
mRNA <strong>an</strong>d catalytic activity were determined in <strong>the</strong> prefrontal cortex of <strong>the</strong> rats.<br />
Eth<strong>an</strong>ol signific<strong>an</strong>tly increased protein levels of GAPDH, TIEG2 <strong>an</strong>d MAO B mRNA <strong>an</strong>d<br />
enzyme activity in <strong>the</strong> prefrontal cortex compared to control rats.<br />
Eth<strong>an</strong>ol increases GAPDH-TIEG2-MAO B brain cell death cascade in <strong>the</strong> rat brain just as it does<br />
in <strong>the</strong> brain-derived cell lines exposed to eth<strong>an</strong>ol, suggesting that <strong>the</strong> GAPDH-TIEG2-MAO Bmediated<br />
signaling pathway may contribute to chronic alcohol-induced brain tissue damage.<br />
Disclosures: X. Ou, None; C. Johnson, None; D. Lu, None; I.A. Paul, None; M.C. Austin,<br />
None; A.H. Iyo, None; J.J. Miguel-Hidalgo, None; J. W<strong>an</strong>g, None; D.B. Sittm<strong>an</strong>, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.11/Y32<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms
Support: NIH Gr<strong>an</strong>t AA010983<br />
Title: Chronic eth<strong>an</strong>ol reduces SK ch<strong>an</strong>nel conduct<strong>an</strong>ce <strong>an</strong>d surface expression in hippocampus<br />
Authors: *P. J. MULHOLLAND, J. J. WOODWARD, H. C. BECKER, L. J. CHANDLER;<br />
Neurosciences, Med. Univ. South Carolina, Charleston, SC<br />
Abstract: Small-conduct<strong>an</strong>ce calcium-activated K+ (SK2) ch<strong>an</strong>nels localize to <strong>the</strong> postsynaptic<br />
density of dendritic spines in hippocampus <strong>an</strong>d amygdala where <strong>the</strong>y <strong>for</strong>m a calcium-mediated<br />
negative feedback loop with synaptic NMDA receptors. Activation of this feedback loop is<br />
thought to regulate spine calcium tr<strong>an</strong>sients <strong>an</strong>d synaptic depolarizations, <strong>an</strong>d recent evidence<br />
suggests that SK2 ch<strong>an</strong>nels critically modulate NMDA receptor-dependent synaptic plasticity in<br />
hippocampus. We have previously demonstrated that chronic eth<strong>an</strong>ol produces <strong>an</strong> activitydependent<br />
homeostatic targeting of NMDA receptors to synaptic sites. Because SK2 ch<strong>an</strong>nels<br />
undergo activity-dependent internalization that contributes to <strong>the</strong> increased excitatory<br />
postsynaptic potentials underlying long-term potentiation, we hypo<strong>the</strong>sized that chronic eth<strong>an</strong>ol<br />
exposure would reduce surface SK2 expression as part of a homeostatic response that would lead<br />
to impairment of <strong>the</strong> SK2 ch<strong>an</strong>nel-NMDA receptor feedback loop. Moreover, disruption of this<br />
feedback loop may contribute to <strong>the</strong> development of toler<strong>an</strong>ce to eth<strong>an</strong>ol <strong>an</strong>d to hyperexcitability<br />
during eth<strong>an</strong>ol withdrawal. We found that chronic eth<strong>an</strong>ol treatment of org<strong>an</strong>otypic hippocampal<br />
slices signific<strong>an</strong>tly reduces surface expression of SK2 ch<strong>an</strong>nels <strong>an</strong>d peak amplitude of apaminsensitive<br />
SK tail currents recorded in CA1 neurons. Activating SK ch<strong>an</strong>nels by bath application<br />
of <strong>the</strong> positive modulator 1-EBIO (400 uM) markedly attenuates acute eth<strong>an</strong>ol withdrawal<br />
hyperexcitability in CA1 neurons. We next examined SK2 ch<strong>an</strong>nel expression using <strong>an</strong> in vivo<br />
mouse model of chronic eth<strong>an</strong>ol inhalation that produces toler<strong>an</strong>ce to eth<strong>an</strong>ol <strong>an</strong>d eth<strong>an</strong>ol<br />
dependence. Chronic eth<strong>an</strong>ol inhalation produces a signific<strong>an</strong>t reduction in SK2 ch<strong>an</strong>nel<br />
expression in hippocampal synaptosomes, <strong>an</strong>d i.p. injection of 1-EBIO (10 mg/kg) at 4 <strong>an</strong>d 8 h<br />
after <strong>the</strong> removal <strong>from</strong> eth<strong>an</strong>ol vapor inhalation signific<strong>an</strong>tly reduces h<strong>an</strong>dling-induced<br />
convulsion scores in mice undergoing acute eth<strong>an</strong>ol withdrawal. These data suggest that<br />
decreases in SK2 ch<strong>an</strong>nels <strong>an</strong>d increases in synaptic NMDA receptors represent a common<br />
homeostatic adaptive response to prolonged reductions in NMDA receptor activity during<br />
chronic eth<strong>an</strong>ol exposure. We fur<strong>the</strong>r suggest this represents a functional uncoupling of <strong>the</strong> SK2<br />
ch<strong>an</strong>nel-NMDA receptor calcium-dependent feedback loop in synapses that may contribute to<br />
toler<strong>an</strong>ce development <strong>an</strong>d to hyperexcitability during eth<strong>an</strong>ol withdrawal. These data on SK2<br />
ch<strong>an</strong>nels provide evidence <strong>for</strong> novel <strong>the</strong>rapeutic targets <strong>for</strong> <strong>the</strong> control of eth<strong>an</strong>ol withdrawal<br />
hyperexcitability <strong>an</strong>d possibly relapse.<br />
Disclosures: P.J. Mulholl<strong>an</strong>d, None; J.J. Woodward, None; H.C. Becker, None; L.J.<br />
Ch<strong>an</strong>dler, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.12/Y33<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: VA Merit Review<br />
NIH AA015086<br />
NIH AA016548<br />
Title: Neural mech<strong>an</strong>isms <strong>for</strong> cross-toler<strong>an</strong>ce between nicotine <strong>an</strong>d alcohol<br />
Authors: *W. R. PROCTOR, P. H. WU;<br />
Psychiatry, VA ECHCS & Univ. Colo Denver, Aurora, CO<br />
Abstract: Acute alcohol intake produces impairment of cognition <strong>an</strong>d/or motor function, <strong>an</strong>d<br />
chronic consumption has been shown to produce toler<strong>an</strong>ce to <strong>the</strong>se effects. Acute administration<br />
of nicotine (smoking) produces cognitive enh<strong>an</strong>cement, <strong>an</strong>d chronic exposure to nicotine<br />
produces toler<strong>an</strong>ce to <strong>the</strong>se effects. In addition, chronic treatment also produces cross-toler<strong>an</strong>ce<br />
to <strong>the</strong> effects of alcohol. O<strong>the</strong>r reports show that alcohol <strong>an</strong>d nicotine are often used <strong>an</strong>d abused<br />
toge<strong>the</strong>r, raising <strong>the</strong> possibility that <strong>the</strong>se two drugs have common sites of action. We<br />
hypo<strong>the</strong>size that brain glutamatergic <strong>an</strong>d GABAergic neurotr<strong>an</strong>smitter systems are common loci<br />
where alcohol <strong>an</strong>d nicotine exert <strong>the</strong>ir actions <strong>an</strong>d are likely interactive sites where toler<strong>an</strong>ce <strong>an</strong>d<br />
cross-toler<strong>an</strong>ce c<strong>an</strong> occur. The acute effects of alcohol <strong>an</strong>d/or nicotine were measured using<br />
whole cell recording <strong>from</strong> rodent pyramidal neurons in hippocampal brain slices.<br />
Pharmacologically isolated NMDA, <strong>an</strong>d GABAA currents were voltage clamped near <strong>the</strong>ir<br />
resting potentials (-60 to -55 mV), <strong>an</strong>d after steady baseline measurements (5-10 min), alcohol<br />
(40-120 mM) <strong>an</strong>d/or nicotine (50-200 nM) were perfused via bath application. For chronic<br />
treatment, alcohol (6%) or nicotine (70 µg/ml) was added to a liquid diet <strong>for</strong> two weeks. Acute<br />
alcohol application inhibits synaptic NMDA currents <strong>an</strong>d enh<strong>an</strong>ces GABAA currents in control<br />
<strong>an</strong>imals, but <strong>an</strong> acute drug challenge in chronically treated <strong>an</strong>imals resulted in diminished<br />
NMDA inhibition while GABAA enh<strong>an</strong>cement is unaltered. Acute nicotine enh<strong>an</strong>ces NMDA<br />
currents, but this enh<strong>an</strong>cement is not observed in chronic nicotine-treated <strong>an</strong>imals. However,<br />
chronic nicotine treatment results in diminished effects of <strong>an</strong> acute alcohol challenge on NMDA<br />
currents, but no ch<strong>an</strong>ge was observed on GABAA currents by this treatment paradigm. These<br />
results suggest that <strong>the</strong> NMDA receptor is a locus <strong>for</strong> <strong>the</strong> action <strong>an</strong>d interaction between nicotine<br />
<strong>an</strong>d alcohol. The development of toler<strong>an</strong>ce <strong>an</strong>d cross-toler<strong>an</strong>ce between nicotine <strong>an</strong>d alcohol<br />
may underlie etiology of concurrent abuse of nicotine <strong>an</strong>d alcohol.<br />
Disclosures: W.R. Proctor, None; P.H. Wu, None.
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.13/Y34<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t AA016959<br />
Title: Partial activation of microglia in a rodent model of <strong>an</strong> alcohol use disorder<br />
Authors: S. A. MARSHALL, M. L. KELSO, *K. NIXON;<br />
Dept Pharmaceut. Sci., Univ. of Kentucky, Lexington, KY<br />
Abstract: Excessive alcohol intake, characteristic of <strong>an</strong> Alcohol Use Disorder (AUD), results in<br />
neurodegeneration <strong>an</strong>d cognitive deficits that may recover in abstinence. Hippocampal recovery<br />
may be due to <strong>an</strong> increase in adult neurogenesis in abstinence, but <strong>the</strong> mech<strong>an</strong>isms that cause this<br />
proliferative burst or alcohol effects on <strong>the</strong> neurogenic niche are not well understood. Recent<br />
discoveries suggest that low-grade, reactive microglia phenotypes or “partial activation” may<br />
promote neurogenesis after damage. There<strong>for</strong>e, this study examines <strong>the</strong> phenotype of microglia<br />
in <strong>the</strong> hippocampus in a rodent model of <strong>an</strong> AUD. Adult male Sprague-Dawley rats were divided<br />
into two groups receiving ei<strong>the</strong>r <strong>an</strong> eth<strong>an</strong>ol or control diet via intragastric gavage every 8 hours<br />
<strong>for</strong> 4 days. The initial dose each rat received was 5g/kg, but subsequent doses were adapted<br />
based on <strong>the</strong>ir level of intoxication. Rats were scored <strong>for</strong> withdrawal severity between 10 <strong>an</strong>d 26<br />
h after <strong>the</strong>ir last dose. In abstinence, 2 days (T48) or 7 days (T168) after <strong>the</strong>ir last dose, brains<br />
were collected <strong>for</strong> immunohistochemical studies or <strong>for</strong> ELISA assays following st<strong>an</strong>dard<br />
procedures. These time points correspond to our previous observations of reactive microgliosis<br />
(T48, microglia proliferation) <strong>an</strong>d increased neurogenesis (T168). Intoxication parameters were<br />
similar between both time points as evident by similar blood eth<strong>an</strong>ol concentrations determined<br />
<strong>from</strong> tail bloods on day 2 of <strong>the</strong> binge <strong>for</strong> <strong>the</strong> T48 (411.3±38.4 mg/dl) <strong>an</strong>d T168 (360.0±41.9)<br />
eth<strong>an</strong>ol treated groups. Fur<strong>the</strong>r, withdrawal behavior, including seizures was similar between<br />
eth<strong>an</strong>ol-treated groups. We have previously reported that no ED1-positive (ED1+) cells were<br />
visible within <strong>the</strong> brain parenchyma at T48 or T168 following binge eth<strong>an</strong>ol exposure. In this<br />
study we replicated those reports <strong>an</strong>d examined whe<strong>the</strong>r <strong>an</strong> immune response marker OX6<br />
(MHC II) was expressed. Similar to ED1, little to no OX6-positive cells were observed in <strong>the</strong><br />
brain parenchyma of <strong>the</strong> hippocampal dentate gyrus at ei<strong>the</strong>r time point. In order to examine <strong>the</strong><br />
functional phenotype of microglia, we examined TNF-alpha (TNFa) protein levels, a proinflammatory<br />
cytokine, in brain tissue homogenates via ELISA at T48 <strong>an</strong>d T168. TNFa levels<br />
were not different between <strong>an</strong>y groups (T48Controls: 38.9± 5.28pg/ml, T48EtOH:<br />
40.9±7.98pg/ml, T168Controls: 42.9±8.58pg/ml, T168EtOH: 45.6±10.8pg/ml). The lack of<br />
increase in TNFa expression coupled with a lack of phagocytic microglia (ED1) or MHC II-
expressing microglia (OX6) observed at ei<strong>the</strong>r time point support that only partial activation is<br />
occurring at <strong>the</strong>se time points following alcohol dependence.<br />
Disclosures: S.A. Marshall, None; M.L. Kelso, None; K. Nixon, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.14/Y35<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: AACP NIP<br />
Title: Effects of cytisine <strong>an</strong>d lobeline on midbrain dopamine in eth<strong>an</strong>ol pretreated mice<br />
Authors: *S. RAHMAN, R. SAJJA;<br />
Pharmaceut. Sci., South Dakota State Univ., Brookings, SD<br />
Abstract: The midbrain dopamine system has been implicated in <strong>the</strong> rewarding effects of m<strong>an</strong>y<br />
drugs of addiction, including alcohol (eth<strong>an</strong>ol) <strong>an</strong>d nicotine. Given <strong>the</strong> expression of nicotinic<br />
acetylcholine receptors (nAChRs) in <strong>the</strong> midbrain regions, it is likely that <strong>the</strong> development of<br />
compounds that target brain nAChRs <strong>an</strong>d regulate midbrain dopamine function may offer new<br />
pharmaco<strong>the</strong>rapeutic strategies <strong>for</strong> eth<strong>an</strong>ol dependence. Previous studies have shown that acute<br />
injections of cytisine <strong>an</strong>d lobeline, a partial agonist <strong>an</strong>d a putative <strong>an</strong>tagonist at nAChRs,<br />
respectively, attenuate eth<strong>an</strong>ol drinking behavior in <strong>an</strong>imal models. The present study examined<br />
<strong>the</strong> effects of cytisine <strong>an</strong>d lobeline on acute eth<strong>an</strong>ol-induced ch<strong>an</strong>ges in midbrain dopamine (DA)<br />
<strong>an</strong>d dihydroxyphenylacetic acid (DOPAC) content in C57BL/6J mice. Animals were killed 30<br />
min after acute eth<strong>an</strong>ol (2.5 g/kg) to determine <strong>the</strong> brain tissue content of DA <strong>an</strong>d DOPAC by<br />
high per<strong>for</strong>m<strong>an</strong>ce liquid chromatography coupled with electrochemical detection. Acute eth<strong>an</strong>ol<br />
injection produced <strong>an</strong> increase in both DA <strong>an</strong>d DOPAC content in <strong>the</strong> ventral striatum (VS).<br />
Pretreatment (30 min prior to eth<strong>an</strong>ol injection) with cytisine (3 mg/kg, sc) signific<strong>an</strong>tly reduced<br />
acute eth<strong>an</strong>ol-induced DA <strong>an</strong>d DOPAC content in <strong>the</strong> VS. Similarly, pretreatment (30 min prior<br />
to eth<strong>an</strong>ol injection) with lobeline (10 mg/kg, sc) signific<strong>an</strong>tly attenuated acute eth<strong>an</strong>ol-induced<br />
DA <strong>an</strong>d DOPAC content in <strong>the</strong> VS. The eth<strong>an</strong>ol-induced DA <strong>an</strong>d DOPAC content in <strong>the</strong> VS<br />
were signific<strong>an</strong>tly reduced by pretreatment with <strong>the</strong> centrally acting non-selective nAChR<br />
<strong>an</strong>tagonist mecamylamine (3 mg/kg, sc). Overall, <strong>the</strong>se results indicate that acute systemically<br />
administered eth<strong>an</strong>ol produced <strong>an</strong> increase in <strong>the</strong> content of DA <strong>an</strong>d DOPAC in VS.<br />
Pretreatment of cytisine <strong>an</strong>d lobeline regulates eth<strong>an</strong>ol-induced DA <strong>an</strong>d DOPAC in this critical
eward relev<strong>an</strong>t region by targeting nAChRs. These findings provide fur<strong>the</strong>r evidence that<br />
nAChRs could be promising targets <strong>for</strong> pharmaco<strong>the</strong>rapy of alcohol dependence. (Supported by<br />
AACP NIP <strong>an</strong>d SD 2010 Res gr<strong>an</strong>t).<br />
Disclosures: S. Rahm<strong>an</strong>, None; R. Sajja, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.15/Z1<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH gr<strong>an</strong>t AA017803<br />
NIH gr<strong>an</strong>t AA013573<br />
NIH gr<strong>an</strong>t AA015148<br />
NIH gr<strong>an</strong>t AA014983<br />
Department of Defense gr<strong>an</strong>t W81XWH-06-1-0158<br />
Title: Activation of central corticotropin-releasing factor type 2 receptors (CRF2R) attenuates<br />
high levels of eth<strong>an</strong>ol consumption associated with “drinking in <strong>the</strong> dark” procedures<br />
Authors: *E. G. LOWERY, M. NAVARRO, A. LYONS, T. THIELE;<br />
Univ. North Carolina, Chapel Hill, NC<br />
Abstract: Binge drinking is a major health concern in <strong>the</strong> United States <strong>an</strong>d worldwide, <strong>an</strong>d has<br />
been linked to <strong>the</strong> development of eth<strong>an</strong>ol dependence. Binge-like eth<strong>an</strong>ol consumption c<strong>an</strong> be<br />
modeled in <strong>an</strong>imals using <strong>the</strong> drinking-in-<strong>the</strong>-dark (DID) procedure, a paradigm which reliably<br />
elicits high voluntary eth<strong>an</strong>ol consumption <strong>an</strong>d pharmacologically relev<strong>an</strong>t blood eth<strong>an</strong>ol<br />
concentrations (BEC). Several neurochemical systems appear to be involved in maintaining<br />
binge-like eth<strong>an</strong>ol consumption observed in <strong>the</strong> DID paradigm, including corticotropin-releasing<br />
factor (CRF). We have recently found evidence suggesting that binge-like eth<strong>an</strong>ol consumption<br />
is modulated by central, extrahypothalamic CRF signaling through <strong>the</strong> CRF1R. However, <strong>the</strong><br />
role of <strong>the</strong> CRF2R in this behavior remains unclear. There<strong>for</strong>e, <strong>the</strong> current experiments used DID<br />
procedures to fur<strong>the</strong>r characterize <strong>the</strong> role of CRF receptor signaling in binge-like eth<strong>an</strong>ol
consumption by C57Bl/6J mice. The role of central CRF2R signaling was assessed with<br />
intracerebroventricular (i.c.v.) infusion of <strong>the</strong> selective CRF2R agonist, urocortin III (U3; 0,<br />
0.05, 0.1, or 0.5 µg/1 µl). Results revealed that pretreatment with a 0.1 or a 0.5 µg dose of U3<br />
signific<strong>an</strong>tly attenuated DID consumption of a 20% eth<strong>an</strong>ol solution relative to vehicle<br />
treatment, but did not signific<strong>an</strong>tly alter 10% sucrose consumption. These results suggest that<br />
central CRF2R signaling modulates binge-like eth<strong>an</strong>ol intake in mice. Experiments are currently<br />
being conducted to fur<strong>the</strong>r characterize <strong>the</strong> role of CRF2R signaling in binge-like eth<strong>an</strong>ol<br />
consumption using immunohistochemistry procedures.<br />
Disclosures: E.G. Lowery, None; M. Navarro, None; A. Lyons, None; T. Thiele, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.16/Z2<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: USPHS gr<strong>an</strong>t AA-12882<br />
Title: Acute <strong>an</strong>d chronic eth<strong>an</strong>ol intake differentially affect peri<strong>for</strong>nical lateral hypothalamic<br />
peptide expression<br />
Authors: *I. YAROSLAVSKY, G.-Q. CHANG, Z. Y. YE, O. KARATAYEV, S. F.<br />
LEIBOWITZ;<br />
Behavioral Neurobio., Rockefeller Univ., New York, NY<br />
Abstract: Recent studies <strong>from</strong> our laboratory support <strong>the</strong> involvement of hypothalamic<br />
orexigenic peptides in eth<strong>an</strong>ol intake. In rats, gal<strong>an</strong>in injection in <strong>the</strong> hypothalamic<br />
paraventricular nucleus (PVN) stimulates eth<strong>an</strong>ol consumption, <strong>an</strong>d both acute <strong>an</strong>d chronic<br />
eth<strong>an</strong>ol exposure increases gal<strong>an</strong>in gene expression in <strong>the</strong> PVN, suggesting a positive feedback<br />
relationship between eth<strong>an</strong>ol intake <strong>an</strong>d peptides in this hypothalamic nucleus. Ano<strong>the</strong>r<br />
hypothalamic region intimately involved in feeding, <strong>the</strong> peri<strong>for</strong>nical lateral hypothalamus (PLH),<br />
contains a dense collection of orexin <strong>an</strong>d mel<strong>an</strong>in-concentrating hormone (MCH) producing<br />
cells. Recent studies suggest that <strong>the</strong> orexin <strong>an</strong>d MCH systems are involved in <strong>the</strong> rewarding<br />
aspects of eth<strong>an</strong>ol intake, although <strong>the</strong>re is little evidence showing <strong>the</strong> effects that eth<strong>an</strong>ol, in<br />
turn, has on <strong>the</strong>se peptide systems. There<strong>for</strong>e, <strong>the</strong> current study was designed to measure orexin<br />
<strong>an</strong>d MCH peptide expression following acute or chronic eth<strong>an</strong>ol intake. In experiment 1, rats<br />
were trained to voluntarily consume a 2% or 9% eth<strong>an</strong>ol solution. Real-time qu<strong>an</strong>titative
polymerase chain reaction (qPCR) <strong>an</strong>alysis showed chronic daily intake of eth<strong>an</strong>ol to reduce<br />
orexin <strong>an</strong>d MCH mRNA in <strong>the</strong> PLH, with this effect more pronounced in <strong>an</strong>imals consuming a<br />
greater amount (2.5g/kg/day) compared to a lower amount (0.75g/kg/day) of eth<strong>an</strong>ol. In<br />
experiment 2, <strong>an</strong>imals received <strong>an</strong> acute gavage of water, 0.75g/kg or 2.5g/kg eth<strong>an</strong>ol. <strong>When</strong><br />
compared to <strong>the</strong> water group, acute eth<strong>an</strong>ol enh<strong>an</strong>ced peptide expression in <strong>the</strong> PLH, with orexin<br />
mRNA increased at <strong>the</strong> lower eth<strong>an</strong>ol dose (0.75g/kg) <strong>an</strong>d MCH mRNA enh<strong>an</strong>ced at <strong>the</strong> higher<br />
dose (2.5g/kg). These acute effects were measured with qPCR <strong>an</strong>d confirmed with radiolabeled<br />
in situ hybridization. Fur<strong>the</strong>r <strong>an</strong>alyses were per<strong>for</strong>med with digoxigenin-labeled in situ<br />
hybridization to better distinguish mRNA expression in <strong>the</strong> peri<strong>for</strong>nical (PF) versus lateral<br />
hypothalamic (LH) regions. The low dose of eth<strong>an</strong>ol (0.75g/kg) increased <strong>the</strong> density of orexinexpressing<br />
cells in <strong>the</strong> LH but not <strong>the</strong> PF region. The increased density of MCH-expressing cells<br />
was also evident only in <strong>the</strong> LH, <strong>an</strong>d it was seen only at <strong>the</strong> higher dose of eth<strong>an</strong>ol (2.5g/kg),<br />
suggesting that orexin neurons in this area are more sensitive to <strong>the</strong> effects of acute eth<strong>an</strong>ol.<br />
Based on <strong>the</strong>se results, we propose that <strong>the</strong> initial increase in orexin <strong>an</strong>d MCH expression<br />
observed with acute oral administration of eth<strong>an</strong>ol triggers central reward mech<strong>an</strong>isms that<br />
encourage fur<strong>the</strong>r eth<strong>an</strong>ol consumption. With chronic eth<strong>an</strong>ol intake, this shifts to a reduction in<br />
peptide mRNA expression, possibly reflecting a negative feedback system regulating <strong>the</strong>se<br />
peptide systems in response to eth<strong>an</strong>ol intake.<br />
Disclosures: I. Yaroslavsky, None; G. Ch<strong>an</strong>g, None; Z.Y. Ye, None; O. Karatayev,<br />
None; S.F. Leibowitz, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.17/Z3<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: USPHS gr<strong>an</strong>t AA-12882<br />
Title: Effect of chronic eth<strong>an</strong>ol on enkephalin expression in <strong>the</strong> hypothalamus <strong>an</strong>d extrahypothalamic<br />
areas<br />
Authors: *J. R. BARSON 1,2 , G.-Q. CHANG 2 , O. KARATAYEV 2 , B. G. HOEBEL 1 , S. F.<br />
LEIBOWITZ 2 ;<br />
1 2<br />
Psychol Prog Neurosci, Princeton Univ., Princeton, NJ; Behavioral Neurobio., The Rockefeller<br />
Univ., New York, NY
Abstract: Eth<strong>an</strong>ol may be consumed <strong>for</strong> reasons such as reward, <strong>an</strong>xiety reduction, or caloric<br />
content, <strong>an</strong>d <strong>the</strong> opioid enkephalin (ENK) appears to be involved in m<strong>an</strong>y of <strong>the</strong>se functions.<br />
Previous studies have demonstrated that ENK in <strong>the</strong> hypothalamic paraventricular nucleus<br />
(PVN) is stimulated by chronic consumption of eth<strong>an</strong>ol, suggesting that this peptide may be<br />
involved in promoting <strong>the</strong> drinking of eth<strong>an</strong>ol. This idea is supported by our recent studies<br />
showing a stimulatory effect of PVN injections of ENK <strong>an</strong>alogues on eth<strong>an</strong>ol intake in Sprague-<br />
Dawley rats. In <strong>the</strong> present study, to broaden our underst<strong>an</strong>ding of <strong>the</strong> function of this peptide,<br />
we measured ENK gene expression in additional brain areas in rats voluntarily drinking eth<strong>an</strong>ol<br />
over several weeks. These areas were <strong>the</strong> ventral tegmental area (VTA), nucleus accumbens shell<br />
(NAcSh) <strong>an</strong>d core (NAcC), central nucleus of <strong>the</strong> amygdala (CeA), <strong>an</strong>d <strong>the</strong> medial prefrontal<br />
cortex (mPFC). Rats drinking water, 1 g/kg/day eth<strong>an</strong>ol, or 3 g/kg/day eth<strong>an</strong>ol were sacrificed,<br />
<strong>an</strong>d <strong>the</strong>ir brains were prepared <strong>for</strong> <strong>an</strong>alysis using real-time qu<strong>an</strong>titative polymerase chain<br />
reaction (qPCR). In rats chronically drinking eth<strong>an</strong>ol, levels of ENK mRNA were found to be<br />
stimulated not only in <strong>the</strong> PVN but also in <strong>the</strong> VTA, NAcSh, NAcC <strong>an</strong>d mPFC, although not <strong>the</strong><br />
CeA. In a second experiment, rats chronically drinking water or 3 g/kg/day eth<strong>an</strong>ol were<br />
sacrificed, <strong>an</strong>d <strong>the</strong>ir brains were prepared <strong>for</strong> <strong>an</strong>alysis using radiolabeled in situ hybridization<br />
<strong>an</strong>d dioxygenin-labeled in situ hybridization. With <strong>the</strong>se techniques, ENK mRNA in rats<br />
drinking eth<strong>an</strong>ol was once again found to be elevated in all areas examined, including <strong>the</strong> CeA.<br />
These findings lead us to propose that this opioid in <strong>the</strong> extra-hypothalamic areas responds to<br />
eth<strong>an</strong>ol with enh<strong>an</strong>ced expression <strong>an</strong>d, in turn, may act through its specific, area-related<br />
functions to promote eth<strong>an</strong>ol intake. These functions include: caloric intake in <strong>the</strong> PVN; reward<br />
<strong>an</strong>d motivation in <strong>the</strong> VTA <strong>an</strong>d NAcSh; response-rein<strong>for</strong>cement learning in <strong>the</strong> NAcC; stress<br />
reduction in <strong>the</strong> CeA; <strong>an</strong>d disinhibition of behavioral control in <strong>the</strong> mPFC. All of <strong>the</strong>se functions<br />
c<strong>an</strong> be factors in determining why <strong>an</strong>imals consume eth<strong>an</strong>ol. There<strong>for</strong>e, <strong>the</strong> enh<strong>an</strong>ced expression<br />
of ENK in <strong>the</strong>se different brain areas may reflect a broader function of this opioid peptide in<br />
driving <strong>an</strong>imals to consume eth<strong>an</strong>ol.<br />
Disclosures: J.R. Barson, None; G. Ch<strong>an</strong>g, None; O. Karatayev, None; B.G. Hoebel,<br />
None; S.F. Leibowitz, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.18/Z4<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: USPHS Gr<strong>an</strong>t AA-12882
Edward H. L<strong>an</strong>e Foundation<br />
Title: Modulation of eth<strong>an</strong>ol intake by GABA in specific hypothalamic nuclei<br />
Authors: *M. BOCARSLY 1 , J. R. BARSON 1,2 , Y.-W. CHEN 1 , S. LEIBOWITZ 2 , B. G.<br />
HOEBEL 1 ;<br />
1 Psychology, Princeton Univ., Princeton, NJ; 2 Rockefeller Univ., New York, NY<br />
Abstract: Neurochemical influences in <strong>the</strong> hypothalamus are import<strong>an</strong>t in regulating various<br />
homeostatic functions, including behavior, as indicated in feeding, drinking <strong>an</strong>d arousal. Inputs<br />
to <strong>the</strong> various hypothalamic nuclei determine behavioral outputs. Peptides within <strong>the</strong><br />
hypothalamus have been shown to modulate eth<strong>an</strong>ol consumption. Thus, it is crucial to<br />
underst<strong>an</strong>d <strong>the</strong> influences of <strong>the</strong> neurochemical inputs on behavioral outputs in regulation of<br />
eth<strong>an</strong>ol intake. It has previously been shown that eth<strong>an</strong>ol increases GABA in <strong>the</strong> hypothalamus.<br />
Here, we explore <strong>the</strong> effects of GABA in two specific hypothalamic nuclei, <strong>the</strong> paraventricular<br />
nucleus (PVN) <strong>an</strong>d lateral hypothalamus (LH), on voluntary eth<strong>an</strong>ol consumption. Based on<br />
studies of food intake, we hypo<strong>the</strong>size that GABA in <strong>the</strong> PVN exerts positive control, as<br />
reflected by <strong>an</strong> increase in eth<strong>an</strong>ol intake, whereas GABA in <strong>the</strong> LH provides a negative control,<br />
decreasing eth<strong>an</strong>ol intake. In order to explore this hypo<strong>the</strong>sis, <strong>the</strong> GABA-A agonist, muscimol,<br />
<strong>an</strong>d <strong>the</strong> GABA-A <strong>an</strong>tagonist, bicuculline, were used. During <strong>the</strong> dark cycle, adult male Sprague-<br />
Dawley rats trained to consume 9% eth<strong>an</strong>ol ad libitum were injected bilaterally in <strong>the</strong> LH with<br />
ei<strong>the</strong>r muscimol (0.1 µg) or bicuculline (0.04 µg) counterbal<strong>an</strong>ced with saline. A similar group<br />
of rats received injections in <strong>the</strong> PVN of saline, muscimol (0.15 µg) or bicuculline (0.04 µg).<br />
Eth<strong>an</strong>ol, food, <strong>an</strong>d water intake were measured during <strong>the</strong> first 4 h post-injection. In <strong>the</strong> LH, <strong>the</strong><br />
GABA agonist decreased eth<strong>an</strong>ol intake <strong>for</strong> <strong>the</strong> first 2 h after injection <strong>an</strong>d decreased food intake<br />
between 2 <strong>an</strong>d 4 h, while having no effect on water intake. Conversely, <strong>the</strong> <strong>an</strong>tagonist in <strong>the</strong> LH<br />
increased eth<strong>an</strong>ol <strong>an</strong>d food intake, <strong>an</strong>d again had no effect on water intake. This suggests that <strong>an</strong><br />
elevation in extracellular GABA in <strong>the</strong> LH inhibits eth<strong>an</strong>ol consumption <strong>an</strong>d that GABA itself<br />
may be tonically active in this region. Results obtained in <strong>the</strong> PVN revealed a very different<br />
pattern. In those rats with elevated baseline eth<strong>an</strong>ol intake in <strong>the</strong> first hour (as determined by<br />
intake after saline injection), <strong>the</strong> GABA agonist increased eth<strong>an</strong>ol intake, while decreasing water<br />
intake at 2 <strong>an</strong>d 4 h <strong>an</strong>d having no effect on food intake. The GABA <strong>an</strong>tagonist signific<strong>an</strong>tly<br />
suppressed water intake, while having no effect on eth<strong>an</strong>ol or food intake. These data suggest<br />
that GABA in <strong>the</strong> LH has inhibitory control of eth<strong>an</strong>ol intake in eth<strong>an</strong>ol drinking rats, similar to<br />
effects observed with food intake in eth<strong>an</strong>ol-naïve rats, while it has <strong>the</strong> opposite effect in <strong>the</strong><br />
PVN.<br />
Disclosures: M. Bocarsly, None; J.R. Barson, None; Y. Chen, None; S. Leibowitz,<br />
None; B.G. Hoebel, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.19/Z5<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: AA10717<br />
AA13517<br />
AA07611<br />
AA10721<br />
Title: Repeated systemic eth<strong>an</strong>ol administration may alter serotonin (5-HT) neurotr<strong>an</strong>smission in<br />
<strong>the</strong> central nucleus of <strong>the</strong> amygdala (CeA) of Wistar, but not P, rats<br />
Authors: E. A. ENGLEMAN 1,4 , A. D. MORAN 1,4 , L. LUMENG 2,5 , *J. M. MURPHY 6,4 , W. J.<br />
MCBRIDE 1,4,3 ;<br />
1 Psychiatry, 2 Med., 3 Biochem. & Mol. Biol., Indi<strong>an</strong>a Univ. Sch. of Med., Indi<strong>an</strong>apolis, IN; 4 Inst.<br />
of Psychiatric Res., Indi<strong>an</strong>apolis, IN; 5 VAMC, Indi<strong>an</strong>apolis, IN; 6 Sch. Sci. - IUPUI, Indi<strong>an</strong>apolis,<br />
IN<br />
Abstract: This study tests <strong>the</strong> hypo<strong>the</strong>sis that differential alterations in <strong>the</strong> dorsal raphe (DR)<br />
serotonin (5-HT) system between alcohol-preferring (P) <strong>an</strong>d Wistar rats occur with repeated<br />
systemic eth<strong>an</strong>ol administration. We have previously shown reductions in basal 5-HT<br />
neurotr<strong>an</strong>smission in <strong>the</strong> central nucleus of <strong>the</strong> amygdale (CeA) <strong>an</strong>d <strong>the</strong> nucleus accumbens<br />
(Acb) after 8 weeks of free-choice consumption of 10% eth<strong>an</strong>ol by P rats. The objective of <strong>the</strong><br />
present study was to determine ch<strong>an</strong>ges in <strong>the</strong> DR 5-HT system following repeated i.p. eth<strong>an</strong>ol<br />
injections in P <strong>an</strong>d Wistar rats. Rats received daily injections (1.0 g/kg, i.p.) of EtOH or saline<br />
<strong>for</strong> five days; <strong>the</strong> no-net-flux qu<strong>an</strong>titative microdialysis procedure was used to determine basal 5-<br />
HT concentrations in <strong>the</strong> CeA of P <strong>an</strong>d Wistar rats. Repeated eth<strong>an</strong>ol administration had no<br />
signific<strong>an</strong>t effect on basal extracellular 5-HT concentrations in <strong>the</strong> CeA of ei<strong>the</strong>r P (0.35 ± 0.05<br />
vs. 0.37 ± 0.03 nM) or Wistar (0.26 ± 0.03 vs. 0.25 ± 0.04 nM) rats, <strong>for</strong> saline vs. eth<strong>an</strong>ol<br />
treatments, respectively. Extraction fractions <strong>for</strong> 5-HT were signific<strong>an</strong>tly elevated in eth<strong>an</strong>ol<br />
treated Wistar (64 ± 3 vs. 93 ± 4%) but not P (76 ± 5 vs. 77 ± 3%) rats. The increased extraction<br />
fraction (clear<strong>an</strong>ce) without a ch<strong>an</strong>ge in extracellular levels suggests that repeated eth<strong>an</strong>ol<br />
injections may increase 5-HT neurotr<strong>an</strong>smission in <strong>the</strong> CeA of Wistar rat, but not in <strong>the</strong> CeA of<br />
P rats. In addition, <strong>the</strong> results suggest that repeated eth<strong>an</strong>ol injections do not produce similar<br />
effects on 5-HT neurotr<strong>an</strong>smission as observed with chronic alcohol drinking.<br />
Disclosures: E.A. Englem<strong>an</strong>, None; A.D. Mor<strong>an</strong>, None; L. Lumeng, None; J.M. Murphy,<br />
None; W.J. McBride, None.
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.20/Z6<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: PRIN<br />
Title: Eth<strong>an</strong>ol stimulates corticotropic releasing hormone (CRH) release <strong>from</strong> rat hypothalamic<br />
expl<strong>an</strong>ts; Role of acetaldehyde<br />
Authors: *C. CANNIZZARO 1 , M. LA BARBERA 2 , F. PLESCIA 2 , S. CACACE 2 , G.<br />
TRINGALI 3 ;<br />
1 Scienze Farmacologiche, Univ. Di Palermo, 90127 Palermo, Italy; 2 Pharmacol. sciences, Univ.<br />
of Palermo, Palermo, Italy; 3 Catholic Univ. of Sacred Heart, Rome, Italy<br />
Abstract: Eth<strong>an</strong>ol is able to activate <strong>the</strong> hypothalamic-pituitary-adrenal (HPA) axis, <strong>an</strong>d to<br />
modify its response to o<strong>the</strong>r stressors, releasing adrenocorticotropic hormone (ACTH), <strong>an</strong>d<br />
glucocorticoid release, (Lee et al 1999, 2000). In <strong>the</strong> brain eth<strong>an</strong>ol is diverted by catalase activity<br />
into acetaldehyde, which is reported to mediate some of its behavioural <strong>an</strong>d neurochemical<br />
effects. (Pe<strong>an</strong>a et al 2008, Melis et al 2007) Thus, to clarifìy <strong>the</strong> mech<strong>an</strong>isms underlying eth<strong>an</strong>ol<br />
activity on <strong>the</strong> HPA axis we investigated :1) eth<strong>an</strong>ol effect on CRH release <strong>from</strong> incubated<br />
hypothalamic expl<strong>an</strong>ts; 2) <strong>the</strong> role of acetaldehyde (ACD), in mediating eth<strong>an</strong>ol activity. To this<br />
aim, rat hypothalamic expl<strong>an</strong>ts were incubated with: 1) medium containing eth<strong>an</strong>ol at 150 mg %;<br />
2) different concentrations of ACD (4.4 I , 13.2 II , 44 III , 132 IV x 10-3 mg%); 3) eth<strong>an</strong>ol plus 3amino-1,2,4-triazole<br />
(3-AT, 269 mg%), <strong>an</strong> inhibitor of cerebral catalase; 4) ACD plus Dpenicillamine<br />
(D-P, 74.6 mg%), <strong>an</strong> ACD-trapping agent. CRH levels were evaluated by a<br />
radioimmunoassay. Our results show that: incubation with eth<strong>an</strong>ol (150 mg %) induced a<br />
signific<strong>an</strong>t increase in CRH secretion (p< 0.01); ACD was able to increase CRH release in a dose<br />
dependent m<strong>an</strong>ner (I=p
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.21/Z7<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: AA015566<br />
AA06420<br />
AA016985<br />
Title: Eth<strong>an</strong>ol dependence induces neuroadaptation of <strong>the</strong> nociceptin <strong>an</strong>d CRF systems at <strong>the</strong><br />
GABAergic synapses in <strong>the</strong> central amygdala<br />
Authors: *M. T. CRUZ, M. BAJO, P. SCHWEITZER, M. ROBERTO;<br />
CNAD, Scripps Res. Inst., La Jolla, CA<br />
Abstract: Behavioral <strong>an</strong>d neurochemical studies indicate that <strong>the</strong> GABAergic system in <strong>the</strong><br />
central nucleus of <strong>the</strong> amygdala (CeA) has <strong>an</strong> import<strong>an</strong>t role in mediating stress <strong>an</strong>d <strong>an</strong>xiety.<br />
Corticotrophin-releasing factor (CRF) <strong>an</strong>d nociceptin/orph<strong>an</strong>in FQ (nociceptin) within <strong>the</strong> CeA<br />
have also been implicated in acute stress response <strong>an</strong>d eth<strong>an</strong>ol dependence. Recently, we<br />
reported that nociceptin moderately decreased basal GABAA receptor-mediated IPSP (IPSPs)<br />
amplitudes <strong>an</strong>d blocked eth<strong>an</strong>ol-induced increases in IPSPs via a decrease in GABA release in<br />
<strong>the</strong> CeA.<br />
Here we used <strong>an</strong> in vitro CeA slice preparation to examine <strong>the</strong> interactions of CRF <strong>an</strong>d<br />
nociceptin on GABAergic tr<strong>an</strong>smission <strong>an</strong>d study <strong>the</strong> neuroadaptation of <strong>the</strong>se systems during<br />
<strong>the</strong> development of alcohol dependence. We found that 200 nM CRF signific<strong>an</strong>tly enh<strong>an</strong>ced<br />
evoked IPSPs, <strong>an</strong>d decreased paired-pulse facilitation (PPF) in neurons of naïve rats, whereas<br />
nociceptin decreased basal IPSP amplitudes <strong>an</strong>d increased PPF, implicating a regulation of<br />
GABA release. Interestingly, nociceptin reversed <strong>the</strong> CRF-induced GABA release. Fur<strong>the</strong>rmore,<br />
pretreatment with nociceptin blocked <strong>the</strong> CRF-induced increase in IPSPs. In neurons <strong>from</strong><br />
eth<strong>an</strong>ol-dependent rats, <strong>the</strong> basal GABA release was elevated <strong>an</strong>d <strong>the</strong> nociceptin-induced<br />
inhibition of IPSPs was increased, indicating <strong>an</strong> enh<strong>an</strong>ced sensitivity to nociceptin. Moreover,<br />
<strong>the</strong> ability of CRF to increase IPSPs was enh<strong>an</strong>ced compared to naïve neurons, <strong>an</strong>d <strong>the</strong> inhibition<br />
of basal IPSPs by nociceptin was greater th<strong>an</strong> that in neurons of naïve rats. Nociceptin also<br />
blocked <strong>the</strong> CRF-induced increase of IPSPs in eth<strong>an</strong>ol-dependent rats.<br />
These findings suggest <strong>an</strong> import<strong>an</strong>t nociceptin-CRF influence on GABAergic tr<strong>an</strong>smission in
<strong>the</strong> CeA that undergoes signific<strong>an</strong>t neuroadaptation in <strong>the</strong> development of eth<strong>an</strong>ol dependence.<br />
Our data implicate a nociceptin-GABA-CRF interaction in <strong>the</strong> regulation of voluntary eth<strong>an</strong>ol<br />
intake <strong>an</strong>d <strong>an</strong>xiety-like behaviors associated with eth<strong>an</strong>ol dependence.<br />
Supported by gr<strong>an</strong>ts <strong>from</strong> NIAAA: AA015566, AA06420, AA016985, <strong>from</strong> Harold L. Dorris<br />
Neurological Research Institute, <strong>an</strong>d <strong>from</strong> <strong>the</strong> Pearson Center <strong>for</strong> Alcoholism <strong>an</strong>d Addiction<br />
Research.<br />
Disclosures: M.T. Cruz, None; M. Bajo, None; P. Schweitzer, None; M. Roberto, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.22/Z8<br />
Topic: C.17.c. Alcohol: Developmental effects<br />
Support: KOSEF <strong>2009</strong>-0058805<br />
BK21<br />
Title: Eth<strong>an</strong>ol exposure on GABAB receptor downstream pathway <strong>an</strong>d intrinsic neuronal<br />
apoptosis: Role of GABAB1 receptor siRNA on FAS-associated microencephaly<br />
Authors: *L. HAE YOUNG 1 , N. NAHA 2 , T.-H. KIM 2 , M. I. NASEER 2 , M.-O. KIM 2 ;<br />
1 2<br />
biol & Applied Life Sci., Jinju, Republic of Korea; biol & Applied Life Sci., Gyeong-S<strong>an</strong>g Nat<br />
Univ., Jinju, Republic of Korea<br />
Abstract: Maternal eth<strong>an</strong>ol exposure during pregn<strong>an</strong>cy causes structural <strong>an</strong>d functional<br />
abnormalities in fetal brain, resulting in FAS. The cerebral cortex <strong>an</strong>d hippocampus are <strong>the</strong> most<br />
susceptible areas in <strong>the</strong> developing brain. As <strong>the</strong> inhibitory neurotr<strong>an</strong>smitter GABA modulates<br />
eth<strong>an</strong>ol consumption <strong>an</strong>d alcohol withdrawal symptoms by interacting with <strong>the</strong> GABAB1<br />
receptor (R), <strong>the</strong>re<strong>for</strong>e <strong>the</strong> present study was undertaken to explore whe<strong>the</strong>r GABAB1R has <strong>an</strong>y<br />
role in <strong>the</strong> downstream signal pathway upon eth<strong>an</strong>ol exposure by using GABAB1R RNAi<br />
(30nM, 48 hr). We also used GABABR’s agonist baclofen (50M) <strong>an</strong>d <strong>an</strong>tagonist phaclofen<br />
(100M) in combination with eth<strong>an</strong>ol (100mM) to elucidate <strong>the</strong> possible reversing roles of<br />
GABAB1R in pharmaco<strong>the</strong>rapy <strong>for</strong> eth<strong>an</strong>ol abuse. Pregn<strong>an</strong>t female rats were sacrificed on GD<br />
17.5, <strong>an</strong>d fetal cortex <strong>an</strong>d hippocampus were collected. The expression patterns of <strong>the</strong> signal<br />
molecules in GABAB1R <strong>an</strong>d apoptosis downstream pathway including PKA-α, CREB, p-CREB,<br />
CaMKII, cytochrome C, caspase 3, <strong>an</strong>d PARP varied signific<strong>an</strong>tly in <strong>the</strong> fetal <strong>an</strong>d <strong>the</strong> maternal
ain tissues of different treated groups during tr<strong>an</strong>sfected <strong>an</strong>d non-tr<strong>an</strong>sfected conditions.<br />
Cytosolic concentration of calcium was also reduced with GABAB1R RNAi. GABAB1R, PKAα<br />
<strong>an</strong>d p-CREB immunoreactivity was decreased after GABAB1R siRNA treatment, which<br />
revealed area-specific localization in <strong>the</strong> fetal cortex <strong>an</strong>d hippocampus. Our results suggest a<br />
very crucial role of GABAB1R by modulating downstream signal pathway upon maternal<br />
eth<strong>an</strong>ol exposure. Moreover, <strong>for</strong> <strong>the</strong> first time our study provides <strong>the</strong> in<strong>for</strong>mation that<br />
GABAB1R RNAi would be <strong>an</strong> effective tool <strong>for</strong> combating pharmacological eth<strong>an</strong>ol abuse<br />
including FAS-associated brain damage.(This work was supported by KOSEF <strong>2009</strong>-0058805 &<br />
BK21).<br />
Disclosures: L. Hae Young, None; N. Naha, None; T. Kim, None; M.I. Naseer, None; M.<br />
Kim, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.23/Z9<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: Harry S. Trum<strong>an</strong> Memorial Veter<strong>an</strong>s Hospital<br />
R03 NS059831<br />
Title: Effect of eth<strong>an</strong>ol on adenosine release <strong>an</strong>d c-Fos activation in <strong>the</strong> cholinergic basal<br />
<strong>for</strong>ebrain<br />
Authors: *M. M. THAKKAR 1,2 , R. SHARMA 1 , S. C. ENGEMANN 1 , K. M. WALSH 1 , P.<br />
SAHOTA 1 ;<br />
1 2<br />
Dept Neurol, Univ. Missouri, Columbia, MO; Harry S. Trum<strong>an</strong> Mem. Veter<strong>an</strong>s Hosp.,<br />
Columbia, MO<br />
Abstract: Acute intake of eth<strong>an</strong>ol (EtOH) promotes sleep. However, cellular mech<strong>an</strong>isms<br />
responsible <strong>for</strong> EtOH’s effect on sleep are unknown. We hypo<strong>the</strong>sized that acute EtOH intake<br />
will increase extracellular adenosine (AD) levels in <strong>the</strong> basal <strong>for</strong>ebrain (BF). Increased AD will<br />
to promote sleep by inhibiting BF wake-promoting neurons. We designed <strong>the</strong> following<br />
experiments to test our hypo<strong>the</strong>ses.<br />
Experiment 1: Male Sprague-Dawley rats (~250 g) were unilaterally impl<strong>an</strong>ted with guide<br />
c<strong>an</strong>nula, targeted toward <strong>the</strong> BF (probe co-ordinates: AP= P0.3, ML= L2.0, DV= V9.0; Paxinos
<strong>an</strong>d Watson 1998) <strong>for</strong> later insertion of microdialysis probe. At dark onset, artificial<br />
cerebrospinal fluid (aCSF) was perfused (flow rate = 0.7 µL/min) <strong>for</strong> 80 min <strong>an</strong>d 4x 20 min<br />
samples were collected. Subsequently, 3 doses of EtOH (30, 100 <strong>an</strong>d 300 mM) were perfused to<br />
evaluate <strong>the</strong> cumulative dose profile of EtOH’s effect on BF AD levels. Each dose was perfused<br />
<strong>for</strong> 80 min <strong>an</strong>d 4 x 20 min samples were collected. The samples were stored in ice until AD<br />
levels were measured by HPLC coupled with UV detector. On completion of <strong>the</strong> experiments,<br />
<strong>the</strong> <strong>an</strong>imals were euth<strong>an</strong>ized <strong>an</strong>d brains were removed <strong>an</strong>d processed <strong>for</strong> choline acetyl<br />
tr<strong>an</strong>sferase (ChAT) immunohistochemistry (IH) to localize <strong>the</strong> perfusion sites in <strong>the</strong> BF <strong>an</strong>d<br />
cresyl violet staining to evaluate potential neurotoxicity caused due to EtOH perfusion.<br />
Experiment 2: Rats were administered ei<strong>the</strong>r EtOH (3g/Kg) or water (controls; 10 mL/Kg)<br />
intragastrically at dark onset <strong>an</strong>d were left undisturbed <strong>for</strong> 2 hr. Subsequently, rats were<br />
euth<strong>an</strong>ized <strong>an</strong>d brains were removed <strong>an</strong>d processed <strong>for</strong> c-Fos <strong>an</strong>d ChAT IH in BF.<br />
Results:<br />
Experiment 1: Local perfusion of EtOH caused a signific<strong>an</strong>t increase (F=3.018; df (total) = 34;<br />
p
Authors: I. G. BARBOSA LUNA, J. M. PÉREZ LUNA, *M. MENDEZ UBACH;<br />
Subdirección de Investigaciones Clínicas, Inst. Nacional De Psiquiatría Ramón De La Fuente,<br />
México D.F., Mexico<br />
Abstract: The dopaminergic mesolimbic system plays a key role in <strong>the</strong> rein<strong>for</strong>cement<br />
mech<strong>an</strong>isms elicited by eth<strong>an</strong>ol, although <strong>the</strong> nigrostriatal pathway is also a neural target of this<br />
drug. Eth<strong>an</strong>ol rein<strong>for</strong>cing properties <strong>an</strong>d high alcohol drinking behaviour may be due, at least in<br />
part, to <strong>the</strong> eth<strong>an</strong>ol-induced activation of <strong>the</strong> endogenous opioid system. Activation of<br />
opioidergic pathways by eth<strong>an</strong>ol may involve specific alterations in opioid syn<strong>the</strong>sis, release,<br />
degradation, <strong>an</strong>d/or binding to opioid receptors. The aim of this work was to investigate <strong>the</strong><br />
effect of chronic eth<strong>an</strong>ol exposure on Methionine-enkephalin (Met-enk) levels in several regions<br />
of <strong>the</strong> rat brain. Male Wistar rats were treated during 30 days with eth<strong>an</strong>ol (10 % solution v/v).<br />
Control rats were treated with sucrose <strong>an</strong>d were paired to <strong>the</strong> eth<strong>an</strong>ol group. An ad libitum group<br />
was also included. Food <strong>an</strong>d liquid consumption were recorded along <strong>the</strong> experiment. Met-enk<br />
content was measured by radioimmunoassay in regions such as <strong>the</strong> prefrontal cortex (PFC), <strong>the</strong><br />
nucleus accumbens (NAcc) <strong>an</strong>d <strong>the</strong> caudate-putamen (CP). Chronic eth<strong>an</strong>ol exposure decreased<br />
Met-enk levels in <strong>the</strong> <strong>an</strong>terior-medial region of <strong>the</strong> CP, in comparison to both <strong>the</strong> ad libitum <strong>an</strong>d<br />
sucrose groups. Eth<strong>an</strong>ol also decreased Met-enk content in <strong>the</strong> PFC when compared to <strong>the</strong><br />
sucrose group. Sucrose treatment increased Met-enk levels in this region. In contrast, chronic<br />
eth<strong>an</strong>ol exposure did not modify peptide levels in <strong>the</strong> NAcc or <strong>the</strong> medial-posterior portion of <strong>the</strong><br />
CP. Our findings indicate that Met-enk syn<strong>the</strong>sis in <strong>the</strong> caudate-putamen <strong>an</strong>d prefrontal cortex is<br />
particularly sensitive to chronic eth<strong>an</strong>ol exposure <strong>an</strong>d sucrose treatment. Our results suggest that<br />
enkephalins in <strong>the</strong>se brain areas could play a relev<strong>an</strong>t role in <strong>the</strong> mech<strong>an</strong>isms underlying eth<strong>an</strong>ol<br />
dependence.<br />
Disclosures: I.G. Barbosa Luna, None; J.M. Pérez Luna, None; M. Mendez Ubach, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.25/Z11<br />
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH gr<strong>an</strong>t AA007474 (JTG)<br />
NIH gr<strong>an</strong>t AA016964 (JHY)<br />
NIH gr<strong>an</strong>t AA013825 (MFO)
Title: Voluntary eth<strong>an</strong>ol consumption increases extracellular glutamate levels in <strong>the</strong> ventral<br />
tegmental area as assessed by enzyme-coated glutamate biosensors<br />
Authors: *J. T. GASS 1 , J.-H. YE 2 , M. OLIVE 1 ;<br />
1 Med. Univ. South Carolina, Charleston, SC; 2 Pharmacol., UMDNJ, Or<strong>an</strong>ge, NJ<br />
Abstract: Excitation of dopamine (DA) producing neurons in <strong>the</strong> ventral tegmental area (VTA)<br />
of <strong>the</strong> midbrain is believed to underlie <strong>the</strong> rein<strong>for</strong>cing properties of eth<strong>an</strong>ol. However, <strong>the</strong> precise<br />
neural mech<strong>an</strong>isms by which eth<strong>an</strong>ol excites VTA DA neurons has remained elusive. Recently<br />
we demonstrated that in midbrain slices containing <strong>the</strong> VTA, local perfusion of low (10-80 mM)<br />
concentrations of eth<strong>an</strong>ol increased <strong>the</strong> amplitude of evoked non-NMDA mediated postsynaptic<br />
currents in VTA DA neurons <strong>an</strong>d increased glutamate overflow via a presynaptic D1-mediated<br />
mech<strong>an</strong>ism (Xiao et al., Neuropsychopharmacology 34:307-18, <strong>2009</strong>). To investigate whe<strong>the</strong>r<br />
this occurs in vivo in <strong>an</strong>imals voluntary consuming alcohol, we impl<strong>an</strong>ted male Wistar rats with<br />
unilateral guide c<strong>an</strong>nula aimed at <strong>the</strong> VTA. Following recovery, <strong>an</strong>imals were allowed to drink a<br />
sweetened eth<strong>an</strong>ol solution containing 10% eth<strong>an</strong>ol, 3% glucose, <strong>an</strong>d 0.125% saccharin <strong>for</strong> 30<br />
min per day, 3 days a week. Following at least 3 weeks of baseline eth<strong>an</strong>ol consumption, <strong>an</strong>imals<br />
were impl<strong>an</strong>ted with pre-calibrated glutamate biosensors (Pinnacle Technologies) <strong>an</strong>d allowed to<br />
recover overnight. The biosensors consisted of Pt-Ir electrodes coated with glutamate oxidase (1<br />
mm active sensing zone) that converts glutamate to alpha-ketoglutaraldehyde <strong>an</strong>d hydrogen<br />
peroxide, <strong>the</strong> latter of which is oxidized at <strong>the</strong> surface of <strong>the</strong> electrode to allow near real-time<br />
assessment of ch<strong>an</strong>ges in extracellular glutamate in <strong>the</strong> living brain. The electrode is also coated<br />
with ascorbate oxidase <strong>an</strong>d Nafion to eliminate or repel o<strong>the</strong>r <strong>an</strong>ionic interfer<strong>an</strong>ts. On <strong>the</strong> test<br />
day, <strong>an</strong>imals were allowed 30 min access to <strong>the</strong> sweetened eth<strong>an</strong>ol solution while biosensor<br />
currents were measured in 1 sec intervals. Animals dr<strong>an</strong>k a me<strong>an</strong> of approximately 0.4 g/kg<br />
eth<strong>an</strong>ol during <strong>the</strong> session, primarily during <strong>the</strong> first 5 minutes. Increases in current <strong>from</strong> <strong>the</strong><br />
biosensor were observed beginning at approximately 10 min into <strong>the</strong> session, <strong>an</strong>d peaked at 20<br />
min into <strong>the</strong> session (equivalent to a ch<strong>an</strong>ge of 5 uM glutamate), <strong>an</strong>d declined to baseline<br />
<strong>the</strong>reafter. Blood samples were taken immediately following <strong>the</strong> drinking session <strong>an</strong>d were<br />
determined to have eth<strong>an</strong>ol contents of approximately 25 mg/dl. These data indicate that eth<strong>an</strong>ol<br />
consumption increases extracellular glutamate levels in <strong>the</strong> VTA, which may provide excitatory<br />
input to mesolimbic DA neurons that mediate <strong>the</strong> rein<strong>for</strong>cing effects of eth<strong>an</strong>ol.<br />
Disclosures: J.T. Gass, None; J. Ye, None; M. Olive, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.26/Z12
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIH/NIAAA gr<strong>an</strong>t AA016650<br />
Title: The Homer2 scaffolding protein is necessary <strong>for</strong> <strong>the</strong> <strong>an</strong>ti-binge drinking effect of NAC<br />
mGluR5 blockade<br />
Authors: *D. K. COZZOLI 1 , W. RAMIREZ 1 , A. L. CARUANA 1 , P. F. WORLEY 2 , K. K.<br />
SZUMLINSKI 1 ;<br />
1 2<br />
Univ. CA S<strong>an</strong>ta Barbara, S<strong>an</strong>ta Barbara, CA; Dept. of Neurosci., Johns Hopkins Univ. Sch. of<br />
Med., Baltimore, MD<br />
Abstract: The Homer2 member of <strong>the</strong> Homer family of postsynaptic scaffolding proteins has<br />
emerged as critical <strong>for</strong> NAC glutamate tr<strong>an</strong>smission <strong>an</strong>d alcohol-induced neuroplasticity in vivo.<br />
Through <strong>an</strong> Ena/VASP1 Homology (EVH1) domain, Homers interact with a proline-rich motif<br />
(PxxF) located on Group 1 metabotropic glutamate receptors (mGluR1/5), as well as on o<strong>the</strong>r<br />
proteins involved in Group 1 mGluR intracellular signaling. Converging behavioral,<br />
neurochemical <strong>an</strong>d immunoblotting data support a necessary <strong>an</strong>d active role <strong>for</strong> Homer2-mGluR<br />
signaling within <strong>the</strong> nucleus accumbens in mediating alcohol intake in binge drinking paradigms.<br />
To fur<strong>the</strong>r examine <strong>the</strong> role <strong>for</strong> Homer2 signaling in excessive binge-like alcohol consumption,<br />
Homer2 knock-out mice were trained to self-administer alcohol using <strong>the</strong> Drinking in <strong>the</strong> Dark<br />
(DID) paradigm. For this, mice were presented with a 50 ml sipper tube containing a 20%<br />
alcohol solution <strong>for</strong> 2 hrs, at 3 hrs into <strong>the</strong> dark cycle, every day, without fluid restriction. To our<br />
surprise, genotypic differences in binge drinking phenotype were not observed within this model.<br />
To test <strong>the</strong> hypo<strong>the</strong>sis that Homer2 signaling is necessary <strong>for</strong> <strong>the</strong> <strong>an</strong>ti-binge alcohol drinking<br />
effects of mGluR5 <strong>an</strong>tagonists, we next assessed <strong>the</strong> capacity of intra-NAC infusions of 3.0<br />
µg/side MPEP to reduce binge drinking with-in <strong>the</strong> DID procedure in wild-type <strong>an</strong>d knock-out<br />
littermates. While wild-type littermates showed attenuation of alcohol drinking following<br />
infusion, intra-NAC MPEP failed to reduce alcohol drinking in Homer2 knock-out <strong>an</strong>imals.<br />
These data fur<strong>the</strong>r support <strong>the</strong> hypo<strong>the</strong>sis that intact signaling through <strong>an</strong> mGluR5-Homer2<br />
complex within <strong>the</strong> NAC shell is necessary <strong>for</strong> MPEP’s <strong>an</strong>ti-binge alcohol-drinking effect.<br />
Disclosures: D.K. Cozzoli, None; W. Ramirez, None; A.L. Caru<strong>an</strong>a, None; P.F. Worley,<br />
None; K.K. Szumlinski, None.<br />
Poster<br />
552. Alcohol: Neural Mech<strong>an</strong>isms II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 552.27/Z13
Topic: C.17.e. Alcohol: Neural mech<strong>an</strong>isms<br />
Support: NIAAA Gr<strong>an</strong>t AA013517<br />
Title: Role of eth<strong>an</strong>ol-activated potassium ch<strong>an</strong>nels in eth<strong>an</strong>ol drinking<br />
Authors: *C. CONTET 1 , B. L. KIEFFER 2 , K. WICKMAN 3 , S. N. TREISTMAN 4 , G. F.<br />
KOOB 1 ;<br />
1 Scripps Resch Inst., La Jolla, CA; 2 Inst. de Genetique et de Biologie Moleculaire et Cellulaire,<br />
Illkirch, Fr<strong>an</strong>ce; 3 Univ. of Minnesota, Minneapolis, MN; 4 Univ. of Puerto Rico, Puerto Rico, PR<br />
Abstract: Among several primary targets in <strong>the</strong> brain, alcohol has been shown to activate two<br />
types of potassium ch<strong>an</strong>nels: <strong>the</strong> G protein-activated inwardly rectifying potassium (GIRK)<br />
ch<strong>an</strong>nel <strong>an</strong>d <strong>the</strong> large conduct<strong>an</strong>ce calcium-activated potassium (BK) ch<strong>an</strong>nel. These ch<strong>an</strong>nels<br />
are both activated by eth<strong>an</strong>ol in vitro at physiologically relev<strong>an</strong>t concentrations (10-100 mM),<br />
but <strong>the</strong>ir contribution to eth<strong>an</strong>ol’s pharmacological effects in vivo is not fully understood yet,<br />
due to <strong>the</strong> lack of selective <strong>an</strong>d brain-penetr<strong>an</strong>t compounds blocking <strong>the</strong>se ch<strong>an</strong>nels. However,<br />
<strong>the</strong> use of knockout mice has enabled specific targeted deletion of <strong>the</strong> different subunits<br />
composing neuronal GIRK tetramers (GIRK1-3) <strong>an</strong>d neuronal BK dimers (alpha, beta1 <strong>an</strong>d beta<br />
4). Previous studies have shown that GIRK2 null mut<strong>an</strong>t mice are less sensitive to <strong>the</strong> <strong>an</strong>xiolytic,<br />
convulsive <strong>an</strong>d rewarding effects of eth<strong>an</strong>ol but <strong>the</strong>se mice also show marked behavioral<br />
abnormalities in <strong>the</strong> absence of eth<strong>an</strong>ol. In <strong>the</strong> present study, we <strong>the</strong>re<strong>for</strong>e focused our attention<br />
on GIRK3, <strong>an</strong>o<strong>the</strong>r neuronal GIRK subunit whose deletion does not alter basal locomotion,<br />
<strong>an</strong>xiety-like or oper<strong>an</strong>t behavior. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, BK beta4-deficient mice develop rapid<br />
toler<strong>an</strong>ce to eth<strong>an</strong>ol’s locomotor effects <strong>an</strong>d drink more th<strong>an</strong> <strong>the</strong>ir wild-type counterparts. Based<br />
on <strong>the</strong>se findings, we explored <strong>the</strong> role GIRK3 <strong>an</strong>d BK beta4 subunits may play in voluntary<br />
eth<strong>an</strong>ol drinking, <strong>an</strong>d more particularly in excessive drinking induced by alcohol dependence. To<br />
this end, we used a model of 2-hour limited access two-bottle choice drinking, alternated with<br />
chronic intermittent exposure to eth<strong>an</strong>ol vapor in inhalation chambers. This paradigm increases<br />
<strong>the</strong> eth<strong>an</strong>ol intake of dependent mice, while non-dependent mice (exposed to air) maintain a<br />
stable consumption. We have found that <strong>the</strong> expression of GIRK <strong>an</strong>d BK ch<strong>an</strong>nel subunits is<br />
differentially modified in eth<strong>an</strong>ol-dependent <strong>an</strong>d non-dependent mice, as compared to eth<strong>an</strong>olnaïve<br />
counterparts. We are currently testing knockout mice <strong>for</strong> GIRK3 <strong>an</strong>d BK beta4 subunits in<br />
our behavioral procedure of alcohol dependence to assess whe<strong>the</strong>r <strong>the</strong> tr<strong>an</strong>scriptional alterations<br />
induced by voluntary drinking <strong>an</strong>d <strong>for</strong>ced exposure to repeated cycles of intoxication <strong>an</strong>d<br />
withdrawal have a functional relev<strong>an</strong>ce. This study will potentially uncover novel mech<strong>an</strong>isms<br />
driving <strong>the</strong> motivation to drink alcohol, <strong>an</strong>d <strong>the</strong>reby new targets <strong>for</strong> <strong>the</strong> pharmaco<strong>the</strong>rapy of<br />
alcoholism.<br />
Disclosures: C. Contet, None; B.L. Kieffer, None; K. Wickm<strong>an</strong>, None; S.N. Treistm<strong>an</strong>,<br />
None; G.F. Koob, None.<br />
Poster
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.1/Z14<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: VA Merit<br />
Title: The dopamine beta-hydroxylase (DβH) inhibitor nepicastat blocks cocaine reward <strong>an</strong>d is<br />
<strong>an</strong>xiolytic<br />
Authors: C. N. HAILE 1,2 , *T. A. KOSTEN 1,2 ;<br />
1 Dept Psychiatry, Baylor Col. of Med., Houston, TX; 2 M E Debakey VA Med. Ctr., Houston, TX<br />
Abstract: Cocaine’s effects are partially mediated through central dopaminergic (DA) <strong>an</strong>d<br />
noradrenergic (NE) mech<strong>an</strong>isms. Dopamine Beta-hydroxylase (DβH) enzymatically converts<br />
dopamine (DA) to norepinephrine (NE) <strong>an</strong>d its inhibition decreases NE <strong>an</strong>d increases DA. The<br />
DβH inhibitor disulfiram (DS) shows efficacy <strong>for</strong> cocaine addiction in hum<strong>an</strong>s but has side<br />
effects that preclude its indication <strong>for</strong> this disease. Nepicastat (NEP) is a potent <strong>an</strong>d specific<br />
peripheral <strong>an</strong>d central DβH inhibitor that may prove a viable alternative <strong>for</strong> cocaine addiction<br />
pharmaco<strong>the</strong>rapy. Here we characterize NEP’s effects on cocaine reward, <strong>an</strong>d compared NEP to<br />
DS on acute locomotor activation, <strong>an</strong>xiety <strong>an</strong>d plasma DβH inhibition in rats (N=4-15/group).<br />
NEP (100mg/kg,IP) blocked cocaine (3mg/kg) place conditioning (p>0.05) whereas cocaine<br />
alone produced robust conditioning (vs. saline p0.05). We tested <strong>an</strong>xiety effects using <strong>the</strong> Elevated Plus Maze (EPM) assay. Acute DS had no<br />
effect on EPM whereas NEP (100mg/kg,IP) signific<strong>an</strong>tly increased open arm time <strong>an</strong>d decreased<br />
time on <strong>the</strong> closed arm (vs.saline,p
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.2/Z15<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: MH066172<br />
DA012736<br />
DA007359<br />
Title: Inducible overexpression of deltaFosB in basal <strong>for</strong>ebrain decreases aversive effects of<br />
kappa opioid receptor activation as measured by intracr<strong>an</strong>ial self-stimulation<br />
Authors: *J. W. MUSCHAMP 1 , C. L. NEMETH 2 , E. J. NESTLER 3 , W. A. CARLEZON, Jr. 1 ;<br />
1 Psychiatry, Harvard Med. Sch., Belmont, MA; 2 Neurosci., Emory Univ., Atl<strong>an</strong>ta, GA;<br />
3 Neurosci., Mount Sinai Sch. of Med., New York, NY<br />
Abstract: Increased expression of <strong>the</strong> tr<strong>an</strong>scription factor deltaFosB accomp<strong>an</strong>ies repeated<br />
exposure to drugs of abuse or to stress, particularly in brain areas associated with reward <strong>an</strong>d<br />
motivation (e.g., nucleus accumbens <strong>an</strong>d dorsal striatum). Long-lasting elevation in striatal<br />
deltaFosB is thought to be responsible <strong>for</strong> enh<strong>an</strong>ced sensitivity to <strong>the</strong> rewarding effects of drugs<br />
of abuse <strong>an</strong>d may underlie <strong>the</strong> tr<strong>an</strong>sition to dysregulated drug-seeking that characterizes<br />
addiction. It may also reduce <strong>an</strong> individual’s vulnerability <strong>for</strong> depression. We previously used<br />
<strong>the</strong> intracr<strong>an</strong>ial self-stimulation (ICSS) paradigm to show that tr<strong>an</strong>sgenic mice with inducible<br />
overexpression of deltaFosB in striatal regions are more sensitive to <strong>the</strong> rewarding effects of<br />
cocaine. Drug exposure also increases striatal expression of dynorphin, <strong>the</strong> endogenous lig<strong>an</strong>d at<br />
kappa opioid receptors (KOR). This neuroadaptation is believed to be compensatory, since<br />
dynorphin is implicated in <strong>the</strong> negative hedonic features of stress, <strong>an</strong>d KOR activation is known<br />
to be aversive. Here, we used ICSS to evaluate <strong>the</strong> effect of induced overexpression of striatal<br />
deltaFosB on <strong>the</strong> <strong>an</strong>hedonic properties of <strong>the</strong> KOR agonist U50,488. Mice impl<strong>an</strong>ted with lateral<br />
hypothalamic stimulating electrodes were trained using <strong>the</strong> ‘rate-frequency’ procedure <strong>for</strong> ICSS<br />
to determine <strong>the</strong> frequency at which stimulation becomes rewarding (threshold). A dose-effect<br />
<strong>an</strong>alysis revealed that, compared to littermate controls, <strong>an</strong>imals with genetically-elevated<br />
deltaFosB show reduced sensitivity to <strong>the</strong> <strong>an</strong>hedonic or aversive (reward threshold-elevating)<br />
effects of U50,488 (0.03-5.5 mg/kg, IP). These data suggest a mech<strong>an</strong>ism by which deltaFosB<br />
may oppose depression, as well as a mech<strong>an</strong>ism by which chronic drug exposure might<br />
desensitize addicted users to <strong>the</strong> adverse consequences that sometimes accomp<strong>an</strong>y drug use.<br />
Disclosures: J.W. Muschamp, None; C.L. Nemeth, None; E.J. Nestler, None; W.A.<br />
Carlezon, None.
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.3/Z16<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: DA17328<br />
DA19754<br />
DA19754<br />
M01RR00058<br />
Title: Repeated N-acetyl cysteine reduces cocaine seeking in rodents <strong>an</strong>d craving in cocainedependent<br />
hum<strong>an</strong>s<br />
Authors: *D. A. BAKER 1 , S. AMEN 2 , L. PIACENTINE 3 , S.-J. LI 4 , J. MANTSCH 1 , R. C.<br />
RISINGER 3 ;<br />
1 2<br />
Dept Biomed. Sci., Marquette Univ., Milwaukee, WI; Dept. of Pharmacol. <strong>an</strong>d Toxicology,<br />
3 4<br />
Dept. of Psychiatry <strong>an</strong>d Behavioral Med., Dept. of Biophysics, Med. Col. of Wisconsin,<br />
Milwaukee, WI<br />
Abstract: Addiction is a chronic relapsing disorder produced by drug-induced plasticity that<br />
renders individuals highly vulnerable to craving-inducing stimuli such as re-exposure to <strong>the</strong> drug<br />
of abuse. Drug-induced plasticity that may result in <strong>the</strong> addiction phenotype includes increased<br />
excitatory signaling within corticostriatal pathways which correlates with craving in hum<strong>an</strong>s <strong>an</strong>d<br />
is necessary <strong>for</strong> cocaine seeking in rodents. Reduced cystine-glutamate exch<strong>an</strong>ge by system xc-<br />
appears to contribute to heightened excitatory signaling within <strong>the</strong> striatum, <strong>the</strong>reby posing this<br />
as a novel target in <strong>the</strong> treatment of cocaine addiction. In <strong>the</strong> present study, we examined <strong>the</strong><br />
impact of repeated administration of N-acetyl cysteine, which is commonly used to activate<br />
cystine-glutamate exch<strong>an</strong>ge, on cocaine-induced reinstatement in rodents <strong>an</strong>d cocaine-induced<br />
craving in non-treatment seeking cocaine-dependent hum<strong>an</strong>s. Interestingly, repeated<br />
administration of (7 days) of N-acetyl cysteine (60 mg/kg, IP) produced a signific<strong>an</strong>t reduction in<br />
cocaine- (10 mg/kg, IP) induced reinstatement, even though rats were tested 12 hrs after <strong>the</strong> last<br />
administration of <strong>the</strong> cysteine prodrug. The reduction in behavior despite <strong>the</strong> absence of <strong>the</strong> Nacetyl<br />
cysteine suggests that repeated N-acetyl cysteine altered drug-induced plasticity that<br />
underlies drug-seeking behavior. In parallel, repeated administration (4 days) of N-acetyl
cysteine (1200-2400 g/day) to cocaine-dependent hum<strong>an</strong> subjects produced a signific<strong>an</strong>t<br />
reduction in craving following <strong>an</strong> experimenter-delivered IV injection of cocaine (20mg/70<br />
kg/60 sec). Collectively, <strong>the</strong>se data demonstrate that repeated N-acetylcysteine diminishes <strong>the</strong><br />
motivational qualities of a cocaine challenge injection possibly by altering pathogenic druginduced<br />
plasticity.<br />
Disclosures: D.A. Baker, NIDA, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); S. Amen, None; L.<br />
Piacentine, None; S. Li, None; J. M<strong>an</strong>tsch, None; R.C. Risinger, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.4/Z17<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Title: Group1 metabotropic glutamate receptors <strong>an</strong>d PDZ-domain scaffolding proteins: Role in<br />
reinstatement of cocaine seeking<br />
Authors: *V. KUMARESAN 1 , D. F. MIERKE 3 , D. H. FARB 2 ;<br />
2 Pharmacol., 1 Boston Univ. Med. Sch., Boston, MA; 3 Chem., Dartmouth Col., H<strong>an</strong>over, NH<br />
Abstract: Cocaine induced maladaptive plasticity of glutamatergic tr<strong>an</strong>smission plays a major<br />
role in drug abuse. Glutamate <strong>an</strong>d its cognate receptors mediate <strong>the</strong> majority of fast tr<strong>an</strong>smission<br />
in <strong>the</strong> brain. Both ionotropic <strong>an</strong>d metabotropic glutamate receptors (mGluRs) are critically<br />
involved in mediating <strong>the</strong> rewarding <strong>an</strong>d rein<strong>for</strong>cing properties of cocaine. Group1 mGluRs<br />
consist of both mGluR1 <strong>an</strong>d mGluR5 receptors that are coupled to signaling cascades through<br />
Homer. Metabotropic glutamate receptor 5 (mGluR5) regulates cocaine-seeking behavior<br />
(Kumares<strong>an</strong> et al. <strong>2009</strong> Behav. Brain Res. In Press). mGluR1 receptors, like mGluR5 receptors,<br />
are widely expressed throughout <strong>the</strong> brain. Here, we asked whe<strong>the</strong>r mGluR1 located in <strong>the</strong> shell<br />
of <strong>the</strong> nucleus accumbens (NAc) may be involved in <strong>the</strong> reinstatement of cocaine-seeking<br />
behavior in rat. We report <strong>the</strong> results <strong>from</strong> intracr<strong>an</strong>ial microinjections of <strong>the</strong> highly potent <strong>an</strong>d<br />
specific negative modulator of mGluR1, YM 298198 (6-Amino-N-cyclohexyl-N,3dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide<br />
hydrochloride) on reinstatement.<br />
Rats were surgically impl<strong>an</strong>ted with <strong>an</strong> Intra-jugular ca<strong>the</strong>ter <strong>for</strong> cocaine self-administration.<br />
C<strong>an</strong>nulae were impl<strong>an</strong>ted bilaterally in <strong>the</strong> NAc (shell). Coordinates <strong>for</strong> <strong>the</strong> ventral ends of <strong>the</strong><br />
guide c<strong>an</strong>nulae relative to bregma, according to <strong>the</strong> Atlas of Paxinos <strong>an</strong>d Watson were as<br />
follows: + 1.00mm A/P; +/- 1.00mm M/L; -5.0mm D/V. Following a post-surgery recovery
period rats were trained to self-administer cocaine on a fixed ratio schedule (FR) (FR1 followed<br />
by FR5) <strong>for</strong> a total of four weeks followed by extinction training. During this phase, cocaine was<br />
substituted <strong>for</strong> saline. Extinction training typically lasted <strong>for</strong> a week. At <strong>the</strong> end of <strong>the</strong> extinction<br />
period, <strong>the</strong> active lever responses were less th<strong>an</strong> 10% of <strong>the</strong> responses on <strong>the</strong> last day of cocaine<br />
self-administration. YM298198, or vehicle (physiological saline) was microinjected bilaterally<br />
into <strong>the</strong> NAc (shell), using a counterbal<strong>an</strong>ced order of injection,10 minutes prior to a systemic,<br />
cocaine priming injection (10mg/ml/kg). Rats were <strong>the</strong>n placed in oper<strong>an</strong>t chambers <strong>for</strong><br />
responding. The results demonstrate that pharmacological blockade of mGluR1 in <strong>the</strong> NAc<br />
(shell) with YM298198, attenuates reinstatement of cocaine-seeking behavior (p
<strong>the</strong> effects of intermittent (0.8 or 1.6 mg/kg, s.c., twice daily) versus continuous (1.6 or 3.2<br />
mg/kg/day via osmotic minipump) MP administration across 4 weeks of adolescent development<br />
in spont<strong>an</strong>eously hypertensive rats, <strong>the</strong> most commonly utilized rat model of ADHD. Dependent<br />
variables include spont<strong>an</strong>eous locomotor activity, impulsive behavior in a T-maze, <strong>an</strong>d oper<strong>an</strong>t<br />
cocaine self-administration (0.25 mg/kg/infusion). Preliminary data indicate that intermittent MP<br />
administration results in decreased hyperactivity compared to saline-treated SHR controls, but<br />
diminished reward sensitivity. Data collection <strong>from</strong> <strong>the</strong> continuous MP condition is underway. It<br />
is <strong>an</strong>ticipated that <strong>the</strong>se results will clarify <strong>the</strong> potential role of psychomotor stimul<strong>an</strong>t<br />
medications such as MP in mediating <strong>the</strong> risk to addiction among ADHD populations.<br />
Disclosures: N. Tiberi, None; R. Griggs, None; M. Southard, None; C. Weir, None; T.E.<br />
Koeltzow, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.6/Z19<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIDA Gr<strong>an</strong>t R01DA025922<br />
Title: The histone deacetylase inhibitor sodium butyrate enh<strong>an</strong>ces conditioning, extinction, <strong>an</strong>d<br />
reconditioning of a cocaine-induced place preference<br />
Authors: *E. J. MCCLEERY 1 , C. L. CUNNINGHAM 1 , M. A. WOOD 2 , K. M. LATTAL 1 ;<br />
1 Oregon Hlth. <strong>an</strong>d Sci. University|910004441|0, Portl<strong>an</strong>d, OR; 2 Dept. of Neurobio. <strong>an</strong>d<br />
Behavior, Univ. of Cali<strong>for</strong>nia, Irvine, Irvine, CA<br />
Abstract: M<strong>an</strong>y recent studies have demonstrated a role <strong>for</strong> epigenetic mech<strong>an</strong>isms in learning<br />
<strong>an</strong>d memory. Drugs that increase histone acetylation have been shown to enh<strong>an</strong>ce memory<br />
<strong>for</strong>mation in a variety of tasks, including <strong>the</strong> conditioned place preference (CPP) preparation. We<br />
investigated <strong>the</strong> effects of injecting C57BL/6 mice with <strong>the</strong> histone deacetylase inhibitor sodium<br />
butyrate on initial conditioning, extinction, <strong>an</strong>d reconditioning following extinction of cocaineinduced<br />
CPP. We used <strong>an</strong> unbiased CPP design, in which one floor type (CS+) was paired with<br />
cocaine <strong>an</strong>d a second floor type (CS-) was paired with saline. In Experiment 1, we found that<br />
two 15-min pairings of 20 mg/kg cocaine produced a strong preference <strong>for</strong> <strong>the</strong> cocaine-paired<br />
floor, revealed during a preference test in which mice had simult<strong>an</strong>eous access to both floor<br />
types. Two pairings of <strong>the</strong> CS+ floor with 5 mg/kg cocaine did not produce CPP; however,
sodium butyrate injected immediately after <strong>the</strong> cocaine conditioning trial induced a signific<strong>an</strong>t<br />
preference <strong>for</strong> <strong>the</strong> cocaine-paired floor. In Experiment 2, after conditioning with 20 mg/kg<br />
cocaine, repeated nonrein<strong>for</strong>ced preference tests (extinction sessions) eliminated CPP. This<br />
preference extinction was more rapid <strong>an</strong>d persistent when sodium butyrate followed each<br />
extinction session. Finally, in Experiment 3, CPP was established <strong>an</strong>d extinguished. Following<br />
<strong>the</strong> complete extinction of CPP, weak rein<strong>for</strong>cement (5 mg/kg) trials followed by sodium<br />
butyrate injections signific<strong>an</strong>tly increased <strong>the</strong> expression of CPP. These findings demonstrate<br />
that sodium butyrate c<strong>an</strong> enh<strong>an</strong>ce conditioning, extinction, <strong>an</strong>d reconditioning of a cocaineinduced<br />
CPP. These data have import<strong>an</strong>t implications <strong>for</strong> <strong>the</strong> potential use of HDAC inhibitors in<br />
addiction <strong>the</strong>rapy.<br />
Disclosures: E.J. McCleery, None; C.L. Cunningham, None; M.A. Wood, None; K.M.<br />
Lattal, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.7/Z20<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: R01 DA025922<br />
R01 MH081004<br />
Title: Modulation of chromatin modification facilitates extinction of cocaine-induced<br />
conditioned place preference<br />
Authors: *M. MALVAEZ 1 , C. SANCHIS-SEGURA 2 , K. M. LATTAL 3 , D. VO 1 , M. A.<br />
WOOD 1 ;<br />
1 Neurobio. <strong>an</strong>d Behavior, Univ. of Cali<strong>for</strong>nia, Irvine, Irvine, CA; 2 Area de Psicobiologia, Univ.<br />
Jaume I, Castellon, Spain; 3 Behavioral Neurosci., Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: Exposure to cocaine triggers molecular events that lead to long lasting ch<strong>an</strong>ges in<br />
brain structure <strong>an</strong>d function. These ch<strong>an</strong>ges c<strong>an</strong> lead to <strong>the</strong> development of persistent <strong>an</strong>d robust<br />
behavioral adaptations that characterize addiction. Recent evidence suggests that epigenetic<br />
mech<strong>an</strong>isms have <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> development of addictive behavior. However, little is<br />
known about <strong>the</strong> role of epigenetic mech<strong>an</strong>isms in <strong>the</strong> extinction of drug-induced behavioral<br />
ch<strong>an</strong>ges. To investigate <strong>the</strong> role of epigenetic mech<strong>an</strong>isms in extinction of context-drug
associated memories we used a cocaine-induced conditioned place preference paradigm, in<br />
which mice learn to associate a context with cocaine <strong>an</strong>d later show a preference <strong>for</strong> that context.<br />
We demonstrate that increasing histone acetylation via administration of a histone deacetylase<br />
(HDAC) inhibitor following a non-rein<strong>for</strong>ced exposure to <strong>the</strong> previously cocaine-paired<br />
environment signific<strong>an</strong>tly decreases expression of cocaine-induced conditioned place preference.<br />
In addition, by facilitating extinction of context-drug associated memories, reinstatement of<br />
cocaine-conditioned place preference was signific<strong>an</strong>tly attenuated. This study provides <strong>the</strong> first<br />
evidence that modulation of chromatin modification c<strong>an</strong> facilitate extinction in a persistent<br />
m<strong>an</strong>ner that prevents reinstatement of drug-induced behavioral ch<strong>an</strong>ges. These findings provide<br />
a potential novel approach to <strong>the</strong> development of treatments that facilitate extinction of drugseeking<br />
behavior.<br />
Disclosures: M. Malvaez, None; C. S<strong>an</strong>chis-Segura, None; K.M. Lattal, None; D. Vo,<br />
None; M.A. Wood, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.8/Z21<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: Europe<strong>an</strong> Commission, EC gr<strong>an</strong>t no: LSHM-CT-2004-005166<br />
Medical Research Council, UK<br />
British Pharmacological <strong>Society</strong> Integretative Pharmacology Fund<br />
Title: Differences in dopamine D1 <strong>an</strong>d D2 binding between CD1 mice that respond <strong>an</strong>d those that<br />
do not respond to cocaine-induced conditioned place preference<br />
Authors: *R. AL-HASANI, A. METAXAS, A. BAILEY, S. HOURANI, I. KITCHEN;<br />
Fac. of Hlth. <strong>an</strong>d Med. Sci., Univ. of Surrey, Guild<strong>for</strong>d, United Kingdom<br />
Abstract: Studies within our laboratory showed that reliable cocaine-induced conditioned place<br />
preference (CPP) was not observed in <strong>the</strong> CD1 strain of mouse. The response to cocaine-induced<br />
CPP was highly variable <strong>an</strong>d we were able to divide <strong>the</strong> <strong>an</strong>imals into responders <strong>an</strong>d nonresponders.<br />
The responders showed conditioning behaviour, spending <strong>the</strong> majority of time in <strong>the</strong><br />
chamber associated with cocaine ra<strong>the</strong>r th<strong>an</strong> <strong>the</strong> chamber associated with saline, whereas <strong>the</strong>
non-responders spent <strong>the</strong> majority of time in <strong>the</strong> chamber associated with saline ra<strong>the</strong>r th<strong>an</strong><br />
cocaine. Equivalent numbers were observed in both groups.<br />
Due to <strong>the</strong> role of <strong>the</strong> dopaminergic system in <strong>the</strong> rewarding effect of cocaine, it was<br />
hypo<strong>the</strong>sised that differences between components of <strong>the</strong> dopaminergic system of responders<br />
compared to non-responders may reflect <strong>the</strong> cocaine induced CPP difference in CD1 mice.<br />
Qu<strong>an</strong>titative autoradiography was carried out to study dopamine D1, D2, DAT binding <strong>an</strong>d<br />
dopamine D2 receptor-stimulated [ 35 S] GTPγS binding in <strong>the</strong> brains of CD1 cocaine responders<br />
<strong>an</strong>d non-responders.<br />
There was no difference in DAT binding <strong>an</strong>d dopamine D2 activity between responders <strong>an</strong>d nonresponders.<br />
Signific<strong>an</strong>tly lower levels of dopamine D1 receptor binding was observed in <strong>the</strong><br />
rostral caudate-putamen of responders compared to non-responders. Moreover, signific<strong>an</strong>tly<br />
lower levels of dopamine D2 receptor binding were found in <strong>the</strong> caudal <strong>an</strong>d rostral parts of <strong>the</strong><br />
caudate-putamen in responders compared to non-responders.<br />
The differences in DA D1 <strong>an</strong>d DA D2 receptor binding between mice responding <strong>an</strong>d nonresponding<br />
to cocaine-induced CPP suggest that <strong>the</strong> ability of cocaine to induce a rewarding<br />
effect may depend on dopamine D1 <strong>an</strong>d D2 receptor density <strong>an</strong>d/or D1 <strong>an</strong>d D2 receptor regulation<br />
by cocaine. This might provide <strong>an</strong> import<strong>an</strong>t insight on <strong>the</strong> role of <strong>the</strong> dopaminergic system in<br />
<strong>the</strong> vulnerability to develop cocaine addiction.<br />
Disclosures: R. Al-Has<strong>an</strong>i, None; A. Metaxas, None; A. Bailey, None; S. Hour<strong>an</strong>i, None; I.<br />
Kitchen, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.9/Z22<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH gr<strong>an</strong>t DA022447<br />
Title: Stimulus-response associations <strong>for</strong>med during cocaine self-administration in response to<br />
discrete discriminative stimuli<br />
Authors: *K. A. KEEFE 1 , R. P. KESNER 2 , G. R. HANSON 1 , M. D. RIEDY 3 ;<br />
1 2 3<br />
Dept Pharmacol & Toxicol, Psychology, Univ. Utah, Salt Lake City, UT; Neurosci., Med.<br />
Univ. of South Carolina, Charleston, SC
Abstract: Drug-seeking behavior elicited by conditioned rein<strong>for</strong>cers (CR) or discriminative<br />
stimuli (DS) associated with drug use contributes to recidivism in drug-addicted individuals.<br />
Fur<strong>the</strong>rmore, recent work has implicated <strong>the</strong> dorsomedial <strong>an</strong>d dorsolateral aspects of <strong>the</strong> dorsal<br />
striatum in action-outcome <strong>an</strong>d stimulus-response (S-R) associations underlying instrumental<br />
behaviors, respectively. However, delineation of <strong>the</strong> associations between drug-associated CRs<br />
<strong>an</strong>d DS within this framework has not hereto<strong>for</strong>e been reported. To begin evaluating <strong>the</strong> nature<br />
of <strong>the</strong> associations <strong>for</strong>med during such drug self-administration training, rats were trained to selfadminister<br />
cocaine in response to a discrete DS (white house light plus 2-kHz, 78-dB tone)<br />
predictive of cocaine availability. Self-administration training in <strong>the</strong>se rats was followed by two<br />
weeks of extinction sessions with no DS exposure <strong>an</strong>d no drug rein<strong>for</strong>cement. After extinction,<br />
rats were tested to determine whe<strong>the</strong>r DS-elicited lever pressing was resist<strong>an</strong>t to outcome<br />
devaluation--a test <strong>for</strong> S-R association. At <strong>the</strong> end of <strong>the</strong> extinction sessions, <strong>the</strong> rats were<br />
returned to <strong>the</strong> oper<strong>an</strong>t chambers daily <strong>for</strong> four days <strong>for</strong> 15-min sessions in which <strong>the</strong> DS was<br />
presented but active lever pressing no longer resulted in cocaine delivery (outcome devaluation).<br />
O<strong>the</strong>r DS-trained rats were placed into <strong>the</strong> chambers but continued with extinction sessions in<br />
which <strong>the</strong> levers were present but <strong>the</strong> DS was not presented. DS-trained rats presented with <strong>the</strong><br />
drug-associated DS signific<strong>an</strong>tly increased active lever pressing in response to DS presentation<br />
<strong>for</strong> <strong>the</strong> first three of <strong>the</strong> four days of initial testing. All rats <strong>the</strong>n underwent <strong>an</strong> additional oneweek<br />
abstinence period in <strong>the</strong>ir home cages be<strong>for</strong>e being returned to <strong>the</strong> oper<strong>an</strong>t chambers <strong>for</strong> a<br />
final test session. For final testing, rats were split into DS-exposed <strong>an</strong>d DS-not exposed groups<br />
counterbal<strong>an</strong>ced <strong>for</strong> <strong>the</strong>ir initial test lever pressing. Rats presented with <strong>the</strong> DS on this last day<br />
continued to exhibit signific<strong>an</strong>t increases in active lever pressing relative to rats that were not<br />
presented with <strong>the</strong> DS. These data suggest that drug-seeking behavior elicited by a DS<br />
previously predictive of cocaine availability reflects <strong>an</strong> S-R association, as DS-elicited lever<br />
pressing was resist<strong>an</strong>t to outcome devaluation, <strong>the</strong> defining characteristic of S-R associations.<br />
The extent to which <strong>the</strong> dorsolateral striatum is involved in <strong>the</strong> establishment of or relapse to<br />
drug-seeking behavior elicited by <strong>the</strong> DS remains to be determined.<br />
Disclosures: K.A. Keefe, None; R.P. Kesner, None; G.R. H<strong>an</strong>son, None; M.D. Riedy, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.10/Z23<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: USHPS DA06013
Title: The effects of adrenalectomy on <strong>the</strong> mainten<strong>an</strong>ce of cocaine self-administration in rats<br />
Authors: *G. F. GUERIN, N. E. GOEDERS;<br />
Pharmacology, Toxicology & Neurosci., LSUHSC, Shreveport, LA<br />
Abstract: Previous research has shown that <strong>the</strong> hypothalamic-pituitary-adrenal axis is involved<br />
in psychostimul<strong>an</strong>t rein<strong>for</strong>cement. Specifically, we have found that bilateral adrenalectomy<br />
completely abolished <strong>the</strong> acquisition of intravenous cocaine self-administration without affecting<br />
food maintained responding. This suppression of self-administration was partially reversed by<br />
adding corticosterone to <strong>the</strong> rats' drinking water. In <strong>an</strong>o<strong>the</strong>r experiment, pretreatment with<br />
metyrapone, which blocks <strong>the</strong> syn<strong>the</strong>sis of corticosterone, resulted in dose-related decreases in<br />
ongoing cocaine self-administration. The experiment presented here was designed to fur<strong>the</strong>r<br />
determine <strong>the</strong> role <strong>for</strong> corticosterone in <strong>the</strong> mainten<strong>an</strong>ce of cocaine rein<strong>for</strong>cement in rats by<br />
decreasing plasma levels of <strong>the</strong> hormone with surgical adrenalectomy. Adult male Wistar rats<br />
were trained to respond under a 2-hour multiple, alternating schedule of food rein<strong>for</strong>cement <strong>an</strong>d<br />
cocaine self-administration (fixed-ratio 4) during alternating 15-min periods. After responding<br />
stabilized, <strong>the</strong> rats were exposed to one week of saline substitution <strong>an</strong>d food extinction to train<br />
<strong>the</strong> <strong>an</strong>imals to stop responding in <strong>the</strong> absence of <strong>the</strong> rein<strong>for</strong>cers. Self-administration was <strong>the</strong>n<br />
reestablished <strong>for</strong> at least one to two weeks <strong>an</strong>d until stable. Adrenalectomy or sham surgeries<br />
were <strong>the</strong>n per<strong>for</strong>med <strong>an</strong>d <strong>the</strong> <strong>an</strong>imals allowed to recover <strong>for</strong> one week be<strong>for</strong>e restarting selfadministration<br />
<strong>for</strong> three weeks. Plasma corticosterone was measured to verify <strong>the</strong><br />
adrenalectomies. We report that bilateral adrenalectomy does not abolish ongoing cocaine selfadministration,<br />
but does lower it by about 25%, without affecting food rein<strong>for</strong>cement. These data<br />
suggest that corticosterone, although necessary <strong>for</strong> <strong>the</strong> acquisition of cocaine rein<strong>for</strong>cement, may<br />
not be necessary <strong>for</strong> <strong>the</strong> mainten<strong>an</strong>ce of cocaine rein<strong>for</strong>cement in rats.<br />
Disclosures: G.F. Guerin, None; N.E. Goeders, PI gr<strong>an</strong>t USPHS DA06013, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); patent, common stock with Embera NeuroTherapeutics, Inc, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); consult<strong>an</strong>t <strong>for</strong> Embera<br />
NeuroTherapeutics, Inc., F. Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.11/Z24<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement
Support: Psi Chi Undergraduate Research Gr<strong>an</strong>t (NT)<br />
Psi Chi Undergraduate Research Gr<strong>an</strong>t (RG)<br />
Bradley University Special Emphasis Gr<strong>an</strong>t (TK)<br />
Psi Chi Undergraduate Reserach Gr<strong>an</strong>t (WW)<br />
Title: Continuous methylphenidate administration during development results in decreased<br />
cocaine self-administration in rats<br />
Authors: *R. A. GRIGGS, N. L. TIBERI, D. K. TAYLOR, W. WAYMAN, K. KOTWICA, C.<br />
WEIR, T. E. KOELTZOW;<br />
Dept. of Psychology, Bradley Univ., Peoria, IL<br />
Abstract: Attention-Deficit/Hyperactivity Disorder (ADHD)is linked to disruptions in dopamine<br />
tr<strong>an</strong>sporter (DAT) function <strong>an</strong>d is often treated with <strong>the</strong> DAT blocker methylphenidate (MP).<br />
Adolescent exposure to repeated, intermittent MP produces long-lasting behavioral ch<strong>an</strong>ges in<br />
rats, including increased acquisition of cocaine self-administration. Whereas intermittent cocaine<br />
injections promote behavioral <strong>an</strong>d neurophysiological adaptations linked to subst<strong>an</strong>ce abuse,<br />
continuous infusion is associated with toler<strong>an</strong>ce to <strong>the</strong>se effects. Thus, <strong>the</strong> ability of stimul<strong>an</strong>t<br />
treatment to promote addictive behavior in rats may depend on <strong>the</strong> route of administration. The<br />
current study assessed <strong>the</strong> effects of intermittent (0.8 or 1.6 mg/kg, s.c., twice daily) versus<br />
continuous (1.6 or 3.2 mg/kg/day via osmotic minipump) MP administration across 4 weeks of<br />
adolescent development in rats. Results indicate <strong>the</strong> intermittent MP promotes hyperlocomotor<br />
responses to a novel open field <strong>an</strong>d in a T-Maze task of impulsivity. However, continuous MP<br />
was associated with increased adaptive responding in <strong>the</strong> T-Maze task. Finally, whereas<br />
intermittent MP resulted in increased cocaine seeking behavior <strong>an</strong>d a sensitized locomotor<br />
response to non-contingent cocaine administration (10 mg/kg), continuously-treated rats did not<br />
show this effect. Taken toge<strong>the</strong>r, <strong>the</strong>se data indicate that sustained release <strong>for</strong>mulations of MP<br />
are likely to elicit toler<strong>an</strong>ce to <strong>the</strong> rein<strong>for</strong>cing properties of psychostimul<strong>an</strong>t medications <strong>an</strong>d are<br />
not likely to promote addiction.<br />
Disclosures: R.A. Griggs, None; N.L. Tiberi, None; D.K. Taylor, None; W. Waym<strong>an</strong>,<br />
None; K. Kotwica, None; C. Weir, None; T.E. Koeltzow, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 553.12/Z25<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: Korea Goverment (KRF-2007-313E00595)<br />
Brain korea 21<br />
Title: Acupuncture attenuates reinstatement of cocaine-seeking behavior via mu-receptors in <strong>the</strong><br />
nucleus accumbens<br />
Authors: H. KIM 1 , B. LEE 2 , S. YOON 1 , K. KIM 1 , C. YANG 1 , *S. CHOI 3 ;<br />
1 2 3<br />
Physiol., Acupuncture, Daegu Ha<strong>an</strong>y Univ., Daegu, Republic of Korea; Anat. & Histology,<br />
Daegu Ha<strong>an</strong>y Univ., Daegu.<br />
Abstract: Opioid receptors play a role in modulating dopamine release in <strong>the</strong> nucleus<br />
accumbens <strong>an</strong>d cocaine-seeking behavior. Our previous study showed that acupuncture at<br />
Shenmen (HT7) points decreased reinstatement of cocaine-seeking behavior <strong>an</strong>d dopamine<br />
release in <strong>the</strong> nucleus accumbens induced by <strong>the</strong> cocaine challenge. The present study was<br />
designed to explore <strong>the</strong> potential role of opioid receptors in acupuncture-mediated inhibition of<br />
reinstatement of cocaine-seeking behavior. Male Sprague-Dawley rats were trained to selfadminister<br />
intravenous cocaine (0.5 mg/kg/injection) in 4-h within-session<br />
extinction/reinstatement paradigm. Once rats showed stable responding (active lever ≤5) during<br />
<strong>the</strong> final 1-h of extinction session, acupuncture was applied at bilateral HT7 <strong>for</strong> 1 min be<strong>for</strong>e<br />
intravenous priming with cocaine (2.0 mg/kg). Bilateral microinjections of <strong>the</strong> mu-opioid<br />
receptor agonist DAMGO (1.5 ng/side) or <strong>the</strong> kappa-opioid receptor <strong>an</strong>tagonist Nor-BNI (10<br />
ng/side) into <strong>the</strong> nucleus accumbens were given be<strong>for</strong>e <strong>the</strong> acupuncture treatment. Results<br />
showed that acupuncture at <strong>the</strong> specific acupoint HT7 signific<strong>an</strong>tly attenuated <strong>the</strong> reinstatement<br />
of cocaine-seeking behavior. Most import<strong>an</strong>tly, our results demonstrated that DAMGO, but not<br />
Nor-BNI blocked <strong>the</strong> inhibition of cocaine-primed reinstatement by acupuncture. These results<br />
suggest that acupuncture attenuates cocaine-seeking behavior via mu-opioid receptors in <strong>the</strong><br />
nucleus accumbens.<br />
Disclosures: H. Kim, None; B. Lee, None; S. Yoon, None; K. Kim, None; C. Y<strong>an</strong>g, None; S.<br />
Choi, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 553.13/Z26<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Title: Effects of <strong>the</strong> dopamine receptor <strong>an</strong>tagonist SKF 83566 on cocaine rein<strong>for</strong>cement <strong>an</strong>d<br />
seeking<br />
Authors: *J. MORRISON 1,2 , F. KAHN 1 , R. RANALDI 1 ;<br />
1 Queens Col, CUNY, Flushing, NY; 2 The Grad. Center, CUNY, New York, NY<br />
Abstract: Most studies on <strong>the</strong> role of dopamine (DA) in cocaine reward have not differentiated<br />
between its role in cocaine rein<strong>for</strong>cement or in cocaine-seeking (i.e., cocaine’s incentivemotivational<br />
effect). In <strong>the</strong> present experiment, we examined <strong>the</strong> effects of a selective DA D1<br />
receptor <strong>an</strong>tagonist (0.08, 0.12 <strong>an</strong>d 0.16 mg/kg SKF 83566, IP) on lever pressing in two<br />
procedures: one where pressing is indicative of cocaine rein<strong>for</strong>cement <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r where it is<br />
indicative of cocaine-seeking. Rats were trained to self-administer intravenous cocaine (1<br />
mg/kg/injection) under a fixed-ratio 1 (FR1) schedule of rein<strong>for</strong>cement. After training, all<br />
<strong>an</strong>imals entered a pre-test phase consisting of 10 daily consecutive sessions where only 1 cocaine<br />
injection per session could be earned under <strong>the</strong> FR1 schedule. This was followed by a test<br />
session in which lever pressing was not rein<strong>for</strong>ced. Groups tested in <strong>the</strong> cocaine rein<strong>for</strong>cement<br />
procedure received IP injections of vehicle or a dose of SKF83566 prior to <strong>the</strong> last 5 pre-test<br />
sessions <strong>an</strong>d no treatment prior to <strong>the</strong> test session. Groups tested in <strong>the</strong> cocaine-seeking<br />
procedure received no treatment prior to <strong>the</strong> pre-test sessions, but <strong>an</strong> IP injection of vehicle or a<br />
dose of SKF 83566 prior to <strong>the</strong> test session. The data show a dose-related reduction in active<br />
lever presses during <strong>the</strong> test session in both <strong>the</strong> rein<strong>for</strong>cement <strong>an</strong>d cocaine-seeking groups<br />
compared to <strong>the</strong> respective vehicle groups. These findings suggest that DA is involved in both<br />
cocaine rein<strong>for</strong>cement <strong>an</strong>d cocaine-seeking.<br />
Disclosures: J. Morrison, None; F. Kahn, None; R. R<strong>an</strong>aldi, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.14/Z27<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH Gr<strong>an</strong>t R01 DA13724
NIH Gr<strong>an</strong>t R01 MH49469<br />
NIH Gr<strong>an</strong>t F31 DA021968<br />
NIH Gr<strong>an</strong>t T32 GM008541<br />
NIH Gr<strong>an</strong>t T32 AG00115<br />
NIH Contract N01 DA-2-8822<br />
PhRMA Foundation Fellowship<br />
Title: Cocaine-priming induced reinstatement of drug seeking is attenuated by pregn<strong>an</strong>olone<br />
hemisuccinate, a negative modulator of glutamate receptor function<br />
Authors: *S. C. DESBIENS, M. H. RATNER, M. T. WHITTAKER, K. KANAGARAYER, R.<br />
ALI, K. R. FAMOUS, H. D. SCHMIDT, S. DOWNING, V. KUMARESAN, R. C. PIERCE, T.<br />
T. GIBBS, D. H. FARB;<br />
Dept of Pharmacol., Boston Univ., Boston, MA<br />
Abstract: The discovery of a nonaddictive <strong>the</strong>rapeutic that diminishes relapse of cocaineseeking<br />
behavior would be a major adv<strong>an</strong>ce in <strong>the</strong> treatment of cocaine addiction. Glutamate<br />
receptor activation mediates reinstatement of cocaine-seeking behavior in rats. Here, we asked<br />
whe<strong>the</strong>r pregn<strong>an</strong>olone hemisuccinate (3α5βHS), a syn<strong>the</strong>tic negatively charged neuroactive<br />
steroid that penetrates <strong>the</strong> blood-brain barrier, could attenuate <strong>the</strong> reinstatement of cocaineseeking<br />
behavior. Our electrophysiological studies demonstrate that 3α5βHS is a negative<br />
modulator of recombin<strong>an</strong>t NMDA <strong>an</strong>d AMPA receptors expressed in Xenopus oocytes,<br />
exhibiting <strong>an</strong> 95- to 250-fold higher potency toward NR1/NR2A <strong>an</strong>d GluR1 as compared with<br />
<strong>the</strong> NR2B, 2C, 2D or GluR3 subtypes. 3α5βHS at a concentration sufficient to negatively<br />
modulate ionotropic glutamate receptor function also inhibits <strong>the</strong> NMDA-induced release of<br />
[ 3 H]dopamine <strong>an</strong>d [ 14 C]glutamate <strong>from</strong> superfused isolated rat striatal nerve terminals. In a rat<br />
model, injection of 3α5βHS (i.p.) does not substitute <strong>for</strong> cocaine in a discriminative stimulus<br />
task, nor does it <strong>an</strong>tagonize discrimination of cocaine. Pretreatment of rats self-administering<br />
cocaine with 10 mg/kg 3α5βHS does not affect <strong>the</strong> <strong>an</strong>imal’s motivation to obtain <strong>the</strong> drug as<br />
determined using a progressive ratio schedule of rein<strong>for</strong>cement. By contrast, 10 mg/kg 3α5βHS<br />
pretreatment reduces drug-seeking behavior in <strong>an</strong>imals undergoing cocaine priming-induced<br />
reinstatement. It will be interesting to determine <strong>the</strong> site of action of 3α5βHS in <strong>the</strong> brain. Taken<br />
toge<strong>the</strong>r, <strong>the</strong>se data indicate that syn<strong>the</strong>tic negatively charged neuroactive steroids, such as<br />
3α5βHS, may be a useful plat<strong>for</strong>m <strong>for</strong> <strong>the</strong> discovery of novel non-addictive <strong>the</strong>rapeutics <strong>for</strong> <strong>the</strong><br />
treatment of cocaine craving.<br />
Disclosures: S.C. Desbiens, None; M.H. Ratner, None; M.T. Whittaker, None; K.<br />
K<strong>an</strong>agarayer, None; R. Ali, None; K.R. Famous, None; H.D. Schmidt, None; S. Downing,<br />
None; V. Kumares<strong>an</strong>, None; R.C. Pierce, None; T.T. Gibbs, None; D.H. Farb, None.
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.15/Z28<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIDA DA017673<br />
NIDA DA017673-S1<br />
Title: Involvement of <strong>the</strong> lateral entorhinal cortex <strong>an</strong>d ventral subiculum in drug context-induced<br />
reinstatement of cocaine-seeking behavior in rats<br />
Authors: *D. R. RAMIREZ, X. XIE, H. C. LASSETER, S. J. KASZYCKI, K. L. PONDS, R.<br />
A. FUCHS;<br />
Psychology Dept., Univ. of North Carolina, Chapel Hill, NC<br />
Abstract: The functional integrity of <strong>the</strong> dorsal (DH) <strong>an</strong>d ventral (VH) subregions of <strong>the</strong><br />
hippocampus is critical <strong>for</strong> drug-context induced reinstatement of cocaine-seeking behavior.<br />
Fur<strong>the</strong>rmore, <strong>an</strong>atomical studies suggest that <strong>the</strong> DH <strong>an</strong>d VH may communicate with <strong>the</strong> rest of<br />
<strong>the</strong> contextual relapse circuitry via different input/output regions, such as <strong>the</strong> lateral entorhinal<br />
cortex (lEnt) <strong>an</strong>d dorsal subiculum, as well as <strong>the</strong> ventral subiculum (vSub) <strong>an</strong>d medial<br />
entorhinal cortex, respectively. Thus, <strong>the</strong> purpose of <strong>the</strong> present study was to begin investigating<br />
<strong>the</strong> contributions of hippocampal input/output regions, specifically <strong>the</strong> lEnt <strong>an</strong>d vSub, to contextinduced<br />
cocaine seeking behavior. Rats were trained to lever press <strong>for</strong> un-signaled cocaine<br />
infusions (0.15 mg/infusion, i.v.) in a distinct environmental context (cocaine-paired context).<br />
Responding was <strong>the</strong>n extinguished in a different context where lever presses had no programmed<br />
consequences (extinction context). Subsequently, rats were re-exposed to ei<strong>the</strong>r <strong>the</strong> cocainepaired<br />
context or <strong>the</strong> extinction context in <strong>the</strong> absence of cocaine rein<strong>for</strong>cement to test <strong>for</strong><br />
cocaine-seeking behavior (i.e., non-rein<strong>for</strong>ced lever responses). Prior to testing, neural activity<br />
was temporarily inhibited in <strong>the</strong> lEnt, vSub, or ectorhinal cortex (Ect; <strong>an</strong>atomical control) using<br />
tetrodotoxin (5 ng/0.5 µl/side) or vehicle (0.5 µl/side). Intra-lEnt or intra-vSub tetrodotoxin<br />
infusions attenuated <strong>the</strong> reinstatement of cocaine-seeking behavior in <strong>the</strong> previously cocainepaired<br />
context without altering responding in <strong>the</strong> extinction context, relative to vehicle. In<br />
contrast, intra-Ect tetrodotoxin infusions failed to alter cocaine-seeking behavior, relative to<br />
vehicle. Toge<strong>the</strong>r, <strong>the</strong>se data suggest that <strong>the</strong> lEnt <strong>an</strong>d vSub, but not <strong>the</strong> Ect, play a critical role in<br />
drug-context-induced cocaine seeking. Thus, <strong>the</strong>se brain regions may indeed facilitate<br />
communication between <strong>the</strong> hippocampus <strong>an</strong>d o<strong>the</strong>r components of <strong>the</strong> contextual relapse<br />
circuitry.
Disclosures: D.R. Ramirez, None; X. Xie, None; H.C. Lasseter, None; S.J. Kaszycki,<br />
None; K.L. Ponds, None; R.A. Fuchs, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.16/Z29<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH Gr<strong>an</strong>t DA21420<br />
NIH Gr<strong>an</strong>t DA11792<br />
NIH Gr<strong>an</strong>t AA16179<br />
NIH Gr<strong>an</strong>t RR00168<br />
Title: Locomotor-activating <strong>an</strong>d o<strong>the</strong>r behavioral effects of long-term cocaine selfadministration<br />
in rhesus monkeys<br />
Authors: *N. M. SHINDAY 1,2 , D. M. PLATT 1 , W. YAO 1,2 , J. K. ROWLETT 1,2 ;<br />
1 Behavioral Biol., New Engl<strong>an</strong>d Primate Res. Center/Harvard Med. Sch., Southborough, MA;<br />
2 Neurosci. <strong>an</strong>d Behavior, Univ. of Massachusetts at Amherst, Amherst, MA<br />
Abstract: Behavioral sensitization (i.e., a progressive increase in locomotor activity as a<br />
consequence of repeated drug exposure) is often used to model certain aspects of addiction, in<br />
particular neural adaptations in <strong>the</strong> brain reward circuitry following long-term drug exposure.<br />
However, research conducted with primates, including hum<strong>an</strong>s, has not consistently reported<br />
behavioral sensitization following repeated cocaine treatment. To explore fur<strong>the</strong>r <strong>the</strong> extent to<br />
which sensitization develops to <strong>the</strong> locomotor-activating effects of cocaine in monkeys, we<br />
examined ch<strong>an</strong>ges in drug induced behaviors by repeated intravenous (i.v.) cocaine selfadministration.<br />
Rhesus monkeys were impl<strong>an</strong>ted with i.v. ca<strong>the</strong>ters <strong>an</strong>d allowed to selfadminister<br />
cocaine (0.03 mg/kg/injection) <strong>for</strong> 1 hour/day or a daily maximum of 3.0 mg/kg (100<br />
injections) under a continuous rein<strong>for</strong>cement schedule <strong>for</strong> approximately 100 days. An additional<br />
group (yoked control) received a passive infusion of saline coinciding with each cocaine<br />
injection. Locomotor activity was measured in 1 minute intervals, 24 hours per day using <strong>the</strong><br />
Minimitter Actiwatch system. In addition, species-typical behaviors were recorded using a<br />
modified frequency scoring system, a qu<strong>an</strong>titative behavioral observation technique. We focused
on observable psychostimul<strong>an</strong>t related effects including stereotypy (repetitive, ritualized pattern<br />
of behavior lacking obvious function), locomotor (two directed steps in <strong>the</strong> horizontal <strong>an</strong>d/or<br />
vertical pl<strong>an</strong>e), <strong>an</strong>d lever press (depression of oper<strong>an</strong>t lever) <strong>an</strong>d tactile/oral (tactile or oral<br />
m<strong>an</strong>ipulation of environment). During self-administration sessions, behavior varied <strong>from</strong> day-today<br />
<strong>from</strong> about 20 to 100 injections per session, although a gradual increase in drug taking was<br />
observed across sessions. The cocaine-exposed <strong>an</strong>imals exhibited no consistent increases in<br />
locomotor activity across daily self-administration sessions, measured by Actiwatch or<br />
observation techniques. Fur<strong>the</strong>rmore, no ch<strong>an</strong>ges in stereotypy, lever press, <strong>an</strong>d tactile/oral<br />
behaviors were observed across sessions, although marked individual differences were observed<br />
in some cases. These findings suggest that non-hum<strong>an</strong> primates may not undergo behavioral<br />
sensitization to motor effects following prolonged cocaine self-administration, raising <strong>the</strong><br />
possibility of <strong>an</strong> import<strong>an</strong>t species difference with respect to <strong>the</strong> role of sensitization in<br />
rein<strong>for</strong>cement processes.<br />
Disclosures: N.M. Shinday, None; D.M. Platt, None; W. Yao, None; J.K. Rowlett, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.17/Z30<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH Gr<strong>an</strong>t DA021460<br />
NIH Gr<strong>an</strong>t DA012513<br />
Title: Intra-infralimbic cortex microinjections of PEPA enh<strong>an</strong>ce <strong>an</strong>d impair <strong>the</strong> consolidation<br />
<strong>an</strong>d expression, respectively, of extinction training of cocaine self-administration<br />
Authors: *R. T. LALUMIERE 1 , K. J. CORSON 2 , P. W. KALIVAS 1 ;<br />
1 Dept Neurosci, Med. Univ. South Carolina, Charleston, SC; 2 Col. of Charleston, Charleston, SC<br />
Abstract: The infralimbic cortex (IL) plays a critical role in <strong>the</strong> consolidation <strong>an</strong>d expression of<br />
extinction memories <strong>for</strong> cocaine-seeking behavior. Whe<strong>the</strong>r increased IL activation with <strong>the</strong><br />
AMPA receptor agonist PEPA enh<strong>an</strong>ces <strong>the</strong> consolidation of extinction training or reduces cueinduced<br />
reinstatement is unknown. There<strong>for</strong>e, <strong>the</strong> current experiments examined <strong>the</strong>se questions.<br />
Male Sprague-Dawley rats (225-250 g) underwent surgeries <strong>for</strong> impl<strong>an</strong>tation of <strong>an</strong> intravenous<br />
ca<strong>the</strong>ter, a double-barreled c<strong>an</strong>nula aimed at <strong>the</strong> IL, <strong>an</strong>d, in some cases, bilateral c<strong>an</strong>nulas aimed
at <strong>the</strong> basolateral amygdala (BLA). In <strong>the</strong> first set of experiments, rats underwent two weeks of<br />
cocaine self-administration (2 hr sessions/day) followed by extinction training. For extinction<br />
days 1-5, rats underwent 15-minute extinction sessions. Immediately after each session on days<br />
1-5, <strong>the</strong> rats received intra-IL microinjections (0.3 µl) of <strong>the</strong> allosteric AMPA receptor agonist<br />
PEPA (30 ng) or vehicle. For day 6 of extinction <strong>an</strong>d beyond, all sessions were 2 hrs <strong>an</strong>d no<br />
microinjections were given. Rats that received PEPA microinjections had enh<strong>an</strong>ced retention of<br />
<strong>the</strong>ir extinction training, as indicated by reduced active lever pressing on days 6-12. This<br />
increased lever pressing compared to vehicle-controls persisted <strong>for</strong> several days. These findings<br />
suggest that stimulation of IL AMPA receptors immediately after each day of extinction training<br />
on days 1-5 enh<strong>an</strong>ced <strong>the</strong> memory consolidation of <strong>the</strong> extinction learning. In a second set of<br />
experiments, rats underwent two weeks of cocaine self-administration, in which cocaine<br />
infusions were paired with a light <strong>an</strong>d tone, <strong>an</strong>d <strong>the</strong>n extinction training (2 hr/day) <strong>for</strong> a<br />
minimum of 12 days. Rats <strong>the</strong>n underwent cue-induced reinstatement. Immediately prior to <strong>the</strong><br />
reinstatement session, rats received intra-IL microinjections of PEPA or vehicle. Those rats<br />
receiving PEPA had decreased active lever presses during <strong>the</strong> reinstatement session, <strong>an</strong> effect<br />
that was reversed by concurrent microinjections (0.3 µl) of dopamine (15 µg) into <strong>the</strong> BLA.<br />
Toge<strong>the</strong>r, <strong>the</strong>se findings indicate that pharmacological activation of <strong>the</strong> IL enh<strong>an</strong>ces memory<br />
consolidation of extinction training after cocaine self-administration <strong>an</strong>d reduces drug-seeking<br />
behavior during cue-induced reinstatement.<br />
Disclosures: R.T. LaLumiere, None; K.J. Corson, None; P.W. Kalivas, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.18/Z31<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: P50 DA15369<br />
T32 DA07288<br />
Title: Activity-regulated cytoskeleton gene expression in <strong>the</strong> dorsal striatum is decreased by<br />
<strong>an</strong>tisense knockdown in cocaine-seeking rats<br />
Authors: *M. C. HEARING, A. J. BACHE, R. E. SEE, J. F. MCGINTY;<br />
Dept Neurosci, MUSC, Charleston, SC
Abstract: The dorsal striatum (DS) has been implicated in stimulus-response (habit) learning,<br />
<strong>an</strong>d accumulating evidence suggests that neuroadaptive ch<strong>an</strong>ges in <strong>the</strong> DS mediate <strong>the</strong><br />
compulsive nature of drug-seeking following chronic cocaine self-administration. Recent<br />
findings <strong>from</strong> our laboratory have demonstrated that re-exposure to <strong>an</strong> oper<strong>an</strong>t chamber<br />
previously associated with cocaine, but not yoked-saline, increases mRNA expression of <strong>the</strong><br />
activity-regulated cytoskeleton gene (arc) within <strong>the</strong> DS following prolonged abstinence. These<br />
increases were fur<strong>the</strong>r correlated with <strong>the</strong> amount of drug-seeking expressed as lever pressing<br />
during <strong>the</strong> relapse test. Thus, in this study, we tested <strong>the</strong> hypo<strong>the</strong>sis that <strong>an</strong>tisense gene<br />
knockdown of arc expression within <strong>the</strong> DS would alter cocaine-seeking. In Experiment 1, a<br />
single infusion of arc <strong>an</strong>tisense oligodeoxynucleotide (AS-ODN; 2 nmol in 1 µl) into <strong>the</strong> lateral<br />
DS signific<strong>an</strong>tly attenuated <strong>the</strong> induction of arc mRNA produced by a single cocaine exposure<br />
(20 mg/kg, i.p.) as compared to scrambled-ODN (S-ODN) -infused controls. However, cocaineinduced<br />
locomotor activity did not signific<strong>an</strong>tly differ between cocaine AS- <strong>an</strong>d S-ODN-treated<br />
rats. In Experiment 2, we investigated <strong>the</strong> effects of a single arc AS-ODN infusion into <strong>the</strong><br />
lateral DS of rats 15 days after <strong>the</strong> end of chronic cocaine self-administration <strong>an</strong>d prior to a 1-h<br />
test of relapse. Arc mRNA was signific<strong>an</strong>tly increased within <strong>the</strong> DS of S-ODN infused rats reexposed<br />
to a previously cocaine-paired oper<strong>an</strong>t chamber (COC-S) when compared to S-ODN<br />
infused yoked-saline controls (SAL-S). Additionally, COC-S rats expressed signific<strong>an</strong>tly greater<br />
Arc mRNA levels in <strong>the</strong> DS th<strong>an</strong> AS-ODN-infused rats with a cocaine history (COC-AS). Arc<br />
AS-ODN did not affect zif268 mRNA expression. Arc protein levels did not signific<strong>an</strong>tly differ<br />
in cocaine-treated vs. yoked-saline rats 1-h after re-exposure to <strong>the</strong> oper<strong>an</strong>t chamber; however,<br />
<strong>the</strong>re was a trend towards a decrease in Arc protein within <strong>the</strong> lateral DS of both cocaine <strong>an</strong>d<br />
yoked-saline AS-ODN-infused rats as compared to S-ODN controls. Fur<strong>the</strong>r <strong>an</strong>alyses will<br />
examine <strong>the</strong> relationship of cocaine-seeking <strong>an</strong>d Arc expression during relapse. This study<br />
indicates <strong>the</strong> feasibility of suppressing cocaine context-induced Arc mRNA expression by<br />
<strong>an</strong>tisense gene knockdown in order to investigate <strong>the</strong> role of striatal Arc in drug-seeking.<br />
Supported by P50 DA15369 <strong>an</strong>d T32 DA07288.<br />
Disclosures: M.C. Hearing, None; A.J. Bache, None; R.E. See, None; J.F. McGinty, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.19/Z32<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH DA014025
Title: Sex differences in cocaine preference of adult rats emerge only after mid-adolescence, <strong>an</strong>d<br />
adult cocaine preference c<strong>an</strong> be decreased by exposure to <strong>an</strong> enriched environment during <strong>the</strong><br />
postnatal period<br />
Authors: J. C. BASSO, *J. I. MORRELL;<br />
Rutgers Univ., Newark, NJ<br />
Abstract: The National Survey on Drug Use <strong>an</strong>d Health indicates that approximately 9.5% of<br />
hum<strong>an</strong> adolescents (ages 12-17), 19.7% of young adults (ages 18-25), <strong>an</strong>d 5.8% of adults (age 26<br />
or older) use illicit drugs. Because of <strong>the</strong>se facts <strong>an</strong>d <strong>the</strong> finding that early drug use is related to<br />
increased levels of drug use in adulthood, finding ways to naturally decrease <strong>the</strong> salience of<br />
drugs of abuse is <strong>an</strong> import<strong>an</strong>t focus <strong>for</strong> preclinical studies. Our laboratory utilized a threechambered<br />
conditioned place preference (CPP) paradigm to investigate <strong>the</strong> rat’s proclivity<br />
towards cocaine, a stimul<strong>an</strong>t drug of abuse, throughout postnatal development. During four 30<br />
minute conditioning sessions, juvenile (p18-22), adolescent (p28-32), <strong>an</strong>d adult (p85-89) male<br />
<strong>an</strong>d female Sprague Dawley rats learned to associate one CPP chamber with saline <strong>an</strong>d a distinct<br />
chamber with cocaine. The preference <strong>for</strong> a cocaine-associated chamber, using a 5.0 mg/Kg<br />
dosage of cocaine given intraperitoneally (IP), increased <strong>from</strong> 18% of <strong>an</strong>imals at p22 to 44% of<br />
<strong>an</strong>imals at p32, with no sex differences emerging at ei<strong>the</strong>r age. By adulthood, a clear sex<br />
difference was seen. Seventy-five percent of adult females preferred <strong>the</strong> cocaine-associated<br />
chamber <strong>for</strong> very low doses of cocaine (0.5-5.0mg/Kg, IP), but males did not show a preference<br />
<strong>for</strong> <strong>the</strong> cocaine-associated chamber with a 0.5, 5.0, or 10.0 mg/Kg dosage of IP cocaine,<br />
indicating that <strong>the</strong>se doses were not of interest to <strong>the</strong> males. Previous research indicates that<br />
enriched environments c<strong>an</strong> induce plastic <strong>an</strong>d <strong>the</strong>rapeutic effects on a variety of <strong>an</strong>imal models,<br />
so we tested whe<strong>the</strong>r postnatal enrichment would decrease cocaine preference in adult rats. After<br />
we<strong>an</strong>ing (p21), rats were group housed with a running wheel <strong>an</strong>d various enrichment objects,<br />
<strong>the</strong>n tested in adulthood <strong>for</strong> cocaine CPP. In marked contrast to those raised in non-enriched<br />
environments (75% cocaine preference), <strong>the</strong>se enriched females did not have a CPP <strong>for</strong> a 0.5<br />
mg/Kg dosage of IP cocaine, suggesting that <strong>the</strong> environmental enrichment may have altered <strong>the</strong><br />
salience of <strong>the</strong> drug, making it no longer <strong>an</strong> attractive or motivating stimulus. We conclude that<br />
in terms of preference <strong>for</strong> cocaine, male <strong>an</strong>d female Sprague Dawley rats show similar<br />
developmental trajectories but have altered motivation <strong>for</strong> <strong>the</strong> drug as adults, <strong>an</strong>d that<br />
environmental enrichment has <strong>the</strong> ability to decrease <strong>the</strong> hedonic properties of this stimul<strong>an</strong>t<br />
drug of abuse.<br />
Disclosures: J.C. Basso, None; J.I. Morrell, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 553.20/Z33<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: MRC Gr<strong>an</strong>t G9536855<br />
Title: The selective noradrenaline re-uptake inhibitor atomoxetine attenuates cocaine-seeking<br />
behaviour in rats<br />
Authors: D. ECONOMIDOU, *D. E. THEOBOLD, J. W. DALLEY, B. J. EVERITT;<br />
Dept Exp Psychol <strong>an</strong>d Behavioural <strong>an</strong>d Clin. Neurosci. Inst., Univ. Cambridge, Cambridge,<br />
United Kingdom<br />
Abstract: Compulsive cocaine-seeking, a hallmark of drug addiction, is strongly influenced by<br />
<strong>the</strong> presence of conditioned rein<strong>for</strong>cers. Hum<strong>an</strong> studies have shown signific<strong>an</strong>tly positive<br />
correlations between <strong>the</strong> impulsivity scores of addicted patients <strong>an</strong>d drug-cue reactivity,<br />
suggesting <strong>an</strong> import<strong>an</strong>t role of impulsive behaviour in cue-controlled drug-seeking. Here, we<br />
investigated whe<strong>the</strong>r <strong>the</strong> selective noradrenaline re-uptake inhibitor, atomoxetine, a drug shown<br />
to reduce impulsivity, may also be effective in reducing cocaine-seeking maintained by<br />
conditioned stimuli.<br />
Rats were trained to self-administer cocaine (0.25 mg/infusion) under a second-order schedule of<br />
rein<strong>for</strong>cement [FI15(FR10:CS)]. Following acquisition of a stable baseline of responding, rats<br />
were IP treated with atomoxetine (0.0, 0.3-3.0 mg/kg), in a within-subject design, <strong>an</strong>d <strong>the</strong>n tested<br />
under <strong>the</strong> second-order schedule of cocaine rein<strong>for</strong>cement. Atomoxetine dose-dependently<br />
induced a signific<strong>an</strong>t decrease in responding during <strong>the</strong> first drug free interval (cue-controlled<br />
cocaine-seeking interval), whereas at <strong>the</strong> highest dose it also signific<strong>an</strong>tly reduced responding<br />
during <strong>the</strong> second interval, that is, after cocaine had been self-administered. This effect of<br />
atomoxetine was selective <strong>for</strong> cocaine-seeking maintained by conditioned rein<strong>for</strong>cers since it had<br />
no signific<strong>an</strong>t effect on responding <strong>for</strong> cocaine under similar conditions (FI15 min) but in <strong>the</strong><br />
absence of <strong>the</strong> conditioned rein<strong>for</strong>cer. Moreover, in subsequent studies we showed that <strong>the</strong>se<br />
effects of atomoxetine were seen at doses that did not affect basal locomotor activity or sucrose<br />
self-administration (FR1 <strong>an</strong>d FR7). In a successive study, we evaluated <strong>the</strong> effects of<br />
methylphenidate, a drug also known to reduce impulsivity in hum<strong>an</strong>s, on <strong>the</strong> second-order<br />
schedule of cocaine rein<strong>for</strong>cement. Methylphenidate did not affect cue-controlled cocaineseeking<br />
behaviour, in rats.<br />
These results toge<strong>the</strong>r with our previous data, suggest that <strong>the</strong> selective noradrenaline re-uptake<br />
inhibitor, atomoxetine, through its particular pharmacological profile, might provide a promising<br />
treatment strategy <strong>for</strong> decreasing stimulus-controlled drug-seeking <strong>an</strong>d relapse.<br />
Disclosures: D. Economidou, None; D.E. Theobold, None; J.W. Dalley, None; B.J. Everitt,<br />
None.<br />
Poster
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.21/Z34<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIH Gr<strong>an</strong>t DA 10460<br />
Title: Incubation of cue-induced cocaine seeking in C57BL/6 mice<br />
Authors: *A. L. NUGENT, E. B. LARSON, D. W. SELF;<br />
UT Southwestern Med. Ctr., Dallas, TX<br />
Abstract: A hallmark of cocaine addiction is <strong>the</strong> high relapse rate after extended periods of<br />
abstinence <strong>from</strong> chronic cocaine use. Currently, it is believed that <strong>the</strong> high relapse rate is due to<br />
drug-induced adaptations in reward-related learning <strong>an</strong>d memory processes which result in<br />
hypersensitivity to cocaine-associated cues. Prior studies have shown that drug seeking behaviors<br />
(i.e. lever pressing, nose pokes) in chronic cocaine self administering rats increases, ra<strong>the</strong>r th<strong>an</strong><br />
dissipates, over time in withdrawal, suggesting that motivation <strong>for</strong> drug incubates over time.<br />
However, this incubation phenomenon has not been demonstrated in mice. Here, we examine <strong>the</strong><br />
effects of withdrawal time on cocaine-seeking behavior in mice. C57BL/6 mice were trained to<br />
self administer cocaine (0.5 mg/kg/injection) on a fixed ratio schedule (FR1) <strong>for</strong> 3 weeks in daily<br />
two hour sessions prior to a period of <strong>for</strong>ced abstinence (withdrawal). Separate groups of <strong>an</strong>imals<br />
were subjected to ei<strong>the</strong>r 1 or 28 days of withdrawal in <strong>the</strong>ir home cage environment. Following<br />
this withdrawal period, drug-seeking behavior was tested by returning <strong>the</strong> <strong>an</strong>imals to <strong>the</strong> initial<br />
drug associated environment under extinction conditions <strong>for</strong> 6 hours. Following extinction,<br />
<strong>an</strong>imals were presented with discrete cues associated with prior drug infusions in a responsecontingent<br />
m<strong>an</strong>ner. Initial studies suggest that cocaine seeking elicited by initial exposure to<br />
extinction conditions (Ext hr 1) failed to differ <strong>from</strong> early to late withdrawal (n = 4/group). This<br />
result differs <strong>from</strong> rats which do exhibit incubation of such context-induced cocaine seeking.<br />
However, we found subst<strong>an</strong>tial increases in cue-induced reinstatement in mice after 1 month<br />
withdrawal when compared to 1 day withdrawal <strong>from</strong> 3 weeks of chronic cocaine selfadministration.<br />
Thus, following extinction to similar response levels in all study groups (Ext hr<br />
6), prominent drug-paired lever responding was induced by response-contingent cocaine<br />
injection cues only after 1 month withdrawal, averaging > 50 responses/hr in mice with little<br />
responding on <strong>the</strong> inactive lever. In contrast, cue reinstatement of cocaine seeking was virtually<br />
absent after only 1 day withdrawal, despite subst<strong>an</strong>tial responding in extinction similar to <strong>the</strong> 1<br />
month withdrawal group. Studies are underway to determine whe<strong>the</strong>r incubation of sucroseseeking<br />
behavior also occurs in mice. To our knowledge, <strong>the</strong>se data are first to demonstrate<br />
incubation of cocaine seeking in mice, <strong>an</strong>d suggest that drug induced neuroadaptations that<br />
underlie enh<strong>an</strong>ced sensitivity to drug related stimuli also occur in this species. Thus, mouse<br />
models may be useful to study incubation of cocaine-seeking behavior.
Disclosures: A.L. Nugent, None; E.B. Larson, None; D.W. Self, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.22/Z35<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Title: Differential molecular neuroadaptations in <strong>the</strong> rat nucleus accumbens after withdrawal<br />
<strong>from</strong> heroin <strong>an</strong>d cocaine self-administration<br />
Authors: *M. T. MCCOY 1 , M. AIRAVAARA 2 , S. A. GOLDEN 3 , C. L. PICKENS 3 , E.<br />
LEHRMANN 4 , W. WOOD, III 4 , K. G. BECKER 4 , Y. SHAHAM 3 , J. L. CADET 1 ;<br />
1 Mol. Neuropsychiatry Res. Br., 2 Cell. Neurobio. Res. Br., 3 Behav Neurosci. Res. Br.,<br />
DHHS/NIH/NIDA/IRP, Baltimore, MD; 4 Gene expression <strong>an</strong>d Genomics Unit,<br />
DHHS/NIH/NIA/IRP, Baltimore, MD<br />
Abstract: Cocaine <strong>an</strong>d heroin are abused throughout <strong>the</strong> world. Although <strong>the</strong>ir<br />
neuropharmacological actions are well known, <strong>the</strong> molecular mech<strong>an</strong>isms of <strong>the</strong>ir addictive<br />
effects remain to be fully elucidated. Towards this end, we have used a microarray method to<br />
study tr<strong>an</strong>scriptional ch<strong>an</strong>ges associated with extended access to cocaine <strong>an</strong>d heroin selfadministration.<br />
Rats were trained to self-administer ei<strong>the</strong>r cocaine (0.75 mg/kg/infusion), or<br />
heroin (0.075 mg/kg/infusion), or saline (a control condition) <strong>for</strong> 6 h per day <strong>for</strong> 10 days under a<br />
fixed-ratio 1 rein<strong>for</strong>cement schedule. After one day of withdrawal, <strong>the</strong> nucleus accumbens was<br />
dissected <strong>an</strong>d its RNA was assessed using Illumina rat bead arrays that contain 22,523 gene<br />
probes. The array data were <strong>an</strong>alyzed using GeneSpring (Agilent technologies) to select genes<br />
whose expression was signific<strong>an</strong>tly affected (p
Disclosures: M.T. McCoy, Department of Health <strong>an</strong>d Hum<strong>an</strong> Services/ National Institutes of<br />
Health/ National Institute on Drug Abuse/ Intramural Research Program<br />
(DHHS/NIH/NIDA/IRP), A. Employment (full or part-time); M. Airavaara, None; S.A.<br />
Golden, None; C.L. Pickens, None; E. Lehrm<strong>an</strong>n, None; W. Wood, None; K.G. Becker,<br />
None; Y. Shaham, None; J.L. Cadet, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.23/Z36<br />
Topic: C.17.j. Cocaine rein<strong>for</strong>cement, seeking <strong>an</strong>d reinstatement<br />
Support: NIDA gr<strong>an</strong>t 1R01DA023579<br />
NIDA gr<strong>an</strong>t R21DA02062<br />
General Clinical Research Center gr<strong>an</strong>t 5-MO1-RR-10710<br />
Title: Choice <strong>for</strong> cocaine stimuli in cocaine addiction is modulated by recent use <strong>an</strong>d awareness<br />
Authors: *S. J. MOELLER 1 , T. MALONEY 2 , M. A. PARVAZ 2 , N. ALIA-KLEIN 2 , P. A.<br />
WOICIK 2 , F. TELANG 2 , G.-J. WANG 2 , N. D. VOLKOW 3 , R. Z. GOLDSTEIN 2 ;<br />
1 2 3<br />
Univ. of Michig<strong>an</strong>, Ann Arbor, MI; Brookhaven Natl. Lab., Upton, NY; Natl. Inst. on Drug<br />
Abuse, Be<strong>the</strong>sda, MD<br />
Abstract: Individuals with cocaine use disorder (CUD) choose cocaine over non-drug rewards.<br />
However, because CUD <strong>for</strong>m a heterogeneous group, here we tested <strong>the</strong> hypo<strong>the</strong>sis that<br />
individual characteristics (e.g., recency of cocaine use, as objectively indicated by cocaine<br />
metabolites in urine at time of study, <strong>an</strong>d behavioral awareness) may modulate this enh<strong>an</strong>ced<br />
drug-related choice. Sixteen CUD testing positive <strong>for</strong> cocaine in urine (CUD+), 26 CUD testing<br />
negative <strong>for</strong> cocaine in urine (CUD-), <strong>an</strong>d 23 healthy controls (HC) completed two choice tasks<br />
that assessed objective preference <strong>for</strong> viewing four types of pictures (pleas<strong>an</strong>t, unpleas<strong>an</strong>t,<br />
neutral, <strong>an</strong>d cocaine). In <strong>the</strong> ‘explicit’ choice task (i.e., task contingencies were fully known),<br />
subjects chose via continued button pressing between two fully-visible side-by-side images. In<br />
<strong>the</strong> ‘implicit’ choice task (i.e., task contingencies were not fully known), subjects selected via a<br />
single button press between pictures hidden under flipped-over decks of cards. The implicit task<br />
concluded by asking subjects to report <strong>the</strong>ir most selected picture type, allowing <strong>for</strong> inspection of<br />
correspondence between <strong>the</strong>se self-reports <strong>an</strong>d behavior (i.e., <strong>the</strong> objectively measured most
selected picture category) to provide <strong>an</strong> index of behavioral awareness. Analyses of both choice<br />
tasks revealed highest cocaine-related choices in CUD, replicating our previous results (Moeller<br />
et al., in press). Here we extended <strong>the</strong>se results to show <strong>the</strong> highest cocaine-related choice in<br />
CUD+, followed respectively by CUD- <strong>an</strong>d HC [as tested with linear contrasts; Fs(1,62)>13.1,<br />
p
Abstract: M<strong>an</strong>g<strong>an</strong>ese enh<strong>an</strong>ced magnetic reson<strong>an</strong>ce imaging (MEMRI) is a tool that c<strong>an</strong> be used<br />
to explore activity dependent plasticity within <strong>the</strong> brain. In our study, we aimed to explore <strong>the</strong><br />
pathways activated by cocaine <strong>an</strong>d methylphenidate, both of which are classified as<br />
psychostimul<strong>an</strong>ts, but are known to exert <strong>the</strong>ir effects through different mech<strong>an</strong>isms in <strong>the</strong> brain.<br />
We also hoped to see whe<strong>the</strong>r or not <strong>the</strong> plastic ch<strong>an</strong>ges occurring with drug usage ch<strong>an</strong>ge<br />
depending on whe<strong>the</strong>r drug administration is over <strong>an</strong> acute or chronic period of time. Male Long<br />
Ev<strong>an</strong>s rats were administered cocaine, methylphenidate, or saline. The groups were fur<strong>the</strong>r<br />
subdivided; chronic administration, where drug or vehicle was given <strong>for</strong> 14 days, versus acute<br />
administration, where drug or vehicle was given <strong>for</strong> 3 days only. Cocaine treated <strong>an</strong>imals were<br />
administered drug 3 times a day, methylphenidate treated <strong>an</strong>imals were administered drug twice<br />
a day, <strong>an</strong>d <strong>the</strong> vehicle treated <strong>an</strong>imals received saline 3 times a day. M<strong>an</strong>g<strong>an</strong>ese was injected<br />
i.c.v. at a volume of 20microliters on <strong>the</strong> first day of drug administration <strong>for</strong> <strong>the</strong> acute groups,<br />
<strong>an</strong>d on day 12 of <strong>the</strong> drug administration in <strong>the</strong> chronic group. On <strong>the</strong> fourth day <strong>for</strong> <strong>the</strong> acute<br />
group, <strong>an</strong>d <strong>the</strong> fifteenth day <strong>for</strong> <strong>the</strong> chronic group, <strong>the</strong> brains were imaged using a T1 weighted<br />
sc<strong>an</strong>. Each brain was registered to a Rat Brain Atlas, <strong>an</strong>d ROI’s were identified. We were most<br />
interested in <strong>the</strong> subst<strong>an</strong>tia nigra <strong>an</strong>d ventral tegmental area due to <strong>the</strong>ir role as major parts of <strong>the</strong><br />
reward pathway, considering <strong>the</strong> pathway is implicated in <strong>the</strong> modulation of <strong>the</strong> rewarding<br />
effects of both drugs. Acute treatment (4 day) with psychostimul<strong>an</strong>ts had no signific<strong>an</strong>t effect on<br />
T1 contrast in <strong>the</strong> VTA or SN as compared to control. However, chronic treatment with both<br />
methylphenidate <strong>an</strong>d cocaine showed enh<strong>an</strong>ced contrast in <strong>the</strong>se dopaminergic nuclei as<br />
compared to control. These data suggest plastic ch<strong>an</strong>ges c<strong>an</strong> occur in <strong>the</strong> dopaminergic systems<br />
projecting to <strong>the</strong> basal g<strong>an</strong>glia <strong>an</strong>d <strong>the</strong> limbic cortex in response to chronic exposure<br />
methylphenidate that are not dissimilar to cocaine.<br />
Disclosures: T.L. Stolberg, None; T.R. Johnson, None; B.J. Smerkers, None; P. Kulkarni,<br />
None; C. Ferris, None.<br />
Poster<br />
553. Cocaine, Methylphenidate, <strong>an</strong>d Rein<strong>for</strong>cement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 553.25/Z38<br />
Topic: F.01.e. Emotion<br />
Support: D<strong>an</strong>ish Medical Research Council<br />
Lundbeck Foundation<br />
Copenhagen University’s focus area “Body <strong>an</strong>d Mind”
Title: Effects of recreational use of ecstasy <strong>an</strong>d hallucinogens on <strong>the</strong> neural circuitry of emotion<br />
Authors: *T. Z. RAMSOY 1 , K. S. MADSEN 2 , K. H. MADSEN 2 , J. S. WEGENER 4 , G. M.<br />
KNUDSEN 5 , A. M. SKIMMINGE 3 , T. L. JERNIGAN 6 , D. ERRITZØE 5 ;<br />
1 Decision Neurosci. Res. Group, Copenhagen Business Sch., Frederiksberg, Denmark; 2 D<strong>an</strong>ish<br />
Res. Ctr. <strong>for</strong> Magnetic Reson<strong>an</strong>ce, 3 Decision Neurosci. Res. Group, Copenhagen Univ. Hosp.<br />
Hvidovre, Hvidovre, Denmark; 4 Decision Neurosci. Res. Group, Copenhagen Business Sch.,<br />
Frederiksberg, Denmark; 5 Ctr. <strong>for</strong> Integrated Mol. Brain Imaging, Copenhagen, Denmark; 6 Lab.<br />
of Cognitive Imaging, UCSD, S<strong>an</strong> Diego, CA<br />
Abstract: Introduction:<br />
3,4-Methylenedioxymethamphetamine (MDMA, or ecstasy) <strong>an</strong>d hallucinogens (Hal) exert <strong>the</strong>ir<br />
main psychoactive effects through actions in <strong>the</strong> serotonin (5-HT) neurotr<strong>an</strong>smitter system, <strong>an</strong>d<br />
in particular via <strong>the</strong> 2A receptor. Fur<strong>the</strong>rmore, recreational use of MDMA has been associated<br />
with cognitive <strong>an</strong>d affective disturb<strong>an</strong>ces. The aim of this study was to assess <strong>the</strong> effects of<br />
MDMA <strong>an</strong>d Hal use on <strong>the</strong> neural circuitry <strong>for</strong> emotion processing. In a sample of recreational<br />
MDMA/Hal users we have found reductions in cerebral serotonin tr<strong>an</strong>sporter (SERT) <strong>an</strong>d 5-<br />
HT2A receptor binding as well as ch<strong>an</strong>ges in brain responses to emotional stimuli. We<br />
hypo<strong>the</strong>sized that in comparison to age- <strong>an</strong>d gender-matched healthy controls, MDMA/Hal users<br />
have structural brain ch<strong>an</strong>ges in <strong>the</strong> neural circuitry underlying emotions <strong>an</strong>d control of<br />
emotional responses: bilateral orbitofrontal cortex (OfC), insula, amygdala, <strong>an</strong>d <strong>an</strong>terior<br />
cingulate cortex (ACC).<br />
Methods:<br />
Twenty-two current MDMA/Hal users (24.8 ± 4.1, 3 F) <strong>an</strong>d 18 non-using control subjects (23.7<br />
± 3.6, 3 F) underwent MRI in a Siemens Magnetom Trio 3T MR sc<strong>an</strong>ner. Subjects were sc<strong>an</strong>ned<br />
with 1) a T1-weighted sequence (TR=1550, TE=3.04, voxel size = 1x1x1 mm), <strong>an</strong>d 2) a<br />
diffusion weighted sequence (DTI, b=1200, directions=61, voxel size = 2.3 x 2.3 x 2.3 mm).<br />
Structural data were <strong>an</strong>alyzed using a voxel-based morphometry (VBM) <strong>an</strong>alysis based on<br />
normalization using DARTEL. The DTI data were <strong>an</strong>alyzed using a combined ROI <strong>an</strong>d tractbased<br />
spatial statistics approach with focus on <strong>the</strong> uncinate fasciculi (UF).<br />
Results:<br />
Using <strong>an</strong> uncorrected threshold of p
Disclosures: T.Z. Ramsoy, None; K.S. Madsen, None; K.H. Madsen, None; J.S. Wegener,<br />
None; G.M. Knudsen, None; A.M. Skimminge, None; T.L. Jernig<strong>an</strong>, None; D. Erritzøe,<br />
None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.1/AA1<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NSF 0642188<br />
U54NS041071<br />
R24DA021471<br />
T32-HL076133<br />
Title: Alpha-synuclein tr<strong>an</strong>siently interacts with <strong>the</strong> dopamine tr<strong>an</strong>sporter<br />
Authors: *C. A. HANSON 1 , J. S. GOODWIN 2 , A. KENWORTHY 4 , H. KHOSHBOUEI 3 ;<br />
2 C<strong>an</strong>cer Biol., 3 Neurobio. <strong>an</strong>d Neurotoxicology, 1 Meharry Med. Col., Nashville, TN; 4 Mol.<br />
Physiol. <strong>an</strong>d Biophysics, V<strong>an</strong>derbilt Univ., Nashville, TN<br />
Abstract: The dopamine tr<strong>an</strong>sporter (DAT) is a neurotr<strong>an</strong>smitter tr<strong>an</strong>sporter essential <strong>for</strong> <strong>the</strong><br />
mainten<strong>an</strong>ce of dopamine (DA) homeostasis in <strong>the</strong> brain. DAT <strong>an</strong>d alpha-synuclein (a-syn) are<br />
both expressed in <strong>the</strong> presynaptic terminals of dopaminergic neurons <strong>an</strong>d it is suggested <strong>the</strong>y<br />
<strong>for</strong>m a heteromeric complex in co-tr<strong>an</strong>sected cells <strong>an</strong>d mesencephalic neurons (Lee et al. 2001;<br />
Wringer <strong>an</strong>d Sidhu 2003). Deregulation of DA homeostasis is implicated in neurodegenerative<br />
diseases, drug addiction <strong>an</strong>d neuropsychiatric disorders. Similarly, a-syn is implicated in multiple<br />
neurodegenerative diseases <strong>an</strong>d drug addiction <strong>an</strong>d thought to be involved in dopaminergic<br />
tr<strong>an</strong>smission. Although a-syn was previously shown to bind DAT, <strong>the</strong> nature of this interaction<br />
<strong>an</strong>d its physiological consequences on DAT activity have yet to be fully understood. Using<br />
Fluorescence Recovery After Photobleaching (FRAP) <strong>an</strong>d Fluorescence Microscopy we found asyn<br />
<strong>an</strong>d DAT tr<strong>an</strong>siently interact. This interaction occurs at <strong>the</strong> plasma membr<strong>an</strong>e. Our<br />
preliminary data indicates psychostimul<strong>an</strong>ts such as methamphetamine alter <strong>the</strong> nature of this<br />
interaction, to mediate co-trafficking of a-syn <strong>an</strong>d DAT. In addition, we examined <strong>the</strong> effect of<br />
this interaction on DAT activity in immortalized DAergic neurons <strong>an</strong>d cells stably expressing
YFP-DAT. Co-expression of a-syn <strong>an</strong>d DAT decreased <strong>the</strong> rate of substrate uptake via DAT.<br />
These findings enh<strong>an</strong>ce our underst<strong>an</strong>ding of molecular mech<strong>an</strong>isms of DAT function <strong>an</strong>d its<br />
regulation by interacting proteins such as a-syn.<br />
Disclosures: C.A. H<strong>an</strong>son, None; J.S. Goodwin, None; A. Kenworthy, None; H.<br />
Khoshbouei, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.2/AA2<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NSF 0642188<br />
U54NS041071<br />
R24DA021471<br />
T32-HL076133<br />
Title: Interaction of alpha-synuclein <strong>an</strong>d <strong>the</strong> dopamine tr<strong>an</strong>sporter<br />
Authors: *J. S. GOODWIN 1 , T. PATEL 2 , A. K. KENWORTHY 3 , H. KHOSHBOUEI 2 ;<br />
2 Neurobio. <strong>an</strong>d Neurotoxicology, 1 Meharry Med. Col., Nashville, TN; 3 Mol. Physiol. <strong>an</strong>d<br />
Biophysics, V<strong>an</strong>derbilt Univ. Med. Ctr., Nashville, TN<br />
Abstract: Neurotr<strong>an</strong>smitter reuptake by tr<strong>an</strong>sporters is a major mech<strong>an</strong>ism <strong>for</strong> terminating<br />
synaptic tr<strong>an</strong>smission. The hum<strong>an</strong> dopamine tr<strong>an</strong>sporter (hDAT) is one of <strong>the</strong> main targets <strong>for</strong><br />
psychostimul<strong>an</strong>ts, <strong>an</strong>d is critical to DA homeostasis. Lipid rafts are specialized membr<strong>an</strong>e<br />
microdomains that serve as org<strong>an</strong>izing centers to regulate different cellular processes such as<br />
neurotr<strong>an</strong>smission <strong>an</strong>d trafficking. To begin to underst<strong>an</strong>d how psychostimul<strong>an</strong>ts,<br />
methamphetamine (METH) <strong>an</strong>d amphetamine (AMPH), affect hDAT microdomain association,<br />
we utilized fluorescence recovery after photobleaching (FRAP) <strong>an</strong>d density-gradient<br />
centrifugation. Our FRAP studies revealed signific<strong>an</strong>t ch<strong>an</strong>ges in <strong>the</strong> rate (D) <strong>an</strong>d extent (Mf) of<br />
<strong>the</strong> fluorescence recovery into <strong>the</strong> bleached region of <strong>the</strong> plasma membr<strong>an</strong>e in cells expressing<br />
YFP-hDAT in <strong>the</strong> presence of METH <strong>an</strong>d AMPH. Substitution of five N-terminal Ser with Ala,<br />
(S/A-YFP-DAT, c<strong>an</strong>not be phosphorylated) or Asp (S/D-YFP-DAT, is pseudo-phosphorylated),
<strong>an</strong>d removal of <strong>the</strong> 22 N-terminal amino acids (trunc-YFP-hDAT) restored <strong>the</strong> diffusion rate of<br />
<strong>the</strong> tr<strong>an</strong>sporter to control levels. Using density-gradient centrifugation, we found that YFP-hDAT<br />
is distributed into both, classically defined, membr<strong>an</strong>e raft <strong>an</strong>d non-raft fractions. Incubating<br />
with METH <strong>an</strong>d AMPH shifted YFP-hDAT <strong>from</strong> non-raft to raft fractions. These data suggest<br />
that METH <strong>an</strong>d AMPH cause hDAT to partition into lipid raft membr<strong>an</strong>e microdomains, <strong>an</strong>d <strong>the</strong><br />
decrease in <strong>the</strong> hDAT diffusion rate evoked by METH- vs. AMPH-occupied DAT could suggest<br />
that N-terminal domain of <strong>the</strong> tr<strong>an</strong>sporter is associated with distinct group of proteins when<br />
exposed to <strong>the</strong>se psychostimul<strong>an</strong>ts.<br />
Disclosures: J.S. Goodwin, None; T. Patel, None; A.K. Kenworthy, None; H. Khoshbouei,<br />
None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.3/AA3<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NSF 0642188<br />
U54NS041071<br />
R24DA021471<br />
T32-HL076133<br />
Title: a-synuclein interaction with <strong>the</strong> dopamine tr<strong>an</strong>sporter stimulates a tr<strong>an</strong>sporter mediated Cl -<br />
current<br />
Authors: S. CHIRWA 1 , S. GOODWIN 2 , J. SWANT 1 , *H. KHOSHBOUEI 3 ;<br />
1 2 3<br />
Dept. Neurobio. <strong>an</strong>d Neurotoxicology, Dept. C<strong>an</strong>cer Biol., Dept. Biomed Sci., Meharry Med.<br />
Coll, Nashville, TN<br />
Abstract: The neuronal plasma membr<strong>an</strong>e dopamine (DA) tr<strong>an</strong>sporter (DAT) is essential <strong>for</strong> <strong>the</strong><br />
mainten<strong>an</strong>ce of DA homeostasis in <strong>the</strong> brain. Both DAT <strong>an</strong>d a-synuclein (a-syn) are expressed in<br />
<strong>the</strong> presynaptic terminals of dopaminergic neurons. a-syn <strong>an</strong>d DAT <strong>for</strong>m a stable heteromeric<br />
complex in co-tr<strong>an</strong>sfected cells <strong>an</strong>d mesencephalic neurons (Lee et al. 2001; Wersinger <strong>an</strong>d<br />
Sidhu 2003). Dysregulation of DA homeostasis is implicated in neurodegenerative diseases, drug
addiction <strong>an</strong>d neuropsychiatric disorders. a-syn is suggested to regulate DA homeostasis.<br />
However, <strong>the</strong> mech<strong>an</strong>ism of a-syn modulation of DAT activity is unknown. We examined how<br />
a-syn affects DAT activity by utilizing dual pipette whole-cell patch clamp recording, <strong>an</strong>d<br />
substrate fluorescence imaging, in immortalized DAergic neurons <strong>an</strong>d cells stably expressing<br />
YFP-DAT. Co-expression of a-syn <strong>an</strong>d DAT decreased <strong>the</strong> uptake rate of a fluorescent substrate,<br />
ASP + , via DAT. Dialysis of 5 or 10 microM a-syn via <strong>the</strong> whole-cell path pipette stimulated a<br />
Na + independent but Cl - sensitive inward current in DAT-expressing cells. This current is absent<br />
when heat inactivated a-syn is dialyzed into <strong>the</strong>se cells. An a-syn-stimulated inward current is<br />
eliminated by DAT blockers such as cocaine <strong>an</strong>d GBR12935. Taken toge<strong>the</strong>r, our findings<br />
indicate that a-syn stimulates a DAT-mediated Cl - current. Fur<strong>the</strong>rmore, our dual pipette whole<br />
cell patches method may serve as a model to study <strong>the</strong> effects of a-syn on DAT function <strong>an</strong>d DA<br />
homeostasis.<br />
Disclosures: S. Chirwa, None; S. goodwin, None; J. Sw<strong>an</strong>t, None; H. Khoshbouei, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.4/AA4<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH Gr<strong>an</strong>t DA022323<br />
NIH Gr<strong>an</strong>t DA016606<br />
NIH Gr<strong>an</strong>t RR00168<br />
Title: A receptor target <strong>for</strong> methamphetamine in brain<br />
Authors: *Z. XIE 1 , G. M. MILLER 2 ;<br />
1 Dept. of Neurosci., 2 Div. of Neurosci., NEPRC of Harvard Med. Sch., Southborough, MA<br />
Abstract: Methamphetamine not only competes with monoamine neurotr<strong>an</strong>smitters <strong>for</strong> reuptake<br />
but also causes non-competitive modulatory effects on monoamine tr<strong>an</strong>sporters to elevate <strong>the</strong><br />
extracellular levels of <strong>the</strong> neurotr<strong>an</strong>smitters. Here, we show that trace amine-associated receptor<br />
1 (TAAR1) acts as a methamphetamine target in brain <strong>an</strong>d serves as a mediator of its noncompetitive<br />
effects. In uptake assays, when brain synaptosomes were loaded with 10 nM<br />
[3H]DA, 20 nM [3H]NE or 20 nM [3H]5-HT plus 100 nM methamphetamine, we observed that
methamphetamine induced more extensive uptake inhibition in [3H]DA uptake by DAT, [3H]NE<br />
uptake by NET or [3H]5-HT uptake by SERT in rhesus monkey <strong>an</strong>d wild type mouse striatal<br />
([3H]DA, [3H]5-HT) or thalamic ([3H]NE) synaptosomes th<strong>an</strong> in TAAR1 knockout mouse<br />
synaptosomes. Parallelly in tr<strong>an</strong>sfected cells (rhTAAR1/hDAT, rhTAAR1/hNET <strong>an</strong>d<br />
rhTAAR1/hSERT versus hDAT, hNET <strong>an</strong>d hSERT), a larger uptake inhibition occurred in <strong>the</strong><br />
presence of TAAR1. <strong>When</strong> <strong>the</strong> same cells <strong>an</strong>d synaptosomes were pretreated with 1 µM<br />
methamphetamine <strong>an</strong>d <strong>the</strong>n loaded with 10 nM [3H]DA, 20 nM [3H]NE or 20 nM [3H]5-HT,<br />
we found that methamphetamine-induced uptake inhibition was TAAR1-dependent. In efflux<br />
assays, when mouse striatal synaptosomes were preloaded with 1 µM [3H]DA <strong>an</strong>d <strong>the</strong>n treated<br />
with 1 µM methamphetamine, we observed a greater [3H]DA efflux via DAT in wild type mouse<br />
synaptosomes th<strong>an</strong> in TAAR1 knockout mouse synaptosomes. <strong>When</strong> <strong>the</strong> same synaptosomes<br />
were preloaded with 10 nM instead of 1 µM [3H]DA, 1 µM methamphetamine induced [3H]DA<br />
efflux in wild type but not in TAAR1 knockout mouse synaptosomes. At <strong>the</strong> loading<br />
concentration of 20 nM of [3H]NE or [3H]5-HT, we also observed that 1 µM methamphetamine<br />
induced [3H]NE efflux via NET <strong>an</strong>d [3H]5-HT efflux via SERT in a TAAR1-dependent m<strong>an</strong>ner<br />
in mouse thalamic or striatal synaptosomes as well as in tr<strong>an</strong>sfected cells. In addition, we<br />
demonstrated that 1 µM methamphetamine induced DAT internalization in TAAR1/DAT cotr<strong>an</strong>sfected<br />
cells <strong>an</strong>d in wild type mouse striatal synaptosomes, whereas no DAT internalization<br />
appeared in DAT only-tr<strong>an</strong>sfected cells <strong>an</strong>d only a weak ch<strong>an</strong>ge was observed in TAAR1<br />
knockout mouse striatal synaptosomes. TAAR1-mediated uptake inhibition was blocked by 10<br />
µM of <strong>the</strong> PKA inhibitor H89 or <strong>the</strong> PKC inhibitor Ro32-0432, <strong>an</strong>d TAAR1-mediated efflux <strong>an</strong>d<br />
dopamine tr<strong>an</strong>sporter internalization was blocked by 10 µM Ro32-0432. Toge<strong>the</strong>r, our data<br />
demonstrate that methamphetamine targets <strong>an</strong>d interacts with TAAR1 to initiate cellular<br />
phosphorylation cascades to regulate tr<strong>an</strong>sporter function <strong>an</strong>d trafficking, which reveals a novel<br />
mech<strong>an</strong>ism <strong>for</strong> methamphetamine action in brain.<br />
Disclosures: Z. Xie, None; G.M. Miller, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.5/AA5<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIDA<br />
MARC
Title: Validation of trace amine-associated receptor 1 as a molecular target of<br />
methamphetamine’s actions<br />
Authors: *E. A. REESE 1 , K. L. SUCHLAND 1 , R. CLEMENS-GRISHAM 1 , J. R. BUNZOW 1 ,<br />
D. K. GRANDY 2 ;<br />
2 Physiol. <strong>an</strong>d Pharmacol., 1 Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR<br />
Abstract: Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor.<br />
Recombin<strong>an</strong>t TAAR1 stimulates cAMP production when activated by <strong>the</strong> trace amines (TA) βphenylethylamine<br />
(PEA) <strong>an</strong>d para-tyramine (p-TYR). These biogenic amines are chemically<br />
related to <strong>the</strong> neurotr<strong>an</strong>smitters (NTs) dopamine, norepinephrine <strong>an</strong>d epinephrine as well as <strong>the</strong><br />
amphetamines (AMPHs). Given <strong>the</strong> structural similarity between <strong>the</strong> TAs, NTs, <strong>an</strong>d AMPHs,<br />
toge<strong>the</strong>r with <strong>the</strong> hum<strong>an</strong> data suggesting imbal<strong>an</strong>ces in TA levels correlate with mood disorders<br />
<strong>an</strong>d drug abuse, we investigated whe<strong>the</strong>r TAAR1 represents a novel target of methamphetamine<br />
(METH). AMPH <strong>an</strong>d METH exist as stereoisomers. The behavioral <strong>an</strong>d clinical literature<br />
suggest d-S(+)-isomers of both drugs are biologically more active th<strong>an</strong> <strong>the</strong> l-R(-) <strong>for</strong>ms.<br />
There<strong>for</strong>e, it was also of interest to explore <strong>the</strong> stereo-sensitivity of TAAR1. Recombin<strong>an</strong>t wild<br />
type (WT) or mutated mouse, rat, <strong>an</strong>d hum<strong>an</strong> TAAR1 (m, r, <strong>an</strong>d hTAAR1) were individually<br />
expressed in separate HEK-293 cell lines <strong>an</strong>d used to generate functional activation profiles (i.e.<br />
cAMP production). Computer-assisted docking of lig<strong>an</strong>ds to 3-D homology models of WT <strong>an</strong>d<br />
mut<strong>an</strong>t TAAR1s based on β2AR crystal structure were used to generate structural expl<strong>an</strong>ations<br />
<strong>for</strong> our empirical results. PEA <strong>an</strong>d p-TYR are potent full agonists of all species of recombin<strong>an</strong>t<br />
wild type TAAR1 tested yet <strong>the</strong>re is a signific<strong>an</strong>t stereoselective preference <strong>for</strong> <strong>the</strong> isomers of<br />
AMPH <strong>an</strong>d METH that is species-dependent. Substituting <strong>the</strong> conserved aspartate (D102) in<br />
tr<strong>an</strong>smembr<strong>an</strong>e (TM) 3 of TAAR1 with al<strong>an</strong>ine resulted in receptors that failed to stimulate<br />
cAMP production after exposure to TAs, AMPH or METH. However, cells expressing mut<strong>an</strong>t<br />
rTAAR1 where methionine (M268) in TM 6 was ch<strong>an</strong>ged to threonine (T), <strong>the</strong> residue found at<br />
<strong>the</strong> corresponding site in mTAAR1, revealed a rightward shift in EC50 compared to <strong>the</strong> WT<br />
response. Characterization of <strong>the</strong> reciprocal mTAAR1 mut<strong>an</strong>t, T269M, revealed EC50 values<br />
signific<strong>an</strong>tly lower th<strong>an</strong> WT. Substitution of residue 287 in TM7 of rTAAR1 (N287Y) <strong>an</strong>d<br />
mTAAR1 (Y287N) reversed <strong>the</strong> stereoselectivity of <strong>the</strong> respective WT receptors. Computer<br />
modeling suggests hydrogen bonding occurs between <strong>the</strong> amine of AMPH <strong>an</strong>d METH <strong>an</strong>d both<br />
D102 <strong>an</strong>d N287 of rTAAR1. The signific<strong>an</strong>t species difference in EC50s <strong>for</strong> d-S(+) <strong>an</strong>d l-R(-<br />
)AMPH <strong>an</strong>d METH at mTAAR1 is likely due to steric hinder<strong>an</strong>ce between <strong>the</strong> methyl group of<br />
l-R(-) AMPH/METH <strong>an</strong>d <strong>the</strong> phenyl ring of Y287 in TM7. These results validate TAAR1 is a<br />
target of AMPH <strong>an</strong>d METH <strong>an</strong>d that <strong>the</strong> integration of functional data <strong>an</strong>d computer modeling<br />
c<strong>an</strong> assist <strong>the</strong> discovery of novel lig<strong>an</strong>ds with potential <strong>the</strong>rapeutic utility.<br />
Disclosures: E.A. Reese, None; K.L. Suchl<strong>an</strong>d, None; R. Clemens-Grisham, None; J.R.<br />
Bunzow, None; D.K. Gr<strong>an</strong>dy, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.6/AA6<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH DA 13519<br />
NIH DA 16176<br />
Title: N-substituted methamphetamine derivatives as novel vesicular monoamine tr<strong>an</strong>sporter<br />
(VMAT2) inhibitors<br />
Authors: *D. B. HORTON 1 , G. ZHENG 1 , J. S. BECKMANN 2 , A. G. DEACIUC 1 , M. T.<br />
BARDO 2 , P. A. CROOKS 1 , L. P. DWOSKIN 1 ;<br />
1 Col. of Pharm., 2 Psychology, Univ. of Kentucky, Lexington, KY<br />
Abstract: Methamphetamine (METH) interacts with <strong>the</strong> dopamine tr<strong>an</strong>sporter (DAT) <strong>an</strong>d <strong>the</strong><br />
vesicular monoamine tr<strong>an</strong>sporter (VMAT2) to increase extrasynaptic dopamine (DA)<br />
concentrations, contributing to its psychostimul<strong>an</strong>t <strong>an</strong>d rewarding effects, as well as its abuse.<br />
METH inhibits DA uptake <strong>an</strong>d evokes DA release <strong>from</strong> vesicles. Lobel<strong>an</strong>e, a defunctionalized<br />
saturated <strong>an</strong>alog of lobeline, inhibits <strong>the</strong> neurochemical <strong>an</strong>d behavioral effects of METH by<br />
acting at VMAT2. The aim of this study was to evaluate a series of acyclic lobel<strong>an</strong>e <strong>an</strong>alogs,<br />
which c<strong>an</strong> be considered as N-substituted METH derivatives, <strong>for</strong> inhibition of<br />
[ 3 H]dihydrotetrabenazine (DTBZ) binding <strong>an</strong>d [ 3 H]DA uptake at VMAT2. These <strong>an</strong>alogs<br />
contain a METH or a homo-METH moiety with methoxyl or bromo substituents on <strong>the</strong> 4<br />
position of <strong>the</strong> phenyl ring, <strong>an</strong>d a phenethyl or a 3-phenylpropyl moiety attached to <strong>the</strong> N atom.<br />
Results show that all of <strong>the</strong> <strong>an</strong>alogs exhibit moderate potency at <strong>the</strong> [ 3 H]DTBZ binding site (Ki =<br />
0.43-29.8 µM), with <strong>the</strong> most potent <strong>an</strong>alog (GZ-819A; containing 4-bromine on <strong>the</strong> phenyl ring<br />
of a METH moiety) exhibiting a 2-fold higher affinity compared to lobel<strong>an</strong>e (Ki = 0.97 µM). All<br />
<strong>an</strong>alogs were more potent in inhibiting VMAT2 function (Ki = 0.008-0.46 µM) th<strong>an</strong> <strong>the</strong>y were<br />
inhibiting [ 3 H]DTBZ binding. The most potent <strong>an</strong>alog inhibiting [ 3 H]DA uptake was GZ-819A<br />
(Ki = 8.67 nM), which was 5-fold more potent th<strong>an</strong> lobel<strong>an</strong>e (Ki = 0.047 µM). Even though<br />
<strong>the</strong>se <strong>an</strong>alogs structurally resemble METH, representative <strong>an</strong>alogs (GZ-813A, GZ-814A, GZ-<br />
815A, <strong>an</strong>d GZ-819A) in this series did not increase locomotor activity when given systemically<br />
(0.1-10 mg/kg; s.c.) in contrast to METH, indicating that <strong>the</strong>y do not have psychostimul<strong>an</strong>t<br />
properties. In summary, by combining structural motifs of METH <strong>an</strong>d lobel<strong>an</strong>e, a novel series of<br />
VMAT2 inhibitors have been generated with potential as <strong>the</strong>rapeutic c<strong>an</strong>didates <strong>for</strong> treating<br />
METH abuse.<br />
Disclosures: D.B. Horton, None; G. Zheng, None; J.S. Beckm<strong>an</strong>n, None; A.G. Deaciuc,<br />
None; M.T. Bardo, None; P.A. Crooks, None; L.P. Dwoskin, None.
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.7/AA7<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: RO1 DA13519<br />
5 T32 DA016176<br />
Title: [ 3 H]Dihydrotetrabenazine binding <strong>an</strong>d [ 3 H]dopamine uptake inhibitory activity of a series<br />
of pyrrolidine <strong>an</strong>alogs at <strong>the</strong> vesicular monoamine tr<strong>an</strong>sporter-2<br />
Authors: *J. NICKELL 1 , A. VARTAK 2 , P. A. CROOKS 2 , L. P. DWOSKIN 2 ;<br />
2 Pharmaceut. Sci., 1 Univ. KY, Lexington, KY<br />
Abstract: Lobeline, <strong>an</strong> alkaloidal constituent of Lobelia inflata, has demonstrated efficacy in <strong>the</strong><br />
attenuation of <strong>the</strong> rewarding properties of methamphetamine (METH) in rodent models. The<br />
neurochemical mech<strong>an</strong>ism underlying this behavioral effect is postulated to be due to <strong>the</strong> ability<br />
of <strong>the</strong> alkaloid to redistribute intravesicular dopamine (DA) content via potent interaction with<br />
<strong>the</strong> vesicular monoamine tr<strong>an</strong>sporter-2 (VMAT2). Lobeline is, however, relatively nonselective,<br />
as it possesses high affinity <strong>for</strong> several nicotinic acetylcholine receptor subtypes, as well as <strong>the</strong><br />
dopamine tr<strong>an</strong>sporter. This issue prompted <strong>the</strong> search <strong>for</strong> a novel <strong>an</strong>alog with high affinity <strong>an</strong>d<br />
selectivity <strong>for</strong> VMAT2. Lobel<strong>an</strong>e, a defunctionalized <strong>an</strong>d saturated <strong>an</strong>alog of lobeline, fulfilled<br />
<strong>the</strong>se criteria, <strong>an</strong>d systematic structural modification of <strong>the</strong> lobel<strong>an</strong>e molecule has since af<strong>for</strong>ded<br />
a limited number of compounds with enh<strong>an</strong>ced affinity <strong>an</strong>d selectivity <strong>for</strong> VMAT2. The present<br />
study aimed to fur<strong>the</strong>r elucidate <strong>the</strong> pharmacophore of <strong>the</strong> lobel<strong>an</strong>e molecule, specifically<br />
determining <strong>the</strong> import<strong>an</strong>ce of <strong>the</strong> central ring structure by investigating <strong>the</strong> effect of a series of<br />
2, 5-disubstituted pyrrolidine <strong>an</strong>alogs as inhibitors of [ 3 H]DA uptake <strong>an</strong>d [ 3 H]DTBZ binding to<br />
rat brain synaptic vesicle preparations. The results indicate that substitution of a phenyleth<strong>an</strong>ol<br />
moiety <strong>for</strong> <strong>the</strong> N-methyl, <strong>an</strong>d addition of methoxy <strong>an</strong>d hydroxyl moieties onto <strong>the</strong> phenyl rings<br />
generally reduce potency <strong>for</strong> inhibition of [ 3 H]DA uptake without appreciably altering <strong>the</strong><br />
binding affinity of <strong>the</strong> pyrrolidine <strong>an</strong>alogs compared to lobel<strong>an</strong>e. The highest [ 3 H]DA uptake<br />
inhibition potencies (0.0071-0.057 µM; 4 <strong>an</strong>alogs) of this series were obtained with pyrrolidine<br />
<strong>an</strong>alogs bearing side chains containing a single methylene unit. This subgroup of <strong>an</strong>alogs<br />
inhibited [ 3 H]DA uptake in a competitive m<strong>an</strong>ner, <strong>an</strong>d stereochemistry <strong>an</strong>d methylation status of<br />
<strong>the</strong>se molecules did not alter this mech<strong>an</strong>ism. These results demonstrate that 2,5-disubstituted<br />
pyrrolidine <strong>an</strong>alogs constitute a promising series of compounds <strong>for</strong> fur<strong>the</strong>r investigation towards<br />
<strong>the</strong> development of a pharmaco<strong>the</strong>rapeutic <strong>for</strong> METH addiction, as m<strong>an</strong>y of <strong>the</strong>se <strong>an</strong>alogs<br />
exhibited greater potency inhibiting VMAT2-mediated [ 3 H]DA uptake compared with lobel<strong>an</strong>e.
Disclosures: J. Nickell, None; A. Vartak, None; P.A. Crooks, None; L.P. Dwoskin, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.8/AA8<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: USUHS Internal Gr<strong>an</strong>t RO70WG<br />
Title: Evidence that <strong>the</strong> methamphetamine induced protein called Shati is <strong>the</strong> biosyn<strong>the</strong>tic<br />
enzyme of N-acetylaspartate<br />
Authors: *P. S. ARIYANNUR 1 , N. PATAABIRAMAN 2 , P. ARUN 1 , J. R. MOFFETT 1 , C. N.<br />
MADHAVARAO 1 , A. NITTA 3 , A. M. A. NAMBOODIRI 1 ;<br />
1 Anatmoy,Physiology& Genet., Uni<strong>for</strong>med Services Univ., Be<strong>the</strong>sda, MD; 2 Dept. of Structural<br />
<strong>an</strong>d Mol. Biol., Walter Reed Army Inst. of Res., Silver Spring, MD; 3 Dept. of<br />
Neuropsychopharm., Nagoya Univ. Grad. Sch. of Med., Nagoya, Jap<strong>an</strong><br />
Abstract: N-Acetylaspartate (NAA) is <strong>an</strong> amino acid derivative that is present abund<strong>an</strong>tly<br />
(approx.10mM) in <strong>the</strong> CNS. Due to its high concentration in <strong>the</strong> hum<strong>an</strong> brain, NAA is used<br />
clinically as a diagnostic <strong>an</strong>d <strong>the</strong>rapeutic marker in various CNS disorders. However, <strong>the</strong><br />
biosyn<strong>the</strong>tic enzyme <strong>for</strong> NAA has not yet been isolated or characterized at <strong>the</strong> molecular level.<br />
Recently identified N-acetyltr<strong>an</strong>sferase enzyme, Shati (Nat8l), expression was increased by <strong>the</strong><br />
treatment of dopamine agonist methamphetamine which complemented our studies on <strong>the</strong><br />
regulation of NAA biosyn<strong>the</strong>sis in SH-SY5Y neuroblastoma cells with dopamine <strong>an</strong>tagonists.<br />
Shati belongs to <strong>the</strong> superfamily of N-acetyltr<strong>an</strong>sferases called GNAT, which includes histone<br />
N-acetyltr<strong>an</strong>sferases <strong>an</strong>d serotonin N-acetyltr<strong>an</strong>sferase in <strong>the</strong> pineal gl<strong>an</strong>d. By<br />
immunohistochemical localization of this enzyme using <strong>an</strong>ti-Shati peptide <strong>an</strong>tisera, we found<br />
that Shati is localized at high levels in neurons <strong>an</strong>d low levels in oligodendrocytes. This pattern<br />
is very similar to <strong>the</strong> cellular localization of NAA in <strong>the</strong> brain as shown by our laboratory with<br />
<strong>an</strong>tibodies to protein-coupled NAA. Based on <strong>the</strong>se observations, we hypo<strong>the</strong>sized that Shati is<br />
aspartate N-acetyltr<strong>an</strong>sferase (Asp-NAT), <strong>the</strong> enzyme that is involved in <strong>the</strong> biosyn<strong>the</strong>sis of<br />
NAA. We have tested this hypo<strong>the</strong>sis by immunoprecipitation of Asp-NAT activity <strong>from</strong><br />
partially purified enzyme preparations using <strong>the</strong> same <strong>an</strong>ti-Shati peptide <strong>an</strong>tiserum that was used<br />
<strong>for</strong> immunohistochemistry. Also, we have found that methamphetamine treatment of SH-SY5Y<br />
hum<strong>an</strong> neuroblastoma cells caused <strong>an</strong> increase in Asp-NAT activity, similar to <strong>the</strong> increase in<br />
expression of Shati in <strong>the</strong> brain. Fur<strong>the</strong>rmore, with homology modeling studies, we identified a
potential binding pocket near <strong>the</strong> acetyl CoA binding pocket at <strong>the</strong> active site that is highly<br />
specific to aspartate. Fur<strong>the</strong>r studies are in progress to confirm <strong>an</strong>d extend <strong>the</strong>se findings.<br />
Disclosures: P.S. Ariy<strong>an</strong>nur, None; N. Pataabiram<strong>an</strong>, None; P. Arun, None; J.R. Moffett,<br />
None; C.N. Madhavarao, None; A. Nitta, None; A.M.A. Namboodiri, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.9/AA9<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Apocynin attenuates methamphetamine-evoked 3 H-dopamine release<br />
Authors: *D. K. MILLER 1 , C. E. OELRICHS 1 , G. Y. SUN 2 , A. SIMONYI 2 ;<br />
1 Psychological Sci., 2 Biochem., Univ. of Missouri, Columbia, MO<br />
Abstract: Apocynin is derived <strong>from</strong> <strong>the</strong> medicinal herb Picrorhiza kurroa <strong>an</strong>d inhibits NADPH<br />
oxidase by preventing assembly of its subunits. Apocynin has been demonstrated to be protective<br />
in <strong>an</strong>imal models of neurodegenerative diseases, including stroke. Methamphetamine abuse <strong>an</strong>d<br />
neurotoxicity is a signific<strong>an</strong>t public health concern, <strong>an</strong>d a research goal is identifying compounds<br />
that could prevent or block <strong>the</strong> stimul<strong>an</strong>t's effects. Presently,we investigated <strong>the</strong> effect of<br />
apocynin on methamphetamine-evoked 3 H-dopamine release <strong>from</strong> preloaded rat striatal slices.<br />
Rats were injected (i.p.) with apocynin (50 mg/kg/day) or vehicle <strong>for</strong> 7 days, <strong>an</strong>d <strong>the</strong>n <strong>the</strong><br />
methamphetamine concentration-response (0.03-60 µM) was determined. There was<br />
signific<strong>an</strong>tly less (~68-78%) drug-evoked 3 H-dopamine release <strong>from</strong> slices <strong>from</strong> apocynintreated<br />
rats th<strong>an</strong> <strong>from</strong> vehicle-treated rats at <strong>the</strong> 3-10 µM methamphetamine concentrations.<br />
There were no signific<strong>an</strong>t differences at lower (< 3 µM) or higher (>10 µM) methamphetamine<br />
concentrations. In contrast, application of apocynin (10 nM-1 mM) to slices did not evoke 3 Hdopamine<br />
release <strong>an</strong>d apocynin (100 nM-1 mM) applied to to slices did not alter<br />
methamphetamine (3 µM)-evoked 3 H-dopamine release. Overall <strong>the</strong>se data demonstrate that in<br />
vivo--but not in vitro--apocynin treatment signific<strong>an</strong>tly alters methamphetamine-evoked<br />
dopamine release. Fur<strong>the</strong>rmore, <strong>the</strong>se findings suggests a role <strong>for</strong> NADPH oxidase to mediate<br />
methamphetamine's effects to evoke neurotr<strong>an</strong>smitter release.<br />
Disclosures: D.K. Miller, None; C.E. Oelrichs, None; G.Y. Sun, None; A. Simonyi, None.
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.10/AA10<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Sigma receptor lig<strong>an</strong>d SA-4503 attenuates methamphetamine-induced 3 H-dopamine<br />
release<br />
Authors: *K. RODVELT 1 , D. K. MILLER 1 , L. R. BLOUNT 2 , K.-H. FAN 2 , J. R. LEVER 3 , S.<br />
Z. LEVER 2 ;<br />
1 2 3<br />
Dept. of Psychological Sci., Dept. of Chem., Dept. of Radiology, Univ. of Missouri,<br />
Columbia, MO<br />
Abstract: Methamphetamine exhibits affinity (Ki value ≈ 2.2 µM) <strong>for</strong> σ1 sigma receptors, <strong>an</strong>d σ1<br />
receptor <strong>an</strong>tagonists attenuate methamphetamine’s behavioral effects. Presently, <strong>the</strong> effects of<br />
BD-1047 (Ki value ≈ 0.9 nM) <strong>an</strong>d SA-4503 (Ki value ≈ 4.6 nM) on methamphetamine-evoked<br />
dopamine release were investigated. SA-4503 was syn<strong>the</strong>sized by our laboratory via a novel<br />
pathway that included fewer syn<strong>the</strong>sis steps, purification without column chromatography,<br />
syn<strong>the</strong>sis via commercially-available chemicals, <strong>an</strong>d a larger yield. In neurochemical<br />
experiments, nei<strong>the</strong>r BD-1047 nor SA-4503 (100 pM - 10 µM) evoked 3 H-dopamine release<br />
<strong>from</strong> preloaded rat striatal slices. Import<strong>an</strong>tly, SA-4503 (1 nM - 1 µM) attenuated (58-87%)<br />
methamphetamine (3 µM)-evoked 3 H-dopamine release in a concentration-dependent m<strong>an</strong>ner.<br />
BD-1047 (10 nM) attenuated (52%) methamphetamine-evoked 3 H-dopamine release, although<br />
higher <strong>an</strong>d lower concentrations did not markedly alter methamphetamine’s effects. Our present<br />
findings exp<strong>an</strong>d <strong>the</strong> literature on sigma receptors <strong>an</strong>d methamphetamine, by indicating <strong>an</strong> in vitro<br />
inhibition of <strong>the</strong> psychostimul<strong>an</strong>t’s primary neurochemical effect, <strong>an</strong>d suggest that sigma<br />
receptors may be a target <strong>for</strong> <strong>the</strong> development of pharmaco<strong>the</strong>rapies <strong>for</strong> methamphetamine abuse<br />
<strong>an</strong>d dependence. Ongoing work is investigating <strong>the</strong> interaction of sigma lig<strong>an</strong>ds <strong>an</strong>d<br />
methamphetamine in <strong>an</strong>imal behavioral assays to develop fur<strong>the</strong>r <strong>the</strong>se initial observations.<br />
Disclosures: K. Rodvelt, None; D.K. Miller, None; L.R. Blount, None; K. F<strong>an</strong>, None; J.R.<br />
Lever, None; S.Z. Lever, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.11/AA11<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH Gr<strong>an</strong>t DA025303<br />
Title: Methamphetamine-induced stereotypy correlates negatively with patch-enh<strong>an</strong>ced<br />
prodynorphin <strong>an</strong>d arc expression in <strong>the</strong> striatum <strong>an</strong>d is attenuated by striatal mu opioid receptor<br />
blockade<br />
Authors: *K. A. HORNER, Y. E. GILBERT;<br />
Mercer Univ. Sch. Med., Macon, GA<br />
Abstract: Methamphetamine (METH) induces stereotypy, which is described as inflexible,<br />
repetitive behavior. Enh<strong>an</strong>ced c-Fos activation in <strong>the</strong> patch compartment of <strong>the</strong> dorsolateral<br />
region of rostral striatum correlates with psychostimul<strong>an</strong>t-induced stereotypy, suggesting that <strong>the</strong><br />
expression of this behavior may be related to preferential activation of this region. Patchenh<strong>an</strong>ced<br />
mRNA expression of prodynorphin (PD) <strong>an</strong>d <strong>the</strong> immediate early gene arc have also<br />
been observed in <strong>the</strong> rostral striatum after a dose of METH that induces stereotypy. However, it<br />
is not known whe<strong>the</strong>r patch-enh<strong>an</strong>ced activation of PD or arc mRNA expression correlates with<br />
METH-induced stereotypy. Fur<strong>the</strong>rmore, <strong>the</strong> mech<strong>an</strong>isms that contribute to patch-enh<strong>an</strong>ced gene<br />
expression during stereotypy are not understood. Mu opioid receptors are densely expressed on<br />
<strong>the</strong> neurons of <strong>the</strong> patch compartment <strong>an</strong>d <strong>the</strong>ir activation has been shown to contribute to<br />
METH-induced PD mRNA expression in <strong>the</strong> patch compartment. Thus, it is possible that<br />
activation of striatal mu opioid receptors during METH treatment contributes to patch-enh<strong>an</strong>ced<br />
gene expression <strong>an</strong>d stereotyped behavior. The goals of <strong>the</strong> present study were to determine if 1.)<br />
patch-enh<strong>an</strong>ced activation of PD or arc mRNA correlates with METH-induced stereotypy 2.)<br />
blockade of striatal mu opioid receptors alters METH-induced PD <strong>an</strong>d arc expression in <strong>the</strong><br />
patch compartment <strong>an</strong>d 3.) blockade of striatal mu opioid receptors alters METH-induced<br />
stereotypy. Animals were bilaterally infused in <strong>the</strong> dorsolateral rostral striatum with <strong>the</strong> mu<br />
opioid receptor <strong>an</strong>tagonist CTAP (10 µg/µl) or vehicle, followed by METH (7.5 mg/kg) or<br />
saline. Animals were placed in locomotor activity chambers <strong>an</strong>d observed <strong>for</strong> 3h, after which<br />
time <strong>the</strong>y were sacrificed <strong>an</strong>d PD <strong>an</strong>d arc mRNA expression examined in <strong>the</strong> rostral patch <strong>an</strong>d<br />
matrix compartments using in situ hybridization <strong>an</strong>d mu opioid receptor immunohistochemistry.<br />
Stereotypy was scored on a scale <strong>from</strong> 1-10. Unexpectedly, METH-induced patch-enh<strong>an</strong>ced PD<br />
<strong>an</strong>d arc mRNA expression in <strong>the</strong> dorsolateral striatum showed a signific<strong>an</strong>t negative correlation<br />
with stereotypy. CTAP pretreatment attenuated METH-induced increases in PD mRNA<br />
expression in <strong>the</strong> patch compartment of dorsolateral rostral striatum, but had no effect on<br />
METH-induced arc expression in this region. CTAP pretreatment also signific<strong>an</strong>tly reduced<br />
METH-induced stereotypy. These data suggest that <strong>the</strong> ability of mu opioid receptor blockade to<br />
reduce stereotypy may be due to mech<strong>an</strong>isms o<strong>the</strong>r th<strong>an</strong> <strong>the</strong> regulation of PD or arc expression in<br />
<strong>the</strong> patch compartment of rostral striatum.
Disclosures: K.A. Horner, None; Y.E. Gilbert, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.12/AA12<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: Research on Pharmaceutical <strong>an</strong>d Medical Safety Supported by Gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Ministry<br />
of Health, Labor <strong>an</strong>d Welfare, Jap<strong>an</strong><br />
Global COE Program (Basic <strong>an</strong>d Tr<strong>an</strong>slational Research Center <strong>for</strong> Global Brain<br />
Science), MEXT, Jap<strong>an</strong><br />
NIDA-IRP/NIH/DHHS to FSH <strong>an</strong>d GRU<br />
Title: Role of serotonin 1B receptor in methamphetamine-induced behavioral sensitization in<br />
mice lacking serotonin tr<strong>an</strong>sporter<br />
Authors: *I. MOE 1 , H.-W. SHEN 2 , Y. HAGINO 2 , S. FUKUSHIMA 1 , K.-P. LESCH 3 , D. L.<br />
MURPHY 4 , F. S. HALL 5 , G. R. UHL 5 , K. IKEDA 2 , I. SORA 1 ;<br />
1 Tohoku Univ. Sch. Med., Sendai, Jap<strong>an</strong>; 2 Dept. of Mol. Psychiatry, Tokyo Inst. of Psychiatry,<br />
Tokyo, Jap<strong>an</strong>; 3 Dept. of Psychiatry und Psycho<strong>the</strong>rapy, Univ. of Wurzburg, Wurzburg,<br />
Germ<strong>an</strong>y; 4 Clin. Neuropharm. Section,Laboratory of Clin. Sci., NIMH,<br />
DennisMurphy@mail.nih.gov, MD; 5 Mol. Neurobio. Br., NIDA-IRP, Baltimore, MD<br />
Abstract: Repeated administration of methamphetamine (METH), as well as o<strong>the</strong>r<br />
psychostimul<strong>an</strong>ts, could induce increased sensitivity <strong>an</strong>d behavioral response to METH called<br />
behavioral sensitization. Contribution of dopaminergic system to develop behavioral<br />
sensitization has been suggested strongly, however clear expl<strong>an</strong>ation has not been provided yet.<br />
Contribution of serotonergic system <strong>an</strong>d its interaction with dopaminergic system to develop<br />
behavioral sensitization have been proposed as well. We examined <strong>the</strong> role of serotonergic<br />
system <strong>for</strong> development of METH-induced behavioral sensitization by using serotonin<br />
tr<strong>an</strong>sporter (SERT) knockout (KO) mice <strong>an</strong>d observed absence of METH-induced behavioral<br />
sensitization. We hypo<strong>the</strong>sized that increased serotonin tr<strong>an</strong>smission in SERT KO mice would<br />
inhibit development of METH-induced behavioral sensitization. Studies using selective serotonin<br />
reuptake inhibitors also supported our hypo<strong>the</strong>sis. Present study attempted to detect serotonergic<br />
tr<strong>an</strong>smission through particular serotonin receptor(s) that is crucial <strong>for</strong> inhibiting development of
METH-induced behavioral sensitization. Role of ei<strong>the</strong>r serotonin 2 or 1B receptor in<br />
psychostimul<strong>an</strong>ts-induced behavioral sensitization has often been assessed by using each lig<strong>an</strong>ds<br />
<strong>an</strong>d knockout mice. In present study we examined which selective serotonin receptor <strong>an</strong>tagonist<br />
disinhibit development of METH-induced behavioral sensitization in SERT KO mice. We<br />
pretreated SERT KO mice with ket<strong>an</strong>serin as a selective serotonin 2 receptor <strong>an</strong>tagonist, or SB<br />
216641 as a selective serotonin 1B receptor <strong>an</strong>tagonist, <strong>an</strong>d examined development of METHinduced<br />
behavioral sensitization. Ket<strong>an</strong>serin did not reverse serotonergic inhibition of METHinduced<br />
behavioral sensitization in SERT KO. SB 216641 successfully reversed development of<br />
METH-induced behavioral sensitization <strong>an</strong>d increased behavioral response was maintained after<br />
7 days drug-free period. Our present result indicated that serotonin 1B receptor plays a crucial<br />
role in METH-induced behavioral sensitization, but serotonin 2 receptors may not be relev<strong>an</strong>t at<br />
least to <strong>the</strong> developmental phase.<br />
Disclosures: I. Moe, None; H. Shen, None; Y. Hagino, None; S. Fukushima, None; K. Lesch,<br />
None; D.L. Murphy, None; F.S. Hall, None; G.R. Uhl, None; K. Ikeda, None; I. Sora, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.13/AA13<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIDA Gr<strong>an</strong>t DA018165<br />
Title: Involvement of <strong>the</strong> corticotropin-releasing factor type-2 receptor in psychomotor<br />
sensitization to methamphetamine<br />
Authors: *W. J. GIARDINO 1 , A. M. J. ANACKER 1 , E. SPANGLER 1 , J. LI 1 , D. M. COTE 1 ,<br />
T. J. PHILLIPS 1,2 , A. E. RYABININ 1 ;<br />
1 2<br />
Behavioral Neurosci., Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR; Dept. of Veter<strong>an</strong>s Affairs<br />
Med. Ctr., Portl<strong>an</strong>d, OR<br />
Abstract: Progressively enh<strong>an</strong>ced sensitivity to <strong>the</strong> locomotor-stimulating effects of drugs of<br />
abuse has been identified as a key behavioral output accomp<strong>an</strong>ying <strong>the</strong> neuroadaptations<br />
underlying addiction <strong>an</strong>d subsequent relapse. Recent evidence indicates a role <strong>for</strong> <strong>the</strong><br />
corticotropin-releasing factor (CRF) neuropeptide system in a number of addiction-related<br />
behavioral <strong>an</strong>d molecular phenomena. In order to address <strong>the</strong> role of <strong>the</strong> type-2 CRF receptor<br />
(CRF-R2) in locomotor sensitivity <strong>an</strong>d sensitization to methamphetamine (MA), we compared
male <strong>an</strong>d female CRF-R2 knockout (KO) <strong>an</strong>d wild-type (WT) mice in a 12-day protocol that<br />
included repeated MA administration. Following two days of saline administration to allow<br />
habituation (Day 1) <strong>an</strong>d measure baseline activity levels (Day 2), locomotor behavior was<br />
measured after 1.0 mg/kg MA (i.p.) on Days 3, 5, 7, 9 <strong>an</strong>d 11, followed by a final saline test on<br />
Day 12. Locomotor activity was measured <strong>for</strong> 15 minutes in sound-attenuated chambers, <strong>an</strong>d <strong>the</strong><br />
total number of photocell beam breaks was used as <strong>the</strong> dependent variable. Repeated measures<br />
ANOVA revealed signific<strong>an</strong>t main effects of Genotype wherein WT mice demonstrated<br />
enh<strong>an</strong>ced locomotor activity in comparison to <strong>the</strong>ir KO littermates across <strong>the</strong> entire experiment<br />
(Days 1-12) (F(1,58) = 3.99; p < .05), as well as during <strong>the</strong> repeated MA days (Days 3, 5, 7, 9, 11)<br />
(F(1,58) = 4.21; p < .05). Fur<strong>the</strong>r <strong>an</strong>alysis of data <strong>from</strong> repeated MA days found a signific<strong>an</strong>t effect<br />
of Day (F(4,232) = 49.39; p < .0001), but a non-signific<strong>an</strong>t interaction between Day <strong>an</strong>d Genotype<br />
(F(4,232) = .29; p = .89), suggesting that sensitization to MA occurred, but did not differ based<br />
upon genotype. Although Day 12 locomotor activity was greater th<strong>an</strong> that on Day 2 (F(1,58) =<br />
26.26; p < .001), this did not interact with Genotype (F(1,58) = .46; p = .50), <strong>an</strong>d <strong>the</strong>re was no<br />
effect of Genotype in <strong>an</strong> <strong>an</strong>alysis including data <strong>from</strong> all saline days (Days 1, 2, 12) (F(1.58) =<br />
1.84; p = .18). There<strong>for</strong>e, <strong>the</strong>se genotypic differences are specific to MA <strong>an</strong>d c<strong>an</strong>not be attributed<br />
to differences in assignment of salience to <strong>the</strong> test conditions (i.e. habituation) or contextual<br />
sensitization. In summary, CRF-R2 KO mice sensitized similarly to <strong>the</strong>ir WT littermates, despite<br />
showing decreased sensitivity to repeated MA administration overall. To <strong>the</strong> best of our<br />
knowledge, <strong>the</strong>se data are <strong>the</strong> first to implicate <strong>the</strong> involvement of CRF-R2 in psychostimul<strong>an</strong>tinduced<br />
locomotor activation. Because CRF has lower affinity <strong>for</strong> CRF-R2 th<strong>an</strong> <strong>the</strong> urocortin<br />
peptides, <strong>the</strong>se data suggest that urocortins may contribute to this effect.<br />
Disclosures: W.J. Giardino, None; A.M.J. Anacker, None; E. Sp<strong>an</strong>gler, None; J. Li,<br />
None; D.M. Cote, None; T.J. Phillips, None; A.E. Ryabinin, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.14/AA14<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: DGAPA gr<strong>an</strong>t IN301809<br />
CONACyT gr<strong>an</strong>t 60872<br />
Title: Effects of acute food deprivation on <strong>the</strong> expression of methamphetamine-induced<br />
locomotor sensitization
Authors: *F. MIRANDA-HERRERA 1,2 , H. SANCHEZ-CASTILLO 2 , D. N. VELAZQUEZ-<br />
MARTINEZ 2 ;<br />
1 Univ. Nacional Autonoma De Mexico, Tl<strong>an</strong>ep<strong>an</strong>tla, Edo Mex, Mexico; 2 Facultad de Psicología,<br />
Univ. Nacional Autónoma de México, Mexico, Mexico<br />
Abstract: Psychostimul<strong>an</strong>t drugs such as amphetamine (AMPH) <strong>an</strong>d methamphetamine<br />
(METH) exert <strong>the</strong>ir actions on <strong>the</strong> Central Nervous System by increasing dopamine (DA)<br />
neurotr<strong>an</strong>smission in <strong>the</strong> brain reward systems. This property relies on its actions at <strong>the</strong> DA<br />
tr<strong>an</strong>sporter (DAT) that is <strong>the</strong> major clear<strong>an</strong>ce mech<strong>an</strong>ism <strong>for</strong> extracellular DA. It is well known<br />
that AMPH <strong>an</strong>d METH increase DAergic signaling in brain reward systems by stimulating <strong>the</strong><br />
release of DA through reversing <strong>the</strong> DAT function. Recently it has been suggested that <strong>the</strong><br />
insulin signaling pathways have <strong>the</strong> ability to modulate <strong>the</strong> DAT function. Some studies have<br />
reported that <strong>the</strong> hypoinsulinemia attenuates <strong>the</strong> DAT functions <strong>an</strong>d, as a consequence, <strong>the</strong><br />
psychostimul<strong>an</strong>t-induced behaviors are reduced. For example, Galici et al. (2003) reported that<br />
<strong>the</strong> depletion of insulin by single injection of streptozotocin reduced AMPH self-administration.<br />
In <strong>the</strong> present study we examined <strong>the</strong> effects of acute food deprivation, which reduces <strong>the</strong> insulin<br />
<strong>an</strong>d glucose levels, on <strong>the</strong> METH-induced locomotor sensitization. Male Wistar rats were<br />
divided in five groups (n=10) <strong>an</strong>d treated with METH or saline during days 1-5 (development of<br />
METH sensitization). On day 8 (expression of METH sensitization) rats were treated with<br />
METH or saline. On days 6-7, <strong>an</strong>imals remained drug-free in <strong>the</strong>ir home cages. Groups M24M<br />
<strong>an</strong>d M0M were treated with METH (1 mg/kg: ip) during development <strong>an</strong>d expression of METH<br />
sensitization; rats of <strong>the</strong> group M24M were deprived of food 24 h be<strong>for</strong>e <strong>the</strong> expression of<br />
METH sensitization, while rats of group M0M were undeprived. Groups S0M, S24S <strong>an</strong>d S0S<br />
were treated with saline on <strong>the</strong> development of METH sensitization. Rats of group S0M were<br />
undeprived but treated with METH (1 mg/kg: ip) on <strong>the</strong> expression of METH sensitization; rats<br />
of group S24S were deprived of food 24 h be<strong>for</strong>e <strong>the</strong> expression of METH sensitization. The<br />
behavioral activity was recorded <strong>for</strong> 60 min on open-field cages <strong>an</strong>d blood glucose concentration<br />
was measured on days 5 <strong>an</strong>d 8 be<strong>for</strong>e <strong>the</strong> experimental session. The results showed that repeated<br />
administration of METH induced a progressive increase in locomotor activity in rats on <strong>the</strong><br />
development of METH sensitization. However, METH administration on <strong>the</strong> expression of<br />
METH sensitization produced a decrease on locomotor activity after 24 h of food deprivation on<br />
group M24M as compared to rats of group M0M. The results also showed that food deprivation<br />
induced a reduction in <strong>the</strong> glucose levels. These results are in line with previous studies <strong>an</strong>d<br />
suggest that food deprivation reduces some behavioral effects of <strong>the</strong> psychostimul<strong>an</strong>ts such as<br />
AMPH <strong>an</strong>d METH.<br />
Disclosures: F. Mir<strong>an</strong>da-Herrera, None; H. S<strong>an</strong>chez-Castillo, None; D.N. Velazquez-<br />
Martinez, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.15/AA15<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH Gr<strong>an</strong>t RR-P20 RR17701<br />
Title: The µ-opioid receptor knockout mice are less sensitive to methamphetamine-induced<br />
decrease in tyrosine hydroxylase but not in monoamine oxidase A expression in <strong>the</strong> caudate<br />
putamen<br />
Authors: S. PARK 1 , *T. MA 1 , Z. HE 2 ;<br />
1 Pharmacology/Toxicology, 2 Pathology, Univ. Mississippi Med., Jackson, MS<br />
Abstract: Tyrosine hydroxylase (TH) <strong>an</strong>d monoamine oxidase A (MAO-A), respectively, are<br />
<strong>the</strong> key enzymes in syn<strong>the</strong>sis <strong>an</strong>d metabolism of dopamine in <strong>the</strong> central nervous system. These<br />
enzymes are targets <strong>for</strong> psychostimul<strong>an</strong>ts as well. Exposure to psychostimul<strong>an</strong>ts c<strong>an</strong> interfere in<br />
syn<strong>the</strong>sis <strong>an</strong>d metabolism of dopamine <strong>an</strong>d, <strong>the</strong>reby, lead to neurotoxicity <strong>an</strong>d/or addiction.<br />
Methamphetamine (METH) is a highly addictive psychostimul<strong>an</strong>t that, when repeatedly<br />
administered (2.5 <strong>an</strong>d 10 mg/kg), may cause behavioral sensitization in mice, a condition<br />
characterized by hyper-locomotor activity <strong>an</strong>d stereotypic behaviors in previous studies. METHinduced<br />
behavioral sensitization was signific<strong>an</strong>tly attenuated in µ-opioid receptor (µ-OR)<br />
knockout mice. Also, repeated METH administration decreased <strong>the</strong> amount of striatal dialysate<br />
3,4-dihydroxyphenylactic acid (DOPAC), a dopamine metabolite <strong>for</strong>med by MAO, in wild-type<br />
mice but not in µ-OR knockout mice. The mech<strong>an</strong>ism responsible <strong>for</strong> this phenomenon remains<br />
unclear. This study is to determine whe<strong>the</strong>r METH exposure produces different effects on TH<br />
<strong>an</strong>d MAO-A in <strong>the</strong> two genotypes of mice. Mice received daily intraperitoneal injections of<br />
METH (0, 0.625, 2.5, or 10 mg/kg) <strong>for</strong> 7 consecutive days to induce sensitization. On<br />
experimental day 11 (4 days after <strong>the</strong> last injection), brain tissues were taken <strong>for</strong><br />
immunohistochemical <strong>an</strong>alysis. The results indicate that <strong>the</strong> optical densities of MAO-A<br />
immunostaining in <strong>the</strong> caudate putamen are signific<strong>an</strong>tly decreased in all METH-sensitized mice,<br />
without a genotype difference. METH (2.5 <strong>an</strong>d 10 mg/kg) decreased optical densities of TH<br />
immunostaining in <strong>the</strong> caudate putamen <strong>an</strong>d positive TH immunostaining cells in <strong>the</strong> ventral<br />
tegmental area in wild-type mice not in µ-OR knockout mice. In conclusion, lack of µ-OR in<br />
mice attenuates <strong>the</strong> inhibitory effect of METH on TH but not on MAO-A in <strong>the</strong> brain. These data<br />
suggest that attenuation of METH-induced decrease in dopamine syn<strong>the</strong>sis in µ-OR knockout<br />
mice is possibly mediated with <strong>the</strong> less behavioral responses to <strong>the</strong> psychostimul<strong>an</strong>t th<strong>an</strong> wildtype<br />
mice.<br />
Disclosures: S. Park, None; T. Ma, None; Z. He, None.<br />
Poster
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.16/AA16<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Neurochemical effects of escalating dose methamphetamine pretreatment versus acute<br />
binge administration in dopamine neurons of <strong>the</strong> nigrostriatal <strong>an</strong>d mesoaccumbens pathways<br />
Authors: C. E. KINNEY 1 , M. SALVATORE 1 , *N. E. GOEDERS 2 ;<br />
1 LSU Hlth. Sci. Ctr., Shreveport, LA; 2 LSU Hlth. Sci. Cntr, Shreveport, LA<br />
Abstract: The use of <strong>the</strong> psychostimul<strong>an</strong>t drug methamphetamine (MA) is known to be both<br />
addictive as well as neurotoxic to neurons in m<strong>an</strong>y regions of <strong>the</strong> brain. Dopaminergic neurons<br />
of <strong>the</strong> nigrostriatal <strong>an</strong>d mesoaccumbens pathways are directly affected by MA <strong>an</strong>d have been<br />
shown to exhibit lasting functional <strong>an</strong>d neurochemical deficits following even acute<br />
administration. MA initially facilitates a subst<strong>an</strong>tial increase in synaptic dopamine (DA) through<br />
reversal of <strong>the</strong> dopamine active tr<strong>an</strong>sporter (DAT), causing overstimulation of post-synaptic<br />
dopamine receptors. Since <strong>the</strong> normal function of synaptic DAT is termination of<br />
neurotr<strong>an</strong>smission by pre-synaptic dopamine reuptake, dopamine is not taken back up <strong>an</strong>d more<br />
must be syn<strong>the</strong>sized. To aid this burden placed on <strong>the</strong> biosyn<strong>the</strong>tic machinery, we propose that<br />
increased phosphorylation of tyrosine hydroxylase (TH), <strong>the</strong> rate-limiting enzyme in dopamine<br />
biosyn<strong>the</strong>sis, is utilized as a compensatory mech<strong>an</strong>ism to increase enzymatic activity. The<br />
present study was designed to evaluate this hypo<strong>the</strong>sis by comparing <strong>the</strong> effects of <strong>an</strong> acute dose<br />
of MA with <strong>the</strong> effects of <strong>the</strong> same dose following pretreatment with <strong>an</strong> escalating dose regimen.<br />
The effect of <strong>the</strong>se treatments on DAT <strong>an</strong>d TH expression will be assessed, as well as <strong>the</strong><br />
expression of vesicular monoamine tr<strong>an</strong>sporter-2 (VMAT-2) which is inhibited by MA. Our<br />
initial findings suggest that <strong>the</strong> two pathways are differentially affected. Acute binge<br />
administration caused a 28% reduction of dopamine recovery in <strong>the</strong> nucleus accumbens, <strong>an</strong>d <strong>an</strong><br />
increase to 146% of control in <strong>the</strong> ventral tegmental area. Signific<strong>an</strong>t effects were not seen in <strong>the</strong><br />
subst<strong>an</strong>tia nigra or <strong>the</strong> striatum. Pretreatment resulted in no signific<strong>an</strong>t differences in <strong>an</strong>y of <strong>the</strong><br />
four regions.<br />
Disclosures: C.E. Kinney, None; M. Salvatore, None; N.E. Goeders, DA06013, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received).<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.17/AA17<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH/NIDA Gr<strong>an</strong>t DA024038<br />
NARSAD<br />
Title: Repeated methamphetamine exposure increases dopamine <strong>an</strong>d glutamate signaling in<br />
mouse prefrontal cortex <strong>an</strong>d accumbens<br />
Authors: *K. D. LOMINAC 1 , K. K. SZUMLINSKI 2 ;<br />
2 Psychology, 1 Univ. Cali<strong>for</strong>nia- S<strong>an</strong>ta Barbara, S<strong>an</strong>ta Barbara, CA<br />
Abstract: Clinical studies have associated long-term methamphetamine (METH) abuse with a<br />
loss of prefrontal cortical grey matter <strong>an</strong>d ultimately, volitional control. Similarly, repeated<br />
exposure to psychostimul<strong>an</strong>ts is associated with a marked increase in sensitivity to <strong>the</strong> drug’s<br />
behavioral activating effects, as well as <strong>the</strong> glutamate <strong>an</strong>d dopamine releasing effects of<br />
subsequent drug administrations. However, <strong>the</strong> molecular mech<strong>an</strong>isms underlying <strong>the</strong>se<br />
alterations remain unclear <strong>an</strong>d under-characterized. Thus, we employed immunoblotting<br />
techniques to assess ch<strong>an</strong>ges in known regulators of glutamate <strong>an</strong>d dopamine tr<strong>an</strong>smission<br />
alongside in vivo microdialysis to elucidate potential proteonomic <strong>an</strong>d functional alterations in<br />
<strong>the</strong> prefrontal cortex <strong>an</strong>d nucleus accumbens following short (24 hours) <strong>an</strong>d long-term (3 weeks)<br />
withdrawal <strong>from</strong> repeated low dose (10 X 2mg/kg) METH or saline administration. With regard<br />
to <strong>the</strong> nucleus accumbens, we observed a tr<strong>an</strong>sient shift to <strong>the</strong> left in <strong>the</strong> dose-glutamate response<br />
function <strong>for</strong> group1 mGluR agonist DHPG following 24 hours withdrawal <strong>from</strong> repeated METH,<br />
which was not present following long-term withdrawal. Fur<strong>the</strong>rmore, <strong>the</strong> increased mGluR1<br />
function was not associated with ch<strong>an</strong>ges in accumbens levels of mGluR5, although a moderate<br />
increase in Homer1b/c protein expression was observed in METH-treated <strong>an</strong>imals following 3<br />
weeks withdrawal. In <strong>the</strong> prefrontal cortex, no ch<strong>an</strong>ge was observed in <strong>the</strong> levels of <strong>an</strong>y<br />
glutamate receptors, Homer proteins or NMDA receptor subunits. However, we found a<br />
signific<strong>an</strong>t elevation in levels of both dopamine tr<strong>an</strong>sporter <strong>an</strong>d D2 receptor expression in METH<br />
treated <strong>an</strong>imals following short, but not long-term withdrawal. Collectively, <strong>the</strong>se data indicate<br />
that a history of low dose METH produces tr<strong>an</strong>sient ch<strong>an</strong>ges in <strong>the</strong> regulators of dopamine <strong>an</strong>d<br />
glutamate tr<strong>an</strong>smission within limbic regions implicated in <strong>the</strong> addictive properties of this drug.<br />
Disclosures: K.D. Lominac, None; K.K. Szumlinski, None.<br />
Poster
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.18/AA18<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: RL1 DA024853 (EDL)<br />
R01 DA020726 (EDL)<br />
P20 DA022539 (EDL)<br />
UCLA GCRC MOI RR 00865<br />
Additional funding provided by <strong>the</strong> endowment <strong>from</strong> <strong>the</strong> Thomas P. And Ka<strong>the</strong>rine K.<br />
Pike Chair in Addiction Studies (EDL)<br />
Title: Impulsive behavior <strong>an</strong>d dopamine D2/3 receptor availability in healthy <strong>an</strong>d MA-dependent<br />
individuals<br />
Authors: *D. G. GHAHREMANI 1 , B. LEE 1 , G. TABIBNIA 1 , J. MONTEROSSO 5 , R. A.<br />
POLDRACK 1,2,3 , E. D. LONDON 1,4,3 ;<br />
1 Psychiatry <strong>an</strong>d Biobehavioral Sci., 2 Psychology, 3 Brain Res. Inst., 4 Mol. <strong>an</strong>d Med. Pharmacol.,<br />
UCLA, Los Angeles, CA; 5 Psychiatry <strong>an</strong>d Biobehavioral Sci., USC, Los Angeles, CA<br />
Abstract: Several lines of evidence suggest that <strong>the</strong> mesolimbic dopamine (DA) pathway is<br />
import<strong>an</strong>t <strong>for</strong> both rein<strong>for</strong>cement learning <strong>an</strong>d cognitive control. Methamphetamine (MA)<br />
abusers are characterized as having abnormal functioning of this pathway in combination with<br />
impulsive behaviors, such as difficulty in inhibiting a pre-potent response (perseveration). To<br />
assess <strong>the</strong> relationship between baseline DA D2/D3 receptor availability <strong>an</strong>d perseverative<br />
responding in both MA abusers <strong>an</strong>d healthy controls (HC), we correlated DA D2/3 receptor<br />
binding potential (BPND, a measure of receptor availability) <strong>an</strong>d perseverative errors made during<br />
<strong>the</strong> reversal stage of a reversal learning task (using age, sex, <strong>an</strong>d smoking status as covariates of<br />
no interest). BPND was assessed using positron emission tomography with [ 18 F]Fallypride, a<br />
D2/D3 receptor radiolig<strong>an</strong>d. In <strong>the</strong> reversal learning task, MA <strong>an</strong>d HC particip<strong>an</strong>ts were trained<br />
on stimulus-response-outcome associations to criteria prior to reversal stages during which<br />
associations were reversed. More perseverative errors were made by MA th<strong>an</strong> HC particip<strong>an</strong>ts,<br />
<strong>an</strong>d number of errors were negatively correlated with D2/D3 BPND in <strong>the</strong> ventral striatum, a<br />
major target of midbrain dopamine neurons. This correlation was stronger in MA th<strong>an</strong> HC<br />
particip<strong>an</strong>ts. These preliminary results suggest that <strong>the</strong> degree of perseverative responding is<br />
related to baseline D2/D3 receptor availability, such that more errors occur with less availability.
The findings support <strong>the</strong> idea that impairment in behavioral control in MA abuse is related to<br />
atypical DA functioning commonly observed in subst<strong>an</strong>ce abuse individuals.<br />
Disclosures: D.G. Ghahrem<strong>an</strong>i, None; B. Lee, None; G. Tabibnia, None; J. Monterosso,<br />
None; R.A. Poldrack, None; E.D. London, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.19/AA19<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: F31 DA025422 (DEP)<br />
P20 DA022539 (EDL)<br />
R01 DA020726 (EDL)<br />
RL1DA024853 (EDL)<br />
Additional funding provided by <strong>the</strong> endowment <strong>from</strong> <strong>the</strong> Thomas P. And Ka<strong>the</strong>rine K.<br />
Pike Chair in Addiction Studies (EDL)<br />
Title: Emotion regulation <strong>an</strong>d aggression during early abstinence <strong>from</strong> methamphetamine<br />
Authors: *D. E. PAYER 1 , M. D. LIEBERMAN 2 , E. D. LONDON 3,4,5 ;<br />
1 Interdepartment Neurosci., 2 Psychology, 3 Psychiatry <strong>an</strong>d Biobehavioral Sci., 4 Mol. <strong>an</strong>d Med.<br />
Pharmacol., 5 Brain Res. Inst., UCLA, Los Angeles, CA<br />
Abstract: Methamphetamine (MA) abuse is linked to violence <strong>an</strong>d aggression, possibly<br />
reflecting deficits in emotion regulation, i.e., poor prefrontal cortical [PFC] control over limbic<br />
targets such as <strong>the</strong> amygdala. We <strong>the</strong>re<strong>for</strong>e investigated amygdala regulation in conjunction with<br />
aggressive behavior in MA-dependent individuals.<br />
Twenty-seven MA-dependent subjects (9 women; 34.6 ± 9.3 yrs; abstinent 7.4 ± 1.7 days) <strong>an</strong>d<br />
21 healthy control (HC) subjects (12 women; 28.4 ± 8.2 yrs) completed: 1) fMRI paired with <strong>an</strong><br />
emotion regulation task involving emotional faces, including a visual condition (“Match”), a<br />
verbal condition (“Label”) previously shown to reduce amygdala activation, <strong>an</strong>d a control<br />
condition (“Shapes”); 2) <strong>an</strong> aggression task (CRT), in which <strong>the</strong>y exposed a fictitious opponent
to aversive noise; <strong>an</strong>d 3) self-report measures: <strong>the</strong> Aggression Questionnaire (AQ) <strong>an</strong>d Symptom<br />
Checklist (SCL-90).<br />
fMRI data were <strong>an</strong>alyzed using <strong>the</strong> general linear model. Amygdala region-of-interest <strong>an</strong>alyses<br />
yielded parameter estimates <strong>for</strong> each condition of <strong>the</strong> task <strong>for</strong> each subject. Repeated-measures<br />
ANOVA of <strong>the</strong>se values revealed a main effect of condition (F(2,88) = 51.0, p < .001), with<br />
amygdala activation during Match higher th<strong>an</strong> during Label (F(1,44) = 4.8, p < .05), <strong>an</strong>d<br />
activation during both higher th<strong>an</strong> during Shapes (F(1,44) = 103.0, p < .05). There was no effect<br />
of group or group x condition interaction. Whole-brain psychophysiological interaction <strong>an</strong>alyses<br />
revealed that <strong>the</strong> reduction in amygdala activity accomp<strong>an</strong>ied greater functional connectivity<br />
with right PFC during Label th<strong>an</strong> during Match (peak x,y,z [mm] = 44, 30, 16; t = 4.96; p < .05,<br />
FDR-corrected); this effect did not differ between groups.<br />
MA subjects scored marginally higher th<strong>an</strong> HC subjects on <strong>the</strong> CRT (t(19) = 2.0, p = .056), <strong>an</strong>d<br />
signific<strong>an</strong>tly higher on <strong>the</strong> AQ (t(36) = 3.1) <strong>an</strong>d <strong>the</strong> Hostility (t(46) = 2.2) <strong>an</strong>d Interpersonal<br />
Sensitivity (t(46) = 3.1) subscales of <strong>the</strong> SCL-90 (all p < .05).<br />
Amygdala ch<strong>an</strong>ge scores (Match - Label activation) correlated negatively with CRT per<strong>for</strong>m<strong>an</strong>ce<br />
after accounting <strong>for</strong> Group <strong>an</strong>d Sex (r = -.505, p < .05, df = 17). In HC subjects, amygdala<br />
ch<strong>an</strong>ge scores also correlated with AQ scores (r = -.642) <strong>an</strong>d SCL-90 Hostility scores (r = -.662,<br />
both p < .05, df = 14). In MA subjects, amygdala ch<strong>an</strong>ge scores did not correlate with self-report<br />
measures.The results indicate that amygdala regulation is intact <strong>an</strong>d predicts aggressive behavior<br />
in both groups. However, amygdala activity predicts self-report of aggression only in HC<br />
subjects. Why MA subjects report <strong>an</strong>d exhibit more aggression in <strong>the</strong> absence of amygdala<br />
dysregulation, <strong>an</strong>d why <strong>the</strong>ir self-report is discord<strong>an</strong>t with amygdala activity, remains to be<br />
determined.<br />
Disclosures: D.E. Payer, None; M.D. Lieberm<strong>an</strong>, None; E.D. London, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.20/AA20<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NSF0642188<br />
U54NS041071<br />
R24DA021471
T32HL076133<br />
Title: The acute effects of methamphetamine on synaptic plasticity in <strong>the</strong> CA1 region of mouse<br />
hippocampus<br />
Authors: *J. M. SWANT;<br />
Neurobiology/Neurotoxicology, Meharry Med. Col., Nashville, TN<br />
Abstract: Addictive drugs alter synaptic plasticity in <strong>the</strong> brain, <strong>an</strong> effect which may contribute<br />
to <strong>the</strong> etiology of drug addiction. For example, cocaine <strong>an</strong>d GBR12935 (both which increase <strong>the</strong><br />
concentration of synaptic dopamine) increase long-term potentiation (LTP) in <strong>the</strong> CA1 area of<br />
<strong>the</strong> hippocampus (Sw<strong>an</strong>t <strong>an</strong>d Wagner 2006; Thompson et al. 2005; Thompson et al. 2004).<br />
Methamphetamine (METH) profoundly increases <strong>the</strong> concentration of extracellular monoamines<br />
dopamine, norepinephrine, <strong>an</strong>d serotonin by redistributing neurotr<strong>an</strong>smitter <strong>from</strong> synaptic<br />
vesicles to <strong>the</strong> cytosol, while also inducing reverse tr<strong>an</strong>sport <strong>an</strong>d competing <strong>for</strong> tr<strong>an</strong>smitter at <strong>the</strong><br />
dopamine, norepinephrine, <strong>an</strong>d serotonin tr<strong>an</strong>sporter, respectively (Sulzer et al. 2005). Here we<br />
study <strong>the</strong> acute effects of methamphetamine on LTP in <strong>the</strong> CA1 region of <strong>the</strong> mouse<br />
hippocampus. In contrast to cocaine, <strong>the</strong> acute application of METH to in vitro hippocampal<br />
slices decreased LTP. Interestingly, <strong>the</strong> application of METH at a concentration of 30 µM<br />
resulted in <strong>an</strong> increase in baseline synaptic tr<strong>an</strong>smission as well as a profound decrease in LTP.<br />
Pretreatment with thapsigargin, verapamil, timolol, or eticlopride did not block <strong>the</strong> effects of 30<br />
µM METH on baseline synaptic tr<strong>an</strong>smission <strong>an</strong>d LTP. However, pretreatment with D1/D5<br />
dopamine receptor <strong>an</strong>tagonist SCH23390 or 5HT-1A serotonin <strong>an</strong>tagonist NAN-190 eliminated<br />
<strong>the</strong> effects of METH both on baseline synaptic tr<strong>an</strong>smission <strong>an</strong>d LTP. These unexpected results<br />
show that unlike cocaine, which c<strong>an</strong> act to increase LTP in <strong>the</strong> CA1 by activation of D2-like<br />
dopamine receptors (Thompson et al. 2005), METH decreases LTP through a process which<br />
requires <strong>the</strong> activation of dopaminergic or serotonergic receptor systems.<br />
Disclosures: J.M. Sw<strong>an</strong>t, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.21/AA21<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Methamphetamine-induced Npas4, a neuronal PAS doamin 4 expression <strong>an</strong>d a possible<br />
role in neuronal differentiation
Authors: *J. YUN, T. NAGAI, Y. HIBI, H. KOIKE, A. NITTA, K. YAMADA;<br />
Nagoya Univ. Grad. Sch. of Med., Nagoya City/Aichi Prefecture, Jap<strong>an</strong><br />
Abstract: Neuronal PAS domain 4 (Npas4), a brain specific basic helix-loop-helix tr<strong>an</strong>scription<br />
factor, has recently been shown to regulate <strong>the</strong> development of GABAergic inhibitory neurons.<br />
The physiological <strong>an</strong>d pathophysiological role of Npas4, however, remains to be determined. We<br />
have previously reported that <strong>the</strong> expression levels of Npas4 mRNA are decreased in <strong>the</strong><br />
hippocampus of socially isolated mice that show impairment of memory <strong>an</strong>d emotion. Because<br />
tr<strong>an</strong>scriptional regulation of synaptic plasticity <strong>an</strong>d remodeling may have a crucial role in<br />
methamphetamine-induced psychosis <strong>an</strong>d dependence, we investigated <strong>the</strong> effect of<br />
methamphetamine on <strong>the</strong> expression level of Npas4 in ICR mice. Repeated but not acute<br />
administration of methamphetamine (2 mg/kg, sc, 10 days) increased <strong>the</strong> Npas4 mRNA <strong>an</strong>d<br />
protein levels in <strong>the</strong> hippocampus, although <strong>the</strong> treatment had no effect in <strong>the</strong> prefrontal cortex<br />
<strong>an</strong>d striatum.<br />
In <strong>the</strong> murine neuroblastoma cell line (Neuro2a cells), LiCl induced neuronal differentiation with<br />
tau-positive neurite outgrowth. Treatment with siRNA of Npas4 signific<strong>an</strong>tly decreased while<br />
Npas4 overexpression potentiated neurite outgrowth induced by LiCl. In addition, NeuN, a<br />
mature neuronal marker was increased by Npas4 overexpression in Neuro2a cells.<br />
These results suggest that repeated administration of methamphetamine increases <strong>the</strong> expression<br />
levels of Npas4 in <strong>the</strong> hippocampus which may have a role in brain synaptic remodeling induced<br />
by methamphetamine.<br />
Disclosures: J. Yun, None; T. Nagai, None; Y. Hibi, None; H. Koike, None; A. Nitta,<br />
None; K. Yamada, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.22/AA22<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Methamphetamine (METH) preconditioning attenuates <strong>the</strong> toxic effects of multiple binge<br />
METH challenges in <strong>the</strong> rat brain<br />
Authors: *A. B. HODGES 1,2 , B. LADENHEIM 2 , M. T. MCCOY 2 , G. BEAUVAIS 2 , N. S.<br />
CAI 2 , I. N. KRASNOVA 2 , J. L. CADET 2 ;<br />
1 Psychology, Morg<strong>an</strong> St Univ., Baltimore, MD; 2 Mol. Neuropsychiatry Res. Br<strong>an</strong>ch, Intramural<br />
Res. Program, Natl. Inst. on Drug Abuse, NIH, DHHS, Baltimore, MD
Abstract: Methamphetamine (METH) abusers show biochemical <strong>an</strong>d structural abnormalities in<br />
<strong>the</strong>ir brains. Rodent models of METH toxicity typically utilize a binge dose model in which large<br />
doses of METH are given multiple times with short intervals within a single day. The toxicity is<br />
characterized by depletion of dopamine (DA), serotonin (5-HT) <strong>an</strong>d of <strong>the</strong>ir metabolites in<br />
various regions of <strong>the</strong> rat brain. However, injections of smaller METH doses have been shown to<br />
prevent or attenuate some of <strong>the</strong> toxic effects of <strong>the</strong> drug. These results are somewhat<br />
contradictory to observations in hum<strong>an</strong> METH abusers who progressively increase <strong>the</strong>ir intake of<br />
METH <strong>an</strong>d yet show signific<strong>an</strong>t depletion of DA <strong>an</strong>d 5-HT in post-mortem studies. In order to<br />
clarify <strong>the</strong>se issues fur<strong>the</strong>r, we have begun to investigate <strong>the</strong> biochemical effects of different<br />
schedules of METH preconditioning. In <strong>the</strong> present study, rats were preconditioned <strong>for</strong> two<br />
weeks with ei<strong>the</strong>r METH or saline <strong>an</strong>d <strong>the</strong>n given a METH challenge (5mg/kg X 6 at 1 hr<br />
intervals) or saline twice within a 72-hour interval on <strong>the</strong> third week. The <strong>an</strong>imals were<br />
euth<strong>an</strong>ized two weeks after <strong>the</strong> last METH challenge. Rats pretreated with saline <strong>an</strong>d challenged<br />
with METH showed marked decreases in DA <strong>an</strong>d 5-HT levels in <strong>the</strong> striatum. They also<br />
experience signific<strong>an</strong>t decreases in DA <strong>an</strong>d 5-HT levels in <strong>the</strong> nucleus accumbens. 5-HT levels<br />
were also decreased in <strong>the</strong> cortex <strong>an</strong>d hippocampus. Saline-pretreated <strong>an</strong>imals that received two<br />
METH challenges did not show <strong>an</strong>y fur<strong>the</strong>r reduction in <strong>the</strong> levels of <strong>the</strong> monoamines. Rats<br />
preconditioned with METH followed by two METH challenges show signific<strong>an</strong>t protection<br />
against METH toxicity. The METH preconditioning model will be useful in studies aimed at<br />
assessing molecular <strong>an</strong>d cellular mech<strong>an</strong>isms by which <strong>the</strong> brain adapts to repeated but not<br />
overwhelming toxic insults.<br />
Disclosures: A.B. Hodges, None; B. Ladenheim, None; M.T. McCoy, None; G. Beauvais,<br />
None; N.S. Cai, None; I.N. Krasnova, None; J.L. Cadet, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.23/AA23<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: Intramural Research Program of <strong>the</strong> DHHS/NIH/National Institute of Drug Abuse.<br />
Title: Methamphetamine preconditioning <strong>an</strong>d tr<strong>an</strong>scriptional responses in <strong>the</strong> striatum <strong>an</strong>d<br />
midbrain of rats after a single-day multiple-dose methamphetamine challenge
Authors: *J. L. CADET, N. CAI, I. KRASNOVA, B. LADENHEIM, G. BEAUVAIS, M. T.<br />
MCCOY, N. WILSON;<br />
Mol. Neuropsychiat Br., NIH/NIDA, Baltimore, MD<br />
Abstract: Methamphetamine (METH) is <strong>an</strong> illicit drug that causes neurotoxic effects in <strong>the</strong> rat<br />
brain. Animal studies have revealed signific<strong>an</strong>t decreases in dopamine (DA) levels in <strong>an</strong>imals<br />
exposed to moderate to large doses of <strong>the</strong> drug given within a short period of time. However,<br />
repeated injections of <strong>the</strong> drug followed by a challenge with toxic METH doses af<strong>for</strong>d signific<strong>an</strong>t<br />
protection against METH-induced DA depletion. The present study was undertaken to test <strong>the</strong><br />
possibility that repeated injections of <strong>the</strong> drug might be accomp<strong>an</strong>ied by ch<strong>an</strong>ges that render <strong>the</strong><br />
nigrostriatal DA system refractory to <strong>the</strong> acute toxic effects of METH. METH preconditioning<br />
provided signific<strong>an</strong>t protection against METH-induced striatal DA depletion. In addition,<br />
qu<strong>an</strong>titative PCR revealed that preconditioning with METH leads to responses in <strong>the</strong> midbrain<br />
that are different <strong>from</strong> those observed in <strong>the</strong> striatum after a METH toxic challenge. These<br />
include METH preconditioning-induced increases in <strong>the</strong> tr<strong>an</strong>scripts <strong>for</strong> CuZn superoxide<br />
dismutase (SOD) <strong>an</strong>d MnSOD, both of which breakdown <strong>the</strong> superoxide radical to hydrogen<br />
peroxide. There were signific<strong>an</strong>t increases in <strong>the</strong> levels of glutathione peroxidase (GPx) mRNA,<br />
<strong>an</strong> enzyme which breaks down hydrogen peroxide. Heme oxygenase-1 (Hmox-1) was also<br />
signific<strong>an</strong>tly increased in <strong>the</strong> midbrain but nor in <strong>the</strong> striatum of METH-pretreated rats. <strong>When</strong><br />
taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that repeated injections of METH led to <strong>the</strong> development of<br />
latent METH toler<strong>an</strong>t brain. The refractory brain is characterized by increased midbrain<br />
<strong>an</strong>tioxid<strong>an</strong>t responses after <strong>the</strong> METH challenge <strong>an</strong>d resist<strong>an</strong>ce to <strong>the</strong> acute METH-induced<br />
striatal DA depletion. Our model is consistent with observations reported in o<strong>the</strong>r models of<br />
brain preconditioning.<br />
Disclosures: J.L. Cadet, None; N. Cai, None; I. Krasnova, None; B. Ladenheim, None; G.<br />
Beauvais, None; M.T. McCoy, None; N. Wilson, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.24/AA24<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: Intramural Research Program of <strong>the</strong> DHHS/NIH/National Institute of Drug Abuse.<br />
Title: Methamphetamine induces time- <strong>an</strong>d dose-dependent increases in <strong>the</strong> levels of mRNAs <strong>for</strong><br />
several tr<strong>an</strong>scription factors in <strong>the</strong> rat striatum
Authors: *N. CAI, B. LADENHEIM, M. T. MCCOY, J. CADET;<br />
NIDA/NIH, Baltimore, MD<br />
Abstract: METH is <strong>an</strong> illicit drug that is abused worldwide. Its addictive <strong>an</strong>d neurodegenerative<br />
effects are mediated by <strong>the</strong> release of presynaptic dopamine (DA) <strong>an</strong>d <strong>the</strong> activation of DA<br />
receptors in <strong>the</strong> basal g<strong>an</strong>glia. We have sought to dissect <strong>the</strong> molecular underpinnings of <strong>the</strong><br />
long-term effects of <strong>the</strong> drug by measuring its impact on gene tr<strong>an</strong>scription in various brain<br />
regions of <strong>the</strong> rodent brain at various times after its acute or chronic administration. In <strong>the</strong><br />
present study, we used qu<strong>an</strong>titative PCR to measure <strong>the</strong> effects of <strong>the</strong> drug on <strong>the</strong> levels of<br />
mRNAs <strong>for</strong> several tr<strong>an</strong>scription factors that are thought to be involved in plastic remodeling of<br />
<strong>the</strong> central nervous system (CNS). We found that METH (20 mg/kg) caused subst<strong>an</strong>tial timedependent<br />
increases in Egr1, Egr2, <strong>an</strong>d Egr3 expression, with Egr2 showing <strong>the</strong> increases (~60fold)<br />
peaking at 1h after <strong>the</strong> drug injection. METH also caused increases in Nr4a1a, Nr4a2, <strong>an</strong>d<br />
Nr4a3 mRNA levels. Nr4a3 showed <strong>the</strong> greatest increases, reaching ~12-fold at 1h after drug<br />
administration. METH-induced increases in <strong>the</strong> expression of Arc, Icer, <strong>an</strong>d Nab2 were of<br />
smaller magnitude <strong>an</strong>d were somewhat more delayed, reaching <strong>the</strong>ir peaks at 2h after <strong>the</strong> drug<br />
injection. Moreover, METH (1, 5, 10, 20, 40 mg/kg) also caused dose-dependent increases in <strong>the</strong><br />
expression of <strong>the</strong>se genes measured at 1h after drug administration. <strong>When</strong> taken toge<strong>the</strong>r, <strong>the</strong>se<br />
observations suggest that <strong>the</strong> long-term neuroplastic <strong>an</strong>d neurotoxic effects of <strong>the</strong> drug are<br />
secondary to <strong>the</strong> activation of genes that are targets <strong>for</strong> <strong>the</strong> tr<strong>an</strong>scription factors identified in our<br />
present experiment.<br />
Disclosures: N. Cai , None; B. Ladenheim, None; M.T. McCoy, None; J. Cadet, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.25/AA25<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Title: Methamphetamine induces dopamine D1 receptor-dependent unfolded protein response in<br />
<strong>the</strong> rat striatum<br />
Authors: *S. JAYANTHI 1 , M. T. MCCOY 1 , G. BEAUVAIS 1 , B. LADENHEIM 1 , W. WOOD,<br />
III 2 , K. BECKER 2 , J. L. CADET 1 ;<br />
1 NIDA, Baltimore, MD; 2 Gene Expression <strong>an</strong>d Genomics Unit, Natl. Inst. of Aging,, Baltimore,,<br />
MD
Abstract: Methamphetamine (METH) is <strong>an</strong> illicit toxic psychostimul<strong>an</strong>t which is widely abused.<br />
Its toxic effects depend on <strong>the</strong> release of excessive levels of dopamine (DA) that activates striatal<br />
DA receptors <strong>an</strong>d <strong>the</strong> DA receptor D1 (DRD1) <strong>an</strong>tagonist, SCH23390, protects against METHinduced<br />
neuronal apoptosis. The initial purpose of <strong>the</strong> present study was to investigate, using<br />
microarray <strong>an</strong>alyses, <strong>the</strong> influence of SCH23390 on tr<strong>an</strong>scriptional responses in <strong>the</strong> rat striatum<br />
caused by a single METH injection at 2 <strong>an</strong>d 4 hours after drug administration. We identified 545<br />
out of a total of 22,227 genes as METH-responsive. These include genes which are involved in<br />
apoptotic pathways, endoplasmic reticulum (ER) stress, <strong>an</strong>d in tr<strong>an</strong>scription regulation, among<br />
o<strong>the</strong>rs. Of <strong>the</strong>se, a total of 172 genes showed SCH23390-induced inhibition of METH-mediated<br />
ch<strong>an</strong>ges. Among <strong>the</strong>se SCH23390-responsive genes were several genes that are regulated during<br />
ER stress, namely ATF3, HSP27, Hmox1, HSP40, <strong>an</strong>d CHOP/Gadd153. The secondary goal of<br />
<strong>the</strong> study was to investigate <strong>the</strong> role of DRD1 stimulation on <strong>the</strong> expression of genes that<br />
participate in <strong>the</strong> ER stress-mediated unfolded protein response (UPR). We thus used<br />
qu<strong>an</strong>titative PCR to confirm ch<strong>an</strong>ges in <strong>the</strong> METH-responsive ER genes identified in <strong>the</strong><br />
microarray <strong>an</strong>alyses. We measured <strong>the</strong> expression of <strong>the</strong>se genes <strong>an</strong>d of ATF4, ATF6,<br />
BiP/GRP78, <strong>an</strong>d of GADD34 over a more extended time course. SCH23390 attenuated or<br />
blocked METH-induced increases in <strong>the</strong> expression of <strong>the</strong> majority of <strong>the</strong>se genes. Western blot<br />
<strong>an</strong>alysis revealed METH-induced increases in <strong>the</strong> expression of <strong>the</strong> <strong>an</strong>tioxid<strong>an</strong>t protein, Hmox-1,<br />
which lasted <strong>for</strong> about 24 hours after <strong>the</strong> METH injection. Additionally, METH caused<br />
SCH23390-dependent tr<strong>an</strong>sit of <strong>the</strong> Hmox-1 regulator protein, NRF2, <strong>from</strong> cytosolic into nuclear<br />
fractions where <strong>the</strong> protein exerts its regulatory functions. <strong>When</strong> taken toge<strong>the</strong>r, <strong>the</strong>se findings<br />
indicate that SCH23390 c<strong>an</strong> provide protection against neuronal apoptosis by inhibiting METHmediated<br />
DRD1 receptor-mediated ER stress in <strong>the</strong> rat striatum. Our data also suggest that<br />
METH-induced toxicity might be a useful model to elucidate molecular mech<strong>an</strong>isms involved in<br />
UPR activation in <strong>the</strong> rodent brain.<br />
Disclosures: S. Jay<strong>an</strong>thi , None; M.T. McCoy, None; G. Beauvais, None; B. Ladenheim,<br />
None; W. Wood, None; K. Becker, None; J.L. Cadet, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.26/AA26<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: TRF-Senior Research Scholar Fellowship<br />
Postdoctoral Fellowship <strong>from</strong> <strong>the</strong> Commission on Higher Education
Royal Gloden Jubilee PhD Program<br />
Title: Methamphetamine induced neurotoxicity in dopaminergic system of <strong>the</strong> neonatal rat brain<br />
Authors: *P. GOVITRAPONG 1,2 , S. MUKDA 1,2 , S. KAEWSUK 1 , K. SAE-UNG 3 , B.<br />
CHESTSAWANG 1 , P. PHANSUWAN-PUJITO 3 ;<br />
1 Neuro-Behavioural Biol. Cent, Inst. of Sci. & Tech, Mahidol Univ., Nakornpathom, Thail<strong>an</strong>d;<br />
2 Ctr. <strong>for</strong> Neuroscience, Fac. of Sci., Mahidol Univ., B<strong>an</strong>gkok, Thail<strong>an</strong>d; 3 Dept. of Anat. <strong>an</strong>d<br />
Neurosci. Res. Center, Fac. of Med., Srinakharinwirot Univ., B<strong>an</strong>gkok, Thail<strong>an</strong>d<br />
Abstract: Methamphetamine (METH) is a most commonly abused drug which damages nerve<br />
terminals by causing <strong>for</strong>mation of reactive oxygen species <strong>an</strong>d finally neuronal damage. The<br />
developing fetus is subst<strong>an</strong>tially deficient in most <strong>an</strong>tioxidative enzymes, <strong>an</strong>d may <strong>the</strong>re<strong>for</strong>e be at<br />
high risk <strong>from</strong> drug-enh<strong>an</strong>ced oxidative stress.The present study attempted to investigate <strong>the</strong><br />
effects of METH induced neurotoxicity in <strong>the</strong> dopaminergic system of <strong>the</strong> neonatal rat brain. The<br />
results showed that in chronic METH administration in postnatal rat, tyrosine hydroxylase<br />
enzyme levels were signific<strong>an</strong>tly decreased in <strong>the</strong> dorsal striatum, prefrontal cortex, nucleus<br />
accumbens <strong>an</strong>d subst<strong>an</strong>tia nigra, whereas synaptophysin levels decreased in <strong>the</strong> striatum <strong>an</strong>d<br />
prefrontal. METH induced a decrease in VMAT-2 immunoreactivity <strong>an</strong>d a decrease in<br />
phosphorylated tyrosine hydroxylase expression. We also observed <strong>an</strong> increase in alphasynuclein<br />
immunoreactivity in striatum of postnatal rat. Dopamine D1 receptor (DRD1) levels<br />
increased in <strong>the</strong> dorsal striatum whereas dopamine D2 receptor (DRD2) levels signific<strong>an</strong>tly<br />
decreased in both <strong>the</strong> prefrontal cortex <strong>an</strong>d <strong>the</strong> dorsal striatum but signific<strong>an</strong>tly increased in <strong>the</strong><br />
nucleus accumbens. DRD1 mRNA levels were signific<strong>an</strong>tly increased in <strong>the</strong> dorsal striatum<br />
whereas DRD2 mRNA levels were signific<strong>an</strong>tly increased in all three brain regions.<br />
Fur<strong>the</strong>rmore, we showed that melatonin, <strong>an</strong> <strong>an</strong>tioxid<strong>an</strong>t agent released <strong>from</strong> pineal gl<strong>an</strong>d,<br />
attenuates <strong>the</strong> alteration of dopaminergic system homeostasis produced by amphetamine. These<br />
results suggest that melatonin provides a protective effect against METH-induced nerve terminal<br />
degeneration in <strong>the</strong> immature rat brain probably via its <strong>an</strong>tioxid<strong>an</strong>t properties.<br />
Acknowledgement: This study was supported by a TRF-Senior Research Scholar Fellowship<br />
<strong>from</strong> <strong>the</strong> Thail<strong>an</strong>d Research Fund <strong>an</strong>d a Mahidol University Research Gr<strong>an</strong>t to PG <strong>an</strong>d a<br />
Postdoctoral Fellowship <strong>from</strong> <strong>the</strong> Commission on Higher Education, Department of Education to<br />
SM.<br />
Disclosures: P. Govitrapong, None; S. Mukda, None; S. Kaewsuk, None; K. Sae-ung,<br />
None; B. Chestsaw<strong>an</strong>g, None; P. Ph<strong>an</strong>suw<strong>an</strong>-Pujito, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 554.27/AA27<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: NIH Gr<strong>an</strong>t T32NS051152<br />
NIH Gr<strong>an</strong>t R01DA02632<br />
NIH Gr<strong>an</strong>t<br />
NIH Gr<strong>an</strong>t S10RR023461<br />
AHA gr<strong>an</strong>t 0635595T<br />
The DANA Foundation Brain&Immuno Imaging Program<br />
Title: Methamphetamine induces decrease in cerebral blood flow in mouse somatosensory<br />
cortex<br />
Authors: *O. POLESSKAYA 1 , C. M. SANFILIPPO 2 , A. SUN 3 , B. PANEPENTO 3 , M. L.<br />
MILLER 3 , J. SHEN 3 , K. A. KASISCHKE 3 , S. M. POLOYAC 4 , S. DEWHURST 3 ;<br />
1 2 3 4<br />
Rochester, NY; Bausch & Lomb, Rochester, NY; Univ. of Rochester, Rochester, NY; Sch. of<br />
Pharm., Univ. of Pittsburgh, Pittsburgh, PA<br />
Abstract: Methamphetamine (MA) causes brain damage by inducing mitochondrial dysfunction<br />
<strong>an</strong>d oxidative stress. MA also produces signific<strong>an</strong>t neurovascular effects. Here we tested <strong>the</strong><br />
hypo<strong>the</strong>sis that MA exposure disrupts cerebral blood flow (CBF) <strong>an</strong>d causes oxygen deprivation<br />
of brain tissue.<br />
Acute exposure of adult c57BL mice to MA (5 <strong>an</strong>d 20 mg/kg i.p.) caused a biphasic ch<strong>an</strong>ge in<br />
CBF. An initial, tr<strong>an</strong>sient (5 min) increase in CBF was followed by a prolonged decrease, lasting<br />
about 30 minutes <strong>an</strong>d reaching a peak decline of about 20%. This CBF depression demonstrated<br />
a striking contrast to <strong>the</strong> o<strong>the</strong>r aspects of <strong>the</strong> acute physiological response to MA with known<br />
increases in blood pressure, heart rate, body temperature <strong>an</strong>d peripheral blood flow, suggesting<br />
that MA exerts a specific effect on <strong>the</strong> cerebral vasculature.<br />
A chronic escalating/binge dose exposure to MA resulted in a more prolonged (at least 60 min)<br />
<strong>an</strong>d more severe decline in CBF, reaching a peak decline of approx. 50%, of baseline CBF<br />
levels. In contrast, <strong>the</strong> magnitude of MA-induced hyper<strong>the</strong>rmia in <strong>the</strong>se chronically exposed<br />
<strong>an</strong>imals was reduced, reflecting tachyphylaxis.<br />
We also examined microregional vasculature architecture <strong>an</strong>d tissue oxygenation in MA-exposed<br />
<strong>an</strong>imals. To visualize microvasculature, mice were injected i.v. with fluorescent dye <strong>an</strong>d imaged<br />
using two-photon microscopy. Acute MA exposure lead to <strong>the</strong> robust decrease of diameters of<br />
arterioles but not venules. Tissue levels of <strong>the</strong> energy intermediate NADH were qu<strong>an</strong>titatively<br />
imaged using two-photon microscopy in layer I of <strong>the</strong> somatosendory cortex. This <strong>an</strong>alysis<br />
revealed a robust rise in NADH fluorescence following acute exposure to MA, contempor<strong>an</strong>eous<br />
with <strong>the</strong> MA-induced inhibition of CBF. These data are consistent with tissue hypoxia.<br />
Finally, mech<strong>an</strong>istic experiments were conducted, to test whe<strong>the</strong>r MA’s effects on CBF might be
mediated by vasoactive eicos<strong>an</strong>oids. The decrease of CBF by acute MA exposure was abolished<br />
by pretreatment with <strong>an</strong> inhibitor of 20-HETE, HET0016. Direct measurement of 20-HETE in<br />
brain tissue showed that although its concentration was decreased by HET0016 (as expected),<br />
MA exposure did not cause 20-HETE to increase. These data suggest that MA’s effects on <strong>the</strong><br />
cerebral vasculature may be mediated by ch<strong>an</strong>ges in vasodilatory eicos<strong>an</strong>oids whose function<br />
opposes that of 20-HETE. An alternative expl<strong>an</strong>ation is that MA produces ch<strong>an</strong>ges in 20-HETE<br />
only at <strong>the</strong> endo<strong>the</strong>lial level, which we c<strong>an</strong>not see when measuring metabolites in entire cortical<br />
section.<br />
Conclusion: Exposure to MA leads to a rapid decrease in CBF, arterial spasm, <strong>an</strong>d hypoxia of<br />
brain tissue. This response is likely mediated by vasomodulatory eicos<strong>an</strong>oids.<br />
Disclosures: O. Polesskaya, None; C.M. S<strong>an</strong>filippo, None; A. Sun, None; B. P<strong>an</strong>epento,<br />
None; M.L. Miller, None; J. Shen, None; K.A. Kasischke, None; S.M. Poloyac, None; S.<br />
Dewhurst, None.<br />
Poster<br />
554. Methamphetamine <strong>an</strong>d Related Drugs: Addiction Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 554.28/AA28<br />
Topic: C.17.k. Amphetamine <strong>an</strong>d related drugs: Neural mech<strong>an</strong>isms of addiction<br />
Support: DA 12065<br />
MH45294<br />
MH64905<br />
Title: HIV <strong>an</strong>d methamphetamine related diffusion effects<br />
Authors: *S. L. ARCHIBALD 1 , M. W. JACOBSON 3 , R. J. THEILMANN 2 , M.<br />
OGASAWARA 1 , S. P. WOODS 1 , I. GRANT 1 , T. L. JERNIGAN 1,3 ;<br />
1 2 3<br />
Psychiatry, Radiology, Univ. of Cali<strong>for</strong>nia S<strong>an</strong> Diego, La Jolla, CA; VA S<strong>an</strong> Diego<br />
Healthcare Syst., S<strong>an</strong> Diego, CA<br />
Abstract: Introduction: HIV disease is characterized by neurodegenerative ch<strong>an</strong>ges in white<br />
matter, basal g<strong>an</strong>glia <strong>an</strong>d cortical structures. In contrast, methamphetamine dependence (METH)<br />
has been associated with a pattern of volume increases in basal g<strong>an</strong>glia <strong>an</strong>d parietal lobe regions.<br />
Some morphometry studies of HIV <strong>an</strong>d METH, which have a high comorbidity, suggest that
opposing effects of atrophy <strong>an</strong>d hypertrophy are superimposed in dually affected individuals,<br />
especially in <strong>the</strong> caudate nucleus. A recent functional MRI study in our laboratory demonstrated<br />
interaction effects of dual HIV <strong>an</strong>d METH exposure beyond <strong>the</strong> single factor effects. Studies<br />
using diffusion imaging have shown ch<strong>an</strong>ges in white matter integrity in both HIV <strong>an</strong>d METH<br />
but little is known about <strong>the</strong>ir combined effects. This study examined white matter<br />
microstructure using diffusion in groups exposed to HIV <strong>an</strong>d METH, alone <strong>an</strong>d in combination.<br />
Subjects: Thirty-seven particip<strong>an</strong>ts were recruited <strong>from</strong> <strong>the</strong> UCSD HIV Neurobehavioral<br />
Research Center <strong>from</strong> 4 groups: community controls with nei<strong>the</strong>r of <strong>the</strong> risk factors, a group with<br />
HIV only, a group with METH only, <strong>an</strong>d a group who had both HIV <strong>an</strong>d METH. METH subjects<br />
met DSM-IV criteria <strong>for</strong> dependence in <strong>the</strong> past 18 months but were abstinent at assessment.<br />
Groups were well matched on age, gender <strong>an</strong>d years of education. Singly <strong>an</strong>d dually affected<br />
groups did not differ signific<strong>an</strong>tly on HIV disease parameters or METH use histories. The 3<br />
patient groups showed signific<strong>an</strong>t cognitive impairment relative to controls.<br />
Methods: Imaging was per<strong>for</strong>med on a 3T GE Excite system. The diffusion weighted sequence<br />
had 15 directions, b=1250, <strong>an</strong>d 32 4mm slices. Diffusion images were <strong>an</strong>alyzed using Tract<br />
Based Spatial Statistics (TBSS). Whole brain measures of me<strong>an</strong> diffusivity (MD) <strong>an</strong>d fractional<br />
<strong>an</strong>isotropy (FA) <strong>from</strong> <strong>the</strong> skeleton produced by TBSS were compared across groups using<br />
<strong>an</strong>alysis of vari<strong>an</strong>ce.<br />
Results: There were no group differences in FA in this small sample. The ANOVA <strong>for</strong> <strong>the</strong> MD<br />
values however was signific<strong>an</strong>t (p
Title: Dendrodendritic tr<strong>an</strong>smission between mitral <strong>an</strong>d tufted cells terminals in <strong>the</strong> glomeruls of<br />
<strong>the</strong> mouse olfactory Bulb: An ultrastructural study<br />
Authors: *M. PIGNATELLI 1 , T. LACHAUD 1 , D. DEBASREE 1 , S. LASSERRE 2 , H.<br />
MARKRAM 1 , A. CARLETON 3 , G. KNOTT 1 ;<br />
1 EPFL, Brain Mind Inst., Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d; 2 Inst. d'architecture et de la ville, EPFL,<br />
Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d; 3 Geneva Neurosci. Ctr., Geneva, Switzerl<strong>an</strong>d<br />
Abstract: The Olfactory system is characterized by <strong>the</strong> convergence of <strong>the</strong> olfactory nerves<br />
terminals expressing <strong>the</strong> same olfactory receptor onto a single glomerulus, where <strong>the</strong> signal is<br />
tr<strong>an</strong>smitted to a limited number of Mitral <strong>an</strong>d Tufted (M/T) cells. These cells interact exclusively<br />
in <strong>the</strong> glomerulus establishing dendrodendritic relations. The dendrodendritic tr<strong>an</strong>smission<br />
between M/T cells is believed to adopt a sophisticated mech<strong>an</strong>ism. The spillover of glutamate<br />
<strong>from</strong> <strong>the</strong> presynaptic terminals c<strong>an</strong> elicit <strong>an</strong> excitatory current in <strong>the</strong> same presynaptic terminals,<br />
which <strong>the</strong>n propagates through gap junctions to <strong>the</strong> postsynaptic compartments. The<br />
consequence at <strong>the</strong> microcircuit level is reflected by <strong>the</strong> presence of single possible interaction<br />
between M/T cells: a reciprocal connection supported by a simult<strong>an</strong>eous electric coupling.<br />
However, different kind of interactions not supported by electric coupling such as non-reciprocal<br />
connections, are routinely observed.<br />
There<strong>for</strong>e, we decided to investigate <strong>the</strong> dendrodendritic interactions between M/T cells<br />
employing <strong>an</strong> ultrastructural approach: tr<strong>an</strong>smission electron microscopy of <strong>the</strong> glomerular<br />
neuropil in presence of M/T dendritic arborization labeled by biocytin <strong>an</strong>d followed by 3D serial<br />
section reconstruction.<br />
M/T cells dendrites were characterized by electron lucent cytoplasm, presence of a signific<strong>an</strong>t<br />
array of neurotubules <strong>an</strong>d regular shape. Asymmetric synapses were observed between M/T cells<br />
dendrites in <strong>the</strong> glomerular neuropil. The synapses were characterized by small number of<br />
vesicles engaging <strong>the</strong> presynaptic membr<strong>an</strong>e, large synaptic cleft <strong>an</strong>d a relev<strong>an</strong>t postsynaptic<br />
electrondense texture.<br />
These observations support <strong>an</strong> alternative mech<strong>an</strong>ism <strong>for</strong> dendrodendritic tr<strong>an</strong>smission based on<br />
direct synaptic contact, while gap junctions c<strong>an</strong> be responsible <strong>for</strong> <strong>the</strong> amplification of <strong>the</strong><br />
synaptic connectivity.<br />
Disclosures: M. Pignatelli, None; T. Lachaud, None; D. debasree, None; S. Lasserre,<br />
None; H. Markram, None; A. Carleton, None; G. Knott, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.2/AA30
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: NIH NIA Gr<strong>an</strong>t P01-028054<br />
Title: Aging in <strong>the</strong> synaptic circuitry of <strong>the</strong> olfactory bulb<br />
Authors: *M. B. RICHARD 1 , S. TAYLOR 2 , C. A. GREER 1,2,3 ;<br />
1 2 3<br />
Neurosurg. Dept., Interdepartmental Neurosci. Program, Neurobio. Dept., Yale Univ. Sch. of<br />
Med., New Haven, CT<br />
Abstract: One of <strong>the</strong> most common complaints among <strong>the</strong> elderly is <strong>the</strong> loss of sensory function.<br />
Behavioral <strong>an</strong>alyses document declines in olfactory acuity, sensitivity <strong>an</strong>d discrimination in aged<br />
hum<strong>an</strong> <strong>an</strong>d rodent populations. However, it remains controversial how alterations of <strong>the</strong><br />
org<strong>an</strong>ization of <strong>the</strong> olfactory system may contribute to age-related declines in <strong>the</strong> olfactory<br />
system function. We investigated <strong>the</strong> effects of aging on <strong>the</strong> cellular <strong>an</strong>d molecular org<strong>an</strong>ization<br />
of <strong>the</strong> mouse olfactory bulb synaptic circuits at 6, 12, 18 <strong>an</strong>d 24 months in order to determine <strong>the</strong><br />
temporal apparition of age-related defects.<br />
Using C57Bl/6 mice, we investigated modifications of <strong>the</strong> cellular org<strong>an</strong>ization in <strong>the</strong> olfactory<br />
bulb. The number <strong>an</strong>d <strong>the</strong> diameter of olfactory glomeruli, <strong>the</strong> site of <strong>the</strong> first primary afferent<br />
synapses, are maintained throughout aging. The density <strong>an</strong>d total number of mitral/tufted cells<br />
(projection neurons) <strong>an</strong>d of gr<strong>an</strong>ule cell interneurons are preserved during aging. Interestingly,<br />
<strong>the</strong> density <strong>an</strong>d <strong>the</strong> total number of periglomerular interneurons signific<strong>an</strong>tly increase between 6<br />
<strong>an</strong>d 12 months, <strong>an</strong>d stabilize <strong>the</strong>reafter.<br />
In <strong>the</strong> olfactory bulb, <strong>the</strong> primary afferent synaptic pathway <strong>an</strong>d <strong>the</strong> modulatory local circuit<br />
synapses are both segregated <strong>an</strong>d easily identified. Using electron microscopy, we showed that<br />
<strong>the</strong> density of synapses in <strong>the</strong> glomerular <strong>an</strong>d in <strong>the</strong> periglomerular layers is maintained through<br />
aging.<br />
A reproducible receptotopic map is established in <strong>the</strong> olfactory bulb by <strong>the</strong> convergence of<br />
homogeneous population of olfactory sensory neuron axons in a restricted number of glomeruli.<br />
This map contributes to odor coding in <strong>the</strong> olfactory bulb, <strong>an</strong>d its perturbation might lead to<br />
olfactory deficits in <strong>the</strong> aged. Preliminary evidence suggested that <strong>the</strong> specific glomerular<br />
targeting <strong>an</strong>d convergence of <strong>the</strong> olfactory sensory neuron axons expressing <strong>the</strong> M72 odor<br />
receptor are maintained through aging.<br />
Overall, our results showed that <strong>the</strong> morphology <strong>an</strong>d <strong>the</strong> cellular components of <strong>the</strong> olfactory<br />
bulb are surprisingly stable during aging. In order to identify more subtle <strong>an</strong>d functional<br />
alterations of <strong>the</strong> synaptic circuitry in <strong>the</strong> olfactory bulb, we are currently assessing <strong>the</strong><br />
molecular composition of synapses during aging.<br />
Disclosures: M.B. Richard, None; S. Taylor, None; C.A. Greer, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.3/AA31<br />
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: NIH Gr<strong>an</strong>t DC008855<br />
Title: Visualizing cholinergic innervation in <strong>the</strong> mouse main olfactory bulb using ChAT-tau-<br />
EGFP BAC tr<strong>an</strong>sgenic <strong>an</strong>imals<br />
Authors: *E. SALCEDO, T. TRAN, X. LY, D. RESTREPO, S. VIJAYARAGHAVAN;<br />
Cell, Devel Biol., Univ. Colorado Denver, Aurora, CO<br />
Abstract: The role that cholinergic innervation of <strong>the</strong> main olfactory system plays in odor<br />
encoding remains to be fully determined. Towards this end, we have developed a tr<strong>an</strong>sgenic line<br />
of mice which express a fusion of <strong>the</strong> microtubule-associated protein, tau, with green<br />
fluorescence protein (GFP) under <strong>the</strong> control of <strong>the</strong> choline acetyltr<strong>an</strong>sferase (ChAT) promoter.<br />
This tau-GFP fusion product allows <strong>for</strong> a remarkably vivid <strong>an</strong>d clear visualization of cholinergic<br />
innervation in <strong>the</strong> main olfactory bulb (MOB) <strong>an</strong>d indeed throughout <strong>the</strong> rest of <strong>the</strong> brain. In<br />
adult <strong>an</strong>imals we find GFP-positive fibers in all layers of <strong>the</strong> bulb, including <strong>the</strong> glomerular,<br />
mitral cell, <strong>an</strong>d gr<strong>an</strong>ule cell layers. Consistent with previous results, we found <strong>the</strong> glomerular<br />
layer to be <strong>the</strong> most densely innervated by GFP-positive fibers. Interestingly, we found this<br />
staining pattern to be somewhat irregular so that GFP-positive fibers appeared to be targeting<br />
specific clusters of glomeruli in both <strong>the</strong> <strong>an</strong>terior <strong>an</strong>d posterior regions of <strong>the</strong> MOB. We partially<br />
confirmed this result by colabeling <strong>the</strong> green fluorescence staining with <strong>an</strong> immunomarker<br />
specific <strong>for</strong> necklace glomeruli found in <strong>the</strong> posterior MOB. Consistent with previous reports, we<br />
found no overlap between <strong>the</strong>se two labels. We also examined <strong>the</strong> green immunofluorescence<br />
patterns during critical developmental periods. Remarkably, we do not find signific<strong>an</strong>t GFP<br />
immunolabeling in <strong>the</strong> MOB until after postnatal days 10 -12. Finally, in tr<strong>an</strong>sgenic <strong>an</strong>imals in<br />
which we occlude one naris, we find a differential GFP staining pattern between <strong>the</strong> contra- <strong>an</strong>d<br />
ipsi- lateral bulbs.<br />
Disclosures: E. Salcedo, None; T. Tr<strong>an</strong>, None; X. Ly, None; D. Restrepo, None; S.<br />
Vijayaraghav<strong>an</strong>, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.4/AA32<br />
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: NSERC<br />
OGS<br />
University of Ottawa<br />
Title: Elucidating <strong>the</strong> neural circuitry involved with sex pheromones signaling in male goldfish<br />
Authors: *W. E. LADO 1 , D. C. SPANSWICK 2 , J. E. LEWIS 1 , V. L. TRUDEAU 1 ;<br />
1 2<br />
Biol., Univ. of Ottawa, Ottawa, ON, C<strong>an</strong>ada; Warwick Med. Sch., Univ. of Warwick,<br />
COVENTRY, United Kingdom<br />
Abstract: The sex pheromones, 17 αλπηα, 20 βετα-dihydroxy-4-pregene-3-one <strong>an</strong>d<br />
prostagl<strong>an</strong>din-F2αλπηα, elicit specific reproductive behaviours in male goldfish. To better<br />
underst<strong>an</strong>d <strong>the</strong> neural circuitry involved with sex pheromones signalling in goldfish<br />
reproduction, we used male goldfish brain expl<strong>an</strong>ts. The adv<strong>an</strong>tage of working with <strong>the</strong> expl<strong>an</strong>ts<br />
is that <strong>the</strong> neural circuitry <strong>from</strong> <strong>the</strong> olfactory bulb (OB) to <strong>the</strong> preoptic area (POA) is preserved.<br />
We electrically stimulated <strong>the</strong> OB <strong>an</strong>d per<strong>for</strong>med whole-cell current clamp recordings <strong>from</strong><br />
neurons in <strong>the</strong> POA to discern <strong>the</strong> functional properties <strong>an</strong>d connections of this circuitry. These<br />
neurons fired action potentials spont<strong>an</strong>eously with some also displaying burst firing. We<br />
demonstrate evidence <strong>for</strong> a monosynaptic connection between <strong>the</strong> OB <strong>an</strong>d POA implying a role<br />
<strong>for</strong> sex pheromones signal tr<strong>an</strong>smission through <strong>the</strong> medial olfactory tract. A major component<br />
of <strong>the</strong> synaptic connection was blocked by DL-2-amino-5-phosphonopent<strong>an</strong>oic acid (AP5), <strong>an</strong><br />
N-Methyl-D-Aspartate (NMDA) receptor <strong>an</strong>tagonist. 6-cy<strong>an</strong>o-7-nitroquinoxaline-2, 3-dione<br />
(CNQX), a non NMDA receptor <strong>an</strong>tagonist, subsequently blocked <strong>the</strong> rest of <strong>the</strong> response.<br />
Overall, <strong>the</strong>se results indicate that <strong>the</strong> functional connection between <strong>the</strong> OB <strong>an</strong>d POA of male<br />
goldfish mediated by glutamatergic neurotr<strong>an</strong>smission is monosynaptic. This is consistent with<br />
<strong>the</strong> major role glutamate plays in <strong>the</strong> hormonal regulation of reproduction in male goldfish.<br />
Disclosures: W.E. Lado, None; D.C. Sp<strong>an</strong>swick, None; J.E. Lewis, None; V.L. Trudeau,<br />
None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.5/AA33<br />
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: NIH Gr<strong>an</strong>t 1F30DC010323-01<br />
NIH Gr<strong>an</strong>t RO1 DC000566<br />
Title: Adrenergic receptor-mediated disinhibition of olfactory bulb mitral cells triggers longterm<br />
enh<strong>an</strong>cement of gamma oscillations<br />
Authors: *S. PANDIPATI 1 , D. H. GIRE 2 , N. E. SCHOPPA 2 ;<br />
1 MSTP, 2 Physiol. & Biophysics, Univ. Colorado Denver - AMC, Aurora, CO<br />
Abstract: Norepinephrine (NE) may be essential <strong>for</strong> m<strong>an</strong>y <strong>for</strong>ms of olfactory learning but <strong>the</strong><br />
underlying cellular mech<strong>an</strong>isms are not known. One potential mech<strong>an</strong>ism was revealed by a<br />
recent study (Gire <strong>an</strong>d Schoppa, 2008) that showed that tr<strong>an</strong>sient exposure of olfactory bulb<br />
(OB) slices to NE could elicit long-term enh<strong>an</strong>cement of gamma oscillations (30-70 Hz) in <strong>the</strong><br />
local field potential (LFP) evoked by olfactory nerve stimulation. Increased gamma oscillations<br />
are linked with olfactory learning (Martin et al., 2004). Here we studied acute actions of NE that<br />
may be responsible <strong>for</strong> NE’s long-term effects using patch-clamp recordings in slices <strong>from</strong> rat<br />
OB. Initial studies were done in young pups (P9-13). One prominent acute effect of NE was<br />
disinhibition of mitral cells, seen as a 51±7% reduction (n=8, p=0.0023) in <strong>the</strong> amplitude of<br />
inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation in <strong>the</strong> gr<strong>an</strong>ule cell layer<br />
(50-200 µA, 100 µsec). This effect appeared to be mediated mainly by alpha2-adrenergic<br />
receptors (ARs), as 5µM clonidine also reduced IPSCs (by 31±13%, n=8, p=0.04). Disinhibition<br />
is a plausible mech<strong>an</strong>ism <strong>for</strong> long-term enh<strong>an</strong>cement of gamma oscillations, since it could lead<br />
to elevated intracellular calcium typically associated with long-term synaptic ch<strong>an</strong>ges. Consistent<br />
with disinhibition triggering enh<strong>an</strong>ced gamma oscillations, we found that tr<strong>an</strong>sient exposure of<br />
slices to a moderate concentration of gabazine (5µM), to directly block GABAA receptors,<br />
mimicked NE’s action in inducing long-term enh<strong>an</strong>cement of gamma LFP oscillations in <strong>the</strong><br />
external plexi<strong>for</strong>m layer (128±37% increase in gamma power, measured 50-60 min following<br />
washout of gabazine, n=4, p=0.041; gabazine co-applied with 12.5µM NMDA). Also, activation<br />
of alpha2-ARs alone with clonidine was sufficient to induce long-term enh<strong>an</strong>cement of <strong>the</strong><br />
oscillations (83±22% increase, n=15, p=0.001). We also examined <strong>the</strong> age-dependence of NE’s<br />
effects on OB circuit properties. Whereas NE effectively induced long-term enh<strong>an</strong>cement of<br />
gamma oscillations in young rat pups, NE was ineffective at doing so in older pups (P18-19;<br />
25±21% decrease, n=5, p=0.25). The difference in NE’s long-term effects with age may be<br />
linked to NE’s acute effects on gr<strong>an</strong>ule-to-mitral cell inhibition. While NE induced disinhibition<br />
in young pups, NE (20 µM) increased inhibition in older pups, as measured by <strong>the</strong> frequency of<br />
spont<strong>an</strong>eous IPSCs in mitral cells (174±39% increase, n=4, p=0.021; see also Nai et al., <strong>2009</strong>).<br />
These results suggest that NE-mediated disinhibition leading to enh<strong>an</strong>ced gamma oscillations<br />
might only occur in a limited age-window in rat pups.
Disclosures: S. P<strong>an</strong>dipati, None; D.H. Gire, None; N.E. Schoppa, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.6/AA34<br />
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: NIH F31 DC009118<br />
NIH RO1 DC006640<br />
Title: Rethinking <strong>the</strong> olfactory glomerulus: Sensory input reaches mitral cells mainly through a<br />
multi-cellular, spill-over-mediated pathway<br />
Authors: *D. H. GIRE 1 , N. E. SCHOPPA 2 ;<br />
1 2<br />
Neurosci. Program, Physiol. <strong>an</strong>d Biophysics, Univ. of Colorado Denver, Anschutz Med.<br />
Campus, Aurora, CO<br />
Abstract: The c<strong>an</strong>onical view of in<strong>for</strong>mation flow in <strong>the</strong> olfactory system is that signals pass<br />
<strong>from</strong> olfactory receptor neuron (ORN) axons directly to mitral cells, which are <strong>the</strong> main output<br />
neurons of <strong>the</strong> olfactory bulb. However, a recent study (De Saint J<strong>an</strong> et al., <strong>2009</strong>) has provided<br />
evidence that part of mitral cell excitation could be due to <strong>an</strong> indirect, multi-cellular pathway<br />
mediated by <strong>an</strong>o<strong>the</strong>r class of excitatory neurons, external tufted (ET) cells. In our studies, done<br />
using patch clamp recordings in rat olfactory bulb slices, we examined <strong>the</strong> relative contribution<br />
of <strong>the</strong> direct <strong>an</strong>d indirect paths of mitral cell activation, as well as underlying mech<strong>an</strong>isms. To<br />
evaluate <strong>the</strong> contribution of <strong>the</strong> direct ORN-to-mitral cell path, we recorded excitatory postsynaptic<br />
currents (EPSCs) while stimulating <strong>the</strong> olfactory nerve (ON) at moderate intensities<br />
(10-50 µA). Under <strong>the</strong>se conditions, we never observed monosynaptic ORN-EPSCs in mitral<br />
cells (n=12 cells), even though we routinely observed <strong>the</strong>m in ET cells (2.9±0.2 ms onset-delay,<br />
n=7). The lack of clear ORN-EPSCs in mitral cells, implying <strong>an</strong> absence of direct ORN-to-mitral<br />
cell connections, was likely not due to dendritic filtering, since fast “lateral” EPSCs that<br />
originate at primary dendrites (<strong>from</strong> o<strong>the</strong>r mitral cells) are readily observable in mitral cells<br />
(Schoppa <strong>an</strong>d Westbrook, 2002). To examine <strong>the</strong> mech<strong>an</strong>isms of indirect tr<strong>an</strong>smission <strong>from</strong><br />
ORNs to mitral cells, we measured ON stimulation-evoked responses during pair recordings<br />
between ET cells <strong>an</strong>d mitral cells. In <strong>the</strong>se studies, we found evoked slow EPSCs in mitral cells<br />
(half width = 46±17 ms, n=3 pairs) that were locked in time (onset-delay=1.8±0.2 ms) to spikes<br />
in ET cells. These EPSCs in mitral cells appeared to be due to glutamate spill-over <strong>from</strong>
synapses between ET cells <strong>an</strong>d GABAergic periglomerular (PG) cells, based on <strong>the</strong> following<br />
evidence. First, in pair PG/mitral cell recordings, we found similar slow EPSCs in mitral cells<br />
(half width = 45±15 ms) that were closely time-locked to fast EPSCs in PG cells (in 4 of 6 pairs;<br />
mitral cell EPSCs initiated within 1 ms of PG cell EPSCs). Second, <strong>the</strong> amplitudes of <strong>the</strong> EPSCs<br />
in PG <strong>an</strong>d mitral cells were strongly correlated across stimulus trials (correlation coefficient =<br />
0.48±0.05, n=4 pairs), as would be expected if <strong>the</strong> two EPSC-types were derived <strong>from</strong> <strong>the</strong> same<br />
synaptic source. Lastly, block of glutamate uptake with DL-threo-b-benzyloxyaspartic acid<br />
(TBOA, 10 µM) increased <strong>the</strong> slow mitral cell EPSC (34 <strong>an</strong>d 80% increases, n=2). Taken<br />
toge<strong>the</strong>r, our experiments suggest that <strong>the</strong> main mode of mitral cell activation is a multi-cellular<br />
mech<strong>an</strong>ism that includes spill-over <strong>from</strong> glutamatergic ET-to-PG cell synapses.<br />
Disclosures: D.H. Gire, None; N.E. Schoppa, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.7/AA35<br />
Topic: D.01.d. Olfaction: Central circuits <strong>an</strong>d neurotr<strong>an</strong>smitters<br />
Support: R01-DC04285<br />
Title: Evoked responses <strong>an</strong>d short-term plasticity of <strong>the</strong> gr<strong>an</strong>ule cell-to-mitral cell inhibitory<br />
synapse in <strong>the</strong> rat olfactory bulb<br />
Authors: *Y. GAO 1 , B. W. STROWBRIDGE 2 ;<br />
2 Dept. of Neurosciences, 1 Case Western Res. Univ., Clevel<strong>an</strong>d, OH<br />
Abstract: Local inhibition plays a crucial role in modulating <strong>the</strong> output of <strong>the</strong> olfactory bulb in<br />
response to sensory input. Despite extensive work on self- <strong>an</strong>d lateral disynaptic inhibition<br />
mediated by reciprocal dendrodendritic synapses between mitral <strong>an</strong>d gr<strong>an</strong>ule cells, less is known<br />
about <strong>the</strong> properties of <strong>the</strong> actual gr<strong>an</strong>ule cell-to-mitral cell synapse that mediates this inhibition.<br />
We defined <strong>the</strong>se synapses using focal microstimulation in <strong>the</strong> gr<strong>an</strong>ule cell layer (GCL), directly<br />
below mitral cell bodies <strong>an</strong>d lateral dendrites visualized using 2-photon microscopy in rat<br />
olfactory bulb slices. Using this method, we evoked both monosynaptic IPSPs that were<br />
insensitive to blockade of ionotropic glutamate receptors with NBQX <strong>an</strong>d APV (onset latency =<br />
2.4 ± 0.29 ms; n = 8) <strong>an</strong>d disynaptic IPSPs that were abolished by <strong>the</strong>se receptor <strong>an</strong>tagonists<br />
(latency = 4.1 ± 0.33 ms; n = 13). Nearly all monosynaptic IPSPs depressed with paired GCL<br />
stimulation (7 of 8; 50 ms ISI) while only most (62%) disynaptic IPSPs facilitated with paired
stimulation. Presumably disynaptic IPSP facilitation reflects stimulation of centrifugal<br />
glutamatergic synapses onto gr<strong>an</strong>ule cells that exhibit paired-pulse facilitation. Mono- <strong>an</strong>d<br />
disynaptic mitral cell IPSPs were often followed by depolarizing responses that triggered spikes<br />
when <strong>the</strong> membr<strong>an</strong>e potential was held near threshold. Both on-beam stimulation in <strong>the</strong> GCL<br />
(
we demonstrate that <strong>the</strong> activation of glomerular nAChRs depolarizes ET cells <strong>an</strong>d subsequently<br />
increases <strong>the</strong>ir firing rates. nAChR activation also increases <strong>the</strong> frequency of spont<strong>an</strong>eous<br />
inhibitory postsynaptic currents (sIPSCs) in a majority of ET <strong>an</strong>d PG cells. This increase in<br />
sIPSC frequency is maintained upon blockade of ionotropic glutamate receptors. In a<br />
subpopulation of PG cells, nAChR activation increases <strong>the</strong> frequency of spont<strong>an</strong>eous excitatory<br />
postsynaptic currents (sEPSCs). Our results suggest that activation of nAChRs leads to <strong>an</strong><br />
increase in <strong>the</strong> release of both glutamate <strong>an</strong>d GABA in <strong>the</strong> glomerulus, ex<strong>citing</strong> <strong>the</strong> output<br />
neurons of <strong>the</strong> olfactory bulb while, at <strong>the</strong> same time, providing timed inhibition onto <strong>the</strong>m.<br />
Disclosures: R. D'Souza, None; S. Vijayaraghav<strong>an</strong>, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.9/AA37<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: THE ISRAEL SCIENCE DOUNDATION gr<strong>an</strong>t 1082/07<br />
08<br />
THE ISRAEL SCIENCE FOUNDATION gr<strong>an</strong>t 1493/07<br />
THE NATIONAL INSTITUTE FOR PSYCHOBIOLOGY IN ISRAEL gr<strong>an</strong>t 237-07-<br />
Title: Long-lasting GABAB mediated inhibition modulates <strong>the</strong> responses of accessory olfactory<br />
bulb mitral cells to sensory stimulation<br />
Authors: *S. WAGNER, G. SHPAK;<br />
Haifa Univ., Haifa, Israel<br />
Abstract: Social behavior in rodents is mainly olfactory guided, <strong>an</strong>d is mediated by two distinct<br />
olfactory systems: <strong>the</strong> main <strong>an</strong>d <strong>the</strong> accessory. Sensory cells of each system project to distinct<br />
areas of <strong>the</strong> olfactory bulb, <strong>the</strong> main (MOB) <strong>an</strong>d <strong>the</strong> accessory (AOB) olfactory bulbs, where<br />
<strong>the</strong>y terminate upon mitral cells, <strong>the</strong> output neurons of <strong>the</strong> bulb. We have previously shown a<br />
signific<strong>an</strong>t difference between <strong>the</strong> responses of MOB <strong>an</strong>d AOB mitral cells to similar stimuli,<br />
with a robust tr<strong>an</strong>sient (1-4 s) MOB response compared to <strong>the</strong> weaker but much longer (10-30 s)<br />
response of <strong>the</strong> AOB. Accordingly we hypo<strong>the</strong>sized that <strong>the</strong>se apparently similar neuronal
networks are built to generate distinct types of sensory in<strong>for</strong>mation processing. Here, we use<br />
patch clamp recordings <strong>an</strong>d voltage imaging in acute olfactory bulb slices to demonstrate that <strong>the</strong><br />
time course of evoked GABAB mediated inhibition markedly differs between both bulbs. While<br />
in <strong>the</strong> MOB this inhibition is strongest at 0.2 s <strong>an</strong>d is over at 2 s post-stimulation, <strong>the</strong> AOB<br />
GABAB mediated inhibition is much slower <strong>an</strong>d longer, peaking at 1 s <strong>an</strong>d lasting <strong>for</strong> at least 15<br />
s.h Moreover, we show that <strong>the</strong> GABAB mediated inhibition affects fast inhibitory synaptic<br />
activity in <strong>the</strong> AOB more th<strong>an</strong> fast excitatory synaptic activity, thus enh<strong>an</strong>cing <strong>the</strong> excitatory<br />
response during its extended time course. We conclude that <strong>the</strong> powerful long-lasting GABAB<br />
mediated inhibition modulates <strong>the</strong> unique slow <strong>an</strong>d long response of AOB mitral cells to sensory<br />
stimuli.<br />
Disclosures: S. Wagner, None; G. Shpak, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.10/AA38<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: NIH DC007876<br />
Title: GABAergic interneurons of <strong>the</strong> superficial external plexi<strong>for</strong>m layer of mouse olfactory<br />
bulb receive tonic inhibitory synaptic inputs<br />
Authors: *K. A. HAMILTON, Y.-F. WANG;<br />
LSU Hlth. Sci. Ctr., Shreveport, LA<br />
Abstract: The superficial external plexi<strong>for</strong>m layer (sEPL) of <strong>the</strong> main olfactory bulb (OB)<br />
contains a variety of GABAergic interneurons, synaptic connections <strong>an</strong>d functions of which are<br />
unknown. We have previously shown that sEPL interneurons exhibit spont<strong>an</strong>eous <strong>an</strong>d<br />
stimulation-evoked EPSCs that are primarily mediated by AMPA receptors. Here, we report<br />
characteristics of spont<strong>an</strong>eous <strong>an</strong>d evoked IPSCs of sEPL interneurons in OB slices <strong>from</strong><br />
tr<strong>an</strong>sgenic mice expressing eGFP under control of <strong>the</strong> promoter <strong>for</strong> <strong>the</strong> GAD65 gene. At -10mV<br />
holding potential, m<strong>an</strong>y eGFP+ sEPL cells exhibited frequent spont<strong>an</strong>eous GABAA receptormediated<br />
IPSCs (sIPSCs), which were blocked by gabazine or bicuculine. In <strong>the</strong> presence of<br />
0.5µM TTX, miniature IPSCs (mIPSCs) were observed; mIPSC frequency was lower th<strong>an</strong> sIPSC<br />
frequency but nei<strong>the</strong>r <strong>the</strong> rising nor decay slopes differed signific<strong>an</strong>tly. Bath application of APV
had no consistent effect on mIPSCs whereas application of CNQX or NBQX signific<strong>an</strong>tly<br />
reduced mIPSC frequency. Bath application of 12mM KCl increased mIPSC frequency, <strong>an</strong>d <strong>the</strong><br />
increase was blocked by bath application of 100µM CdCl2. These results suggest that sEPL<br />
interneurons are tonically inhibited by GABAergic presynaptic cells, <strong>an</strong>d that AMPA receptors<br />
are import<strong>an</strong>t in effecting GABA release at <strong>the</strong> synapses. Because NMDA receptors play a major<br />
role in mitral/tufted cell-mediated excitation of gr<strong>an</strong>ule cell (GC) distal dendritic synapses in <strong>the</strong><br />
sEPL, <strong>the</strong> results also suggest that sEPL interneurons might tonically inhibit each o<strong>the</strong>r in <strong>the</strong><br />
absence of GC-mediated inhibition. In normal bath solution, near-threshold stimulation of <strong>the</strong><br />
glomerular layer or internal plexi<strong>for</strong>m layer evoked short-latency compound IPSCs in GFP+<br />
cells. We <strong>the</strong>re<strong>for</strong>e postulate that sEPL interneurons tonically inhibit each o<strong>the</strong>r in response to<br />
ongoing or weak mitral/tufted cell-mediated excitation, whereas <strong>the</strong> interneurons are inhibited by<br />
GCs in response to stronger mitral/tufted cell-mediated excitation of those cells.<br />
Disclosures: K.A. Hamilton, None; Y. W<strong>an</strong>g, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.11/BB1<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: NIDCD R03 DC008874<br />
NIDCD R01 DC000086<br />
NIDCD P01 DC004732<br />
Title: Mitral <strong>an</strong>d tufted cell connections to gr<strong>an</strong>ule cells of <strong>the</strong> rat olfactory bulb are sparse <strong>an</strong>d<br />
non-stochastic<br />
Authors: M. E. PHILLIPS, D. H. KIM, A. Y. CHANG, H. K. PATEL, G. M. SHEPHERD,<br />
*D. C. WILLHITE;<br />
Dept Neurobiol, Yale Univ., New Haven, CT<br />
Abstract: The mammali<strong>an</strong> olfactory bulb (OB) processes chemosensory stimuli through<br />
modular units, <strong>the</strong> glomeruli. Previous viral tr<strong>an</strong>ssynaptic tracing results suggested that <strong>the</strong><br />
modules extend to <strong>the</strong> gr<strong>an</strong>ule cell layer <strong>an</strong>d <strong>for</strong>m columnar units. Fur<strong>the</strong>rmore, <strong>the</strong> columnar
patterns in <strong>the</strong> gr<strong>an</strong>ule cell layer <strong>from</strong> a focal viral injection in <strong>the</strong> glomerular layer were widely<br />
distributed in space within <strong>the</strong> ipsilateral half of <strong>the</strong> OB. This finding led to <strong>the</strong> interpretation<br />
that Mitral <strong>an</strong>d Tufted (M/T) cell lateral dendrites are only connected to specific columns of<br />
gr<strong>an</strong>ule cells. However, <strong>the</strong> connective extent of <strong>the</strong> individual gr<strong>an</strong>ule cells within <strong>the</strong> columnar<br />
modules to separate spatial populations of M/T cells remains unknown. To investigate this<br />
question we probed <strong>the</strong> connectivity of <strong>the</strong> lateral field in <strong>the</strong> OB with two focal, glomerular<br />
layer injections of retrograde viral tr<strong>an</strong>ssynaptic tracers encoding red <strong>an</strong>d green fluorescent<br />
proteins.<br />
Based on <strong>the</strong> distributed nature of <strong>the</strong> columnar labeling <strong>from</strong> a single, glomerular layer injection<br />
we expected to see a mix of segregated (red or green) <strong>an</strong>d purely convergent (yellow) columns.<br />
However, <strong>the</strong> observed convergent columns showed a segregated interdigitation of red <strong>an</strong>d green<br />
gr<strong>an</strong>ule cells mixed with <strong>an</strong> apparently minor population of convergently connected GCs. We<br />
<strong>the</strong>n tested <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong>se dendrodendritic connections between GC <strong>an</strong>d M/T cells are<br />
stochastically <strong>for</strong>med. Labeled populations of columns were counted <strong>an</strong>d compared to <strong>the</strong> total<br />
neuron count in <strong>the</strong> column volume. Results showed that <strong>the</strong> total cells labeled generally<br />
represent less th<strong>an</strong> 10% of <strong>the</strong> total neurons, <strong>an</strong>d that <strong>the</strong> observed number of convergent neurons<br />
signific<strong>an</strong>tly exceeded <strong>the</strong> expected value of stochastically connected convergent neurons.<br />
In addition, we explore <strong>the</strong> dist<strong>an</strong>ce-dependence of this non-stochastic connectivity. The results<br />
indicate both <strong>the</strong> local <strong>an</strong>d distal lateral networks of <strong>the</strong> OB are generally segregated, but show a<br />
tendency toward convergence. Implications <strong>for</strong> olfactory in<strong>for</strong>mation processing, including<br />
putative inhibitory gating mech<strong>an</strong>isms are discussed.<br />
Disclosures: M.E. Phillips, None; D.H. Kim, None; A.Y. Ch<strong>an</strong>g, None; H.K. Patel,<br />
None; G.M. Shepherd, None; D.C. Willhite, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.12/BB2<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: NIH Gr<strong>an</strong>t DC 008962<br />
Title: Different profiles of main <strong>an</strong>d accessory olfactory bulb mitral cell projections revealed in<br />
mice using <strong>an</strong> <strong>an</strong>terograde tracer <strong>an</strong>d a flattened cortex preparation
Authors: *N. KANG 1 , M. J. BAUM 2 , J. A. CHERRY 3 ;<br />
2 Biol., 3 Psychology, 1 Boston Univ., Boston, MA<br />
Abstract: In<strong>for</strong>mation about social odor<strong>an</strong>ts detected by <strong>the</strong> main <strong>an</strong>d accessory olfactory<br />
systems converges in <strong>the</strong> rodent medial amygdala (Me) be<strong>for</strong>e being conveyed to <strong>the</strong><br />
hypothalamus. We previously showed that mitral cells in <strong>the</strong> main <strong>an</strong>d accessory olfactory bulbs<br />
project to separate, abutting laminae of <strong>the</strong> mouse Me. In order to trace <strong>the</strong> pathways of main<br />
versus accessory olfactory bulb (MOB; AOB) mitral cell axons to <strong>the</strong>ir medial amygdalar <strong>an</strong>d/or<br />
cortical targets, <strong>the</strong> <strong>an</strong>terograde tracer, PHAL was injected into ei<strong>the</strong>r <strong>the</strong> AOB or different<br />
portions of <strong>the</strong> MOB, <strong>an</strong>d axonal projections were later mapped using a whole mount flattened<br />
mouse brain preparation. Axonal collaterals <strong>from</strong> <strong>the</strong> AOB <strong>an</strong>d MOB overlapped in <strong>the</strong> <strong>an</strong>terior<br />
cortical amygdala (ACo) <strong>an</strong>d Me. , Surprisingly, AOB mitral cells sent 3-fold more collaterals to<br />
<strong>the</strong> ACo th<strong>an</strong> did MOB mitral cells, <strong>an</strong>d as expected, sent m<strong>an</strong>y more (> 7-fold) collaterals to <strong>the</strong><br />
Me. AOB mitral cells extended no collaterals to piri<strong>for</strong>m cortex whereas numerous cortical<br />
collaterals extended <strong>from</strong> MOB mitral cell axons. Ventral MOB mitral cells sent 8- <strong>an</strong>d 2.5-fold<br />
more collaterals to <strong>the</strong> Me th<strong>an</strong> did dorsal <strong>an</strong>d dorsal-lateral MOB mitral cells, respectively.<br />
Inputs to different populations of MOB mitral cells may play distinct roles in <strong>the</strong> processing of<br />
different types of social odor<strong>an</strong>ts.<br />
Disclosures: N. K<strong>an</strong>g, None; M.J. Baum, None; J.A. Cherry, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.13/BB3<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: EUSynapse project; LSHM-CT-2005-019055<br />
ESF Europe<strong>an</strong> Young Investigator Award<br />
Title: Neurochemical diversity of deep short-axon cells of <strong>the</strong> rat main olfactory bulb<br />
Authors: *M. D. EYRE, K. KERTI, Z. NUSSER;<br />
Inst. Exptl. Med., Budapest, Hungary
Abstract: In m<strong>an</strong>y brain areas, <strong>the</strong> most diverse cell populations are local circuit GABAergic<br />
interneurons. In order to determine <strong>the</strong>ir place in <strong>the</strong> network <strong>an</strong>d underst<strong>an</strong>d <strong>the</strong>ir roles, it is<br />
crucial to be able to selectively identify <strong>an</strong>d study <strong>the</strong>m. Interneurons have been classified based<br />
on <strong>the</strong>ir morphologies, neurochemical content <strong>an</strong>d electrical properties <strong>an</strong>d this has greatly<br />
adv<strong>an</strong>ced our underst<strong>an</strong>ding of <strong>the</strong>ir roles. We recently identified three major subtypes of deep<br />
short-axon cells (dSACs) of <strong>the</strong> main olfactory bulb (MOB) based on <strong>the</strong>ir axo-dendritic<br />
distributions <strong>an</strong>d synaptic connectivity. Here, we investigated <strong>the</strong> molecular diversity of dSACs<br />
<strong>an</strong>d asked whe<strong>the</strong>r distinct dSAC subtypes selectively express certain molecules.<br />
Immunofluorescent colocalization experiments revealed that <strong>the</strong> most commonly used molecular<br />
markers of dSACs (i.e. VIP, SOM, CB, NOS) label only very small subpopulations (
NIH Gr<strong>an</strong>t DC006972<br />
Title: The location of mitral cells in <strong>the</strong> mouse olfactory bulb is determined by time of origin<br />
Authors: *F. IMAMURA 1 , C. A. GREER 2 ;<br />
1 Dept. Neurosurg., 2 Dept. Neurosurg. <strong>an</strong>d Neurobio., Yale Univ,Sch Med., New Haven, CT<br />
Abstract: The olfactory epi<strong>the</strong>lium <strong>an</strong>d glomerular layer (GL) of mammali<strong>an</strong> olfactory bulb<br />
(OB) c<strong>an</strong> be divided into several domains according to molecular expression patterns. The most<br />
prominent division is dorsomedial (D) <strong>an</strong>d ventrolateral (V) domains that are defined by OCAMnegative<br />
or OCAM-positive olfactory sensory neuron axons, respectively. However, whe<strong>the</strong>r<br />
<strong>the</strong>se domains extend into <strong>the</strong> deeper layers of <strong>the</strong> OB is not known. Here, we report that <strong>the</strong><br />
mitral cells born at different time points localize to different regions of <strong>the</strong> mitral cell layer<br />
(MCL) that correspond, at least in part, with D <strong>an</strong>d V domains of GL.<br />
During development, more th<strong>an</strong> 80% of mitral cells are born during embryonic days (E) 10-12.<br />
They are born within <strong>the</strong> ventricular proliferative zone, <strong>an</strong>d migrate radially toward <strong>the</strong> surface<br />
of <strong>the</strong> OB. To test <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong>re is relationship between time of origin <strong>an</strong>d final<br />
destination of mitral cells, we labeled <strong>the</strong>m with intraperitoneal injections of thymidine <strong>an</strong>alogs<br />
(BrdU, CldU, or IdU) into pregn<strong>an</strong>t mo<strong>the</strong>rs at E10, 11, or 12, <strong>an</strong>d <strong>an</strong>alyzed <strong>the</strong> position of<br />
labeled cells in <strong>the</strong> MCL at postnatal day (P) 20. We found that <strong>the</strong> distribution of mitral cells in<br />
<strong>the</strong> MCL varied according to <strong>the</strong>ir time of origin. The density of mitral cells born at E10 was<br />
higher in <strong>the</strong> dorsomedial MCL th<strong>an</strong> ventrolateral MCL. In contrast, mitral cells born at E12<br />
were preferentially found in <strong>the</strong> ventrolateral MCL, while E11 born mitral cells were evenly<br />
distributed in <strong>the</strong> MCL. To determine when <strong>the</strong> time of origin dependent localization of mitral<br />
cells is established, we examined <strong>the</strong> distribution of mitral cells at P0 <strong>an</strong>d E15. At P0, we found<br />
<strong>the</strong> same distribution pattern of mitral cells as at P20. At E15, however, E10 born mitral cells<br />
were evenly distributed in <strong>the</strong> MCL. In contrast, most E12 born mitral cells have not reached to<br />
<strong>the</strong> MCL at E15, <strong>an</strong>d were specifically localized in <strong>the</strong> ventrolateral region of <strong>the</strong> OB. This<br />
suggests that specific targeting of E12 born mitral cells to <strong>the</strong> ventrolateral region also causes <strong>the</strong><br />
differential distribution of E10 born mitral cells by decreasing <strong>the</strong>ir density in <strong>the</strong> ventrolateral<br />
MCL. Moreover, since we did not find evidence of mitral cell death at <strong>an</strong>y prenatal age, we<br />
concluded that <strong>the</strong>re is no overproduction of mitral cells during development.<br />
In summary, we found that mitral cells born at E10 <strong>an</strong>d E12 are preferentially localized in <strong>the</strong><br />
dorsomedial <strong>an</strong>d ventrolateral MCL, respectively. This implies that, in <strong>the</strong> mouse OB, D <strong>an</strong>d V<br />
domain segregation c<strong>an</strong> be extended into <strong>the</strong> MCL. Our results fur<strong>the</strong>r suggest that specific<br />
targeting of E12 born mitral cells into <strong>the</strong> ventrolateral region is <strong>an</strong> underlying mech<strong>an</strong>ism of <strong>the</strong><br />
time of origin dependent localization of mitral cells.<br />
Disclosures: F. Imamura, None; C.A. Greer, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.15/BB5<br />
Topic: D.01.c. Olfaction: Olfactory coding--higher-order regions <strong>an</strong>d integrative studies<br />
Support: NIH DC005798<br />
Title: Temporal response properties of mitral cells <strong>an</strong>d <strong>the</strong>ir targets in higher olfactory centers<br />
Authors: *A.-M. M. OSWALD, N. N. URBAN;<br />
Dept. of Biol. Sciences, Ctr. Neural Basis of Cognition, Carnegie Mellon Univ., Pittsburgh, PA<br />
Abstract: Within <strong>the</strong> olfactory bulb, <strong>the</strong> processing of odor<strong>an</strong>t in<strong>for</strong>mation occurs on multiple<br />
timescales that reflect <strong>the</strong> dynamics of <strong>the</strong> stimulus, <strong>the</strong> breathing cycle of <strong>the</strong> <strong>an</strong>imal, <strong>an</strong>d <strong>the</strong><br />
ensuing network activity that may last <strong>for</strong> seconds after stimulus offset. We investigated <strong>the</strong><br />
temporal response properties of mitral cells (MC) <strong>an</strong>d <strong>the</strong>ir putative target neurons in <strong>the</strong> <strong>an</strong>terior<br />
olfactory nucleus (AON) <strong>an</strong>d piri<strong>for</strong>m cortex (PC). Neurons were directly stimulated <strong>for</strong> 100 s<br />
by a subthreshold bias current (
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.16/BB6<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: NIH Gr<strong>an</strong>t DC003195<br />
NIH Gr<strong>an</strong>t DC009049<br />
NIH Gr<strong>an</strong>t NS058850<br />
NIH Gr<strong>an</strong>t DA021194<br />
Title: Detection of tr<strong>an</strong>sient receptor potential (TRP) ch<strong>an</strong>nel mRNA in <strong>the</strong> mouse main<br />
olfactory bulb (MOB)<br />
Authors: *H. DONG 1 , S.-Y. DING 2 , Q. NAI 1 , F.-M. ZHOU 2 , Y.-H. ZHANG 1 , W.-L. ZHANG 1 ,<br />
M. ENNIS 1 ;<br />
1 Dept Anat. & Neurobiol, 2 Pharmacol., Univ. Tennessee, HSC, Memphis, TN<br />
Abstract: TRP ch<strong>an</strong>nels are a large family of cation ch<strong>an</strong>nels with wide distribution in <strong>the</strong> CNS.<br />
TRP ch<strong>an</strong>nels are involved in sensory processing in <strong>the</strong> visual, gustatory, olfactory, auditory <strong>an</strong>d<br />
somatosensory pathways. Currently, 28 TRP gene subtypes have been identified <strong>an</strong>d subdivided<br />
into 6 families (TRPC1-7, TRPV1-6, TRPM1-8, TRPP2-3, 5, TRPML1-3 <strong>an</strong>d TRPA2). The<br />
MOB has been reported to express several TRP ch<strong>an</strong>nel subtypes. However, <strong>the</strong> full expression<br />
profile of <strong>the</strong> TRP ch<strong>an</strong>nel family in <strong>the</strong> MOB has not been investigated, <strong>an</strong>d very little is known<br />
about TRP ch<strong>an</strong>nel expression in different MOB cell types. In present study, we employed MOB<br />
tissue <strong>an</strong>d single-cell RT-PCR methods to investigate <strong>the</strong> expression of 28 TRP ch<strong>an</strong>nel mRNAs<br />
in <strong>the</strong> mouse MOB. Our results showed that TRPC1-7, TRPV1-4, 6, TRPM1-4, 6-8, TRPP2-3, 5<br />
TRPML1-3 <strong>an</strong>d TRPA1 mRNA were expressed in <strong>the</strong> MOB. To date, TRPC3-5 was detected in<br />
electrophysiologically identified external cells <strong>an</strong>d periglomerular cells whereas TRPC3,<br />
TRPC5, TRPV3-4 <strong>an</strong>d TRPM2, 8 were detected in electrophysiologically identified mitral cells.<br />
Disclosures: H. Dong, None; S. Ding, None; Q. Nai, None; F. Zhou, None; Y. Zh<strong>an</strong>g,<br />
None; W. Zh<strong>an</strong>g, None; M. Ennis, None.
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.17/BB7<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: NIH Gr<strong>an</strong>t F31DC009376-03<br />
Title: Development of functional circuitry in <strong>the</strong> accessory olfactory bulb<br />
Authors: *K. HOVIS, N. URBAN;<br />
Biol Sci., Carnegie Mellon Univ., Pittsburgh, PA<br />
Abstract: The accessory olfactory system in mice, which includes <strong>the</strong> vomeronasal org<strong>an</strong> <strong>an</strong>d<br />
<strong>the</strong> accessory olfactory bulb (AOB) plays <strong>an</strong> essential role in detecting pheromones, recognizing<br />
conspecifics <strong>an</strong>d mediating stereotypic sexual behaviors. Here we describe <strong>the</strong> development of<br />
functional circuitry in <strong>the</strong> AOB of mice over <strong>the</strong> first two postnatal weeks. AOB principal<br />
neurons (mitral cells) receive excitatory inputs <strong>from</strong> axons of vomeronasal sensory neurons<br />
(VSNs). The output of mitral cells is controlled, in large part, by recurrent dendrodendritic<br />
inhibition between excitatory mitral cells <strong>an</strong>d axonless inhibitory gr<strong>an</strong>ule cells. Previous work in<br />
our lab has shown that during <strong>the</strong> first two weeks of post-natal development, VSN axons<br />
coalescence into well-defined glomeruli <strong>an</strong>d mitral cell dendritic arbors are pruned, presumably<br />
establishing proper sensory neuron to mitral cell connections. In addition, we have shown that in<br />
adult mice, activation of group I metabotropic glutamate receptors evokes gr<strong>an</strong>ule cell mediated<br />
inhibition of mitral cells.<br />
Here we describe <strong>the</strong> development of recurrent inhibition onto mitral cells during this critical<br />
period, using direct activation of mGluRs via application of <strong>the</strong> group I mGluR agonist (S)-3,5dihydroxyphenylglycine<br />
(DHPG). We found that during <strong>the</strong> first three days of post-natal<br />
development, DHPG had no effect on ei<strong>the</strong>r inward or outward going synaptic events (n=5).<br />
However, by post-natal day three, application of DHPG induced a large increase in <strong>the</strong><br />
spont<strong>an</strong>eous rate of inward going synaptic events with <strong>the</strong> rate increasing <strong>from</strong> <strong>an</strong> average of<br />
0.027 Hz +/- 0.018 to 2.43 Hz +/- 1.08 (n=3, p=0.09) which were blocked using <strong>the</strong> GABAAspecific<br />
<strong>an</strong>tagonist gabazine (SR95531). Interestingly, by post-natal nine, application of DHPG<br />
had <strong>the</strong> opposite effect, resulting in more th<strong>an</strong> a 10 fold increase in <strong>the</strong> rate of spont<strong>an</strong>eous<br />
outward synaptic events with <strong>the</strong> event rate increasing <strong>from</strong> <strong>an</strong> average of 0.41Hz +/- 0.20 to<br />
5.86 Hz +/- 1.26 (n=7, p< 0.01). These results suggest that mGluR I expression may affect<br />
inhibition during early post-natal development <strong>an</strong>d that this along with <strong>the</strong> GABAergic
excitatory to inhibitory shift between post natal day three <strong>an</strong>d nine may play <strong>an</strong> integral role in<br />
mitral cell dendritic pruning <strong>an</strong>d <strong>the</strong> establishment of proper connections in <strong>the</strong> accessory<br />
olfactory bulb.<br />
Disclosures: K. Hovis, None; N. Urb<strong>an</strong>, None.<br />
Poster<br />
555. Olfactory Bulb: Synapses <strong>an</strong>d Circuits<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 555.18/BB8<br />
Topic: D.01.b. Olfaction: Olfactory coding--second order regions (olfactory bulb <strong>an</strong>d <strong>an</strong>tennal<br />
lobe)<br />
Support: JSPS Project #19208029<br />
Title: Morphological <strong>an</strong>d histochemical studies of <strong>the</strong> nasal cavity <strong>an</strong>d fused olfactory bulb of<br />
<strong>the</strong> brown-eared bulbul (Hysipetes amaurotis)<br />
Authors: *M. YOKOSUKA 1 , T. R. SAITO 1 , M. AOYAMA 2 , S. SUGITA 2 , M. ICHIKAWA 3 ;<br />
1 Nippon Vet & Life Sci. Univ., Musashino, Tokyo, Jap<strong>an</strong>; 2 Sch. of Agr., Utsunomiya Univ.,<br />
Utsunomiya, Jap<strong>an</strong>; 3 Neurosci. Basic Techniques, Tokyo Metropolit<strong>an</strong> Inst. <strong>for</strong> Neurosci.,<br />
Tokyo, Jap<strong>an</strong><br />
Abstract: The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Jap<strong>an</strong> where it<br />
is regarded as a harmful bird that causes damage to agricultural products. Few studies have<br />
investigated <strong>the</strong> sensory apparatus of this bird, so little is known of <strong>the</strong> types of senses that it<br />
uses. We <strong>an</strong>alyzed <strong>the</strong> <strong>an</strong>atomical <strong>an</strong>d histological properties of <strong>the</strong> nasal cavity <strong>an</strong>d olfactory<br />
bulb of <strong>the</strong> bulbul to investigate its functional level of <strong>the</strong> olfaction. In <strong>the</strong> nasal cavity of <strong>the</strong><br />
bulbul, although <strong>the</strong> <strong>an</strong>terior <strong>an</strong>d maxillary conchae were observed, <strong>the</strong>re was no structure<br />
equivalent to <strong>the</strong> posterior concha. The ratio of <strong>the</strong> olfactory bulb size to that of <strong>the</strong> cerebral<br />
hemisphere was very small. The left <strong>an</strong>d right olfactory bulbs were completely fused <strong>an</strong>d located<br />
on <strong>the</strong> ventral side of <strong>the</strong> <strong>an</strong>terior extremity of <strong>the</strong> cerebrum. Several types of lectin, which bind<br />
<strong>the</strong> olfactory nerve of vertebrates, were positively bound to <strong>the</strong> olfactory nerve layer <strong>an</strong>d<br />
glomerular layer of <strong>the</strong> olfactory bulb. Fur<strong>the</strong>rmore, <strong>the</strong> glomerular layer was immunopositive<br />
<strong>for</strong> vesicular glutamate tr<strong>an</strong>sporter 2 (VGLUT2) <strong>an</strong>tibody. These results suggest that <strong>the</strong> bulbul<br />
has a “limited-sense of olfaction”, which is not as developed as that of mammals <strong>an</strong>d o<strong>the</strong>r avi<strong>an</strong><br />
species. Moreover, <strong>the</strong> “fused” olfactory bulb may offer a unique model <strong>for</strong> studying <strong>the</strong><br />
evolution <strong>an</strong>d development of <strong>the</strong> vertebrate olfactory system.
Disclosures: M. Yokosuka, Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research (Project #19208029) of JSPS,<br />
B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well<br />
as gr<strong>an</strong>ts already received); T.R. Saito, None; M. Aoyama, None; S. Sugita, None; M.<br />
Ichikawa, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.1/BB9<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: Wellcom Trust Gr<strong>an</strong>t<br />
Title: Amplitude modulation tr<strong>an</strong>sfer functions determining BOLD response across auditory<br />
areas in <strong>the</strong> macaque<br />
Authors: *S. BAUMANN, A. REES, C. I. PETKOV, L. SUN, A. THIELE, T. D. GRIFFITHS;<br />
Inst. of Neurosci., Newcastle Univ., Newcastle Upon Tyne, United Kingdom<br />
Abstract: The temporal envelope of natural sounds c<strong>an</strong> be represented as a set of sinusoidal<br />
amplitude modulations (AM) with different modulation frequencies. Single unit<br />
electrophysiological recordings in <strong>the</strong> auditory system of various <strong>an</strong>imals have found individual<br />
neurons that show a preference <strong>for</strong> different AM frequencies [1]. This work investigates<br />
differences between <strong>the</strong> modulation rate preference of multiple subcortical <strong>an</strong>d cortical areas.<br />
Whilst tonotopic mapping within subcortical <strong>an</strong>d cortical areas is established, <strong>an</strong>d used as a basis<br />
to define those areas, <strong>the</strong> org<strong>an</strong>isation of areas based on modulation frequency is not well<br />
understood.<br />
We characterised <strong>the</strong> amplitude modulation tr<strong>an</strong>sfer function at <strong>the</strong> level of <strong>the</strong> inferior colliculus<br />
(IC), <strong>the</strong> medial geniculate body (MGB) <strong>an</strong>d <strong>the</strong> auditory cortex in both hemispheres of <strong>the</strong><br />
macaque with fMRI. Stimuli consisted of broadb<strong>an</strong>d noise carriers amplitude modulated at one<br />
of six modulation frequencies between 0.5 - 512 Hz. We recorded fMRI data in multiple sessions<br />
at <strong>an</strong> in-pl<strong>an</strong>e resolution of 1 x 1 mm 2 <strong>an</strong>d a slice thickness of 2 mm using a sparse sampling<br />
paradigm to avoid interference of <strong>the</strong> sc<strong>an</strong>ner noise. Within <strong>the</strong> auditory cortex, <strong>the</strong> auditory core<br />
fields A1, R <strong>an</strong>d RT were distinguished by co-localising <strong>the</strong> recorded data with tonotopic maps<br />
generated by <strong>the</strong> presentation of b<strong>an</strong>dpass sounds at frequencies between 0.5 - 16 kHz.<br />
The contrast between stimulus <strong>an</strong>d silence demonstrated robust BOLD activation in <strong>the</strong> IC <strong>an</strong>d<br />
<strong>the</strong> auditory cortex in response to all AM frequencies presented. The response in <strong>the</strong> MGB was<br />
less robust. In agreement with previous electrophysiology experiments in various species, <strong>the</strong>
<strong>an</strong>ge of preferred AM frequencies decreased progressively at higher levels of <strong>the</strong> auditory<br />
pathway. The maximal responses in <strong>the</strong> IC were found between 128 - 512 Hz, <strong>for</strong> <strong>the</strong> MGB<br />
between 32 Hz - 512 Hz <strong>an</strong>d <strong>for</strong> <strong>the</strong> auditory cortex between 8 Hz - 32 Hz. A pattern of local<br />
areas showing selectivity <strong>for</strong> <strong>the</strong> different AM frequencies was identified within IC, MGB <strong>an</strong>d<br />
auditory cortex.<br />
fMRI provides a method <strong>for</strong> <strong>the</strong> simult<strong>an</strong>eous mapping of activation due to AM sounds in<br />
multiple subcortical <strong>an</strong>d cortical areas that c<strong>an</strong>not be achieved by electrophysiology. The<br />
macaque model is ideal <strong>for</strong> bridging <strong>the</strong> gap between <strong>the</strong> large body of electrophysiological data<br />
in <strong>an</strong>imals with functional imaging data <strong>from</strong> hum<strong>an</strong>s [2]. High resolution imaging in macaque<br />
reveals <strong>an</strong> org<strong>an</strong>isation within subcortical <strong>an</strong>d cortical areas based on AM preference in addition<br />
to <strong>the</strong> established tonotopic mapping that defines <strong>the</strong> fields <strong>the</strong>mselves.<br />
1. Joris PX, Schreiner CE, Rees A (2004). Physiological Reviews 84: 541-577.<br />
2. Harms MP & Melcher JR (2002). J Neurophysiol 88: 1433-1450.<br />
Disclosures: S. Baum<strong>an</strong>n, None; A. Rees, None; C.I. Petkov, None; L. Sun, None; A. Thiele,<br />
None; T.D. Griffiths, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.2/BB10<br />
Topic: D.02.e. Auditory cortex: Perception, cognition, <strong>an</strong>d action<br />
Support: NIH DC02514<br />
NIH F31DC008935-03<br />
McDonnell Foundation<br />
Title: Decision- <strong>an</strong>d state- related activity in primary auditory cortex (A1) of rhesus macaques<br />
per<strong>for</strong>ming amplitude modulation (AM) discrimination<br />
Authors: *M. NIWA 1 , J. S. JOHNSON 2 , E. A. MARSHALL 2 , K. N. O'CONNOR 2 , M. L.<br />
SUTTER 2 ;<br />
2 Ctr. <strong>for</strong> Neurosci., 1 Univ.Calif Davis, Davis, CA<br />
Abstract: We found that single (SU) <strong>an</strong>d multiple-unit (MU) firing rate in A1 was predictive of<br />
<strong>the</strong> <strong>an</strong>imal’s behavioral response during <strong>an</strong> auditory discrimination task. We also found that
neural sensitivity, based on firing rate, <strong>for</strong> detecting <strong>the</strong> presence of AM improved when <strong>the</strong><br />
<strong>an</strong>imal was discriminating AM (behaving condition) th<strong>an</strong> when he was not (passive condition).<br />
In <strong>the</strong> discrimination task, <strong>the</strong> <strong>an</strong>imals pressed a lever to initiate a trial. If <strong>the</strong> target sound was<br />
AM (6-100% depth) he should release <strong>the</strong> lever (lever release = response). If <strong>the</strong> sound was<br />
unmodulated (0% depth), he should continue to hold <strong>the</strong> lever down. To <strong>an</strong>alyze <strong>the</strong> relationship<br />
between unit activity <strong>an</strong>d <strong>the</strong> <strong>an</strong>imals’ trial-by-trial ‘decisions’, we used Sender Operating<br />
Characteristic (SOC or choice probability) <strong>an</strong>alysis. At each modulation depth, <strong>the</strong> SOC<br />
statistically compares a unit’s firing rate on response trials <strong>an</strong>d non-response trials. Because <strong>the</strong><br />
stimuli are identical on <strong>the</strong>se trials, <strong>an</strong>y differences in activity must be decision ra<strong>the</strong>r th<strong>an</strong><br />
stimulus related. <strong>When</strong> we compared <strong>the</strong> activity starting <strong>from</strong> <strong>the</strong> beginning of <strong>the</strong> stimulus up<br />
to <strong>the</strong> <strong>an</strong>imal’s behavioral response, 34.4% of MUs <strong>an</strong>d 16.3% of <strong>the</strong> SUs had signific<strong>an</strong>tly<br />
greater firing rate on response trials th<strong>an</strong> non-response trials. <strong>When</strong> activity only during <strong>the</strong><br />
stimulus was used 14.1% of MUs <strong>an</strong>d 10.9% of SUs had signific<strong>an</strong>tly greater firing rate on<br />
response trials th<strong>an</strong> on non-response trials.<br />
Receiver Operating Characteristic (ROC) <strong>an</strong>alysis was used to determine how well each unit<br />
discriminated AM <strong>from</strong> unmodulated sounds. ROC <strong>an</strong>alysis statistically compares trial-by-trial<br />
responses to AM <strong>an</strong>d unmodulated stimuli. AM sensitivity was determined in both behaving <strong>an</strong>d<br />
passive conditions <strong>for</strong> each unit. Both MU <strong>an</strong>d SU sensitivity to modulation was better in <strong>the</strong><br />
behaving th<strong>an</strong> in <strong>the</strong> passive condition. This was observed as statistically improved ROC area at<br />
modulation depths >= 28% <strong>for</strong> MUS <strong>an</strong>d >= 60% <strong>for</strong> SUs. It is interesting that <strong>the</strong> maximum<br />
improvement occurred at <strong>the</strong> intermediate depths (40% <strong>for</strong> MU <strong>an</strong>d 60% <strong>for</strong> SU).This might be<br />
due to a ceiling effect at <strong>the</strong> highest depths or might also be due to <strong>an</strong> effect of attention, because<br />
<strong>the</strong> higher modulation depths are easier to discriminate <strong>from</strong> unmodulated sound th<strong>an</strong> <strong>the</strong><br />
intermediate depths.<br />
Disclosures: M. Niwa, None; J.S. Johnson, None; E.A. Marshall, None; K.N. O'Connor,<br />
None; M.L. Sutter, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.3/BB11<br />
Topic: D.02.e. Auditory cortex: Perception, cognition, <strong>an</strong>d action<br />
Support: NIH Gr<strong>an</strong>t DC008358<br />
Title: Behavioral state modulation of responses to conspecific song in <strong>the</strong> auditory <strong>for</strong>ebrain of<br />
awake Europe<strong>an</strong> starlings
Authors: *D. P. KNUDSEN 1 , T. Q. GENTNER 2 ;<br />
1 Neurosciences Grad. Program, 2 Psychology Dept., Univ. of Cali<strong>for</strong>nia S<strong>an</strong> Diego, La Jolla, CA<br />
Abstract: Neurons in <strong>the</strong> auditory <strong>for</strong>ebrain of oscine songbirds, in regions <strong>an</strong>alogous to<br />
mammali<strong>an</strong> auditory cortex, are responsive to complex auditory signals including conspecific<br />
song. Multiple lines of evidence support <strong>the</strong> conclusion that <strong>the</strong>se responses are modified by <strong>an</strong><br />
<strong>an</strong>imal's experience with different songs, leading to a representational topography in which<br />
single neurons in secondary auditory regions respond to behaviorally relev<strong>an</strong>t songs. For<br />
example, in <strong>an</strong>es<strong>the</strong>tized Europe<strong>an</strong> starlings <strong>the</strong> spiking responses of single neurons in <strong>the</strong><br />
caudomedial mesopallium (CMM) are tied to <strong>the</strong> phasic components that appear in songs that<br />
birds have learned to recognize, but not to similar components in unfamiliar songs. Little is<br />
known, however, about <strong>the</strong> online encoding of behaviorally relev<strong>an</strong>t auditory in<strong>for</strong>mation <strong>from</strong><br />
which <strong>the</strong>se experience-dependent responses emerge. In order to underst<strong>an</strong>d <strong>the</strong> real-time<br />
dynamics of auditory processing during <strong>the</strong> recognition of complex natural communication<br />
signals, we are recording <strong>from</strong> <strong>the</strong> auditory <strong>for</strong>ebrain of Europe<strong>an</strong> starlings while <strong>the</strong>y are<br />
actively engaged in learned song recognition. In one condition ('engaged'), <strong>the</strong> bird triggers<br />
playback of a number of familiar conspecific songs <strong>an</strong>d classifies <strong>the</strong>m into two groups using <strong>an</strong><br />
oper<strong>an</strong>t apparatus. In a second condition ('passive'), <strong>the</strong> oper<strong>an</strong>t apparatus is turned off <strong>an</strong>d <strong>the</strong><br />
same songs are presented to <strong>the</strong> bird as it rests in a state of quiet wakefulness, <strong>an</strong>d without<br />
oper<strong>an</strong>t contingencies. Preliminary <strong>an</strong>alyses suggest that a subset of <strong>the</strong> neurons show a<br />
modulation in <strong>the</strong>ir auditory responsiveness as a function of behavioral state. Cells show ch<strong>an</strong>ges<br />
in spont<strong>an</strong>eous firing rate between <strong>the</strong> engaged <strong>an</strong>d passive conditions, as well as differences in<br />
<strong>the</strong> me<strong>an</strong> firing rates elicited by <strong>the</strong> same stimulus under <strong>the</strong> different conditions. In addition, <strong>the</strong><br />
degree of modulation above (activation) or below (suppression) <strong>the</strong> spont<strong>an</strong>eous firing rate<br />
appears to be different in <strong>the</strong> two conditions within a single cell. These results suggest that<br />
neurons in <strong>the</strong> auditory <strong>for</strong>ebrain of Europe<strong>an</strong> starlings do not passively encode auditory stimuli,<br />
but ch<strong>an</strong>ge <strong>the</strong>ir response properties based on <strong>the</strong> behavioral state of <strong>the</strong> <strong>an</strong>imal.<br />
Disclosures: D.P. Knudsen, None; T.Q. Gentner, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.4/BB12<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH gr<strong>an</strong>t DC03180
Title: Spatial auditory processing in auditory cortex of sleeping marmosets<br />
Authors: *E. D. REMINGTON, X. WANG;<br />
Johns Hopkins Univ. SOM, Baltimore, MD<br />
Abstract: In sleep, <strong>the</strong> auditory system is open to external stimuli <strong>an</strong>d remains alert to<br />
behaviorally relev<strong>an</strong>t sounds. Recent work shows that in <strong>the</strong> primary auditory cortex <strong>an</strong>d its<br />
adjacent lateral belt areas of marmoset monkeys, neural tunings to sound attributes such as<br />
frequency, level, <strong>an</strong>d amplitude modulation are qualitatively similar to those recorded in <strong>the</strong><br />
awake state (Issa <strong>an</strong>d W<strong>an</strong>g, 2008). No studies have addressed <strong>the</strong> question of spatial hearing in<br />
a sleeping <strong>an</strong>imal. Previous studies in <strong>the</strong> <strong>an</strong>es<strong>the</strong>tized <strong>an</strong>imals report broadly tuned spatial<br />
receptive fields in <strong>the</strong> auditory cortex. In contrast, spatial tuning in <strong>the</strong> awake condition tends to<br />
be sharper <strong>an</strong>d contains both onset <strong>an</strong>d sustained phases of responses. This suggests that <strong>the</strong><br />
arousal level of <strong>the</strong> cortex may influence <strong>the</strong> neural integration process <strong>for</strong> spatial tuning. Here,<br />
we explored to what extent sleep affects <strong>the</strong> spatial tuning properties in <strong>the</strong> auditory cortex. Our<br />
hypo<strong>the</strong>sis is that spatial tuning is preserved in sleep <strong>an</strong>d more closely resembles tuning in <strong>the</strong><br />
awake condition, similar to frequency tuning (Issa <strong>an</strong>d W<strong>an</strong>g, 2008).<br />
We used a free-field speaker setup covering 360 degrees of azimuth <strong>an</strong>d 180 degrees of vertical<br />
space to study single-unit responses to different sound locations in <strong>the</strong> primary auditory cortex<br />
<strong>an</strong>d lateral belt area in <strong>the</strong> common marmoset (Callithrix Jacchus). In a majority of units, tuning<br />
to sound location is modulated by sleep in some way. A minority of neurons displayed<br />
signific<strong>an</strong>t shifting of spatial tuning functions in slow wave or rapid eye-movement sleep. Some<br />
neurons also displayed broadening of tuning curves. These data suggest that in sleep <strong>the</strong> neural<br />
circuits responsible <strong>for</strong> spatial selectivity of cortical neurons remain largely intact, but <strong>the</strong>ir<br />
output c<strong>an</strong> be affected by network activity in sleep. Functionally, this implies that spatial<br />
in<strong>for</strong>mation could be used to drive behavioral responses in a sleeping <strong>an</strong>imal.<br />
Disclosures: E.D. Remington, None; X. W<strong>an</strong>g, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.5/BB13<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH Gr<strong>an</strong>t DC009688 (PC)<br />
NIH Gr<strong>an</strong>t DC03180 (XW)
Title: Laminar dependent representation of time-varying sounds<br />
Authors: *P. A. CRUM, X. WANG;<br />
Biomed Engin., Johns Hopkins Univ. Sch. Med., Baltimore, MD<br />
Abstract: The neural representation of time-varying signals is of particular import<strong>an</strong>ce to our<br />
underst<strong>an</strong>ding of complex <strong>an</strong>d biologically import<strong>an</strong>t sounds such as speech <strong>an</strong>d music. How<br />
sub-cortical <strong>an</strong>d cortical regions differentially encode temporal patterns of acoustic stimuli offers<br />
insight to <strong>the</strong> crucial properties of <strong>the</strong> stimulus being represented as well as <strong>the</strong> functional role of<br />
<strong>the</strong> neural region. It is well known that <strong>the</strong> neural representation of time-varying signals in most<br />
sub-cortical regions is typically synchronous <strong>an</strong>d time-locked to stimulus features. In contrast,<br />
previous studies of awake primate auditory cortex have shown both neurons with stimulussynchronized<br />
phasic responses as well as neurons with non-synchronized tonic responses (Lu et<br />
al, 2001). Here, we show that in <strong>the</strong> awake marmoset monkey a central stage in <strong>the</strong> cortical<br />
tr<strong>an</strong>s<strong>for</strong>mation to a firing rate-based response occurs across <strong>the</strong> cortical laminae. Neurons in<br />
upper cortical laminae show both weaker synchronization th<strong>an</strong> neurons in middle thalamorecipient<br />
laminae (layer IIIb/IV), as well as a signific<strong>an</strong>t shift in <strong>the</strong> percentage of neurons<br />
responsive to time-varying sounds but having no clear time-locked response features. In our<br />
experiments, laminar location of single units was identified through registration with <strong>the</strong> currentsource-density<br />
(CSD) measured within <strong>the</strong> same penetration. All measurements of <strong>the</strong> CSD were<br />
generated <strong>from</strong> local-field-potential (LFP) recordings made across depth <strong>an</strong>d sp<strong>an</strong>ning <strong>the</strong><br />
cortical laminae using <strong>the</strong> same tungsten electrode used to record single-unit responses. A shift<br />
to a firing rate-based representation of time varying signals in upper cortical laminae suggests a<br />
ch<strong>an</strong>ge in <strong>the</strong> stimulus features critical to encoding at successively higher processing stages in<br />
auditory cortex.<br />
Disclosures: P.A. Crum, None; X. W<strong>an</strong>g, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.6/BB14<br />
Topic: D.02.e. Auditory cortex: Perception, cognition, <strong>an</strong>d action<br />
Support: JSPS Gr<strong>an</strong>t<br />
Title: Auditory stream segregation: Time-frequency filtering <strong>an</strong>d response variability in <strong>the</strong><br />
auditory cortex
Authors: *T. NODA 1 , R. KANZAKI 2 , H. TAKAHASHI 2 ;<br />
2 Res. Ctr. <strong>for</strong> Adv<strong>an</strong>ced Sci. <strong>an</strong>d Technol., 1 The Univ. of Tokyo, Tokyo, Jap<strong>an</strong><br />
Abstract: Auditory system c<strong>an</strong> separate sounds into several perceptual objects, termed auditory<br />
stream, which is thought to allow both hum<strong>an</strong>s <strong>an</strong>d <strong>an</strong>imals to select essential environmental<br />
in<strong>for</strong>mation. However, <strong>the</strong> neural mech<strong>an</strong>ism behind <strong>the</strong> perceptual ability remains poorly<br />
understood. Psychophysical evidence reveals that when ABA-tone sequence with various<br />
frequency differences between A <strong>an</strong>d B tones (∆F) <strong>an</strong>d inter tone intervals (ITI) are presented,<br />
sequences with larger ∆F <strong>an</strong>d smaller ITI tend to induce stream segregation. This study, in <strong>the</strong><br />
first part, examined characteristic responses to ABA-tone sequence based on <strong>the</strong> time-frequency<br />
filter property of <strong>the</strong> auditory cortex, <strong>an</strong>d in <strong>the</strong> second part, examined <strong>the</strong> response variability<br />
depended on ∆F / ITI conditions. Local field potentials (LFPs) of rat auditory cortex were<br />
mapped, <strong>an</strong>d <strong>the</strong> responses at A-tone <strong>an</strong>d B-tone-evoked local foci were characterized<br />
respectively. Firstly, <strong>the</strong> amplitude in response to B tone at A-tone-evoked local foci varied nonmonotonically<br />
as a function of ∆F, <strong>an</strong>d had a local maximum when ∆F was 50-60% of A-tone<br />
frequency. The result suggests that this enh<strong>an</strong>ced representation of B tone leads to ambiguous<br />
perception of A tone. Fur<strong>the</strong>rmore, <strong>the</strong> response to ABA-tone sequence was relatively greater <strong>for</strong><br />
<strong>the</strong> larger ∆F at each tone-evoked local focus th<strong>an</strong> summation of responses to pure tone<br />
sequences as control stimuli of ABA-tone. Lastly, coefficient of variation (CV) of <strong>the</strong> amplitude<br />
<strong>an</strong>d vari<strong>an</strong>ce of <strong>the</strong> latency of <strong>the</strong> response evoked by <strong>the</strong> second A tone decreased <strong>for</strong> <strong>the</strong> larger<br />
∆F <strong>an</strong>d longer ITI. These results suggest that <strong>the</strong>se test conditions leading to <strong>the</strong> reduction of <strong>the</strong><br />
amplitude <strong>an</strong>d temporal variability of <strong>the</strong> response stabilize perception of A tone <strong>an</strong>d maintain<br />
<strong>the</strong> perception of temporal order of A-B tones.<br />
Disclosures: T. Noda, None; R. K<strong>an</strong>zaki, None; H. Takahashi, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.7/BB15<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Title: A population code <strong>for</strong> sound localization in <strong>the</strong> auditory cortex<br />
Authors: *K. A. RAZAK;<br />
Univ. Cali<strong>for</strong>nia, Riverside, Riverside, CA
Abstract: The auditory cortex is involved in sound localization behavior. The spatial receptive<br />
fields (SRF) of m<strong>an</strong>y cortical neurons tend to be large, <strong>an</strong>d increase fur<strong>the</strong>r with intensity. A<br />
spatiotopic map has not been found in <strong>the</strong> auditory cortex. This suggests that <strong>the</strong> cortex uses<br />
population codes to represent space. However, <strong>the</strong> nature of such codes remains unknown.<br />
Auditory SRFs are shaped by binaural sensitivity <strong>an</strong>d frequency-specific ear directionality. A<br />
consistent observation is <strong>the</strong> clustered representation of binaural cues in <strong>the</strong> cortex. How binaural<br />
clusters underlie population codes <strong>for</strong> space remains unclear.<br />
These issues were addressed in <strong>the</strong> auditory cortex of <strong>the</strong> pallid bat. The pallid bat uses passive<br />
hearing to localize prey-generated noise. Its auditory cortex contains a relatively large region<br />
selective <strong>for</strong> noise over tones. This noise-selective region contains two binaural clusters based on<br />
sensitivity to interaural intensity differences (IID). One cluster contains binaurally inhibited<br />
neurons with a systematic map of IID sensitivity. The second cluster contains neurons with<br />
peaked IID sensitivity with a systematic map of preferred IID. These clusters predict a<br />
population code in which <strong>the</strong> extent of activated cortex ch<strong>an</strong>ges systematically with azimuth<br />
sound location. This prediction was tested in this study. Using single unit recordings <strong>an</strong>d<br />
sequential dichotic <strong>an</strong>d free-field stimulation, <strong>the</strong> IID sensitivity, frequency tuning <strong>an</strong>d azimuth<br />
SRF were mapped across <strong>the</strong> noise-selective region. In free-field, as <strong>the</strong> sound moved <strong>from</strong> ipsilateral<br />
locations to more contra-lateral locations, <strong>the</strong> region of activated cortex started in lateral<br />
cortical locations to systematically include more medial locations in <strong>the</strong> noise-region. The area of<br />
activated cortex increased almost linearly as sound moved <strong>from</strong> ipsi- to contra-lateral locations.<br />
The activation patterns c<strong>an</strong> be predicted based on closed-loop studies suggesting that <strong>the</strong> two IID<br />
clusters create a substrate <strong>for</strong> <strong>the</strong> observed ch<strong>an</strong>ges in activity patterns <strong>for</strong> different sound<br />
locations. The activity patterns do not ch<strong>an</strong>ge with sound intensity. These data provide cues on<br />
how neurons with a broad r<strong>an</strong>ge of spatial selectivity act toge<strong>the</strong>r to represent space <strong>an</strong>d how<br />
accurate representation of locations c<strong>an</strong> occur with broadly tuned neurons.<br />
Disclosures: K.A. Razak, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.8/BB16<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH DC007172<br />
NIH DC009224
Title: Neural activity in <strong>the</strong> superior temporal gyrus during a discrimination task reflects<br />
stimulus category<br />
Authors: *J. LEE 1 , J. TSUNADA 2 , Y. E. COHEN 2 ;<br />
1 Dartmouth Col., H<strong>an</strong>over, NH; 2 Otorhinoliryngology, Univ. of Pennsylv<strong>an</strong>ia, Philadeplphia, PA<br />
Abstract: One of <strong>the</strong> most import<strong>an</strong>t conceptual models in neuroscience is that spatial <strong>an</strong>d nonspatial<br />
in<strong>for</strong>mation are processed in parallel ch<strong>an</strong>nels In <strong>the</strong> auditory system, a ventral nonspatial<br />
processing stream originates in <strong>the</strong> auditory cortex <strong>an</strong>d is fur<strong>the</strong>r defined by a series of<br />
projections that includes <strong>the</strong> <strong>an</strong>terior belt of <strong>the</strong> auditory cortex <strong>an</strong>d regions of <strong>the</strong> prefrontal<br />
cortex, specifically <strong>the</strong> ventrolateral prefrontal cortex (vPFC) (Rom<strong>an</strong>ski et al.,1999a, Rom<strong>an</strong>ski<br />
et al., 1999b, Rauschecker <strong>an</strong>d Ti<strong>an</strong>, 2000). Our group has recently demonstrated that <strong>the</strong><br />
ventrolateral prefrontal cortex (vPFC) plays <strong>an</strong> import<strong>an</strong>t role in components of executive<br />
function such as decision-making (Russ et. al. 2008, Lee et. al, Submitted). Specifically, we<br />
found that, during a task in which rhesus monkeys discriminated between pairs of auditory<br />
stimuli, vPFC neurons coded <strong>the</strong> monkeys’ behavioral reports (i.e., <strong>the</strong>ir actual choices).<br />
Do <strong>the</strong>se computations occur in <strong>the</strong> vPFC? Or does vPFC reflect computations that occur earlier<br />
in <strong>the</strong> ventral-processing pathway? To address this question, we recorded <strong>from</strong> neurons in <strong>the</strong><br />
superior temporal gyrus (STG), a region of <strong>the</strong> auditory cortex that is part of this ventral pathway<br />
<strong>an</strong>d a cortical region that projects directly to <strong>the</strong> vPFC. While recording STG neurons, monkeys<br />
participated in <strong>the</strong> “same-different” task. This task required monkeys to discriminate between a<br />
reference <strong>an</strong>d test stimuli <strong>an</strong>d to indicate whe<strong>the</strong>r <strong>the</strong>se stimuli were <strong>the</strong> same or different. The<br />
stimuli were <strong>the</strong> prototype hum<strong>an</strong> spoken words bad <strong>an</strong>d dad or morphs of <strong>the</strong>se words.<br />
As we did with our previous vPFC study (Lee et. al, Submitted), we tested how well a trained<br />
multi-layered perceptron could decode three different components of <strong>the</strong> same-different task: (1)<br />
<strong>the</strong> perceptual category of test-stimulus (i.e., <strong>the</strong> test-stimulus category), (2) <strong>the</strong> relationship<br />
between <strong>the</strong> reference <strong>an</strong>d test stimuli (i.e., what <strong>the</strong> monkeys should choose), <strong>an</strong>d (3) <strong>the</strong><br />
monkeys’ actual choice (i.e., <strong>the</strong>ir behavioral reports or decisions). We found that STG neurons<br />
code, on average, <strong>the</strong> test-stimulus category better th<strong>an</strong> <strong>the</strong>se o<strong>the</strong>r two components of <strong>the</strong> samedifferent<br />
task; that is, STG activity does not reflect <strong>the</strong> monkeys’ behavioral reports. Taken<br />
toge<strong>the</strong>r with our results <strong>from</strong> <strong>the</strong> vPFC, it suggests that <strong>the</strong> pathway between <strong>the</strong> STG <strong>an</strong>d <strong>the</strong><br />
vPFC are hierarchically related <strong>an</strong>d that neural correlates of decision-making on our samedifferent<br />
task originate after <strong>the</strong> STG.<br />
Disclosures: J. Lee, None; J. Tsunada, None; Y.E. Cohen, NIH-NIDCD, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received).<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.9/BB17<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH-R01 NS052494 (JPR,YK)<br />
NIH (MM, BH)<br />
Title: Neuronal representation of harmonics in monkey auditory cortex<br />
Authors: *Y. KIKUCHI 1 , M. LAWSON 1 , B. HORWITZ 2 , M. MISHKIN 3 , J.<br />
RAUSCHECKER 1 ;<br />
1 Physiol & Biophysics, Georgetown Univ. Med., Washington, DC; 2 NIDCD, Be<strong>the</strong>sda, MD;<br />
3 NIMH, Be<strong>the</strong>sda, MD<br />
Abstract: Animals <strong>an</strong>d hum<strong>an</strong>s must rapidly detect <strong>an</strong>d extract specific features of incoming<br />
sounds. M<strong>an</strong>y speech sounds <strong>an</strong>d <strong>an</strong>imal vocalizations, <strong>for</strong> inst<strong>an</strong>ce, contain components,<br />
commonly referred to as complex tonal stimuli, which consist of a fundamental frequency (f0)<br />
<strong>an</strong>d higher harmonics. We perceive <strong>the</strong> pitch of a complex tonal stimulus by resolving <strong>the</strong><br />
overtone harmonics to its fundamental frequency (f0) ra<strong>the</strong>r th<strong>an</strong> perceiving each frequency<br />
separately. We hypo<strong>the</strong>sized that <strong>the</strong> neural mech<strong>an</strong>ism of such harmonic processing lay close to<br />
tonotopically org<strong>an</strong>ized auditory areas <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e recorded <strong>from</strong> core <strong>an</strong>d belt areas in rhesus<br />
monkey auditory cortex.<br />
Using simult<strong>an</strong>eous single- <strong>an</strong>d multi-unit techniques, we recorded neurons <strong>from</strong> different<br />
subdivisions of core <strong>an</strong>d lateral belt in two monkeys while <strong>the</strong>y per<strong>for</strong>med <strong>an</strong> auditory<br />
discrimination task. To examine whe<strong>the</strong>r <strong>the</strong>se tonotopically org<strong>an</strong>ized auditory areas are best<br />
characterized by frequency or pitch, we used 84 pure-tone stimuli in semitone steps (110 Hz to<br />
13.3k Hz), <strong>an</strong>d 56 pitch-shifted monkey vocalizations (coos) that consisted of complex-tonal<br />
segments, such that f0 was matched to each pure-tone stimulus. Once we identified <strong>the</strong> neuron’s<br />
best frequency (BF), acoustic stimuli with <strong>the</strong> same center frequency but varying timbre were<br />
introduced by subtracting f0 <strong>an</strong>d/or harmonics <strong>from</strong> <strong>the</strong> stimuli. Based on <strong>the</strong> neuron’s BF <strong>an</strong>d<br />
responsiveness to tones <strong>an</strong>d b<strong>an</strong>dpass noise, <strong>the</strong> recording sites were assigned to <strong>the</strong> primary <strong>an</strong>d<br />
rostral core fields (A1 <strong>an</strong>d R) or <strong>the</strong> middle- <strong>an</strong>d <strong>an</strong>terior-lateral belt fields (ML <strong>an</strong>d AL). The<br />
latencies to BF tone stimuli were signific<strong>an</strong>tly different across <strong>the</strong> four regions (BF latency: N =<br />
105, A1: 30ms; R: 50 ms; ML: 53 ms; AL: 67 ms, P< 0.01). Fur<strong>the</strong>rmore, <strong>the</strong> latency to BF tone<br />
stimuli was signific<strong>an</strong>tly shorter th<strong>an</strong> <strong>the</strong> latency to best f0 harmonic stimuli in A1 (P< 0.02) but<br />
not in o<strong>the</strong>r areas, suggesting a pure-tone frequency representation in A1, whereas pitch may be<br />
represented in <strong>the</strong> o<strong>the</strong>r areas. Based on <strong>the</strong> neuron’s frequency-tuning response function, <strong>the</strong><br />
relations of multiple-peak responses were <strong>an</strong>alyzed. The number of peaks in fields R <strong>an</strong>d AL<br />
were greater th<strong>an</strong> in A1 <strong>an</strong>d ML, often exhibiting harmonic intervals between peaks. These<br />
neurons may serve as high-resolution filters that extract specific harmonic features based on <strong>the</strong><br />
frequency relationship between <strong>the</strong> peaks <strong>an</strong>d <strong>the</strong> fundamental frequency. Such neurons were<br />
found primarily in <strong>an</strong>terior core <strong>an</strong>d lateral belt <strong>an</strong>d were sometimes also responsive to complex
tonal stimuli even when <strong>the</strong>ir fundamental frequencies were removed (‘missing fundamental’).<br />
They may thus play <strong>an</strong> import<strong>an</strong>t role in harmonic processing.<br />
Disclosures: Y. Kikuchi, None; M. Lawson, None; B. Horwitz, None; M. Mishkin, None; J.<br />
Rauschecker, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.10/BB18<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH NINDS R01NS054898<br />
NSF BCS-0547760<br />
NIH R90 DA023419-02<br />
Title: Encoding of natural habitat scenes in <strong>the</strong> primary auditory cortex of monkeys<br />
Authors: *C. CHANDRASEKARAN 1,2 , H. K. TURESSON 1,2 , C. H. BROWN 4 , A. A.<br />
GHAZANFAR 1,2,3 ;<br />
1 Dept. of Psychology, 2 Princeton Neurosci. Inst., 3 Dept. of Ecology <strong>an</strong>d Evolutionary Biol.,<br />
Princeton Univ., Princeton, NJ; 4 Dept. of Psychology, Univ. of South Alabama, Mobile, AL<br />
Abstract: Org<strong>an</strong>isms are typically embedded in auditory scenes composed of sounds <strong>from</strong><br />
several signalers, abiotic noise <strong>an</strong>d acoustic reflections. Our underst<strong>an</strong>ding of how such a<br />
complex scene is encoded at <strong>the</strong> level of primary auditory cortex (A1) is rudimentary. We<br />
characterized <strong>the</strong> spectrotemporal statistics of auditory scenes <strong>from</strong> two primate habitats: riverine<br />
<strong>for</strong>est <strong>an</strong>d sav<strong>an</strong>nah <strong>for</strong>est. The spectral structure of both habitat scenes was primarily in <strong>the</strong> 5 - 6<br />
kHz r<strong>an</strong>ge due to bird vocalizations <strong>an</strong>d insect chirps. The predomin<strong>an</strong>t temporal modulation of<br />
<strong>the</strong> envelope <strong>for</strong> <strong>the</strong> riverine <strong>for</strong>est scene was at 25 Hz with a smaller modulation at 50 Hz. The<br />
sav<strong>an</strong>nah <strong>for</strong>est scene contained nested temporal modulations: repetitions of fast modulations at<br />
a slow rate. The temporal structure of sav<strong>an</strong>nah <strong>for</strong>est scene contained peaks at 7, 14, 18, <strong>an</strong>d 23<br />
Hz <strong>an</strong>d smaller peaks at higher frequencies.<br />
We tested <strong>the</strong> degree to which A1 cortex represented <strong>the</strong>se spectrotemporal statistics by<br />
recording local field potential (LFP) <strong>an</strong>d spiking activity (single unit <strong>an</strong>d multi-unit clusters)<br />
<strong>from</strong> 91 cortical sites in two macaque monkeys as <strong>the</strong>y listened to <strong>the</strong> long sequences of riverine
<strong>an</strong>d sav<strong>an</strong>nah <strong>for</strong>est scenes (60 seconds per sound sample, 3 samples per scene, 5 repetitions per<br />
sample). Neural activity <strong>from</strong> several sites actively tracked <strong>the</strong> dynamics of <strong>the</strong> sound. We used<br />
coherence to qu<strong>an</strong>tify <strong>the</strong> degree of locking between <strong>the</strong> dynamics of <strong>the</strong> sound <strong>an</strong>d <strong>the</strong> neural<br />
activity since its magnitude is function of a consistent phase relationship between <strong>the</strong> two<br />
signals. We observed high coherence between neural activity <strong>an</strong>d <strong>the</strong> sound envelope at <strong>the</strong> 25<br />
Hz temporal modulation peak <strong>for</strong> <strong>the</strong> riverine <strong>for</strong>est scene <strong>an</strong>d, in some cortical sites, even at <strong>the</strong><br />
50 Hz peak. Coherence between sav<strong>an</strong>nah scene <strong>an</strong>d neural activity revealed peaks centered at 7,<br />
14, 18, <strong>an</strong>d 23 Hz. However, not all sites showed such phase-locked activity. High coherence<br />
was only present when <strong>the</strong> spectral selectivity of a particular cortical site overlapped with <strong>the</strong><br />
spectral structure of <strong>the</strong> sound. Thus, <strong>the</strong> fine (spectral) structure of <strong>the</strong> stimulus is encoded by<br />
<strong>the</strong> position along <strong>the</strong> tonotopic axis, whereas <strong>the</strong> dynamics are encoded by <strong>the</strong> locking of neural<br />
activity to <strong>the</strong> temporal modulations of <strong>the</strong> stimulus. This is <strong>an</strong> import<strong>an</strong>t extension to recent<br />
“phase of firing” coding models <strong>an</strong>d suggests that a distributed model is a more accurate one <strong>for</strong><br />
<strong>the</strong> encoding of auditory scenes.<br />
Disclosures: C. Ch<strong>an</strong>drasekar<strong>an</strong>, None; H.K. Turesson, None; C.H. Brown, None; A.A.<br />
Ghaz<strong>an</strong>far, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.11/BB19<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH Gr<strong>an</strong>t R01DC005779<br />
NIH Gr<strong>an</strong>t F32DC008453<br />
Title: Noise robust representation of speech in <strong>the</strong> primary auditory cortex<br />
Authors: N. MESGARANI 1 , S. DAVID 1 , J. FRITZ 1 , *S. A. SHAMMA 2 ;<br />
1 Univ. of Maryl<strong>an</strong>d, College Park, MD; 2 Univ. Maryl<strong>an</strong>d, College Park, MD<br />
Abstract: It is well known that hum<strong>an</strong>s c<strong>an</strong> robustly perceive phonemes despite subst<strong>an</strong>tial<br />
variability across speakers, context <strong>an</strong>d natural distortions such as noise <strong>an</strong>d reverberation. This<br />
robustness is attributed to a rich <strong>an</strong>d invari<strong>an</strong>t representation of perceptually import<strong>an</strong>t features<br />
of speech. Here, we studied this problem by examining <strong>the</strong> responses of neurons in primary<br />
auditory cortex (A1) to phonetically labeled speech stimuli in cle<strong>an</strong> <strong>an</strong>d noisy conditions. We
presented cle<strong>an</strong> speech <strong>an</strong>d speech with added white noise (6dB <strong>an</strong>d 0dB Signal-To-Noise Ratio)<br />
to awake, passively listening ferrets while recording <strong>the</strong> activity of isolated A1 neurons (n=100).<br />
We used <strong>an</strong> optimal linear decoder (Bialek 1991) to reconstruct <strong>the</strong> input stimulus spectrogram<br />
<strong>from</strong> <strong>the</strong> population response. We compared <strong>the</strong> reconstructed spectrogram to <strong>the</strong> original to<br />
discover which features were preserved or enh<strong>an</strong>ced <strong>an</strong>d to asses <strong>the</strong> accuracy of <strong>the</strong>ir encoding.<br />
We observed that spectrograms reconstructed <strong>from</strong> <strong>the</strong> neural responses to noisy speech were<br />
closer to <strong>the</strong> original cle<strong>an</strong> spectrograms th<strong>an</strong> to <strong>the</strong> noisy ones. This indicates that sound<br />
representations in A1 serve to enh<strong>an</strong>ce in<strong>for</strong>mation about natural speech signals relative to noise,<br />
thus extracting signal <strong>from</strong> noise. Examining <strong>the</strong> average reconstructed phoneme spectrograms in<br />
cle<strong>an</strong> <strong>an</strong>d noisy speech revealed a remarkable robustness in <strong>the</strong> encoding of features import<strong>an</strong>t<br />
<strong>for</strong> discrimination of different phonemes, such as energy at higher frequencies. In addition, we<br />
found that <strong>the</strong> strict linear spectro-temporal receptive field (STRF) model of A1 neurons is<br />
insufficient to explain <strong>the</strong> noise robustness observed in <strong>the</strong> neural data. However, when we<br />
integrated non-linear synaptic depression into <strong>the</strong> inputs <strong>for</strong> <strong>the</strong> STRF model, we observed a<br />
noise reduction in <strong>the</strong> reconstructed spectrograms similar to what we observed with <strong>the</strong> actual<br />
neural data.<br />
Disclosures: N. Mesgar<strong>an</strong>i, None; S. David, None; J. Fritz, None; S.A. Shamma, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.12/BB20<br />
Topic: D.02.e. Auditory cortex: Perception, cognition, <strong>an</strong>d action<br />
Support: Tesas Adv<strong>an</strong>ced Research Program<br />
Title: Bilateral auditory cortex lesions impair discrimination of brief speech sounds<br />
Authors: *B. A. PORTER, T. R. ROSENTHAL, J. WOLF, A. C. REED, W. A. VRANA, E.<br />
M. RENFROE, S. K. RAM, M. P. KILGARD;<br />
Univ. Texas at Dallas, Richardson, TX<br />
Abstract: Rats tested with a variety of behavioral methods are able to discriminate hum<strong>an</strong><br />
speech sounds (Toro, et al., 2003; Engineer, et al., 2008; Floody, et al., 2007). Bilateral primary<br />
auditory cortex ablations do not result in <strong>the</strong> inability of rats to per<strong>for</strong>m speech discrimination,<br />
categorization tasks, speech discrimination in noise, frequency discrimination, or to learn of a<br />
new discrimination task following <strong>the</strong> lesion (Porter, et al., 2008). Previous papers suggest that
conson<strong>an</strong>t discriminations c<strong>an</strong> be per<strong>for</strong>med with in<strong>for</strong>mation <strong>from</strong> just <strong>the</strong> first 40 ms of a word<br />
(Reed et al., 2003; Blumstein et al., 1979; Jongm<strong>an</strong>, 1989). A study by Engineer, et al. (2008)<br />
demonstrated a signific<strong>an</strong>t correlation between oper<strong>an</strong>t speech discrimination ability <strong>an</strong>d primary<br />
auditory cortical recordings binned every millisecond <strong>from</strong> <strong>the</strong> first <strong>for</strong>ty milliseconds of a word,<br />
suggesting that <strong>the</strong> primary auditory cortex may be import<strong>an</strong>t <strong>for</strong> discrimination of short stimuli.<br />
We hypo<strong>the</strong>sized that <strong>an</strong>imals with bilateral primary auditory cortex lesions would not be able to<br />
discriminate speech stimuli that were restricted to <strong>the</strong> first 40 ms of <strong>the</strong> word. To test this<br />
hypo<strong>the</strong>sis, 16 rats were trained on <strong>an</strong> oper<strong>an</strong>t training task discriminating six shortened speech<br />
sounds <strong>from</strong> a single shortened target speech sound. After training <strong>for</strong> 40 sessions, half of <strong>the</strong><br />
rats received bilateral lesions to <strong>the</strong> primary auditory cortex <strong>an</strong>d <strong>the</strong> remaining half received<br />
sham lesions. Rats <strong>the</strong>n retrained on <strong>the</strong> shortened stimuli <strong>for</strong> 30 sessions. Lesion <strong>an</strong>imals<br />
showed a signific<strong>an</strong>t initial impairment on <strong>the</strong> task that had little improvement following <strong>the</strong><br />
lesions while sham <strong>an</strong>imals showed no impairment. Ongoing testing will evaluate <strong>the</strong> ability of<br />
lesioned rats to discriminate long speech sounds, short tones, <strong>an</strong>d long tones.<br />
Disclosures: B.A. Porter, None; T.R. Rosenthal, None; J. Wolf, None; A.C. Reed,<br />
None; W.A. Vr<strong>an</strong>a, None; E.M. Renfroe, None; S.K. Ram, None; M.P. Kilgard, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.13/BB21<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: GACR 309/07/1336<br />
LC 554<br />
AVOZ50390512<br />
Title: Coding of communication calls in <strong>the</strong> auditory cortex of guinea pig<br />
Authors: *D. SUTA, J. POPELAR, J. BURIANOVA, J. SYKA;<br />
Inst. of Exp Med., Prague, Czech Republic<br />
Abstract: Coding of communication calls as a class of behaviorally relev<strong>an</strong>t complex acoustical<br />
stimuli was studied using typical calls of <strong>the</strong> guinea pig vocalization repertoire. Recently we<br />
described neuronal responses in two subcortical nuclei of <strong>the</strong> auditory pathway - <strong>the</strong> inferior
colliculus (Suta et al., J Neurophysiol, 2003) <strong>an</strong>d <strong>the</strong> medial geniculate body (Suta et al., Exp<br />
Brain Res, 2007). In this study we evaluated responses of neurons in <strong>the</strong> auditory cortex to four<br />
typical vocalizations. Activity of cortical neurons was recorded in ketamine-xylazine<br />
<strong>an</strong>es<strong>the</strong>tized guinea pigs using a 16-ch<strong>an</strong>nel electrode adv<strong>an</strong>ced orthogonally to <strong>the</strong> cortical<br />
surface. Acoustical stimuli were presented in free-field conditions in <strong>an</strong> <strong>an</strong>echoic, sound-proof<br />
chamber. The localization of individual neurons was identified within <strong>the</strong> primary AC (AI), <strong>the</strong><br />
dorso-caudal field (DC) <strong>an</strong>d <strong>the</strong> ventral <strong>an</strong>d dorsal belt areas of <strong>the</strong> AC. Irrespective of <strong>the</strong>ir<br />
localization in individual subdivisions, neurons typically responded to several calls or all calls.<br />
The response patterns were strongly correlated, although not identical, in <strong>the</strong> case of neurons<br />
recorded <strong>from</strong> <strong>the</strong> same sh<strong>an</strong>k of <strong>the</strong> multielectrode, i.e. in neurons localized within <strong>the</strong> same<br />
cortical column. The correlation between response patterns of neurons recorded <strong>from</strong> separate<br />
sh<strong>an</strong>ks was weaker <strong>an</strong>d decreased with increased dist<strong>an</strong>ce of electrodes. Whistle (a long-lasting<br />
call of a broad frequency r<strong>an</strong>ge) evoked different types of response patterns in individual<br />
neurons, because <strong>the</strong>ir response pattern reflected energy of <strong>the</strong> whistle which corresponded with<br />
<strong>the</strong> characteristic frequency of <strong>the</strong> neuron. Population response of AC neurons to whistle was<br />
less homogenous th<strong>an</strong> in <strong>the</strong> subcortical nuclei. Responses of individual neurons to purr <strong>an</strong>d<br />
chutter (calls with a rhythmic repetition of similar components) demonstrated rhythmic firing<br />
with a various level of a decline of <strong>the</strong> firing during <strong>the</strong> rhythmic repetition of call phrases.<br />
Several neurons displayed very different strengths in <strong>the</strong>ir responses to purr <strong>an</strong>d chutter, despite<br />
<strong>the</strong> similar spectral features of <strong>the</strong>se two calls. Neurons with higher firing rates <strong>an</strong>d higher<br />
correlation between <strong>the</strong> stimulus <strong>an</strong>d <strong>the</strong> response pattern were found in <strong>the</strong> ventral region of <strong>the</strong><br />
AI <strong>an</strong>d DC. In combination with <strong>the</strong> data <strong>from</strong> <strong>the</strong> inferior colliculus <strong>an</strong>d medial geniculate body,<br />
cortical data demonstrate gradual processing of spectral <strong>an</strong>d temporal features of complex<br />
acoustical stimuli <strong>an</strong>d modification of <strong>the</strong> response patterns in individual stages of <strong>the</strong> auditory<br />
pathway.<br />
Disclosures: D. Suta, None; J. Popelar, None; J. Buri<strong>an</strong>ova, None; J. Syka, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.14/BB22<br />
Topic: D.02.d. Auditory cortex: Binaural interaction<br />
Support: University of Connecticut Research Foundation<br />
Title: Representation of sound level cues in two ventral belt auditory cortices of <strong>the</strong> rat
Authors: *N. HIGGINS 1 , D. A. STORACE 2 , M. A. ESCABI 3 , H. L. READ 4 ;<br />
1 3 4<br />
Univ. of Connecticut, storrs, CT; Electrical <strong>an</strong>d Biomed. Engin., Psychology <strong>an</strong>d Biomed.<br />
Engin., 2 Univ. of Connecticut, Storrs, CT<br />
Abstract: In <strong>the</strong> rat, reflection of sound in <strong>the</strong> ‘mid-frequency’ r<strong>an</strong>ge of 8 to 32 kHz creates <strong>an</strong><br />
interaural sound level difference (ILD) cue that ch<strong>an</strong>ges systematically with sound position in<br />
<strong>the</strong> azimuth dimension of acoustic space (Koka et al., 2008). Preliminary studies suggest that<br />
Ventral (VAF) <strong>an</strong>d suprarhinal (SRAF) auditory fields respond more to mid-frequency sounds<br />
whereas primary auditory cortex (A1) responds relatively more to low frequency sound (
Abstract: Primary auditory cortex (A1) <strong>an</strong>d ventral auditory field (VAF) have been considered a<br />
single cortical field due to <strong>the</strong> fact that <strong>the</strong>y share a continuous cochleotopic org<strong>an</strong>ization in rat<br />
temporal cortex. However, high-resolution imaging <strong>an</strong>d multi-unit mapping studies find that A1<br />
has: 1) broader tone response b<strong>an</strong>dwidths, 2) coarser cochleotopy <strong>an</strong>d 3) more monotonic <strong>an</strong>d<br />
higher sound level intensity dependence th<strong>an</strong> VAF (Kalatsky et al., 2005; Polley et al., 2007).<br />
These physiologic distinctions could be inherited, as A1 <strong>an</strong>d VAF receive projections <strong>from</strong><br />
rostral <strong>an</strong>d caudal ends of <strong>the</strong> medial geniculate body (MGB), respectively.<br />
The present study examines whe<strong>the</strong>r <strong>the</strong>se receptive field properties have additional <strong>an</strong>atomical<br />
correlates. Specifically, whe<strong>the</strong>r thalamocortical neurons projecting to A1 <strong>an</strong>d VAF have distinct<br />
frequency org<strong>an</strong>ization. Cochleotopic org<strong>an</strong>ization was mapped in four to five cortical regions<br />
with high resolution Fourier intrinsic optical imaging during continuous presentation of tone<br />
staircase sequences (Kalatsky et al., 2005). Retrograde tracer choleratoxin beta subunit (CTβ)<br />
<strong>an</strong>d its gold conjugated <strong>for</strong>m, CTβG were injected into neighboring iso-frequency contours<br />
separated by 2, 2.5 <strong>an</strong>d 3 octaves (i.e., 4-16kHz, 4-24kHz <strong>an</strong>d 4-32kHz) in both A1 <strong>an</strong>d VAF.<br />
Brains were sliced in <strong>the</strong> coronal pl<strong>an</strong>e <strong>an</strong>d <strong>the</strong> positions of retrograde labeled neurons were<br />
plotted in Stereo Investigator. Cell density was computed per 100x100µm bin of <strong>the</strong> MGB.<br />
In a previous study, we found that <strong>the</strong> highest densities of neurons projecting to <strong>the</strong> 8 kHz isofrequency<br />
contour in A1 <strong>an</strong>d VAF were located in rostral <strong>an</strong>d caudal halves of <strong>the</strong> ventral<br />
division of MGB (MGBv), respectively. Thus, we examined MGBv label only in <strong>the</strong> present<br />
study. A dorsal-to-ventral low-to-high ‘frequency’ gradient of thalamocortical projection neurons<br />
was observed in MGBv. Neurons projecting to low frequency (4kHz) sites in A1 <strong>an</strong>d VAF were<br />
located dorsally near <strong>the</strong> border of MGBv <strong>an</strong>d dorsal division of MGB. Neurons projecting to<br />
high frequency VAF <strong>an</strong>d A1 were always located more ventrally. Frequency separation was<br />
estimated by measuring <strong>the</strong> dist<strong>an</strong>ce between peak densities <strong>for</strong> low versus high frequency<br />
projection neurons <strong>for</strong> each section in a caudal-rostral series. Magnification factor (MF,<br />
mm/octave) of best frequency was signific<strong>an</strong>tly higher in caudal MGB (0.14 ± 0.01) versus<br />
rostral MGB (0.11 ± 0.02) (t(8) = 2.55, p < .05) suggesting a caudal-to-rostral gradient <strong>for</strong><br />
cochleotopic resolution. Thus, <strong>the</strong> cochleotopic resolution or MF differences <strong>for</strong> VAF versus A1<br />
may stem <strong>from</strong> <strong>the</strong>se distinct thalamocortical projections.<br />
Disclosures: D.A. Storace, None; N.C. Higgins, None; H.L. Read, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.16/BB24<br />
Topic: D.02.c. Auditory cortex: Neural processing
Title: Local field potentials <strong>an</strong>d single unit spiking activity: Comparison of spectro-temporal<br />
tuning properties in A1<br />
Authors: *S. ATIANI 1 , S. V. DAVID 2 , N. MALAVAL 3 , S. SHAMMA 2 ;<br />
1 Neurosci. <strong>an</strong>d Cognitive Sci., Univ. of Maryl<strong>an</strong>d, College Park, MD; 2 Institue <strong>for</strong> Systems Res.,<br />
Univ. Of Maryl<strong>an</strong>d, College Park, MD; 3 Natl. Inst. <strong>for</strong> Applied Sci., Lyon, Fr<strong>an</strong>ce<br />
Abstract: Most studies of auditory cortex measure <strong>the</strong> tuning <strong>an</strong>d o<strong>the</strong>r functional properties of<br />
one or multiple single neurons. Local field potentials (LFPs) are <strong>the</strong> integrated activity of m<strong>an</strong>y<br />
hundreds or thous<strong>an</strong>ds of neurons in <strong>the</strong> vicinity of <strong>the</strong> recording site. The LFP has been used to<br />
measure large-scale oscillations <strong>an</strong>d coherences between brain areas, very few studies have<br />
compared <strong>the</strong> tuning of LFP <strong>an</strong>d single neurons directly. Underst<strong>an</strong>ding <strong>the</strong> functional<br />
relationship between LFPs <strong>an</strong>d single spiking activity c<strong>an</strong> provide insight into large-scale<br />
representations of auditory stimuli <strong>an</strong>d behaviorally driven ch<strong>an</strong>ges in <strong>the</strong>se representations.<br />
To address this issue we measured <strong>the</strong> tuning responses of single neurons <strong>an</strong>d different frequency<br />
b<strong>an</strong>ds of LFPs recorded at <strong>the</strong> same location in primary auditroy cortex of awake, passively<br />
listening ferrets. To account <strong>for</strong> <strong>the</strong> possibility that nearby spiking events dominate <strong>the</strong> LFP<br />
signal, we also compared <strong>the</strong>se measurements to a corrected LFP in which events that<br />
correspond to nearby spiking events were removed. We use broadb<strong>an</strong>d, b<strong>an</strong>d pass <strong>an</strong>d narrow<br />
b<strong>an</strong>d stimuli to measure <strong>the</strong> tuning properties (best frequency, spectral <strong>an</strong>d temporal b<strong>an</strong>dwidth)<br />
<strong>an</strong>d compare <strong>the</strong>m between <strong>the</strong> different neural signals. We find that <strong>the</strong> best frequency of single<br />
unit <strong>an</strong>d LFP generally correspond. However on average we find LFPs to be tuned to a lower<br />
spectral modulation frequency th<strong>an</strong> spiking events, when <strong>the</strong> correlated spiking events are<br />
removed this difference becomes larger. This difference between <strong>the</strong> signals indicates that LFP<br />
signals provide in<strong>for</strong>mation about acoustic representations complementary to more commonly<br />
measured single unit activity.<br />
Disclosures: S. Ati<strong>an</strong>i, None; S.V. David, None; N. Malaval, None; S. Shamma, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.17/BB25<br />
Topic: D.02.e. Auditory cortex: Perception, cognition, <strong>an</strong>d action<br />
Support: NIDCD Gr<strong>an</strong>t DCO2514<br />
J. S. McDonnell Foundation
Title: Detection of amplitude modulated noise as <strong>an</strong> elevation cue by a rhesus macaque<br />
Authors: *K. N. O'CONNOR 1,2 , J. S. JOHNSON 1 , E. A. MARSHALL 1 , M. L. SUTTER 1,2 ;<br />
1 Ctr. <strong>for</strong> Neurosci., 2 Neurobiology, Physiol. <strong>an</strong>d Behavior, UC Davis, Davis, CA<br />
Abstract: We trained a rhesus macaque to indicate <strong>the</strong> source of <strong>an</strong> amplitude-modulated (AM)<br />
noise burst (target) in <strong>the</strong> presence of <strong>an</strong> unmodulated noise burst (masker), when <strong>the</strong> target <strong>an</strong>d<br />
masker were simult<strong>an</strong>eously played <strong>from</strong> two different speakers separated by 60 deg in elevation<br />
along <strong>the</strong> frontal midline. The subject was initially trained to horizontally deflect a verticallymounted<br />
joystick to present a target stimulus (100% broadb<strong>an</strong>d AM noise; 5 Hz - 2 kHz AM<br />
frequency; 400 ms duration) <strong>from</strong> a speaker 30 deg above or below ear position, <strong>the</strong>n move <strong>the</strong><br />
joystick up or down in <strong>the</strong> direction of <strong>the</strong> target source. We gradually introduced a simult<strong>an</strong>eous<br />
unmodulated noise masker on <strong>the</strong> opposite speaker. Masker intensity was slowly incremented<br />
over trials until <strong>the</strong> subject was able to reliably report <strong>the</strong> position of <strong>the</strong> 100% modulated target<br />
at better th<strong>an</strong> 90% correct when <strong>the</strong> target <strong>an</strong>d masker were matched in intensity. We <strong>the</strong>n added<br />
targets at six additional modulation levels (6 - 80%) <strong>an</strong>d determined thresholds (d’ = 1) <strong>for</strong><br />
modulation source detection. The relationship between AM frequency <strong>an</strong>d threshold exhibited<br />
<strong>the</strong> typical U-shaped <strong>for</strong>m, with a minimum threshold of ~30% modulation at ~30-120 Hz, <strong>an</strong>d<br />
maximum of ~70% modulation at 2 kHz. Comparison of <strong>the</strong>se results to macaque AM thresholds<br />
previously determined using a ‘go/no-go’ oddball task (O’Connor et. al., J Comp Physiol. A,<br />
186, 903-912, 2000) revealed <strong>the</strong> AM source-location thresholds to be higher by a factor of ~2 at<br />
low to mid-r<strong>an</strong>ge frequencies (< 250 Hz), while thresholds at <strong>the</strong> highest frequencies (> 500 Hz)<br />
were elevated only by a factor of ~0.2. This suggests that <strong>the</strong> effects of a vertically-displaced<br />
masking sound on detection of <strong>an</strong> AM modulated source depend on modulation frequency. Our<br />
results are encouraging <strong>for</strong> <strong>the</strong> use of this choice method <strong>for</strong> <strong>the</strong> behavioral <strong>an</strong>d physiological<br />
investigation of complex auditory scene <strong>an</strong>alysis in rhesus macaques.<br />
Disclosures: K.N. O'Connor, None; J.S. Johnson, None; E.A. Marshall, None; M.L. Sutter,<br />
None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.18/BB26<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIMH DIRP
Title: Neuronal response latency <strong>an</strong>d selectivity in rostral auditory cortex of awake macaques<br />
Authors: *B. H. SCOTT 1 , H. VINAL 1 , P. YIN 2 , M. MISHKIN 1 ;<br />
1 2<br />
Lab. Neuropsychol, NIMH, Be<strong>the</strong>sda, MD; Inst. <strong>for</strong> Systems Res., Univ. of Maryl<strong>an</strong>d, College<br />
Park, MD<br />
Abstract: Regions of <strong>the</strong> rostral superior temporal cortex in primates, downstream <strong>from</strong> <strong>the</strong><br />
primary auditory area, are hypo<strong>the</strong>sized to continue <strong>the</strong> cortical processing of sound quality<br />
begun in area A1 (Kikuchi et al, Soc. Neurosci. Abstr. 2007). Extrapolating <strong>from</strong> <strong>the</strong> difference<br />
in latency between <strong>the</strong> primary <strong>an</strong>d rostral fields of <strong>the</strong> core auditory cortex, regions far<strong>the</strong>r<br />
rostral on <strong>the</strong> supratemporal pl<strong>an</strong>e (STP) may be expected to show longer response latencies th<strong>an</strong><br />
those observed in caudal auditory cortex. Concomit<strong>an</strong>t with a longer time scale of temporal<br />
processing, one may also predict <strong>an</strong> enh<strong>an</strong>ced selectivity in <strong>the</strong>se neurons <strong>for</strong> <strong>the</strong> spectral <strong>an</strong>d<br />
temporal properties of <strong>the</strong> acoustic stimulus. We recorded single-unit activity across <strong>the</strong> rostral<br />
STP, including rostral parts of <strong>the</strong> auditory core, in awake rhesus macaques. The stimulus set<br />
consisted of 21 sounds, each ~300 ms in length, including syn<strong>the</strong>sized sounds, <strong>an</strong>imal<br />
vocalizations (rhesus <strong>an</strong>d o<strong>the</strong>r species), <strong>an</strong>d environmental sounds. The set was presented in <strong>the</strong><br />
context of a working memory task, <strong>an</strong>d response latency <strong>an</strong>d selectivity were measured during<br />
<strong>the</strong> “sample” presentation that beg<strong>an</strong> each trial. (This stimulus set, as well as pure tones <strong>an</strong>d<br />
b<strong>an</strong>d-passed noise, were also presented as <strong>the</strong> <strong>an</strong>imal sat passively). At more rostral locations,<br />
latencies were longer overall, <strong>an</strong>d more variable between neurons. Response selectivity was<br />
assessed by several metrics, including sparseness (Rolls <strong>an</strong>d Tovee 1995) <strong>an</strong>d kurtosis of <strong>the</strong><br />
firing rate distribution. All such metrics were highly variable along <strong>the</strong> rostral-caudal axis, <strong>an</strong>d<br />
no clear trend toward greater selectivity among rostral neurons was evident, though this may be<br />
due to <strong>the</strong> limited number of sounds that were presented. An increase in latency is consistent<br />
with models of serial processing <strong>from</strong> core regions to <strong>the</strong> rostral STP, but whe<strong>the</strong>r neurons in<br />
rostral fields exhibit enh<strong>an</strong>ced selectivity awaits fur<strong>the</strong>r study.<br />
Disclosures: B.H. Scott, None; H. Vinal, None; P. Yin, None; M. Mishkin, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.19/BB27<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIH NRSA 1F32DC009165
NOHR Research Award<br />
Title: Development of auditory cortical responses underlying backward masking in awake<br />
gerbils<br />
Authors: *M. J. ROSEN 1 , D. H. SANES 2 ;<br />
2 Ctr. Neural Sci., 1 New York Univ., New York, NY<br />
Abstract: The emergence of mature temporal processing is essential <strong>for</strong> a broad r<strong>an</strong>ge of<br />
auditory tasks, <strong>from</strong> sound localization to speech recognition. A simple test of temporal<br />
processing is backward masking, in which detection of a brief tone c<strong>an</strong> be masked by <strong>the</strong><br />
subsequent presentation of noise. In children up to ten years old, backward masking thresholds<br />
are higher th<strong>an</strong> in adults. The neural substrate of backward masking has never been examined in<br />
awake <strong>an</strong>imals, <strong>an</strong>d <strong>an</strong>y developmental ch<strong>an</strong>ges that might underlie this slow maturation of<br />
thresholds have not been explored. We have addressed this issue by recording <strong>from</strong> single<br />
neurons in <strong>the</strong> auditory cortex (ACx) of awake restrained gerbils prior to sexual maturation<br />
(postnatal days 30-38) <strong>an</strong>d in adults (>P60). A brief (10 ms) tone at each neuron’s best frequency<br />
(BF) was followed immediately by a const<strong>an</strong>t-amplitude narrowb<strong>an</strong>d noise masker (500 ms)<br />
centered at BF. Tones were presented at increasing amplitudes to allow <strong>for</strong> threshold<br />
measurements. Neural correlates of backward masking were assessed by measuring several<br />
aspects of ACx neuron responses. First, firing rate was measured in <strong>the</strong> time window where <strong>the</strong><br />
tone alone elicited <strong>the</strong> maximal response <strong>for</strong> each cell (usually 20msec width). A decrease in this<br />
response was observed in <strong>the</strong> presence of <strong>the</strong> noise masker despite greater total signal energy,<br />
indicating a potential correlate of backward masking. Second, to assess each cell’s tone-evoked<br />
threshold with <strong>an</strong>d without noise, firing rate was obtained over <strong>the</strong> maximum stimulus period<br />
(500msec). The threshold of tones with noise were referenced to noise-alone trials, while <strong>the</strong><br />
thresholds of tones presented alone were referenced to spont<strong>an</strong>eous activity. On average,<br />
backward masking produced higher thresholds. Preliminary data indicate a trend towards a<br />
greater masking effect in juvenile <strong>an</strong>imals compared with adults, <strong>an</strong>d this was associated with<br />
larger noise-evoked firing rates in juveniles. This larger noise-evoked response may generate a<br />
ceiling effect <strong>an</strong>d thus prevent a tone-evoked response <strong>from</strong> emerging in juvenile <strong>an</strong>imals.<br />
Finally, to determine whe<strong>the</strong>r temporal pattern c<strong>an</strong> reliably distinguish between tone+noise<br />
versus noise-alone responses, we will present a spike-dist<strong>an</strong>ce <strong>an</strong>alysis over a r<strong>an</strong>ge of time<br />
scales. Our data indicate that backward masking is visible in ACx at <strong>the</strong> level of individual<br />
neurons, <strong>an</strong>d suggest a trend towards a larger masking effect in juveniles, consistent with <strong>the</strong><br />
hum<strong>an</strong> literature.<br />
Disclosures: M.J. Rosen, None; D.H. S<strong>an</strong>es, None.<br />
Poster<br />
556. Auditory Cortex: Neural Coding <strong>an</strong>d Perception<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 556.20/BB28<br />
Topic: D.02.c. Auditory cortex: Neural processing<br />
Support: NIDCD R21 DC009454-01 to POK<br />
NIH R01 DC009607 to POK<br />
ISR Seed gr<strong>an</strong>t to SAS <strong>an</strong>d POK<br />
Title: Local <strong>an</strong>d large-scale org<strong>an</strong>ization of auditory cortex probed with in vivo Ca imaging<br />
Authors: *S. BANDYOPADHYAY, S. A. SHAMMA, P. O. KANOLD;<br />
Univ. Maryl<strong>an</strong>d, College Park, MD<br />
Abstract: Encoding of stimuli in <strong>the</strong> primary auditory cortex (A1) is thought to be sparse, with a<br />
multitude of complex overlaid maps of stimulus features. The domin<strong>an</strong>t feature is a tonotopic<br />
map, which is best seen at near-threshold sound levels. One limiting factor in determining<br />
cortical maps is <strong>the</strong> challenge to record <strong>from</strong> large numbers of neurons simult<strong>an</strong>eously. In vivo<br />
2-photon Ca2+ imaging allows <strong>for</strong> <strong>the</strong> functional probing of large numbers of neurons with<br />
single cell resolution.<br />
Thus, to probe <strong>for</strong> <strong>the</strong> existence of multiple maps in mouse A1, we bulk loaded A1 neurons with<br />
<strong>the</strong> Ca2+ indicators <strong>an</strong>d <strong>the</strong>n imaged auditory evoked Ca2+ responses with in vivo 2-photon<br />
Ca2+ imaging. We find that in vivo 2-photon Ca2+ imaging reliably detects auditory responses.<br />
As expected, A1 neurons were found to be frequency selective. However, during repeated<br />
presentations of <strong>the</strong> same stimulus we find that responses were unreliable <strong>from</strong> trial to trial. As<br />
expected, A1 neurons were found to be frequency selective. We were able to identify different<br />
fields in <strong>the</strong> mouse ACX <strong>an</strong>d detect <strong>the</strong> presence of gross tonotopic org<strong>an</strong>ization. However,<br />
locally neurons were found to be heterogeneously org<strong>an</strong>ized <strong>an</strong>d tonotopy on a local scale was<br />
more inhomogeneous th<strong>an</strong> previously seen with extra cellular recordings. Hence columnar<br />
org<strong>an</strong>ization of neurons based on frequency selectivity in <strong>the</strong> mouse ACX is doubtful.<br />
Disclosures: S. B<strong>an</strong>dyopadhyay, None; S.A. Shamma, None; P.O. K<strong>an</strong>old, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 557.1/BB29<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: CIHR<br />
NSERC<br />
GRUM-FRSQ<br />
CIHR-Rx&D Scholar award<br />
Harl<strong>an</strong>d S<strong>an</strong>ders Chair <strong>for</strong> Visual Science<br />
Title: Localization of <strong>the</strong> endoc<strong>an</strong>nabinoid system in <strong>the</strong> retina of <strong>the</strong> St. Kitts green monkey<br />
(Chlorocebus sabaeus): Peripheral vs. central vision<br />
Authors: *J. M. BOUSKILA 1 , M. W. BURKE 2 , S. ZANGENEHPOUR 2 , J.-F. BOUCHARD 2 ,<br />
M. PTITO 2 ;<br />
1 Montreal, QC, C<strong>an</strong>ada; 2 Sch. of Optometry, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: The role of <strong>the</strong> c<strong>an</strong>nabinoid signalling system in <strong>the</strong> normal <strong>an</strong>d pathological<br />
functioning of <strong>the</strong> brain has been <strong>the</strong> subject of intense investigation. C<strong>an</strong>nabinoid receptor type<br />
1 (CB1R) is widely expressed in <strong>the</strong> brain <strong>an</strong>d mediates <strong>the</strong> vast majority of <strong>the</strong> effects produced<br />
by <strong>the</strong> endoc<strong>an</strong>nabinoids (eCBs). Recent work on <strong>the</strong> expression pattern of CB1R has provided<br />
evidence <strong>for</strong> its presence in <strong>the</strong> retina. However, <strong>the</strong> details of CB1R expression pattern across<br />
<strong>the</strong> retina, particularly with regards to <strong>the</strong> center vs. periphery, remain unknown. In <strong>the</strong> current<br />
study, we used immunohistochemical methods to locate CB1R, c<strong>an</strong>nabinoid receptor type 2<br />
(CB2R), fatty acid amide hydrolase (FAAH; a catabolic enzyme <strong>for</strong> eCBs), <strong>an</strong>d N-acyl<br />
phosphatidyleth<strong>an</strong>olamine phospholipase D (NAPE-PLD; <strong>the</strong> enzyme implicated in <strong>the</strong> syn<strong>the</strong>sis<br />
of <strong>an</strong><strong>an</strong>damide, a CB1R lig<strong>an</strong>d), throughout <strong>the</strong> primate retina. Our results show that CB1R,<br />
CB2R <strong>an</strong>d NAPE-PLD have low expression in <strong>the</strong> photoreceptor (PR) <strong>an</strong>d outer nuclear layers<br />
(ONL) with moderate to high expression levels in <strong>the</strong> outer plexi<strong>for</strong>m, inner nuclear, inner<br />
plexi<strong>for</strong>m <strong>an</strong>d retinal g<strong>an</strong>glion cell layers (OPL, INL, IPL, <strong>an</strong>d RGCL, respectively), in both <strong>the</strong><br />
peripheral <strong>an</strong>d central retina. FAAH has moderate to high expression levels in <strong>the</strong> ONL <strong>an</strong>d<br />
RGCL <strong>an</strong>d shows low to no expression in o<strong>the</strong>r layers. However, in <strong>the</strong> central retina, FAAH has<br />
high to moderate expression levels in all layers except in <strong>the</strong> INL <strong>an</strong>d PR layer, where staining is<br />
low to nil. The Henle fiber layer does not stain <strong>for</strong> <strong>an</strong>y of <strong>the</strong> mediators of <strong>the</strong> eCB system. All<br />
eCB mediators were found in <strong>the</strong> RGCL with higher intensity th<strong>an</strong> o<strong>the</strong>r layers. These data<br />
suggest that <strong>the</strong> eCB system is present in <strong>the</strong> central <strong>an</strong>d peripheral retina <strong>an</strong>d may play a role in<br />
<strong>the</strong> mediation of some of <strong>the</strong> known effects of pharmacological administration of c<strong>an</strong>nabinoids,<br />
such as <strong>the</strong> reported delay in glare recovery, reduction in Vernier <strong>an</strong>d Snellen acuity, increase<br />
photosensitivity <strong>an</strong>d alterations in color discrimination related to c<strong>an</strong>nabis consumption.<br />
Sponsored by: CIHR; NSERC. J.M. B. is supported by a GRUM-FRSQ studentship, J.-F. B. by a<br />
CIHR-Rx&D Scholar award <strong>an</strong>d M.P. holds <strong>the</strong> Harl<strong>an</strong>d S<strong>an</strong>ders Chair <strong>for</strong> Visual Science.
Disclosures: J.M. Bouskila, None; M.W. Burke, None; S. Z<strong>an</strong>genehpour, None; J.<br />
Bouchard, None; M. Ptito, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.2/BB30<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Tul<strong>an</strong>e Research Enh<strong>an</strong>cement Fund<br />
Title: Nitric oxide synthase inhibitor, L-NAME reduced <strong>the</strong> light sensitivity of retinal g<strong>an</strong>glion<br />
cells by inhibiting cone glutamate release<br />
Authors: *J. P. NEMARGUT, III, W. HUANG, G.-Y. WANG;<br />
Structural & Cell Biol, Tul<strong>an</strong>e Univ., New Orle<strong>an</strong>s, LA<br />
Abstract: We have recently demonstrated that eliminating <strong>the</strong> neuronal nitric oxide synthase<br />
(nNOS) gene or blocking NOS reduces <strong>the</strong> sensitivity of retinal g<strong>an</strong>glion cells (RGCs) to<br />
scotopic stimuli in <strong>the</strong> dark-adapted mouse retina (W<strong>an</strong>g et al., 2007). In <strong>the</strong> current study, we<br />
focused on how inhibiting NOS affects <strong>the</strong> visual responses of mouse RGCs under light<br />
adaptation. Our results with current- <strong>an</strong>d voltage-clamp recordings demonstrated that NOS<br />
inhibitor, L-NAME reduced <strong>the</strong> sensitivity of light-adapted RGCs to light. These observations<br />
were seen in On, Off, <strong>an</strong>d On-Off RGCs, as identified physiologically <strong>an</strong>d morphologically with<br />
intracellular Lucifer Yellow labeling. The findings in <strong>the</strong> current study are in line with those of<br />
our previous study (W<strong>an</strong>g et al., 2007). We recorded <strong>the</strong> inhibitory postsynaptic currents (IPSCs)<br />
<strong>an</strong>d excitatory postsynaptic currents (EPSCs) <strong>from</strong> RGCs <strong>an</strong>d pharmacologically ‘dissected’ <strong>the</strong><br />
visual pathways to <strong>the</strong> RGC to locate <strong>the</strong> sites causing <strong>the</strong> reduction of RGC sensitivity to light.<br />
The results showed that L-NAME did not increase <strong>the</strong> amplitude or frequency of IPSCs of<br />
RGCs, indicating that L-NAME is not reducing <strong>the</strong> sensitivity via increasing <strong>the</strong> inhibitory<br />
activity in <strong>the</strong> retina. Interestingly, after eliminating inhibitory neurotr<strong>an</strong>smission to <strong>the</strong> RGC, L-<br />
NAME reduced <strong>the</strong> amplitude <strong>an</strong>d frequency of spont<strong>an</strong>eous EPSCs (sEPSCs), clearly<br />
demonstrating that L-NAME primarily inhibits <strong>the</strong> glutamate release in <strong>the</strong> retina to reduce <strong>the</strong><br />
sensitivity of RGCs to light. To determine if <strong>the</strong> g<strong>an</strong>glion cell itself was <strong>the</strong> site of <strong>the</strong> reduced<br />
sensitivity, DL-2-amino-phosphonobutyric acid (APB) was used to hyperpolarize <strong>the</strong> On cone<br />
bipolar cell. APB dramatically reduced <strong>the</strong> frequency of sEPSCs of RGCs, but no fur<strong>the</strong>r<br />
ch<strong>an</strong>ges were seen in <strong>the</strong> frequency or amplitude of sEPSCs following L-NAME treatment,<br />
indicating L-NAME is not primarily acting on <strong>the</strong> g<strong>an</strong>glion cell excitability. Hydroxylamine
(HA) was used to eliminate <strong>the</strong> glutamate tr<strong>an</strong>smission <strong>from</strong> cones to cone bipolar cells. HA<br />
alone dramatically reduced <strong>the</strong> amplitude <strong>an</strong>d frequency of sEPSCs of RGCs, but <strong>the</strong>re were no<br />
fur<strong>the</strong>r ch<strong>an</strong>ges after L-NAME, showing L-NAME is not acting on <strong>the</strong> glutamate release <strong>from</strong><br />
cone bipolar cells. Thus, cones are <strong>the</strong> only sites where L-NAME inhibits glutamate release in<br />
<strong>the</strong> retina. Collectively, <strong>the</strong>se results suggest that L-NAME primarily inhibits glutamate release<br />
<strong>from</strong> cones to reduce <strong>the</strong> sensitivity of RGCs to light.<br />
Disclosures: J.P. Nemargut, None; W. Hu<strong>an</strong>g, None; G. W<strong>an</strong>g, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.3/BB31<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Tul<strong>an</strong>e Research Enh<strong>an</strong>cement Fund<br />
Title: C<strong>an</strong>nabinoid <strong>an</strong>tagonist differentially modulated light-evoked responses of mouse retinal<br />
g<strong>an</strong>glion cells under light adaptation<br />
Authors: *J. N. PAHNG, J. YANG, G.-Y. WANG;<br />
Structural <strong>an</strong>d Cell. Biol., Tul<strong>an</strong>e Univ. Sch. of Med., New Orle<strong>an</strong>s, LA<br />
Abstract: Endoc<strong>an</strong>nabinoids (eCBs) in <strong>the</strong> brain act as retrograde messengers that bind to<br />
presynaptic c<strong>an</strong>nabinoid receptors causing inhibition of neurotr<strong>an</strong>smitter release <strong>from</strong> <strong>the</strong><br />
presynaptic terminal. In <strong>the</strong> mouse <strong>an</strong>d hum<strong>an</strong> retina, c<strong>an</strong>nabinoid receptors have been found in<br />
<strong>the</strong> synaptic terminals of <strong>the</strong> photoreceptors, <strong>the</strong> inner plexi<strong>for</strong>m layer, <strong>an</strong>d retinal g<strong>an</strong>glion cells.<br />
The functional role of eCBs <strong>an</strong>d <strong>the</strong>ir receptors in modulating light-evoked responses of retinal<br />
g<strong>an</strong>glion cells are unknown, however. It was hypo<strong>the</strong>sized that eCBs in <strong>the</strong> retina act like <strong>the</strong>y do<br />
in <strong>the</strong> brain as retrograde messengers, by inhibiting <strong>the</strong> release of neurotr<strong>an</strong>smitter <strong>from</strong> <strong>the</strong><br />
photoreceptors to <strong>the</strong> bipolar cells (Reviewed by Yazulla, 2008). Thus, blocking <strong>the</strong> effects of<br />
eCBs with a c<strong>an</strong>nabinoid receptor 1 (CB1) <strong>an</strong>tagonist was hypo<strong>the</strong>sized to increase <strong>the</strong><br />
amplitude of light responses. As <strong>the</strong> initial study toward underst<strong>an</strong>ding how eCBs modulate<br />
visual responses under light adaptation, we per<strong>for</strong>med whole-cell voltage clamp recordings in<br />
mouse retinal g<strong>an</strong>glion cells in a bath perfused with <strong>the</strong> CB1R <strong>an</strong>tagonist AM251. The recorded<br />
cells were labeled with Lucifer Yellow <strong>from</strong> <strong>the</strong> recording electrodes. Our preliminary results<br />
showed that bath application of AM251 increased <strong>the</strong> amplitude of light-evoked excitatory<br />
postsynaptic currents (EPSCs) in some g<strong>an</strong>glion cells under light adaptation. This finding
favored <strong>the</strong> hypo<strong>the</strong>sis that eCBs function as retrograde messengers by inhibiting presynaptic<br />
glutamate release in <strong>the</strong> retina. However, our results also showed that AM251 decreased <strong>the</strong><br />
amplitude of light-evoked EPSCs in o<strong>the</strong>r retinal g<strong>an</strong>glion cells. Moreover, <strong>the</strong> modulation in<br />
amplitude of light-evoked EPSCs was found over a wide r<strong>an</strong>ge of light intensities. Based on our<br />
limited number of samples, a correlation between <strong>the</strong> differential effects of AM251 <strong>an</strong>d<br />
morphological cell types c<strong>an</strong>not be determined. More samples are needed to draw solid<br />
conclusions. Taken toge<strong>the</strong>r <strong>the</strong>se data suggest that eCBs in <strong>the</strong> retina act not only as retrograde<br />
messengers inhibiting <strong>the</strong> light-evoked release of glutamate in some cells, but also facilitate <strong>the</strong><br />
light-evoked release of glutamate in o<strong>the</strong>r cells. Fur<strong>the</strong>r experiments are needed to elucidate <strong>the</strong><br />
mech<strong>an</strong>isms by which eCBs differentially modulate light responses in <strong>the</strong> retina in light<br />
adaptation.<br />
Disclosures: J.N. Pahng, None; J. Y<strong>an</strong>g, None; G. W<strong>an</strong>g, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.4/BB32<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH/NEI #EY- 14050<br />
Title: ACh release in <strong>an</strong> in vivo model of glaucoma<br />
Authors: *K. IWAMOTO 1 , P. J. BIRKHOLZ 1 , D. M. LINN 3 , C. L. LINN 2 ;<br />
1 2 3<br />
Dept. of Biol. Sci., Western Michig<strong>an</strong> Univ., Kalamazoo, MI; Dept. of Biomed. Sci., Gr<strong>an</strong>d<br />
Valley State Univ., Allendale, MI<br />
Abstract: Neuroprotection is a process where neurons are protected <strong>from</strong> injury or degeneration.<br />
In retinal g<strong>an</strong>glion cells (RGC), <strong>an</strong> excess of <strong>the</strong> neurotr<strong>an</strong>smitter glutamate c<strong>an</strong> lead to<br />
apoptosis. This excitotoxic effect of glutamate may be involved in glaucoma, a progressive<br />
retinal disease leading to a loss of RGCs. In previous in vitro studies using isolated cultured<br />
RGCs, ACh was shown to have neuroprotective properties against glutamate-induced<br />
excitotoxicity through Bgt-sensitive nicotinic ACh receptors, but <strong>an</strong>y physiological<br />
neuroprotective effect of ACh in <strong>the</strong> in vivo retina is unclear. In this study, we use <strong>an</strong> in vivo rat<br />
model of glaucoma to determine if ACh release <strong>from</strong> <strong>the</strong> retina ch<strong>an</strong>ges under glaucoma-like<br />
conditions. To induce glaucoma in adult Long Ev<strong>an</strong>s rats, 0.5 ml of 2M hypertonic saline was<br />
injected into <strong>the</strong> episcleral vein of each <strong>an</strong>imal’s right eye. Hypertonic injections cause scar
tissue that reduces aqueous humor outflow, thus causing <strong>an</strong> increase in intraocular pressure<br />
leading to a loss of RGCs. Loss of RGCs was signific<strong>an</strong>t one month after injection. To measure<br />
release of ACh under glaucoma-like conditions, eyecups were removed <strong>from</strong> each rat <strong>an</strong>d retinal<br />
cells were incubated with 20 micro-Ci of 3H-choline <strong>for</strong> 30 minutes. During <strong>the</strong> incubation<br />
period, <strong>the</strong> retina was stimulated with 3-Hz flashing light to maximize choline uptake. The<br />
eyecup was <strong>the</strong>n perfused with Ames solution at 1.5 ml/min <strong>for</strong> 8 minutes to reduce <strong>the</strong><br />
background radioactivity to a steady level. One minute fractions were collected directly into vials<br />
<strong>an</strong>d prepared <strong>for</strong> liquid scintillation counting (Packard CA2400) by <strong>the</strong> addition of 2 ml<br />
scintillation fluid. To ensure that physiological ACh release could be obtained, perfused eyecups<br />
were stimulated with a white LED light <strong>for</strong> 4 minutes. In o<strong>the</strong>r experiments, 100 micromolar KA<br />
was applied to <strong>the</strong> eyecups to demonstrate pharmacological release of 3H-choline after receptor<br />
activation. Lastly, to obtain a maximal response, 20 mM KCl was perfused through eyecups. In<br />
response to flashing light, <strong>the</strong> release of 3H-choline increased 2-3 times <strong>the</strong> basal efflux in<br />
control untreated eyecups. However, <strong>the</strong> release of 3H-choline signific<strong>an</strong>tly decreased in<br />
glaucoma-induced eyecups. Pharmacological m<strong>an</strong>ipulations produced signific<strong>an</strong>tly larger<br />
responses as 100 micromolar KA demonstrated a 5 fold increase of 3H-choline <strong>from</strong> basal levels<br />
<strong>an</strong>d 20 mM KCl elicited a maximal response correlating to a 7 fold increase of 3H-choline <strong>from</strong><br />
basal levels. These results support <strong>the</strong> hypo<strong>the</strong>sis that ACh release in <strong>the</strong> retina may be<br />
compromised under glaucoma-like conditions.<br />
Disclosures: K. Iwamoto, None; P.J. Birkholz, None; D.M. Linn, None; C.L. Linn, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.5/BB33<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Legacy Good Samarit<strong>an</strong> Foundation<br />
Sears Medical Trust<br />
1R21EY017345-01<br />
Title: Qu<strong>an</strong>titative proteomic <strong>an</strong>alyses of ischemic toler<strong>an</strong>ce in rat retina<br />
Authors: *C. STOWELL 1,2 , B. ARBOGAST 3 , L. WANG 1 , J. LAN 2 , C. BURGOYNE 1 , A.<br />
ZHOU 2 ;
1 2 3<br />
Devers Eye Inst., Portl<strong>an</strong>d, OR; Robert S. Dow Neurobio., Portl<strong>an</strong>d, OR; Oregon State Univ.,<br />
Corvallis, OR<br />
Abstract: Neuronal ischemic toler<strong>an</strong>ce, induced by exposure to a short duration of non-lethal<br />
ischemia prior to <strong>an</strong> o<strong>the</strong>rwise lethal ischemia, has been shown to protect <strong>the</strong> brain against<br />
ischemia similar to what occurs in a stroke. Glaucoma is thought to produce, at least in part, <strong>an</strong><br />
ischemic injury in <strong>the</strong> retina. The induction of toler<strong>an</strong>ce in <strong>the</strong> retina may protect against<br />
ischemic damage to <strong>the</strong> retinal cells. To identify effectors of retina ischemic toler<strong>an</strong>ce, retinal<br />
ischemia was induced in rats by tr<strong>an</strong>siently increasing <strong>the</strong> intraocular pressure (IOP), <strong>an</strong>d <strong>an</strong><br />
ischemic toler<strong>an</strong>ce paradigm was modeled in three groups of <strong>an</strong>imals as follows: 1) Ischemic:<br />
110 mmHg <strong>for</strong> 60 minutes followed by 24 hours reperfusion; 2) Preconditioned: 110 mmHg <strong>for</strong><br />
8 minutes <strong>an</strong>d 48 hour reperfusion; 3) Ischemic-toler<strong>an</strong>t: preconditioning treatment followed by<br />
<strong>an</strong>o<strong>the</strong>r 60 minute of 110 mmHg <strong>an</strong>d 24 hour reperfusion. Sham control eyes were treated with<br />
20 mmHg <strong>an</strong>d time-matched reperfusions. Tryptic digests of proteins extracted <strong>from</strong> retinas of<br />
<strong>the</strong> a<strong>for</strong>e-mentioned conditions were <strong>an</strong>alyzed with a label-free qu<strong>an</strong>titative mass spectrometry<br />
system, <strong>an</strong>d protein qu<strong>an</strong>tities in each treatment condition was compared with that of <strong>the</strong> sham<br />
condition. Both up- <strong>an</strong>d down-regulated proteins were found in each of <strong>the</strong> pre-conditioned,<br />
ischemic <strong>an</strong>d ischemic-toler<strong>an</strong>t retinas. MetaCore gene ontology <strong>an</strong>alysis showed a general up<br />
regulation of proteins involved in chromatin <strong>for</strong>mation <strong>an</strong>d mainten<strong>an</strong>ce in <strong>the</strong> pre-conditioned<br />
retinas, <strong>an</strong>ti-apoptotic proteins in <strong>the</strong> toler<strong>an</strong>t retinas, <strong>an</strong>d complement protein inflamation<br />
response in <strong>the</strong> injurious retinas among o<strong>the</strong>rs. Condition-specific ch<strong>an</strong>ges were also detected <strong>for</strong><br />
proteins that previously have not been reported <strong>for</strong> <strong>the</strong> retina. Some of <strong>the</strong>se condition-specific<br />
ch<strong>an</strong>ges may provide very useful in<strong>for</strong>mation <strong>for</strong> determining a mech<strong>an</strong>ism of ischemic toler<strong>an</strong>ce<br />
in <strong>the</strong> retina.<br />
Disclosures: C. Stowell, None; B. Arbogast, None; L. W<strong>an</strong>g, None; J. L<strong>an</strong>, None; C.<br />
Burgoyne, None; A. Zhou, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.6/BB34<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH EY 012204<br />
Title: CLC chloride tr<strong>an</strong>sporters in <strong>the</strong> avi<strong>an</strong> retina
Authors: *E. A. MCMAINS 1 , J. M. COLONNA 2 , V. S. KRISHNAN 1 , E. L. GLEASON 1 ;<br />
1 2<br />
Biol. Sci., Louisi<strong>an</strong>a State Univ., Baton Rouge, LA; Intitute <strong>for</strong> Neurosci., Univ. of Texas,<br />
Austin, TX<br />
Abstract: Previous work in our lab has shown that nitric oxide (NO) releases Cl - <strong>from</strong> <strong>an</strong><br />
internal compartment in cultured retinal amacrine cells. This NO-induced release of Cl - is<br />
dependent upon a NO-mediated decrease in cytosolic pH. Multiple endosomal compartments<br />
maintain high lumenal [Cl - ] <strong>an</strong>d [H + ] through <strong>the</strong> activity of proton pumps <strong>an</strong>d CLC Cl -<br />
tr<strong>an</strong>sporters. CLCs are ubiquitous in neural tissue, <strong>an</strong>d mutations in several CLC genes result in<br />
hum<strong>an</strong> diseases. ClC-3 <strong>an</strong>d ClC-7 knockouts exhibit marked CNS neurodegeneration, including<br />
deterioration of <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong> retina. To begin to elucidate <strong>the</strong> role of CLCs in <strong>the</strong><br />
effects of NO, we examined <strong>the</strong> distribution of internally expressed CLC family members (ClC-3<br />
- ClC-7) in cultured chick retinal amacrine cells <strong>an</strong>d in sections of adult chicken retina. We<br />
labeled retinal cultures with commercially available, polyclonal <strong>an</strong>tibodies raised against rat<br />
ClC-3, ClC-4, ClC-5, ClC-6, <strong>an</strong>d ClC-7. ClC-3, ClC-4, ClC-5, <strong>an</strong>d ClC-7 were expressed in<br />
cultured amacrine cell bodies <strong>an</strong>d processes to varying degrees, with <strong>an</strong>tibodies against ClC-5<br />
displaying <strong>the</strong> most intense labeling <strong>an</strong>d those against ClC-3 <strong>the</strong> least. ClC-6 <strong>an</strong>tibody signal was<br />
indiscernible. We also labeled chicken retinal sections with <strong>the</strong> <strong>an</strong>tibodies to ClC-3 - ClC-7. In<br />
retinal sections, relative <strong>an</strong>tibody labeling intensities were similar to that found in retinal<br />
cultures. Antibodies against ClC-4, ClC-5, <strong>an</strong>d ClC-7 gave distinctive labeling patterns, with<br />
ClC-5 <strong>an</strong>d ClC-7 labeling in both nuclear <strong>an</strong>d plexi<strong>for</strong>m retinal layers <strong>an</strong>d ClC-4 labeling only<br />
nuclear layers. ClC-3 <strong>an</strong>d ClC-6 were not detectable in retinal tissue. The distinctive expression<br />
patterns of CLC proteins in <strong>the</strong> retina, along with <strong>the</strong>ir import<strong>an</strong>ce <strong>for</strong> retinal development<br />
suggested by CLC knockouts, motivates fur<strong>the</strong>r physiological investigation. Fur<strong>the</strong>rmore, <strong>the</strong><br />
presence of CLC proteins in cultured retinal amacrine cells supports <strong>the</strong>ir potential role in <strong>the</strong><br />
NO-induced release of internal Cl - .<br />
Disclosures: E.A. McMains, None; J.M. Colonna, None; V.S. Krishn<strong>an</strong>, None; E.L.<br />
Gleason, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.7/BB35<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH/NEI #EY-14050
Title: Tropisetron as a neuroprotective agent against glutamate-induced excitotoxicity<br />
Authors: M. M. SWARTZ 1 , *C. L. LINN 2 ;<br />
1 Dept. of Biol. Sci., 2 Western Michig<strong>an</strong> Univ., Kalamazoo, MI<br />
Abstract: Glutamate-induced excitotoxicity, defined as apoptotic cell death resulting <strong>from</strong><br />
excessive amounts of <strong>the</strong> neurotr<strong>an</strong>smitter glutamate, has been implicated in <strong>the</strong> pathology of a<br />
number of CNS diseases. A number of previous studies have <strong>an</strong>alyzed mech<strong>an</strong>isms that prevent<br />
cells <strong>from</strong> dying by excessive glutamate. Previous studies <strong>from</strong> this lab have demonstrated that<br />
acetylcholine (ACh) has a neuroprotective effect against glutamate-induced excitotoxicity.<br />
Ano<strong>the</strong>r pharmacological agent, tropisetron, is best known as a selective 5-HT3 receptor<br />
<strong>an</strong>tagonist used to treat nausea <strong>an</strong>d emesis in c<strong>an</strong>cer patients undergoing chemo<strong>the</strong>rapy <strong>an</strong>d<br />
radiation. However, some studies have suggested that tropisetron acts as <strong>an</strong> alpha7 nicotinic ACh<br />
receptor (nAChR) agonist as well. To explore this possibility, we examined <strong>the</strong> neuroprotective<br />
effect of tropisetron on glutamate-induced excitotoxicity in isolated adult pig retinal g<strong>an</strong>glion<br />
cells (RGCs), to determine if tropisetron mimics ACh-induced neuroprotection. In dose response<br />
studies, various concentrations of tropisetron were added to isolated <strong>an</strong>d cultured pig RGCs<br />
be<strong>for</strong>e application of a large glutamate insult that normally leads to signific<strong>an</strong>t cell death. After 3<br />
days in culture, live cells were labeled with calcein <strong>an</strong>d counted. The number of cells surviving<br />
in treated conditions was compared to <strong>the</strong> number of surviving cells counted under control<br />
conditions. To determine if tropisetron’s effect was mediated through alpha7 nAChRs, alpha-Bgt<br />
or methyllycaconitine (MLA), were added to cultured RGCs be<strong>for</strong>e application of tropisetron<br />
<strong>an</strong>d glutamate. To ensure that tropisetron’s neuroprotection was not acting through 5-HT3<br />
receptors, control studies using <strong>the</strong> 5-HT3 agonist SR 57227, was substituted <strong>for</strong> tropisetron <strong>an</strong>d<br />
neuroprotection against glutamate excitotoxicity was assessed. Lastly, <strong>an</strong>o<strong>the</strong>r 5-HT3 <strong>an</strong>tagonist,<br />
ond<strong>an</strong>setron, found to have a low binding affinity <strong>for</strong> alpha7 nAChRs was used in studies to<br />
ensure that binding of tropisetron to 5-HT3 receptors did not contribute to neuroprotection of<br />
RGCs. The results <strong>from</strong> <strong>the</strong>se pharmacological studies demonstrated that low concentrations of<br />
tropisetron protected a signific<strong>an</strong>t percentage of RGCs against glutamate-induced excitotoxicity.<br />
In <strong>the</strong> presence of specific inhibitors of alpha7 nAChRs, tropisetron’s neuroprotective effects<br />
were eliminated. However, activation or inhibition of 5-HT3 receptors played no role in this<br />
neuroprotection. These results suggest that tropisetron could be used as a neuroprotective agent<br />
against diseases involving excitotoxicity through alpha7 nAChRs.<br />
Disclosures: M.M. Swartz, None; C.L. Linn, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 557.8/BB36<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NEI EY05725<br />
Title: Sweet taste receptors in rat retinal neurons<br />
Authors: *J. YANG 1 , J. MYERS 1 , M. M. SLAUGHTER 2 ;<br />
1 Neurosci., SUNY Buffalo, Amherst, NY; 2 SUNY buffalo, buffalo, NY<br />
Abstract: Purpose: Metabotropic glutamate <strong>an</strong>d GABA receptors are members of a G-proteincoupled<br />
family containing more th<strong>an</strong> 50 receptors. Exploring retinal mRNA of family members,<br />
we found expression of TR1 <strong>an</strong>d TR2 taste<br />
receptors. We explored <strong>the</strong> molecular <strong>an</strong>d physiological characteristics of sweet taste receptors<br />
(TR2) in rat retina.<br />
Methods: Acutely dissociated or cultured retinal cells <strong>from</strong> 10 - 16 day Sprague-Dawley rats<br />
were tested using various sweet taste lig<strong>an</strong>ds. Internal calcium was monitored using a fluo3based<br />
imaging while calcium currents were measured using whole cell patch clamp techniques.<br />
RT-PCR <strong>an</strong>d RACE (rapid amplification of cDNA Ends) were used to determine TR expression.<br />
Results: RT-PCR revealed that <strong>the</strong> TR1&2 genes are expressed in rat retina. By RT-PCR <strong>an</strong>d<br />
RACE, we pulled out <strong>the</strong> full sequences, which were identical to tongue taste receptors.<br />
Aspartame application raised internal calcium in a subset of isolated retinal neurons. If cells were<br />
depolarized by 50 mM KCl, <strong>the</strong>n aspartame reduced internal calcium. The EC50 <strong>for</strong> suppression<br />
of elevated calcium was 13uM aspartame, but <strong>the</strong> EC50 <strong>for</strong> <strong>the</strong> enh<strong>an</strong>cement of basal calcium<br />
was 1.7mM aspartame. Glutamate metabotropic receptor agonists produced similar effects<br />
through activation of different receptors.<br />
Aspartame suppressed high-voltage activated calcium currents. O<strong>the</strong>r TR2 agonists (2mM Dtryptoph<strong>an</strong>,<br />
1mM D-phenylal<strong>an</strong>ine, 1mM saccharine, <strong>an</strong>d 50mM glucose) also enh<strong>an</strong>ced<br />
intracellular calcium levels. The calcium elevation was inhibited when voltage-gated calcium<br />
ch<strong>an</strong>nels were blocked by nimodipine or cadmium. It was also blocked by caffeine depletion of<br />
internal stores.<br />
Conclusions: Sweet taste receptors are expressed in retina. Aspartame produced a biphasic<br />
modulation of internal calcium: raising calcium in resting neurons <strong>an</strong>d reducing calcium in<br />
stimulated neurons. Similar effects were observed using ACPD, a metabotropic glutamate<br />
agonist, indicating this type of modulation exists among various metabotropic receptors.<br />
Bimodal modulation implies that metabotropic receptors c<strong>an</strong> activate opposing biological<br />
cascades in response to a single agonist.<br />
Disclosures: J. Y<strong>an</strong>g, None; J. Myers, None; M.M. Slaughter, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.9/BB37<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NEI EY06472<br />
Title: Localization of histamine receptor 2 in <strong>the</strong> macaque retina<br />
Authors: *D. W. MARSHAK 1 , A. VILA 2 , J. O’BRIEN 3 , R. JANZ 3 , S. C. MASSEY 3 , D. R.<br />
MARSHAK 4 ;<br />
2 Dept. of Neurobio. <strong>an</strong>d Anat., 3 Dept. of Ophthalmology <strong>an</strong>d Visual Science, Univ. of Texas<br />
Med. Sch. at Houston, Ho, 1 Univ. Texas Med. Sch., Houston, TX; 4 Perkin Elmer Inc., Waltham,<br />
MA<br />
Abstract: The primate retina receives input <strong>from</strong> neurons in <strong>the</strong> posterior hypothalamus that<br />
contain histamine, but it is uncertain how histamine influences <strong>the</strong> processing of visual<br />
in<strong>for</strong>mation. To address this question, we are identifying <strong>the</strong> cells that express <strong>the</strong> 3 types of<br />
histamine receptors (HR). Antisera were raised in goats against a KLH conjugate of a syn<strong>the</strong>tic<br />
peptide corresponding to part of <strong>the</strong> intracellular domain of HR2 near <strong>the</strong> C-terminus. Some of<br />
<strong>the</strong> <strong>an</strong>tiserum was purified by affinity chromatography using protein G. Antibody specificity was<br />
confirmed using Western blots against a GST fusion protein containing <strong>the</strong> intracellular portion<br />
of <strong>the</strong> C-terminus of HR2 <strong>an</strong>d with absorption controls <strong>for</strong> <strong>the</strong> immunolabeling. Macaque retinal<br />
tissue was fixed in 4% para<strong>for</strong>maldehyde with or without 0.1% glutaraldehyde. Vibratome<br />
sections or whole mounts were permeabilized with graded eth<strong>an</strong>ol solutions, incubated with <strong>an</strong>ti-<br />
HR2 <strong>an</strong>d <strong>an</strong>tibodies to markers <strong>for</strong> retinal neurons; <strong>the</strong> double <strong>an</strong>d triple labeled retinas were<br />
<strong>an</strong>alyzed by confocal microscopy. HR2-IR puncta were found in <strong>the</strong> outer plexi<strong>for</strong>m layer. The<br />
HR2 receptors were localized to cone pedicles, identified using monoclonal <strong>an</strong>tibody 7G6<br />
against cone arrestin; no puncta were associated with rods. The puncta were closely associated<br />
with synaptic ribbons labeled using <strong>an</strong>tibody to ribeye, but <strong>the</strong> two molecules were not<br />
colocalized. HR2 was associated with dendrites of ON bipolar cells labeled with mGluR6,<br />
though <strong>the</strong> two were not colocalized. To determine whe<strong>the</strong>r HR2 was expressed by horizontal<br />
cell dendrites, type I horizontal cells were visualized with DAPI, filled with Neurobiotin <strong>an</strong>d<br />
labeled with avidin Cy-3. Puncta labeled with <strong>an</strong>ti-HR2 were associated with Neurobiotin-filled<br />
dendrites but not colocalized. Retinas were labeled using <strong>an</strong>tibody to GluR4, <strong>an</strong> AMPA receptor<br />
subunit on dendrites of horizontal cells. HR2-IR puncta were found above <strong>the</strong> b<strong>an</strong>d of GluR4-IR<br />
puncta, a finding suggesting that <strong>the</strong>y are in cones, not horizontal cells. Syntaxin 3<br />
immunolabeling was confined to <strong>the</strong> plasma membr<strong>an</strong>e of photoreceptor terminals. Cones double<br />
labeled with HR2 <strong>an</strong>d Syntaxin 3 showed clusters of both types of puncta that were closely<br />
associated, but <strong>the</strong> HR2 puncta were located slightly above <strong>the</strong>m. Previously, we showed that<br />
HR3 was localized to <strong>the</strong> tips of ON bipolar cell dendrites. Because <strong>the</strong> axons containing<br />
histamine terminate in <strong>the</strong> inner plexi<strong>for</strong>m layer, <strong>the</strong>se findings suggest that histamine acts in <strong>the</strong><br />
primate retina via volume tr<strong>an</strong>smission.
Disclosures: D.W. Marshak, None; A. Vila, None; J. O’Brien, None; R. J<strong>an</strong>z, None; S.C.<br />
Massey, None; D.R. Marshak, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.10/BB38<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Kakenhi Gr<strong>an</strong>t 20500294<br />
Title: Immunohistochemical <strong>an</strong>d physiological <strong>an</strong>alyses of histamine <strong>an</strong>d histamine receptors on<br />
g<strong>an</strong>glion cells in <strong>the</strong> developing gerbil retina<br />
Authors: H. IMADA 1 , M. OHKUMA 1 , M. KOKUBO 2 , T. KATO 3 , *E.-I. MIYACHI 1 ;<br />
1 Dept Physiol, Fujita Hlth. Univ. Sch. Med., Toyoake, Aichi, Jap<strong>an</strong>; 2 Joint Res. Lab., Fujita Hlth.<br />
Univ., Toyoake, Jap<strong>an</strong>; 3 Clin. Lab. Sci., Fujita Hlth. Univ. Coll, Toyoake, Aichi, Jap<strong>an</strong><br />
Abstract: In <strong>the</strong> central nervous system, histamine is known to act on three major types of Gprotein-coupled<br />
receptors; histamine H1 receptor, histamine H2 receptor <strong>an</strong>d histamine H3<br />
receptor. We examined <strong>the</strong> localization of histamine <strong>an</strong>d histamine receptors in <strong>the</strong> developing<br />
gerbil retina using immunohistochemical method. The gerbils were perfused intra-cardially with<br />
a mixture of 4% para<strong>for</strong>maldehyde in 0.1 M sodium phosphate buffer. The removed eyeballs<br />
were frozen with <strong>the</strong> liquid nitrogen. Then <strong>the</strong>y were cut with section of 16 µm using a cryostat.<br />
The sections were examined using ABC (avidin-biotin-peroxidase complex)<br />
immunohistochemical staining method. In <strong>the</strong> adult retinal section, histamine positive line in <strong>the</strong><br />
g<strong>an</strong>glion cell layer was observed. H1 receptor was labeled in <strong>the</strong> g<strong>an</strong>glion cells <strong>from</strong> p (postnatal<br />
day) 2 to p 350 (adult) in <strong>the</strong> gerbil retinal sections. At p 14, H1 receptor was strongly stained in<br />
<strong>the</strong> g<strong>an</strong>glion cell layer. The distal portions of <strong>the</strong> g<strong>an</strong>glion cell somata were more strongly<br />
stained th<strong>an</strong> <strong>the</strong> proximal portions. The axon of each g<strong>an</strong>glion cell was weakly stained. H2<br />
receptor was labeled in <strong>the</strong> g<strong>an</strong>glion cells after p 8. After p 26, <strong>the</strong> g<strong>an</strong>glion cells were hardly<br />
stained. And H3 receptor was labeled in <strong>the</strong> g<strong>an</strong>glion cells after p 6. After p 70, <strong>the</strong> g<strong>an</strong>glion<br />
cells were weakly stained, <strong>an</strong>d most of <strong>the</strong>m were not stained at p 350. H2 <strong>an</strong>d H3 receptors were<br />
strongly stained at <strong>the</strong> distal portions of <strong>the</strong> g<strong>an</strong>glion cell somata at p 14. The fura-2 based<br />
calcium imaging was also per<strong>for</strong>med at <strong>the</strong> gerbil retina. The preparations were superfused at 1<br />
ml/min with 100 µM histamine-containing solution. Histamine increased intracellular Ca 2+ in<br />
g<strong>an</strong>glion cells. And some retinal g<strong>an</strong>glion cells were excited by betahistine, <strong>an</strong> agonist of <strong>the</strong> H1<br />
receptor. Dimaprit, <strong>an</strong> agonist of <strong>the</strong> H2 receptor, also increased intracellular Ca 2+ in <strong>the</strong>
premature g<strong>an</strong>glion cells. These results suggest that histamine <strong>an</strong>d histamine H1 receptor exist in<br />
<strong>the</strong> gerbil retina, <strong>an</strong>d that H2 <strong>an</strong>d H3 receptors were expressed on develpoing gerbil g<strong>an</strong>glion<br />
cells.<br />
Disclosures: H. Imada, None; M. Ohkuma, None; M. Kokubo, None; T. Kato, None; E.<br />
Miyachi , None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.11/CC1<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t R01 EY004864<br />
NIH Gr<strong>an</strong>t R01 EY014764<br />
NIH Gr<strong>an</strong>t T32 EY007092<br />
NIH Gr<strong>an</strong>t P30 EY006360<br />
Research to Prevent Blindness<br />
Title: Tr<strong>an</strong>scriptional profiling of dopamine D4 receptor inactivation in <strong>the</strong> mouse retina<br />
Authors: *C. R. JACKSON 1 , N. POZDEYEV 1 , M. IUVONE 2 ;<br />
2 Ophthalmology, 1 Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: The dopamine D4 receptor (D4R) controls aspects of mammali<strong>an</strong> retinal physiology.<br />
Activating this receptor promotes light adaptation, suppresses a light-sensitive pool of cyclic<br />
AMP in photoreceptor cells, <strong>an</strong>d regulates diurnal rhythms of protein phosphorylation in<br />
photoreceptors. Moreover, D4R activation controls cAMP not only by negatively coupling to <strong>the</strong><br />
type 1 adenylyl cyclase (AC1), but it also regulates expression of <strong>the</strong> gene encoding AC1, Adcy1.<br />
These data support <strong>the</strong> hypo<strong>the</strong>sis that D4Rs c<strong>an</strong> regulate gene expression in <strong>the</strong> mammali<strong>an</strong><br />
retina. To investigate this hypo<strong>the</strong>sis fur<strong>the</strong>r, Affymetrix microarray <strong>an</strong>alysis was pre<strong>for</strong>med on<br />
mice lacking <strong>the</strong> D4R (Drd4 -/- ) <strong>an</strong>d compared with WT littermate controls. Using conservative<br />
filtering criteria with a false discovery rate of
encode proteins involved in signal tr<strong>an</strong>sduction (e.g., Adcy1-positive control, Rgs20, Guca1b),<br />
gene expression (e.g., Hdac9), alternative mRNA splicing (e.g., Mbnl2), <strong>an</strong>d synaptic function<br />
(e.g., Syngap1). Confirmation of <strong>the</strong>se ch<strong>an</strong>ges in expression is underway using qu<strong>an</strong>titative realtime<br />
PCR. To our knowledge this study is <strong>the</strong> first to show that lack of dopamine D4 receptor<br />
activity results in signific<strong>an</strong>t ch<strong>an</strong>ges in retinal gene expression.<br />
Disclosures: C.R. Jackson, None; N. Pozdeyev, None; M. Iuvone, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.12/CC2<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Title: Baclofen effects in <strong>the</strong> retina adapted to <strong>the</strong> darkness <strong>an</strong>d monochromatic light<br />
Authors: *N. S. GARINA, V. G. ERCHENKOV;<br />
Moscow State Univ., Moscow, Russi<strong>an</strong> Federation<br />
Abstract: In <strong>the</strong> vertebrate retina GABAb-receptors modulate b-wave in <strong>the</strong> electroretinogram<br />
(ERG) <strong>an</strong>d light responses of <strong>the</strong> amacrine <strong>an</strong>d g<strong>an</strong>glion cells. G<strong>an</strong>glion cell response<br />
modulation provokes ch<strong>an</strong>ges in <strong>the</strong> light-evoked potentials of <strong>the</strong> primary visual centers. The<br />
goal of <strong>the</strong>se experiments is to investigate effects of GABAb-receptors activation in <strong>the</strong> rode-<br />
<strong>an</strong>d cone-dominated conditions.<br />
In <strong>the</strong> carp (Ciprynus Carpio L.) ERG <strong>an</strong>d tectal evoked potentials (EP) to white light stimuli<br />
(duration 1 ms) were simult<strong>an</strong>eously registered in <strong>the</strong> following conditions: darkness, const<strong>an</strong>t<br />
red (λ = 658nm) <strong>an</strong>d const<strong>an</strong>t green (λ = 528nm) light. After 40 minutes adaptation to <strong>the</strong><br />
darkness or const<strong>an</strong>t light retinal surface was superfused with baclofen solution (10-4 M; general<br />
dose 0,15 mkM). Amplitudes of b-wave ERG <strong>an</strong>d tectal EP to light ON-set, as reflections of<br />
bipolar <strong>an</strong>d, correspondingly, g<strong>an</strong>glion sell activities, were <strong>an</strong>alyzed.<br />
All fishes were <strong>an</strong>es<strong>the</strong>tized <strong>an</strong>d immobilizaied in accord<strong>an</strong>ce with <strong>the</strong> NIH guideline on <strong>the</strong><br />
Care <strong>an</strong>d Use of Animals in Research <strong>for</strong> minimizing pain <strong>an</strong>d distress.<br />
In darkness b-wave amplitudes grow up <strong>an</strong>d reached maximum (100%) through 30-40 minutes<br />
of dark adaptation. In this conditions retinal superfusion with baclofen solution diminished bwave<br />
amplitude to 30-50% <strong>from</strong> maximal level in darkness. Const<strong>an</strong>t green or red light<br />
diminished b-wave so that this ERG component was 30-40% <strong>from</strong> dark-adapted level. Baclofen<br />
decreased (on 10-15%) b-wave amplitudes when <strong>the</strong> retina was adapted to <strong>the</strong> red light, but this<br />
drug was slightly effective in <strong>the</strong> green light adaptation conditions.
Tectal EP amplitudes did not signific<strong>an</strong>t ch<strong>an</strong>ge through dark or light adaptation. Retinal<br />
baclofen superfusion increased EP amplitude up to 30-90% in <strong>the</strong> all adaptation conditions.<br />
Tectal EP amplitudes increased early or simult<strong>an</strong>eously with b-wave amplitude ch<strong>an</strong>ges.<br />
These findings suggest that retinal GABAb-receptors mainly modulate rod component in b-wave<br />
ERG, but this receptors similar affect cone <strong>an</strong>d rod signals in retinal output.<br />
Disclosures: N.S. Garina, None; V.G. Erchenkov, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.13/CC3<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NSF Gr<strong>an</strong>t DMS-0718308<br />
Title: Computational study of cat retinal cone-horizontal cell interaction<br />
Authors: S. CHANG, *S. M. BAER, S. M. CROOK, C. L. GARDNER, C. RINGHOFER;<br />
Arizona State Univ., Tempe, AZ<br />
Abstract: Small-spot flicker responses of cat retinal horizontal cells c<strong>an</strong> be amplified upon <strong>the</strong><br />
onset of dim background (Pflug et al. 1990). This phenomenon is called background-induced<br />
flicker enh<strong>an</strong>cement, <strong>an</strong>d is believed to be caused by <strong>an</strong> increased calcium flow into <strong>the</strong> cone<br />
pedicles in response to rod-induced hyperpolarization of <strong>the</strong> horizontal cells (Pflug et al. 1990,<br />
Nelson et al. 1990, Verweij et al. 1996). However, <strong>the</strong> underlying mech<strong>an</strong>ism that relates <strong>the</strong><br />
hyperpolarization of <strong>the</strong> horizontal cells to <strong>the</strong> rise of calcium current in <strong>the</strong> cones remains<br />
unclear. Two hypo<strong>the</strong>ses have received <strong>the</strong> most attention. One hypo<strong>the</strong>sis states that <strong>the</strong><br />
dynamics of a calcium-blocking agent (most likely gamma-aminobutyric acid (GABA)) plays a<br />
major role in this process (Nelson et al. 1990). The o<strong>the</strong>r hypo<strong>the</strong>sis argues that <strong>the</strong> mech<strong>an</strong>ism is<br />
GABA-independent <strong>an</strong>d is purely ephaptic in nature (Kamerm<strong>an</strong>s et al. 2004). To explore <strong>the</strong>se<br />
two hypo<strong>the</strong>ses, a two-cell ordinary differential equation (ODE) model is proposed, which<br />
describes <strong>the</strong> synaptic interaction between a cone <strong>an</strong>d a horizontal cell <strong>an</strong>d incorporates both <strong>the</strong><br />
GABA <strong>an</strong>d ephaptic mech<strong>an</strong>isms. To capture <strong>the</strong> spatial features of <strong>the</strong> enh<strong>an</strong>cement effect <strong>an</strong>d<br />
to make <strong>the</strong> modeling more realistic, <strong>the</strong> ODE model is extended into a partial differential<br />
equation (PDE) model of a reaction-diffusion type on two spatial dimensions with alterable<br />
flicker stimulus regions. Simulation results of <strong>the</strong> two models suggest a combined mech<strong>an</strong>ism<br />
involving both <strong>the</strong> GABA <strong>an</strong>d ephaptic effects.
Disclosures: S. Ch<strong>an</strong>g, None; S.M. Baer, None; S.M. Crook, None; C.L. Gardner, None; C.<br />
Ringhofer, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.14/CC4<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: EY11105<br />
Title: Modulation of <strong>the</strong> light-mediated cation ch<strong>an</strong>nel in retinal ON bipolar cells by G-protein<br />
subunits<br />
Authors: *Y. XU, N. VARDI;<br />
Neurosci., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: In darkness, glutamate released <strong>from</strong> photoreceptors hyperpolarizes retinal ON bipolar<br />
cells by activating <strong>the</strong> heterotrimeric G-protein Go <strong>an</strong>d closing non-selective cation ch<strong>an</strong>nels.<br />
Theoretically, ei<strong>the</strong>r Gαo1 or Gβγ c<strong>an</strong> carry <strong>the</strong> cascade to mediate ch<strong>an</strong>nel closure.<br />
To test which subunit mediates ch<strong>an</strong>nel closure, we per<strong>for</strong>med whole-cell patch recordings <strong>from</strong><br />
mouse rod bipolar cells, <strong>an</strong>d dialyzed <strong>the</strong> cell with ei<strong>the</strong>r Gαo1 subunit or a peptide (SIGK) that<br />
releases Gβγ <strong>from</strong> <strong>the</strong> trimeric complex without affecting <strong>the</strong> Gα nucleotide-binding state.<br />
Cesium was included in <strong>the</strong> pipette solution to block potassium <strong>an</strong>d BAPTA to reduce response<br />
run down. To eliminate chloride currents, strychnine <strong>an</strong>d picrotoxin were added to <strong>the</strong> external<br />
solution <strong>an</strong>d cells were clamped at -60 mV. For Gαo dialysis, cells were dark-adapted, <strong>an</strong>d a light<br />
pulse (50 ms) was given every 30 sec. We found that <strong>the</strong> holding inward current in control cells<br />
(no Gαo added, n=11) was relatively const<strong>an</strong>t at ~ -40 pA up to 3 minutes after break in, but that<br />
of cells dialyzed with 100 nM Gαo (n=12) increased continuously; <strong>an</strong>d at 2 min, it was<br />
signific<strong>an</strong>tly larger th<strong>an</strong> <strong>the</strong> initial value (-75 ± 11 pA vs. -29 ± 6 pA, p
esponses over time. The initial light response was greater in <strong>the</strong> SIGK-dialyzed cells (47± 10<br />
pA, n=9) compared to control (33 ± 8 pA, n=4), <strong>an</strong>d so was <strong>the</strong> holding current under light<br />
adapted conditions (-41 ± 9 pA vs. -9 ± 1 pA). As dialysis proceeded, <strong>the</strong> holding inward current<br />
<strong>an</strong>d <strong>the</strong> conduct<strong>an</strong>ce decreased. Correspondingly, <strong>the</strong> light OFF response decreased over time.<br />
The holding current <strong>an</strong>d OFF response in <strong>the</strong> control cells remained stable (up to about 5 min).<br />
Although <strong>the</strong> interpretation of <strong>the</strong>se data is not straight-<strong>for</strong>ward, it is consistent with <strong>the</strong> idea that<br />
Gβγ closes <strong>the</strong> cation ch<strong>an</strong>nel. <strong>When</strong> Gα (in ei<strong>the</strong>r state) enters <strong>the</strong> cell in excess, it c<strong>an</strong> scavenge<br />
Gβγ <strong>an</strong>d allows <strong>the</strong> cation ch<strong>an</strong>nels to open.<br />
Disclosures: Y. Xu, None; N. Vardi, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.15/CC5<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NEI<br />
Title: On bipolar cell desensitization makes <strong>the</strong> post-synaptic responses tr<strong>an</strong>sient<br />
Authors: *T. KAUR, S. NAWY;<br />
Neurosci., Albert Einstein Col. of Med., Bronx, NY<br />
Abstract: Light depolarizes retinal On bipolar cells by inactivating <strong>the</strong> mGluR6-mediated<br />
signaling cascade <strong>an</strong>d opening a non-selective cation ch<strong>an</strong>nel, believed to be TRPM1. We have<br />
previously shown that a prolonged simulated light stimulus results in a Ca2+-dependent<br />
depression of <strong>the</strong> tr<strong>an</strong>sduction current underlying <strong>the</strong> light response in On bipolar cells, which<br />
we have referred to as desensitization. Desensitization of <strong>the</strong> tr<strong>an</strong>sduction current results in<br />
repolarization of <strong>the</strong> On bipolar cell membr<strong>an</strong>e potential. However, it is unclear whe<strong>the</strong>r<br />
membr<strong>an</strong>e repolarization is rapid <strong>an</strong>d large enough to reduce tr<strong>an</strong>smitter release <strong>from</strong> On bipolar<br />
cell terminals, perhaps sparing <strong>the</strong>m <strong>from</strong> vesicle depletion. Here, we test this possibility by<br />
recording <strong>from</strong> g<strong>an</strong>glion cells while simult<strong>an</strong>eously stimulating On bipolar cells. Our results<br />
suggest that desensitization of On bipolar cells limits <strong>the</strong> duration of g<strong>an</strong>glion cell responses (see<br />
also Awatram<strong>an</strong>i <strong>an</strong>d Slaughter, 2000) <strong>an</strong>d may also contribute to <strong>the</strong> conservation of synaptic<br />
vesicles at <strong>the</strong> On bipolar cell terminal.<br />
Disclosures: T. Kaur, None; S. Nawy, None.
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.16/CC6<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH 5R01NS029169-19<br />
Title: Identification <strong>an</strong>d characterization of non-glycinergic, non-GABAergic amacrine cells in<br />
mouse retina<br />
Authors: *P. E. VOINESCU, J. N. KAY, J. R. SANES;<br />
Mol. <strong>an</strong>d Cell. Biol., Ctr. <strong>for</strong> Brain Science, Harvard Univ., Cambridge, MA<br />
Abstract: Although retinal amacrine cells are diverse in structure <strong>an</strong>d function, <strong>the</strong>y are often<br />
divided into two main groups depending on whe<strong>the</strong>r <strong>the</strong>y use GABA or glycine as <strong>the</strong>ir<br />
neurotr<strong>an</strong>smitter. However, using markers of all amacrines (syntaxin-1), glycinergic cells<br />
(glycine tr<strong>an</strong>sporter <strong>an</strong>d glycine) <strong>an</strong>d GABAergic cells (glutamate decarboxylase <strong>an</strong>d GABA),<br />
we found that about 15% of amacrines in mouse retina are nei<strong>the</strong>r GABA-ergic nor glycinergic;<br />
we call <strong>the</strong>m NGNG amacrines. We assayed tr<strong>an</strong>sgenic mouse lines <strong>for</strong> expression in <strong>the</strong>se cells,<br />
<strong>an</strong>d found one in which integration site-dependent expression of a cy<strong>an</strong> fluorescent protein (CFP;<br />
Misgeld et al., 2007) selectively labels <strong>the</strong> NGNG amacrines. Expression of CFP allowed us to<br />
<strong>an</strong>alyze <strong>the</strong> morphology of NGNG amacrines; most have narrow arbors that project to <strong>the</strong> most<br />
distal <strong>an</strong>d central sublaminae of <strong>the</strong> inner plexi<strong>for</strong>m layer (S1 <strong>an</strong>d S3). Finally, we used CFP to<br />
isolate <strong>the</strong> NGNG amacrines by fluorescence activated cell sorting (FACS), <strong>the</strong>n per<strong>for</strong>med<br />
cDNA microarray <strong>an</strong>alysis on <strong>the</strong>se <strong>an</strong>d four o<strong>the</strong>r amacrine subtypes, plus on several g<strong>an</strong>glion<br />
cell <strong>an</strong>d bipolar cell subtypes to identify genes selectively expressed in <strong>the</strong> NGNG population.<br />
Genes identified in this way may allow us to obtain endogenous markers <strong>for</strong> <strong>the</strong> NGNG<br />
amacrines, <strong>an</strong>d to <strong>for</strong>mulate hypo<strong>the</strong>ses about <strong>the</strong>ir development <strong>an</strong>d functional properties.<br />
Disclosures: P.E. Voinescu, None; J.N. Kay, None; J.R. S<strong>an</strong>es, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.17/CC7<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t EY013333<br />
Title: The AII amacrine circuit mediates asymmetrical rectification in On <strong>an</strong>d Off pathways<br />
Authors: *Z. LIANG, M. A. FREED;<br />
Neurosci., Univ. of Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: On <strong>an</strong>d Off neurons have opposite responses to <strong>an</strong>y given contrast; additionally <strong>the</strong>ir<br />
responses show marked rectification, so that On cells respond to positive contrasts (brightening)<br />
<strong>an</strong>d Off cells respond to negative contrast (dimming). To measure rectification at <strong>the</strong> synaptic<br />
output of bipolar cells, we recorded excitatory currents <strong>from</strong> g<strong>an</strong>glion cells in <strong>an</strong> in vitro intact<br />
preparation of <strong>the</strong> guinea pig retina. We stimulated with photopic full-field white noise <strong>an</strong>d used<br />
a linear-nonlinear model to plot output versus contrast (static nonlinearity). We also measured<br />
tonic current associated with a const<strong>an</strong>t intensity, i.e., zero contrast. We found that <strong>the</strong> On<br />
bipolar cell devoted most of its output to signaling negative contrasts <strong>an</strong>d some to signaling<br />
negative contrasts (rectification index, RI = 59 ± 5%) but <strong>the</strong> Off bipolar cell devoted almost all<br />
of its output to signaling negative contrasts ( RI = 81 ± 2%). The On bipolar had a subst<strong>an</strong>tial<br />
tonic current associated with zero contrast but <strong>the</strong> Off bipolar cell’s tonic current was<br />
signific<strong>an</strong>tly less (43 ± 17 vs 9 ± 4 pA, p < 0.05).<br />
The On bipolar cell is known to cross inhibit <strong>the</strong> Off bipolar cell via <strong>the</strong> AII amacrine cell.<br />
Severing cross inhibition, with L-AP4 or strychnine, decreased rectification index by 28 ± 3%<br />
(L-AP4) or 10 ± 7% (strychnine) <strong>an</strong>d increased tonic current at <strong>the</strong> Off bipolar cell’s output by<br />
32 ± 11 pA. Augmenting <strong>the</strong> On bipolar cell response with <strong>the</strong> GABAc <strong>an</strong>tagonist TPMPA<br />
increased rectification at <strong>the</strong> Off bipolar cell’s out-put by 10 ± 2 %.<br />
We conclude that <strong>the</strong> On bipolar cell, via <strong>the</strong> AII amacrine circuit, sets <strong>the</strong> membr<strong>an</strong>e potential<br />
of <strong>the</strong> Off bipolar cell close to threshold <strong>for</strong> tr<strong>an</strong>smitter release. Thus <strong>the</strong> Off bipolar's tonic<br />
release is insufficient to signal positive contrast. The ON bipolar cell, however, has sufficient<br />
tonic current to signal positive <strong>an</strong>d negative contrast by increasing <strong>an</strong>d decreasing tonic current.<br />
This asymmetrical rectification of On <strong>an</strong>d Off cells may be <strong>an</strong> adaptation to natural scenes which<br />
have a skewed distribution of contrasts. Thus equalization of contrasts between On <strong>an</strong>d Off<br />
would require that On cells signal some negative contrasts.<br />
Disclosures: Z. Li<strong>an</strong>g, None; M.A. Freed, None.<br />
Poster
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.18/CC8<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH EY 012204<br />
Title: The bal<strong>an</strong>ce of calcium-dependent mech<strong>an</strong>isms regulating L-type calcium ch<strong>an</strong>nel<br />
function in retinal amacrine cells<br />
Authors: *M. TEKMEN, E. GLEASON;<br />
LSU, Baton Rouge, LA<br />
Abstract: Multiple classes of retinal amacrine cells use L-type voltage-gated Ca 2+ ch<strong>an</strong>nels to<br />
mediate synaptic tr<strong>an</strong>smission. We have previously shown that mitochondrial calcium uptake<br />
(MCU) c<strong>an</strong> affect Ca 2+ ch<strong>an</strong>nel function. L-type Ca 2+ ch<strong>an</strong>nels are known to be inactivated by a<br />
Ca 2+ /calmodulin-dependent mech<strong>an</strong>ism <strong>an</strong>d enh<strong>an</strong>ced by protein kinase A (PKA)<br />
phosphorylation. Because both of <strong>the</strong>se regulators c<strong>an</strong> be Ca 2+ /calmodulin-dependent, we ask<br />
how MCU influences <strong>the</strong> regulatory bal<strong>an</strong>ce of amacrine cell L-type Ca 2+ ch<strong>an</strong>nels.<br />
Amacrine cells were initially recorded in <strong>the</strong> per<strong>for</strong>ated patch configuration <strong>an</strong>d cells were<br />
stepped <strong>from</strong> -70mV to 0mV to activate L-type Ca 2+ ch<strong>an</strong>nels. Carbonyl cy<strong>an</strong>ide p-<br />
(trifluromethoxy) phenylhydrazone (FCCP, 1µM, <strong>an</strong> MCU inhibitor), caused a reversible<br />
reduction (46%) in <strong>the</strong> Ca 2+ current (ICa) amplitude. Replacing external Ca 2+ with Ba 2+<br />
minimized <strong>the</strong> effects of FCCP (22% reduction). These observations are consistent with <strong>the</strong><br />
hypo<strong>the</strong>sis that Ca 2+ influx through <strong>the</strong>se ch<strong>an</strong>nels is normally regulated by MCU. If incoming<br />
Ca 2+ is normally sequestered by MCU, <strong>the</strong>n <strong>the</strong> level of cytosolic Ca 2+ buffering should affect<br />
<strong>the</strong> FCCP-dependent inactivation of L-type Ca 2+ ch<strong>an</strong>nels. Patch pipets were filled with ei<strong>the</strong>r<br />
1.1mM or 14.0mM EGTA <strong>for</strong> ruptured patch recordings. Blocking MCU caused a greater<br />
reduction in <strong>the</strong> ICa amplitude in 1.1mM EGTA (39%) th<strong>an</strong> in 14mM EGTA (13%).<br />
Interestingly, with 10mM BAPTA in <strong>the</strong> pipet, FCCP caused <strong>an</strong> increase (24%) ra<strong>the</strong>r th<strong>an</strong> a<br />
decrease in <strong>the</strong> ICa amplitude. We hypo<strong>the</strong>sized that <strong>the</strong> fast buffering capability of BAPTA was<br />
minimizing <strong>the</strong> Ca 2+ -dependent inactivation of <strong>the</strong> ch<strong>an</strong>nels, revealing a Ca 2+ -dependent<br />
enh<strong>an</strong>cement, possibly through PKA activity promoted by <strong>the</strong> action of <strong>the</strong> Ca 2+ /calmodulindependent<br />
adenylate cyclase, AC1. To test <strong>for</strong> <strong>the</strong> involvement of PKA, we used <strong>the</strong> inhibitor<br />
H89 (1µM) on BAPTA-loaded cells. H89 alone caused a decrease in <strong>the</strong> ICa amplitude (43%),<br />
<strong>an</strong>d this effect was enh<strong>an</strong>ced by co-application of FCCP (62%). 3-Isobutyl-1-methylx<strong>an</strong>thine<br />
(IBMX, 100µM), a phosphodiesterase 1 inhibitor, was used to increase cytosolic cAMP. IBMX<br />
lead to enh<strong>an</strong>cement of <strong>the</strong> current consistent with <strong>the</strong> involvement of PKA. These results<br />
indicate that mitochondria are critical to maintaining <strong>the</strong> availability of L-type Ca 2+ ch<strong>an</strong>nels <strong>for</strong><br />
depolarization-dependent signaling in amacrine cells. Fur<strong>the</strong>rmore, it appears that Ca 2+ current
amplitude is set by a bal<strong>an</strong>ce of Ca 2+ /calmodulin-dependent inactivation <strong>an</strong>d phosphorylation.<br />
.<br />
Disclosures: M. Tekmen, None; E. Gleason, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.19/CC9<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: China "973" Gr<strong>an</strong>t 2006CB500805<br />
China NSFC Gr<strong>an</strong>t 30870803<br />
China "973" Gr<strong>an</strong>t 2007CB512205<br />
Sh<strong>an</strong>ghai Puji<strong>an</strong>g Talent Program 08PJ14016<br />
Title: Melatonin potentiates glycine currents through distinct PC-PLC/PKC signaling pathway in<br />
rat retinal g<strong>an</strong>glion cells<br />
Authors: *Z. WANG, W.-J. ZHAO, M. ZHANG, X.-L. YANG;<br />
Fud<strong>an</strong> Univ., Sh<strong>an</strong>ghai, China<br />
Abstract: In vertebrate retina, melatonin regulates various physiological functions. Our previous<br />
work demonstrated potentiation of glycine currents by melatonin in rat retinal g<strong>an</strong>glion cells<br />
(RGCs) via activating melatonin MT2 receptors. In <strong>the</strong> present work we investigated <strong>the</strong><br />
mech<strong>an</strong>isms underlying <strong>the</strong> melatonin effects by whole-cell patch-clamp techniques.<br />
Immunofluorescence double labeling showed that rat RGCs were solely immunoreactive to MT2<br />
receptors. Melatonin potentiates glycine currents <strong>an</strong>d promoted <strong>the</strong>ir desensitization, which were<br />
reversed by 4-P-PDOT, a specific <strong>an</strong>tagonist of MT2 receptors. These effects of melatonin were<br />
blocked by intracellular dialysis of GDP-β-S, a nonhydrolyzable inhibitor of G-proteins, <strong>an</strong>d<br />
<strong>an</strong>tibodies agonist regulator of G-protein signaling (RGS) 2 <strong>an</strong>d 4 proteins. In addition,<br />
preincubation with pertussis toxin (PTX) eliminated <strong>the</strong> melatonin-induced potentiation of<br />
glycine currents. All <strong>the</strong>se results suggest <strong>the</strong> melatonin effects were mediated by PTX-sensitive<br />
Gi/o proteins. Fur<strong>the</strong>rmore, D609, a phosphatidylcholine (PC)-specific phospholipase C (PLC)<br />
inhibitor, but not U73122, a phosphatidylinositol (PI)-PLC Iinhibitor, blocked <strong>the</strong> melatonin
effects. The protein kinase C (PKC) activator PMA potentiates <strong>the</strong> glycine currents, thus<br />
mimicking <strong>the</strong> effects of melatonin, <strong>an</strong>d in <strong>the</strong> presence of PMA melatonin no longer caused<br />
fur<strong>the</strong>r potentiation of <strong>the</strong> currents, whereas application of <strong>the</strong> PKC inhibitor<br />
bisindolylmaleimide IV (Bis IV) abolished <strong>the</strong> melatonin effects. The melatonin-induced<br />
potentiation of glycine currents was calcium-independent, as evidenced by <strong>the</strong> fact that <strong>the</strong><br />
effects were persisted when intracellular Ca 2+ were chelated by BAPTA. As shown by calcium<br />
imaging, melatonin induced no increase in intracellular Ca 2+ . Nei<strong>the</strong>r cAMP-PKA nor cGMP-<br />
PKG signaling pathways seemed to be involved because application of 8-Br-cAMP or 8-BrcGMP<br />
failed to mimic <strong>the</strong> melatonin effects. And both <strong>the</strong> PKA inhibitor H-89 <strong>an</strong>d <strong>the</strong> PKG<br />
inhibitor KT5823 didn’t block <strong>the</strong> melatonin-induced potentiation. In consequence, we conclude<br />
that <strong>the</strong> distinct PC-PLC/PKC signaling pathway is most likely involved in <strong>the</strong> melatonininduced<br />
potentiation of glycine currents of rat RGCs when Gi/o-coupled <strong>an</strong>d RGS2/4-regulated<br />
MT2 receptor are activated.<br />
Disclosures: Z. W<strong>an</strong>g, None; W. Zhao, None; M. Zh<strong>an</strong>g, None; X. Y<strong>an</strong>g, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.20/CC10<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Wayne State University<br />
Title: The role of Kv4.2 potassium ch<strong>an</strong>nels in retinal g<strong>an</strong>glion cells<br />
Authors: *M. BERI, L. CHEN, J. QU, K. MYHR;<br />
Biol. Sci., Wayne State Univ., Detroit, MI<br />
Abstract: Kv4.2 ch<strong>an</strong>nels carry <strong>the</strong> tr<strong>an</strong>sient A-type outward current, which recovers very<br />
rapidly <strong>from</strong> inactivation. Kv4.2 ch<strong>an</strong>nels are regulated by catecholamines, such as epinephrine<br />
<strong>an</strong>d dopamine. Kv4.2 currents regulate action potentials, <strong>an</strong>d when dendritically expressed also<br />
regulate <strong>the</strong> integration of inputs on <strong>the</strong> dendrites. In <strong>the</strong> retina, subtypes of retinal g<strong>an</strong>glion cells<br />
(RGCs) express Kv4.2. Kv4.2 is disproportionately expressed in RGCs with small dendritic<br />
fields <strong>an</strong>d highly br<strong>an</strong>ched dendrites <strong>an</strong>d in <strong>the</strong> intrinsically photosensitive retinal g<strong>an</strong>glion cells<br />
(ipRGCs). There<strong>for</strong>e Kv4.2 may regulate both <strong>for</strong>m <strong>an</strong>d non-<strong>for</strong>m visual functions in <strong>the</strong> brain<br />
by altering <strong>the</strong> processing of inputs <strong>an</strong>d by controlling <strong>the</strong> action potential outputs of RGCs. The<br />
purpose of this work is to determine <strong>the</strong> role of Kv4.2 in subtypes of RGCs. The expression of
Kv4.2 was determined by immunohistochemistry in morphologically identified mouse RGCs.<br />
RGC morphology was determined in flat-mounted retinas by patch-filling g<strong>an</strong>glion cells with<br />
dye after recording <strong>the</strong> electrical properties, or by YFP fluorescence. The properties of currents<br />
were determined via whole-cell <strong>an</strong>d per<strong>for</strong>ated-patch recordings in flat-mounted retinas. Action<br />
potentials were assayed in current-clamp configuration <strong>an</strong>d currents were assayed in voltageclamp<br />
configuration. Kv4.2 currents were blocked by 400 µM BaCl2, which specifically blocks<br />
Kv4.2 currents. BaCl2 decreased <strong>the</strong> average action potential firing frequency in response to onesecond<br />
current injections. The BaCl2 reduced <strong>the</strong> A-type current, but not <strong>the</strong> sustained K +<br />
current. In conclusion, subsets of RGCs that underlie <strong>for</strong>m vision or non-<strong>for</strong>m vision express<br />
Kv4.2. Blocking Kv4.2 ch<strong>an</strong>nels with 400 µM BaCl2 reduces action potential frequencies,<br />
supporting <strong>the</strong> hypo<strong>the</strong>sis that Kv4.2 regulates firing rates in some RGCs.<br />
Disclosures: M. Beri, None; L. Chen, None; J. Qu, None; K. Myhr, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.21/CC11<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t EY13528<br />
NSF IOS0818983<br />
Title: Characterization of retinal directional circuits during development<br />
Authors: *J. ELSTROTT, W. WEI, M. FELLER;<br />
Neurosci., UC Berkeley, Berkeley, CA<br />
Abstract: In <strong>the</strong> visual system, image motion is encoded by specific types of retinal g<strong>an</strong>glion<br />
cells referred to as direction selective g<strong>an</strong>glion cells (DSGCs). DSGCs respond preferentially to<br />
motion in <strong>the</strong> preferred but not <strong>the</strong> null direction. To target recordings <strong>from</strong> DSGCs prior to <strong>the</strong><br />
development of <strong>the</strong> light response, we used bacterial artificial chromosome (BAC) tr<strong>an</strong>sgenic<br />
mice expressing GFP in <strong>the</strong> subtype of ON-OFF DSGCs that prefer motion in <strong>the</strong> posterior<br />
visual direction. We confirmed that all GFP-expressing cells have bistratified dendritic<br />
architectures that costratify with <strong>the</strong> cholinergic plexuses in <strong>the</strong> ON <strong>an</strong>d OFF sublayer of inner<br />
plexi<strong>for</strong>m layer (IPL), which is characteristic of ON-OFF DSGCs. Fur<strong>the</strong>rmore, we per<strong>for</strong>med<br />
two photon targeted recordings in GFP-positive cells to examine <strong>the</strong>ir light responses, <strong>an</strong>d found
that nearly all <strong>the</strong> labeled cells have a strong directional preference <strong>for</strong> images moving towards<br />
<strong>the</strong> posterior direction. Last, we will describe recent progress using targeted recordings <strong>from</strong><br />
<strong>the</strong>se mice to examine <strong>the</strong> development of <strong>the</strong> directional circuits prior to eye opening.<br />
Disclosures: J. Elstrott, None; W. Wei, None; M. Feller, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.22/CC12<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: Korea Science <strong>an</strong>d Engineering Foundation Gr<strong>an</strong>t F01-2008-000-10060-0<br />
Title: The synaptic pattern of nicotinic acetylcholine receptor subunits α7 <strong>an</strong>d ß2 upon <strong>the</strong><br />
direction-selective retinal g<strong>an</strong>glion cells in developing mouse retina<br />
Authors: H.-J. KIM 1 , J.-Y. LEE 1 , K.-P. LEE 1 , *W. SONG 2 , C.-J. JEON 1 ;<br />
1 2<br />
Biol., Kyungpook Natl. Univ., Daegu, Republic of Korea; Inst. Brain Sci. & Technol., Inje<br />
Univ., Bus<strong>an</strong>, Republic of Korea<br />
Abstract: Direction-selective (DS) retinal g<strong>an</strong>glion cells (RGCs) respond strongly when a<br />
stimulus moves in <strong>the</strong>ir preferred direction, but respond little or not at all in <strong>the</strong> opposite<br />
direction. Starburst amacrine cells make cholinergic synapses upon DS RGCs <strong>an</strong>d <strong>the</strong> DS RGCs<br />
are known to sensitive to acetylcholine. In <strong>the</strong> search <strong>for</strong> <strong>an</strong>isotropies that might contribute to a<br />
directional preference of DS RGCs, we studied <strong>the</strong> distributions of nicotinic cholinergic receptor<br />
subtypes α7 <strong>an</strong>d ß2 upon <strong>the</strong> dendritic arbors of DS RGCs of <strong>the</strong> developing mouse [5, 10, 15<br />
days postnatal (PN)] retina with <strong>an</strong>tibody immunocytochemistry. DS RGCs were injected with<br />
Lucifer yellow <strong>an</strong>d <strong>the</strong> cells were identified by <strong>the</strong>ir characteristic morphology. The triplelabeled<br />
images of DS RGCs’ dendrites, α7 or ß2 receptors, <strong>an</strong>d postsynaptic density protein-95<br />
were visualized using confocal microscopy <strong>an</strong>d were reconstructed <strong>from</strong> high-resolution<br />
confocal images. Our results show evidence of asymmetry in α7 or ß2 receptor subunits<br />
examined on <strong>the</strong> dendritic arbors of both On <strong>an</strong>d Off layers of DS RGCs in all three<br />
developmental ages. The number of receptors was similar in all three ages. Thus, <strong>the</strong> spatial<br />
density of α7 or ß2 receptor subunits is decreased in older (15 PN) retina th<strong>an</strong> in younger (5 <strong>an</strong>d<br />
10 PN) ones. The data suggest that <strong>the</strong> directional selective may be equipped at early post natal<br />
stage <strong>an</strong>d <strong>the</strong> asymmetrical distribution suggests that <strong>the</strong> possibility of excitatory responds of<br />
preferred direction to acetylcholine depending on <strong>the</strong> direction of movement.
Disclosures: H. Kim, None; J. Lee, None; K. Lee, None; W. Song, None; C. Jeon, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.23/CC13<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t EY002048<br />
Title: Functional <strong>an</strong>d morphological maps of alpha retinal g<strong>an</strong>glion cell receptive fields<br />
Authors: *A. J. GARTLAND, P. B. DETWILER;<br />
Physiol. <strong>an</strong>d Biophysics, Univ. Washington, Seattle, WA<br />
Abstract: The receptive field (RF) of a retinal g<strong>an</strong>glion cell (RGC) is a map of visual space that<br />
shows where a stimulus will evoke a response. It is dependent upon several factors including <strong>the</strong><br />
intrinsic properties of <strong>the</strong> cell, <strong>the</strong> cell’s dendritic morphology <strong>an</strong>d <strong>the</strong> distribution of excitatory<br />
<strong>an</strong>d inhibitory synaptic inputs <strong>from</strong> bipolar cells <strong>an</strong>d amacrine cells, respectively. To evaluate <strong>the</strong><br />
contribution of <strong>the</strong>se factors to <strong>the</strong> <strong>for</strong>mation of mouse “alpha” RGC RFs, functional maps of <strong>the</strong><br />
RFs were constructed <strong>an</strong>d compared to <strong>the</strong> cell’s morphology recorded using “live” two-photon<br />
imaging.<br />
Functional maps were created by recording <strong>the</strong> spike responses to a small square of light (40 x<br />
40 µm) presented <strong>for</strong> 500 ms at different spatial locations. The position, size <strong>an</strong>d shape of <strong>the</strong> RF<br />
map were qu<strong>an</strong>tified <strong>an</strong>d compared to those of <strong>the</strong> dendritic arbor. Preliminary results show that<br />
<strong>the</strong> shapes of RFs are not well fit by a difference of Gaussi<strong>an</strong>s. Instead <strong>the</strong> RF is highly<br />
correlated with <strong>the</strong> dendritic arbor. This is demonstrated best by cells with highly asymmetric<br />
dendritic arbors which often have asymmetric RFs that reflect <strong>the</strong> dendritic morphology.<br />
To better underst<strong>an</strong>d <strong>the</strong> role of <strong>the</strong> intrinsic properties of <strong>the</strong> dendrites <strong>an</strong>d soma in <strong>the</strong><br />
<strong>for</strong>mation of <strong>the</strong> RF we recorded <strong>the</strong> synaptic currents to <strong>the</strong> cell <strong>an</strong>d created distinct functional<br />
RF maps of <strong>the</strong> excitatory <strong>an</strong>d inhibitory inputs. The preliminary results show that <strong>the</strong> synaptic<br />
currents also <strong>for</strong>m RF maps that correlate with <strong>the</strong> dendritic morphology, but <strong>the</strong> exact<br />
relationships between <strong>the</strong> RF maps <strong>an</strong>d <strong>the</strong> morphology await fur<strong>the</strong>r <strong>an</strong>alysis <strong>an</strong>d supportive<br />
data. Our hope is that by comparing <strong>the</strong> RF maps of both <strong>the</strong> synaptic inputs <strong>an</strong>d spike outputs<br />
<strong>from</strong> a single cell we will be able to make connections between <strong>the</strong> biophysical mech<strong>an</strong>isms of<br />
dendritic integration <strong>an</strong>d its functional role in <strong>the</strong> <strong>for</strong>mation of <strong>the</strong> cell’s RF.<br />
Disclosures: A.J. Gartl<strong>an</strong>d, None; P.B. Detwiler, None.
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.24/CC14<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t EY018204<br />
Title: Physiological <strong>an</strong>d <strong>an</strong>atomical characterization of ground squirrel retinal g<strong>an</strong>glion cells<br />
Authors: *S. H. LINDSTROM, S. H. DEVRIES;<br />
NU-FSM-Ophthalmology, Northwestern Univ., Chicago, IL<br />
Abstract: Parallel processing is <strong>an</strong> import<strong>an</strong>t strategy used throughout <strong>the</strong> central nervous<br />
system. In <strong>the</strong> retina, parallel processing begins at <strong>the</strong> cone synapse. In <strong>the</strong> ground squirrel<br />
(Spermophilus tridecemlineatus), <strong>the</strong> cone signal diverges to approximately 10 different types of<br />
cone bipolar cells (CBP), m<strong>an</strong>y of which have distinct responses to a light flash. We are<br />
interested in how <strong>the</strong> parallel pathways established by <strong>the</strong>se different CBP types contribute to <strong>the</strong><br />
responses of <strong>the</strong> ~20 different types of g<strong>an</strong>glion cells (GC). As a first step, we are classifying <strong>the</strong><br />
GCs of <strong>the</strong> ground squirrel retina by determining <strong>the</strong>ir light responses <strong>an</strong>d <strong>an</strong>atomy.<br />
GCs in flat-mounted retinas were recorded under dark-adapted conditions using whole-cell<br />
voltage clamp in <strong>the</strong> presence of QX314 (<strong>an</strong> internal Na ch<strong>an</strong>nel blocker). A LED stimulator<br />
provided full-field light flashes. Cell-attached recordings were used to classify each cell as ON,<br />
OFF, or ON-OFF <strong>an</strong>d, within <strong>the</strong>se subtypes, tr<strong>an</strong>sient or sustained. A whole cell recording was<br />
<strong>the</strong>n established, using a VHold of -70 mV to isolate excitatory inputs. During recording, cells<br />
were filled with Lucifer yellow or neurobiotin. Afterwards, <strong>the</strong> tissue was fixed, <strong>an</strong>d<br />
counterstained with <strong>an</strong>tibodies against calbindin (a marker of b2 <strong>an</strong>d b5 CBP), recoverin (a<br />
marker of b3 CBP), <strong>an</strong>d/or choline acetyltr<strong>an</strong>sferase (ChAT, a marker of starburst amacrine<br />
cells).<br />
We identified GCs that responded to <strong>the</strong> onset (ON; n=8 cells), offset (OFF; n=9), or both (ON-<br />
OFF; n=14) of a light step. Most GCs had tr<strong>an</strong>sient light responses (n=20); however, a few<br />
examples of sustained light responses were observed in all classes (n=9). As expected, <strong>the</strong><br />
dendrites of ON <strong>an</strong>d OFF GCs stratified in sublamina b <strong>an</strong>d sublamina a, respectively. GCs that<br />
costratified with calbindin-labeled CBP terminals typically had tr<strong>an</strong>sient light responses. There<br />
was no consistent trend in <strong>the</strong> stratification of ON sustained GCs. OFF sustained GCs frequently<br />
costratified with recoverin-labeled b3 CBP terminals. Most ON-OFF GCs had tr<strong>an</strong>sient<br />
responses at both onset <strong>an</strong>d offset of light <strong>an</strong>d had bistratified dendritic arbors costratifying with<br />
ChAT. Thus <strong>the</strong> ON-OFF cells are likely direction selective GCs.
We have previously identified <strong>the</strong> calbindin-labeled b2 <strong>an</strong>d b5 CBPs as carrying a tr<strong>an</strong>sient<br />
signal <strong>from</strong> cones to <strong>the</strong> inner retina. Our current results are consistent with <strong>the</strong> idea that some<br />
tr<strong>an</strong>siently responding GCs have dendrites that costratify with <strong>the</strong> terminals of <strong>the</strong>se CBPs. The<br />
recoverin positive OFF CBP has been identified as carrying a sustained signal, <strong>an</strong>d has axon<br />
terminals that costratify with some OFF sustained GCs.<br />
Disclosures: S.H. Lindstrom, None; S.H. DeVries, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.25/CC15<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH/NEI EY12141<br />
Multidisciplinary Visual Sciences Training Program, 5 T32 EY07128<br />
Title: White noise <strong>an</strong>alysis of in<strong>for</strong>mation tr<strong>an</strong>sfer at a mammali<strong>an</strong> cone terminal reveals<br />
different representations of <strong>the</strong> cone signal in different bipolar cell types<br />
Authors: *C. P. RATLIFF, S. H. DEVRIES;<br />
Ophthalmology, Nu-fsm-Ophthalmology, Chicago, IL<br />
Abstract: The brain’s computational power derives <strong>from</strong> its massively parallel in<strong>for</strong>mation<br />
pathways. In <strong>the</strong> retina, <strong>the</strong>se parallel pathways emerge at <strong>the</strong> cone terminal: a single cone<br />
releases glutamate onto 10 or more types of bipolar cell, <strong>an</strong>d each cell type <strong>for</strong>ms <strong>an</strong> independent<br />
array with a different representation of <strong>the</strong> visual image. The cone’s ribbon synapses are highly<br />
specialized structures with a specific goal: to represent membr<strong>an</strong>e voltage via exocytosis. On <strong>the</strong><br />
o<strong>the</strong>r h<strong>an</strong>d, different bipolar cell arrays may serve different goals. Indeed, we show that each<br />
bipolar cell type produces a distinct pattern of responses to <strong>the</strong> same cone signal.<br />
To study signal tr<strong>an</strong>sfer at <strong>the</strong> cone terminal, we recorded in whole cell voltage clamp <strong>from</strong> pairs<br />
of synaptically connected cones <strong>an</strong>d bipolar cells in slices of ground squirrel retina. During <strong>an</strong><br />
experiment, cone membr<strong>an</strong>e voltage was modulated with a spectrally white stimulus, <strong>an</strong>d <strong>the</strong><br />
resulting currents were recorded in <strong>the</strong> cone <strong>an</strong>d a post-synaptic bipolar cell. The 15-30 second<br />
stimuli were repeated to measure signal-to-noise ratio, <strong>an</strong>d coherence rates between stimulus <strong>an</strong>d<br />
bipolar cell response were also computed. Using reverse correlation techniques, we measured <strong>the</strong><br />
impulse-response function, or tr<strong>an</strong>sfer function, of <strong>the</strong> cone <strong>an</strong>d bipolar cell currents.
We found that both cone <strong>an</strong>d bipolar cell currents were high-pass filtered versions of <strong>the</strong><br />
stimulus, <strong>an</strong>d that some bipolar cell currents were also low-pass filtered. Moreover, <strong>the</strong> impulseresponse<br />
function of each bipolar cell type followed a different time course. Since different<br />
bipolar cell types exhibit different response patterns, we investigated <strong>the</strong> temporal frequencies at<br />
which each type tr<strong>an</strong>smits in<strong>for</strong>mation. We found that some bipolar cells could tr<strong>an</strong>smit reliably<br />
up to <strong>the</strong> maximum frequency used in <strong>the</strong> white noise stimulus: 500Hz. We conclude that<br />
different representations of <strong>the</strong> visual image emerge at <strong>the</strong> cone terminal.<br />
Disclosures: C.P. Ratliff, None; S.H. DeVries, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.26/CC16<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: EY012141<br />
Title: Glutamate spillover between mammali<strong>an</strong> cone photoreceptors<br />
Authors: *B. A. SZMAJDA, S. H. DEVRIES;<br />
Ophthalmology, Northwestern Univ., Chicago, IL<br />
Abstract: Direct cone-to-cone chemical synapses have not been described in <strong>the</strong> mammal.<br />
However, cones release a prodigious amount of glutamate at <strong>the</strong> end of a light step (~400<br />
vesicles c<strong>an</strong> fuse at <strong>the</strong> base of a 3-5 µm diameter terminal), <strong>an</strong>d also express glutamate<br />
tr<strong>an</strong>sporters at <strong>the</strong>ir terminals (detectable by <strong>the</strong>ir associated <strong>an</strong>ion conduct<strong>an</strong>ce). Though<br />
neighboring cones are isolated by Muller glial cell sheaths, we hypo<strong>the</strong>sized that <strong>the</strong> glutamate<br />
released by one cone could produce a response in neighboring cones via this tr<strong>an</strong>sporter<br />
conduct<strong>an</strong>ce, <strong>an</strong>d fur<strong>the</strong>r that such crosstalk may be a component of signal processing under<br />
physiological conditions.<br />
Adjacent cones were voltage-clamped in retinal slices or wholemounts obtained <strong>from</strong> conedomin<strong>an</strong>t<br />
ground squirrel. One cone, <strong>the</strong> ‘donor’, was stepped <strong>from</strong> -70 to 0 mV to elicit<br />
glutamate release. The current responses in <strong>the</strong> adjacent ‘acceptor’ cone were typically measured<br />
at a holding potential of -70 mV. Patch pipette solutions contained ei<strong>the</strong>r Cl - or SCN - , <strong>an</strong>d also<br />
Gramicidin D during experiments in which it was necessary to maintain <strong>the</strong> intracellular Cl -<br />
concentration. Light flashed produced by <strong>an</strong> LED stimulator had intensities between 15000 <strong>an</strong>d<br />
450,000 photon/µm 2 /s at 570 nm. Values are me<strong>an</strong> ± S.D.
Depolarizing a ‘donor’ cone elicited a two-component response in a neighboring cone. The fast<br />
component was due to electrical coupling, <strong>an</strong>d <strong>the</strong> slow component could be blocked in three<br />
ways: by Co 2+ <strong>an</strong>d Cd 2+ -containing external solution (n = 3), by substitution of Cs + <strong>for</strong> Na + in <strong>the</strong><br />
external solution (n = 2), or by glutamate tr<strong>an</strong>sporter blockers (TBOA: 76.0 ± 9.7% block, n =<br />
11; THA: 95.6 ± 9.8%, n = 5). The results suggest that <strong>the</strong> slow component is mediated by Ca 2+ -<br />
dependent release <strong>from</strong> <strong>the</strong> donor cone, which spills out of <strong>the</strong> synaptic cleft to activate a<br />
glutamate tr<strong>an</strong>sporter Cl - conduct<strong>an</strong>ce in <strong>the</strong> acceptor cone. In experiments on wholemounts with<br />
matched internal <strong>an</strong>d external Cl - , acceptor cone currents had me<strong>an</strong> amplitudes of -8.5 ± 7.2 pA<br />
(r<strong>an</strong>ge = 0-31 pA, n = 39), 20-80% rise times of 5.9 ± 1.9 ms, <strong>an</strong>d were often bidirectional.<br />
Acceptor cone currents were increased 4-8x when SCN - was substituted <strong>for</strong> Cl - in <strong>the</strong> pipette<br />
solution. During per<strong>for</strong>ated patch recordings, <strong>the</strong> tr<strong>an</strong>sporter current reversed at -26.8 ± 14.0 mV<br />
(n = 10). Finally, with SCN - in our pipette solutions, we have observed cone-cone glutamate<br />
spillover in response to light stimulation.<br />
In summary, we have observed a crosstalk between cone photoreceptors that is mediated by a<br />
glutamate tr<strong>an</strong>sporter Cl - conduct<strong>an</strong>ce. A tr<strong>an</strong>sporter reversal potential of approximately -25 mV<br />
is consistent with <strong>an</strong> excitatory role <strong>for</strong> cone-cone chemical tr<strong>an</strong>smission.<br />
Disclosures: B.A. Szmajda, None; S.H. DeVries, None.<br />
Poster<br />
557. Retinal Circuitry: Receptors <strong>an</strong>d Synaptic Interactions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 557.27/CC17<br />
Topic: D.04.b. Retinal circuitry: Receptors <strong>an</strong>d synaptic interactions<br />
Support: NIH Gr<strong>an</strong>t EY12141<br />
Title: Expression of AMPA <strong>an</strong>d kainate receptors in OFF bipolar cells of <strong>the</strong> ground squirrel<br />
retina<br />
Authors: *D. G. RYAN, S. H. DEVRIES;<br />
Ophthalmology, Northwestern Univ., Chicago, IL<br />
Abstract: Parallel processing begins in <strong>the</strong> mammali<strong>an</strong> visual system when a cone releases<br />
glutamate onto 8-10 <strong>an</strong>atomically distinct types of bipolar cells. The cone bipolar cells c<strong>an</strong> be<br />
divided functionally into On <strong>an</strong>d Off cell types expressing metabotropic <strong>an</strong>d ionotropic (non-<br />
NMDA) glutamate receptors, respectively. In <strong>the</strong> cone domin<strong>an</strong>t retina of <strong>the</strong> ground squirrel, 3<br />
of <strong>the</strong> 4 identified Off bipolar cell types use kainate receptors to receive <strong>the</strong> cone signal, <strong>the</strong> o<strong>the</strong>r
uses AMPA receptors. The predomin<strong>an</strong>ce of kainate receptors is surprising since, unlike AMPA<br />
receptors which recovery rapidly, kainate receptors recovery slowly following a desensitizing<br />
pulse of glutamate. In order to better underst<strong>an</strong>d <strong>the</strong> function of <strong>the</strong> different types of Off bipolar<br />
cells, we are characterizing <strong>the</strong> subunit composition of glutamate receptors in <strong>the</strong>se cells. As a<br />
first step toward this goal, we searched <strong>the</strong> database of ground squirrel genomic sequences with<br />
hum<strong>an</strong> <strong>an</strong>d rat mRNAs of each of <strong>the</strong> AMPA (GluR1-4) <strong>an</strong>d kainate (GluR5-7, KA1,2) receptor<br />
subunits. Exonic sequences were extracted <strong>an</strong>d spliced toge<strong>the</strong>r to produce longer contiguous<br />
tr<strong>an</strong>scripts which were aligned against <strong>the</strong>ir hum<strong>an</strong> <strong>an</strong>d rat homologues. PCR primers were<br />
designed to discriminate between each of <strong>the</strong> nine subunits in <strong>the</strong> ground squirrel. RNA prepared<br />
<strong>from</strong> <strong>the</strong> ground squirrel retina was used as template in a series of RT-PCR tests <strong>for</strong> <strong>the</strong> presence<br />
of each of <strong>the</strong> receptor subunits. PCR reaction products of <strong>the</strong> expected size were cloned <strong>an</strong>d<br />
sequenced. Multiple <strong>an</strong>notations containing individual exonic sequences of each of <strong>the</strong> nine<br />
subunits were found in <strong>the</strong> ground squirrel database. Compared to <strong>the</strong> hum<strong>an</strong> genome, <strong>the</strong> exonintron<br />
boundaries were well conserved. PCR reactions with retinal cDNA generated products of<br />
<strong>the</strong> expected size (0.5-1.7 kbp) <strong>for</strong> all but one (KA1) of <strong>the</strong> subunits. Tr<strong>an</strong>slations of <strong>the</strong> cloned<br />
sequences revealed >98% homology to <strong>the</strong>ir rat <strong>an</strong>d hum<strong>an</strong> counterparts. With respect to <strong>the</strong><br />
kainate receptor subunits, qualitative <strong>an</strong>alyses of <strong>the</strong> PCR reactions indicated <strong>an</strong> abund<strong>an</strong>ce of<br />
messages <strong>for</strong> GluR5, GluR6 <strong>an</strong>d KA2 in retinal tissue. The complete open reading frame of one<br />
of <strong>the</strong>se, GluR5, was obtained. The predomin<strong>an</strong>t <strong>for</strong>m contained <strong>the</strong> 15 aa insert in its N-terminal<br />
region <strong>an</strong>d <strong>the</strong> ‘b’ spliced <strong>for</strong>m at its C-terminus. Q/R editing was detected in ~50% of <strong>the</strong><br />
tr<strong>an</strong>scripts. Both <strong>the</strong> abund<strong>an</strong>ce of GluR5, GluR6, <strong>an</strong>d KA2 receptor subunits <strong>an</strong>d <strong>the</strong> paucity of<br />
KA1 are in concord<strong>an</strong>ce with <strong>the</strong> literature. The pharmacological diversity of <strong>the</strong> 3 types of<br />
kainate receptor-containing Off bipolar cells most likely results <strong>from</strong> combinations of GluR5,<br />
GluR6, <strong>an</strong>d KA2 subunits, although we c<strong>an</strong>not rule out <strong>the</strong> involvement of GluR7.<br />
Disclosures: D.G. Ry<strong>an</strong>, None; S.H. DeVries, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.1/CC18<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Australi<strong>an</strong> NH&MRC Gr<strong>an</strong>t 457337<br />
Australi<strong>an</strong> NH&MRC Gr<strong>an</strong>t 511967
Title: The impact of temporal frequency on motion integration by neurons in <strong>the</strong> middle<br />
temporal area of <strong>the</strong> marmoset<br />
Authors: *S. G. SOLOMON 1 , C. TAILBY 2 , N. J. COOREY 1 , A. J. CAMP 1 ;<br />
1 2<br />
Bosch Institute, Univ. of Sydney, Camperdown, Australia; Natl. Vision Res. Inst., Melbourne,<br />
Australia<br />
Abstract: Some cells in <strong>the</strong> middle temporal area (MT) of macaque are selective <strong>for</strong> <strong>the</strong> overall<br />
motion of a visual pattern, while o<strong>the</strong>rs respond to <strong>the</strong> motion of <strong>the</strong> components of that pattern.<br />
MT cells have broad temporal frequency (TF) tuning, yet it is not known how <strong>the</strong> motion<br />
integration depends on TF. We addressed this by making extracellular recordings <strong>from</strong> 53 cells<br />
in <strong>the</strong> middle-temporal region of two opiate-<strong>an</strong>aes<strong>the</strong>tised marmosets (Callithrix jacchus);<br />
direction tuning curves were measured <strong>for</strong> brief presentations of 50% contrast sinusoidal gratings<br />
drifting at each of four TF (3, 6, 12, & 25 Hz), <strong>an</strong>d plaids made of two gratings that drifted at <strong>the</strong><br />
same TF in directions 120 degrees apart. From <strong>the</strong>se responses we calculated a pattern-selectivity<br />
index [Smith MA, Majaj NJ, Movshon JA (2005) Dynamics of motion signaling by neurons in<br />
macaque area MT. Nat Neurosci, 8:220-8]. We first established that <strong>the</strong> pattern/component<br />
distinction holds in <strong>the</strong> marmoset, a diurnal New World primate: of 37 cells that responded to <strong>the</strong><br />
lowest TF stimuli, 19 were component-selective, 7 were pattern-selective, <strong>an</strong>d 3 responded to<br />
plaids but not <strong>the</strong> component gratings. Similar distributions of pattern-selectivity indices were<br />
found at <strong>the</strong> highest TF: of <strong>the</strong> 24 cells that responded, 14 were component selective <strong>an</strong>d 4 were<br />
pattern selective. Among individual cells motion integration was generally stable across <strong>the</strong> TF<br />
to which <strong>the</strong> cell responded - <strong>the</strong> index at 3 Hz predicted that obtained at 25 Hz (r = 0.86).<br />
However, 4 cells classified as pattern cells at 3 Hz were tuned to low TF <strong>an</strong>d were unresponsive<br />
at 25 Hz; 2 cells responsive but unclassifiable at 3 Hz preferred high TF <strong>an</strong>d were classified as<br />
pattern-selective cells at 25 Hz. We conclude that as a population MT cells are able to signal<br />
pattern motion at low <strong>an</strong>d high TF, but probably through <strong>the</strong> activity of different cells. Finally we<br />
note that <strong>the</strong> speed of <strong>the</strong> plaid ‘intersections’ is twice that of <strong>the</strong> component gratings: <strong>for</strong> plaids<br />
whose components drifted at 3 Hz <strong>the</strong> TF of <strong>the</strong> comparison grating (3 or 6 Hz) did not matter,<br />
but as TF increased <strong>the</strong> distinction became more import<strong>an</strong>t. The upshot was that more cells<br />
became pattern-selective if we made <strong>the</strong> classification with gratings that drifted at <strong>the</strong> same<br />
speed as <strong>the</strong> plaid.<br />
Disclosures: S.G. Solomon, None; C. Tailby, None; N.J. Coorey, None; A.J. Camp, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.2/CC19
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Michael Smith Foundation <strong>for</strong> Health Research<br />
Hum<strong>an</strong> Early Learning Partnership<br />
C<strong>an</strong>adi<strong>an</strong> Optometric Education Trust Fund<br />
Title: The cortical basis of attentive tracking deficits in amblyopia: An fMRI study<br />
Authors: *J. SECEN 1 , J. C. CULHAM 3 , D. GIASCHI 1,2 ;<br />
1 Dept. of Psychology, 2 Dept. of Ophthalmology, Univ. of British Columbia, V<strong>an</strong>couver, BC,<br />
C<strong>an</strong>ada; 3 Dept. of Psychology, Univ. of Western Ontario, London, ON, C<strong>an</strong>ada<br />
Abstract: Objectives: Unilateral amblyopia is a developmental visual disorder affecting<br />
approximately 3% of <strong>the</strong> population. It is characterized by reduced visual acuity in one eye, <strong>an</strong>d<br />
normal visual acuity in <strong>the</strong> o<strong>the</strong>r, fellow eye. In addition, deficits in several aspects of motion<br />
perception, including multiple-object tracking, have been reported. These deficits are found<br />
when stimuli are viewed not only through <strong>the</strong> amblyopic eye, but also through <strong>the</strong> fellow eye.<br />
The purpose of this project was to examine <strong>the</strong> cortical basis <strong>for</strong> <strong>the</strong>se multiple-object tracking<br />
deficits using functional magnetic reson<strong>an</strong>ce imaging (fMRI).<br />
Methods: Particip<strong>an</strong>ts with <strong>an</strong>d without amblyopia per<strong>for</strong>med a multiple-object tracking task<br />
inside a 3T Philips sc<strong>an</strong>ner. Particip<strong>an</strong>ts viewed a rear-projection screen through a mirror <strong>an</strong>d<br />
were instructed to fixate a central cross throughout each run. A run beg<strong>an</strong> with 9 identical balls<br />
moving r<strong>an</strong>domly around <strong>the</strong> display at a speed of 6 deg/s. Two seconds later, some of <strong>the</strong> balls<br />
(0, 1, 2 or 4) ch<strong>an</strong>ged colour briefly during movement to indicate that <strong>the</strong>y were <strong>the</strong> targets to be<br />
tracked. After 12 seconds of tracking, one of <strong>the</strong> nine balls was high-lighted <strong>an</strong>d <strong>the</strong> particip<strong>an</strong>t<br />
pressed a button to indicate whe<strong>the</strong>r or not this was one of <strong>the</strong> balls <strong>the</strong>y had been tracking. The<br />
task was completed monocularly using red <strong>an</strong>d green filters to m<strong>an</strong>ipulate which eye viewed <strong>the</strong><br />
display throughout each run. Two or three runs were collected <strong>for</strong> each eye. A whole-brain<br />
<strong>an</strong>alysis was used to compare <strong>the</strong> cortical areas activated by passive viewing (0 balls tracked)<br />
versus tracking (1, 2 or 4 balls tracked) in all particip<strong>an</strong>ts. This was used to guide post-hoc<br />
region of interest (ROI) <strong>an</strong>alyses to determine <strong>the</strong> percent signal ch<strong>an</strong>ge in each particip<strong>an</strong>t as a<br />
function of <strong>the</strong> number of balls tracked.<br />
Results: Whole-brain <strong>an</strong>alysis <strong>for</strong> <strong>the</strong> attentive tracking contrast revealed bilateral activation in<br />
area MT <strong>an</strong>d in posterior parietal cortex (PPC), including <strong>the</strong> intraparietal sulcus <strong>an</strong>d superior<br />
parietal lobule. In <strong>the</strong> PPC ROI, as <strong>the</strong> number of balls tracked increased, <strong>the</strong> percent signal<br />
ch<strong>an</strong>ge increased <strong>for</strong> each particip<strong>an</strong>t (p < .05). In <strong>the</strong> amblyopia group, however, <strong>the</strong> average<br />
percent signal ch<strong>an</strong>ge was less th<strong>an</strong> in <strong>the</strong> control group (p < .05), except in <strong>the</strong> passive viewing<br />
condition. There was no difference between amblyopic <strong>an</strong>d fellow eyes. In <strong>the</strong> MT ROI, <strong>the</strong>re<br />
were no signific<strong>an</strong>t differences between groups or as a function of <strong>the</strong> number of balls tracked.<br />
Conclusions: These results implicate <strong>the</strong> PPC in <strong>the</strong> multiple-object tracking deficit in<br />
amblyopia, <strong>an</strong>d suggest that <strong>the</strong> deficits found in amblyopia extend to higher-level processing in<br />
extrastriate cortex.<br />
Disclosures: J. Secen, None; J.C. Culham, None; D. Giaschi, None.
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.3/CC20<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH CTSA 5 TL1 RR024135-02<br />
NIH Gr<strong>an</strong>t EY10287<br />
NIA Gr<strong>an</strong>t AG17596<br />
Title: MST neuronal responses to optic flow during a global motion steering task: Does<br />
behavioral signific<strong>an</strong>ce trump stimulus selectivity?<br />
Authors: *M. JACOB, C. DUFFY;<br />
Univ. of Rochester, Rochester, NY<br />
Abstract: We have previously shown that attention has subst<strong>an</strong>tial effects on MST neuronal<br />
responses to optic flow altering both response amplitude <strong>an</strong>d stimulus selectivity. We now report<br />
<strong>the</strong> preliminary results of studies in which MST neurons are recorded while <strong>the</strong> monkey<br />
per<strong>for</strong>ms a delayed match to sample active steering task. In this task, <strong>the</strong> monkey must pl<strong>an</strong> <strong>an</strong>d<br />
execute a path of simulated self-movement that tr<strong>an</strong>s<strong>for</strong>ms its heading direction to match that in<br />
a remembered stimulus.<br />
The monkey maintains centered fixation while viewing one of 16 optic flow sample stimuli (500<br />
ms) with eight inward or outward heading directions distributed around <strong>the</strong> fixation point. The<br />
sample is followed by a 1000 ms delay period <strong>an</strong>d <strong>the</strong>n a new optic flow stimulus containing one<br />
of <strong>the</strong> o<strong>the</strong>r heading directions. After 500 ms <strong>the</strong> monkey is allowed free viewing as it uses a<br />
rotational joystick to steer <strong>the</strong> heading direction to match <strong>the</strong> location in <strong>the</strong> sample stimulus.<br />
During <strong>the</strong> steering task <strong>the</strong> monkey must select <strong>the</strong> most efficient path around <strong>the</strong> circle <strong>from</strong><br />
<strong>the</strong> starting position to <strong>the</strong> sample position, <strong>an</strong>d <strong>the</strong>n halt rotation when <strong>the</strong> display matches <strong>the</strong><br />
sample position.<br />
After 18 months of training, <strong>the</strong> monkey per<strong>for</strong>ms this task at 60% correct (ch<strong>an</strong>ce per<strong>for</strong>m<strong>an</strong>ce<br />
is 12.5%). Most errors are wrong decisions about <strong>the</strong> initial rotation direction. The monkey<br />
learned to use <strong>the</strong> global motion pattern, demonstrated by responding correctly at <strong>the</strong> starting<br />
position in 72% <strong>an</strong>d 71% of trials <strong>for</strong> inward <strong>an</strong>d outward optic flow, respectively. Response<br />
latency was longer in trials with steering by inward optic flow, 410 ms versus 357 ms <strong>for</strong><br />
outward optic flow.
We have recorded neurons in one hemisphere of one rhesus monkey <strong>an</strong>d observed a robust<br />
activation of neuronal activity at <strong>the</strong> onset of <strong>the</strong> steering stimulus. This activation does not<br />
depend on <strong>the</strong> heading stimulus presented in <strong>the</strong> sample or <strong>the</strong> steering start position. The<br />
activation of MST neurons at <strong>the</strong> beginning of steering, when <strong>the</strong> task dem<strong>an</strong>ds both perceptual<br />
decision making <strong>an</strong>d action pl<strong>an</strong>ning, suggests that MST neurons are involved in guiding<br />
behavioral responses to optic flow stimuli. Thus, MST may play a role in motor control as well<br />
as perceptual processing.<br />
Disclosures: M. Jacob, None; C. Duffy, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.4/CC21<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH Gr<strong>an</strong>t EY10287<br />
NIH Gr<strong>an</strong>t AG17596<br />
Title: Attentional modulation of mst neuronal response interactions with combined optic flow<br />
<strong>an</strong>d object motion cues about self-movement<br />
Authors: *C. J. DUFFY, S. A. KISHORE, N. SATO, N. HORNICK, W. K. PAGE;<br />
Univ. Of Rochester Med. Ctr., Rochester, NY<br />
Abstract: Self-movement is guided by in<strong>for</strong>mation derived <strong>from</strong> <strong>the</strong> global pattern of visual<br />
motion in optic flow <strong>an</strong>d <strong>the</strong> visual motion of salient objects generated by relative movement.<br />
Previous studies in macaque medial superior temporal cortex (MST) demonstrated that MST<br />
neurons are sensitive to <strong>an</strong>d selective <strong>for</strong> <strong>the</strong> self-movement heading simulated by both optic<br />
flow (Duffy <strong>an</strong>d Wurtz, 1995) <strong>an</strong>d object motion (Log<strong>an</strong> <strong>an</strong>d Duffy, 2006). We have now<br />
focused on <strong>the</strong> attentional modulation of responsiveness to naturalistically combined optic flow<br />
<strong>an</strong>d object motion stimuli.<br />
.<br />
We recorded single neuron activity in macaque area MST while <strong>the</strong> <strong>an</strong>imal maintained centered<br />
fixation on a 90 X 90 deg. rear projection screen. In <strong>the</strong> first experiment, <strong>the</strong> monkey viewed<br />
optic flow alone, object motion alone, <strong>an</strong>d a systematic set of 16 combinations of <strong>the</strong> optic flow<br />
<strong>an</strong>d object motion. We compared <strong>the</strong> summed firing rates evoked by optic flow alone <strong>an</strong>d object
motion alone to that evoked by <strong>the</strong> corresponding combined stimuli. The wide variety of<br />
combination stimuli used in <strong>the</strong>se studies yielded sub-additive interactions whenever subst<strong>an</strong>tial<br />
excitatory or inhibitory responses were combined.<br />
.<br />
In <strong>the</strong> second experiment, we engaged <strong>the</strong> monkey in actively steering optic flow, object motion,<br />
or combined stimuli <strong>from</strong> <strong>an</strong> eccentric heading toward <strong>the</strong> center of <strong>the</strong> screen. In this steering<br />
task, response interactions shifted toward greater additivity: when both cues were attended<br />
additivity was greater <strong>the</strong>n when only one of <strong>the</strong> cues was attended.<br />
.<br />
In <strong>the</strong> third experiment, we engaged <strong>the</strong> monkey in actively steering <strong>the</strong> simulated heading of<br />
self-movement based on ei<strong>the</strong>r optic flow or object motion. In this task, individual neurons<br />
showed selective activation, similar to a winner-take-all mech<strong>an</strong>ism. Most commonly, neurons<br />
were activated during attention to <strong>the</strong> cue that results in greater responsiveness when <strong>the</strong> two<br />
cues were presented alone.<br />
.<br />
We conclude that MSTd neurons integrate visual cues about self-movement in a m<strong>an</strong>ner that<br />
reflects stimulus <strong>an</strong>d task attributes as well as <strong>the</strong> intrinsic response properties of each neuron.<br />
We see a continuum of neuronal response interactions <strong>from</strong> integrative (additive) to<br />
discriminative (WTA) under behavioral-task control.<br />
Disclosures: C.J. Duffy, Cerebral Assessment Systems, Inc, A. Employment (full or parttime);<br />
S.A. Kishore, None; N. Sato, None; N. Hornick, None; W.K. Page, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.5/CC22<br />
Topic: D.04.n. Visual motion: Behavioral studies<br />
Support: NIH Gr<strong>an</strong>t AG17596<br />
NIH Gr<strong>an</strong>t EY10287<br />
NIH CTSA 5TL1 RR024135-02<br />
Title: Attention suppresses visual motion evoked potentials in older adults: Neurophysiological<br />
evidence <strong>for</strong> age-related narrowing of <strong>the</strong> attentional spotlight
Authors: *A. M. MONACELLI, M. S. JACOB, C. J. DUFFY;<br />
Univ. Rochester, Rochester, NY<br />
Abstract: The large-field pattern of visual motion in optic flow in<strong>for</strong>ms us about our heading of<br />
self-movement. Optic flow <strong>an</strong>alysis relies on large receptive field neurons in dorsal extrastriate<br />
visual cortex identified in monkeys <strong>an</strong>d is reflected in posterior predomin<strong>an</strong>t scalp recorded<br />
visual motion evoked potentials (VMEPs) in hum<strong>an</strong>s. We previously showed that VMEPs are<br />
delayed in aging <strong>an</strong>d diminished in early Alzheimer’s disease. We have now studied VMEPs in<br />
young (YN) <strong>an</strong>d older (ON) normal subjects during a selective attention task to assess whe<strong>the</strong>r<br />
impaired attentional control of perceptual processing might contribute to cognitive aging.<br />
EPs were recorded in 4 stimulus paradigms. 1) Alternating presentation of radial optic flow (90<br />
X 90 deg) simulating a straight ahead heading, <strong>an</strong>d r<strong>an</strong>dom dot motion, with 20% catch trials<br />
consisting of interleaved FOE offsets 20 degrees left or right of fixation requiring a left or right<br />
button press. 2) Alternating presentation of central visual three-letter words (12 X 4 degrees),<br />
<strong>an</strong>d a r<strong>an</strong>dom dot masking stimulus, with 20% catch trials consisting of <strong>an</strong> interleaved left or<br />
right-sided non-letter symbol requiring a left or right button press. 3) Both optic flow <strong>an</strong>d word<br />
paradigms superimposed at two different alternation frequencies (1.57 Hz <strong>an</strong>d 1.11 Hz) <strong>an</strong>d<br />
requiring ei<strong>the</strong>r a bush button response to <strong>the</strong> optic flow only, or 4) to <strong>the</strong> word stimuli only.<br />
Both <strong>the</strong> optic flow <strong>an</strong>d word stimuli evoked N200 responses in posterior recording sites with<br />
peak responses obtained <strong>from</strong> ON subjects having smaller amplitudes <strong>an</strong>d delayed latencies. In<br />
all subjects, <strong>the</strong> largest response amplitudes are seen when ei<strong>the</strong>r optic flow or word trigrams are<br />
presented alone, with slightly smaller responses to <strong>the</strong> attended stimulus in <strong>the</strong> combined<br />
conditions. In YN subjects, responses to <strong>the</strong> unattended stimuli are only slightly smaller th<strong>an</strong><br />
those evoked by <strong>the</strong> attended stimuli. In contrast, ON subjects do not show cortical responses to<br />
<strong>the</strong> unattended optic flow stimulus while attending to <strong>the</strong> word stimulus, whereas <strong>the</strong>y do show<br />
responses to <strong>the</strong> unattended word stimulus while attending to optic flow.<br />
We speculate that ON subjects’ lack of N200 responses to optic flow while <strong>the</strong>y are attending to<br />
word stimuli reflects <strong>the</strong> attentional suppression of peripheral motion responses. This may be<br />
linked to a narrowing of <strong>the</strong> attentional response field in aging.<br />
Disclosures: A.M. Monacelli, None; M.S. Jacob, None; C.J. Duffy, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.6/CC23<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms
Support: NIH Gr<strong>an</strong>t AG17596<br />
NIH Gr<strong>an</strong>t AG20647<br />
Title: Impaired visual cue integration in Alzheimer's disease: All roads lead to roam<br />
Authors: *M. E. MAPSTONE, M. ABROMS, C. J. DUFFY;<br />
Neurol., Univ. of Rochester Sch. of Med., Rochester, NY<br />
Abstract: Independent navigation relies on <strong>the</strong> visual integration of self-movement cues in optic<br />
flow <strong>an</strong>d <strong>the</strong> relative movement of discrete objects in support of heading perception. We have<br />
previously shown that aging impairs <strong>the</strong> perception of heading <strong>from</strong> object motion <strong>an</strong>d that AD<br />
patients are fur<strong>the</strong>r impaired by <strong>an</strong> inability to effectively combine optic flow <strong>an</strong>d object motion<br />
cues. We now test <strong>the</strong> hypo<strong>the</strong>sis that incongruent object motion, simulating <strong>the</strong> movement of <strong>an</strong><br />
<strong>an</strong>imate object, confounds self-movement perception in AD by altering <strong>the</strong> perception of heading<br />
<strong>from</strong> optic flow.<br />
We studied 132 subjects. Ninety-nine served as control subjects; 33 young (YNC), 27 middle<br />
aged (MNC), <strong>an</strong>d 39 older adults (ONC), 20 had mild cognitive impairment (MCI), <strong>an</strong>d 13 had<br />
early Alzheimer’s disease (EAD). All subjects were administered psychophysical measures of<br />
optic flow <strong>an</strong>d object motion perception as well as a real-world navigational test battery.<br />
Subjects also per<strong>for</strong>med self-movement heading estimation <strong>from</strong> object motion, optic flow, both<br />
stimuli combined as congruent cues indicating <strong>the</strong> same heading, or as incongruent stimuli<br />
indicating different headings as seen when <strong>an</strong>imate objects cross <strong>the</strong> path of a moving observer.<br />
Subjects used a steering wheel to move a marker to indicate <strong>the</strong> location of <strong>the</strong> perceived<br />
heading.<br />
All control groups accurately indicate heading using object motion or optic flow alone <strong>an</strong>d in<br />
congruently superimposed combinations; <strong>the</strong> EAD group shows signific<strong>an</strong>t difficulty using <strong>the</strong><br />
object motion (p
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.7/CC24<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Penn State SSRI<br />
Title: Spatiotemporal tuning of cortical responses to motion in children with<br />
amblyopia/strabismus <strong>an</strong>d cortical visual impairment<br />
Authors: *R. O. GILMORE 1 , J. M. WEINSTEIN 2 , L. M. TASHIMA 2 , J. D. FESI 1 ;<br />
1 Penn State Univ., University Park, PA; 2 Ophthalmology, Penn State Univ., Hershey, PA<br />
Abstract: Sensitivity to directionally coherent global motion emerges early in development but<br />
has a prolonged developmental time course. We examined visual evoked potentials (VEPs) in<br />
children with cortical visual impairment (CVI) in response to dynamic r<strong>an</strong>dom dot motion<br />
patterns that varied in coherence at a predetermined frequency. The goal was to compare <strong>the</strong><br />
spatiotemporal tuning of <strong>the</strong>se responses to those collected <strong>from</strong> 2 control samples: (a) a<br />
neurologically <strong>an</strong>d visually normal group <strong>an</strong>d, (b) neurologically normal patients with mild<br />
strabismus/amblyopia. Binocular responses were recorded over <strong>the</strong> occipital midline (Oz) <strong>an</strong>d<br />
two lateral sites (O1 <strong>an</strong>d O2). Analysis centered on phase- locked responses at <strong>the</strong> coherence<br />
modulation frequency (F1: 0.8 Hz) <strong>an</strong>d <strong>the</strong> dot update frequency (F2: 20 or 30 Hz). Coherence<br />
modulation responses in normal controls were robust at both slow (2 deg/s) <strong>an</strong>d moderate (8<br />
deg/s) speeds. Dot update responses increased at larger speeds <strong>an</strong>d with higher update rates, in<br />
accord with previous data <strong>from</strong> normal inf<strong>an</strong>ts <strong>an</strong>d adults. In contrast, patients with CVI showed<br />
diminished responses at <strong>the</strong> both <strong>the</strong> coherence modulation frequency <strong>an</strong>d <strong>the</strong> dot update<br />
frequency. In <strong>the</strong> neurologically normal strabismus/amblyopia group, motion responses were<br />
diminished at <strong>the</strong> coherence modulation frequency but less so at <strong>the</strong> dot update frequency. Taken<br />
toge<strong>the</strong>r, <strong>the</strong> data suggest that cortical responses to motion are altered in CVI <strong>an</strong>d<br />
amblyopia/strabismus in complex ways. Both global <strong>an</strong>d local motion responses are impaired in<br />
patients with CVI, while local processing is relatively spared in amblyopia/strabismus. A more<br />
detailed underst<strong>an</strong>ding of how visual disorders alter cortical spatiotemporal tuning <strong>for</strong> motion<br />
may contribute to improved diagnosis <strong>an</strong>d treatment.<br />
Disclosures: R.O. Gilmore, None; J.M. Weinstein, None; L.M. Tashima, None; J.D. Fesi,<br />
None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.8/CC25<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Wellcome Trust<br />
Title: Distinguishing mech<strong>an</strong>isms of direction selectivity in population models of V1 using<br />
cross-correlation<br />
Authors: *P. M. BAKER, W. BAIR;<br />
Dept of Physiology, Anat. <strong>an</strong>d Genet., Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom<br />
Abstract: Direction selectivity (DS) is a fundamental physiological property of neurons in<br />
primary visual cortex of <strong>the</strong> macaque. Despite intensive study, basic questions remain<br />
un<strong>an</strong>swered: what is <strong>the</strong> nature of <strong>the</strong> subunits or pre-synaptic inputs? How m<strong>an</strong>y functional<br />
inputs drive one DS complex cell? What is <strong>the</strong> nature of <strong>the</strong> nonlinearity, <strong>for</strong> example, is it<br />
facilitatory or suppressive? To develop experimental techniques <strong>for</strong> <strong>an</strong>swering <strong>the</strong>se questions,<br />
we have implemented <strong>an</strong> ensemble of plausible models of direction selectivity based on spiking<br />
cells in <strong>the</strong> visual pathway. We show how cross-correlation c<strong>an</strong> succeed, <strong>an</strong>d sometimes fail, to<br />
distinguish <strong>the</strong>se models.<br />
We built network models starting <strong>from</strong> spiking LGN cells <strong>an</strong>d leading to complex DS cells. The<br />
models varied in network architecture, i.e., which physiological classes of cells were involved<br />
(non-orientation-tuned, orientation-tuned simple, or DS simple cells) <strong>an</strong>d <strong>the</strong> nature of <strong>the</strong> DS<br />
mech<strong>an</strong>ism (<strong>the</strong> type <strong>an</strong>d location of <strong>the</strong> time delay <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong> nonlinearity was facilitatory<br />
or suppressive). In one model, complex DS cells were built directly <strong>from</strong> pairs of spatially offset<br />
LGN cells that had temporally offset responses, using a multiplicative non-linearity. In <strong>an</strong>o<strong>the</strong>r,<br />
<strong>the</strong>y were built using <strong>an</strong> intervening layer of simple cells where <strong>the</strong> temporal offsets were<br />
introduced late, in nonlinear subunits attributed to <strong>the</strong> dendrites of <strong>the</strong> complex DS cell. For each<br />
model, we computed shuffle-corrected cross-correlograms (CCGs) of spike trains <strong>from</strong> various<br />
pairs of units that would in principle be accessible to extracellular recording. Visual stimuli were<br />
typically slowly drifting sinusoidal gratings.<br />
We found that cross-correlation could distinguish models where o<strong>the</strong>r methods, such as<br />
computing 2D spatial interaction maps, failed. In particular, facilitatory <strong>an</strong>d suppressive DS<br />
mech<strong>an</strong>isms generated similar 2D maps but distinct shapes in <strong>the</strong> CCGs. Also, cross-correlation<br />
is well-suited to assess where <strong>the</strong> DS time delay arises. In models where temporal offsets (or<br />
phase differences) are already present in <strong>the</strong> pre-synaptic inputs to <strong>the</strong> DS unit, peaks in <strong>the</strong><br />
CCGs between spike trains <strong>for</strong> all inputs <strong>an</strong>d <strong>the</strong> DS output occur at <strong>the</strong> same time, with a slight<br />
offset <strong>from</strong> zero, related to <strong>the</strong> synaptic integration time. In models where <strong>the</strong> delay was assigned<br />
to dendritic subunits within <strong>the</strong> DS cell, <strong>the</strong> peak of <strong>the</strong> CCGs <strong>for</strong> some of <strong>the</strong> inputs was shifted<br />
to reflect <strong>the</strong> delay. We will address potential challenges to employing <strong>the</strong>se techniques in<br />
electrophysiological recording.
Disclosures: P.M. Baker, None; W. Bair, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.9/CC26<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Howard Hughes Medical Institute<br />
NIH Gr<strong>an</strong>t EY03878<br />
Title: Temporal dynamics <strong>an</strong>d stimulus dependency of spike timing correlation in cortical area<br />
MT<br />
Authors: *X. HUANG 1 , S. G. LISBERGER 2 ;<br />
1 2<br />
Dept. of Physiol., Univ. of Wisconsin, Madison, WI; Dept. of Physiology, Keck Ctr., UCSF;<br />
HHMI, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: To gain a better underst<strong>an</strong>ding of <strong>the</strong> dynamic properties of functional circuitry in<br />
cortical networks, we examined <strong>the</strong> time-course <strong>an</strong>d stimulus-dependency of spike train crosscorrelogram<br />
(CCG) in visual area MT. We used multiple electrodes to record simult<strong>an</strong>eously<br />
<strong>from</strong> 2 to 5 MT neurons in awake fixating monkeys. Visual stimuli were ei<strong>the</strong>r textures of<br />
r<strong>an</strong>dom dots or square-wave gratings that were stationary <strong>for</strong> ~250 ms <strong>an</strong>d <strong>the</strong>n moved <strong>for</strong> 500<br />
ms. To <strong>an</strong>alyze <strong>the</strong> time-course of spike timing correlation, we computed a series of CCGs based<br />
on spike trains within a 60-ms time window sliding in 10-ms steps. M<strong>an</strong>y MT neuron pairs<br />
displayed a positive CCG that peaked at or near <strong>the</strong> zero time-lag between <strong>the</strong> spike trains of <strong>the</strong><br />
two neurons. The central CCG peak based on <strong>the</strong> neural activity ~150 ms be<strong>for</strong>e <strong>the</strong> motion<br />
response onset was wider th<strong>an</strong> that based on <strong>the</strong> tr<strong>an</strong>sient responses to <strong>the</strong> onset of <strong>the</strong> stationary<br />
stimulus; <strong>the</strong> CCG peak narrowed again following <strong>the</strong> motion response onset. As <strong>the</strong> 60-ms<br />
<strong>an</strong>alysis window progressed through <strong>the</strong> initial motion response period of ~100 ms, <strong>the</strong> CCG<br />
gradually developed negative fl<strong>an</strong>ks around <strong>the</strong> positive central peak; after full development, <strong>the</strong><br />
negative fl<strong>an</strong>ks remained stable throughout <strong>the</strong> motion response period. To <strong>an</strong>alyze <strong>the</strong> stimulus<br />
dependency of CCG, we computed CCG based on MT responses during <strong>the</strong> 500-ms motion<br />
presentation period. MT CCGs were stimulus-dependent <strong>an</strong>d usually had <strong>the</strong> narrowest <strong>an</strong>d, in<br />
some cases, <strong>the</strong> highest central peak when MT neurons were activated by <strong>the</strong>ir preferred visual<br />
stimuli. As <strong>the</strong> stimulus direction shifted to <strong>the</strong> least preferred direction, <strong>the</strong> width of <strong>the</strong> CCG<br />
peak increased monotonically; <strong>the</strong> height of <strong>the</strong> CCG peak initially decreased <strong>an</strong>d <strong>the</strong>n increased.
Fur<strong>the</strong>r, <strong>the</strong> central CCG peak was narrower when MT neurons were 1) activated by r<strong>an</strong>dom dots<br />
moving at <strong>the</strong> pair-preferred speed versus <strong>the</strong> less preferred speeds; 2) driven by moving r<strong>an</strong>dom<br />
dots versus firing spont<strong>an</strong>eously in <strong>the</strong> dark; or 3) stimulated by high- versus low-contrast<br />
drifting gratings. Our results suggest that <strong>the</strong> functional connectivity of neurons in area MT<br />
undergoes dynamic ch<strong>an</strong>ges in response to visual stimuli. The temporal evolution of negative<br />
fl<strong>an</strong>ks in CCG implies that cortical inhibition is sometimes shared by neurons in MT; <strong>the</strong><br />
inhibition is recruited following a delay after motion response onset <strong>an</strong>d helps to fur<strong>the</strong>r enh<strong>an</strong>ce<br />
<strong>the</strong> temporal precision of correlated spikes.<br />
Disclosures: X. Hu<strong>an</strong>g, None; S.G. Lisberger, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.10/CC27<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH gr<strong>an</strong>ts EY019087<br />
NIH gr<strong>an</strong>ts EY016178<br />
Title: Noise correlations in area MSTd during multi-sensory heading perception<br />
Authors: *D. E. ANGELAKI 1 , Y. GU 1 , S. FOK 2 , A. SUNKARA 2 , S. LIU 1 , G. C.<br />
DEANGELIS 3 ;<br />
1 Dept Anat Neurobiol, Washington Univ. Sch. Med., Saint Louis, MO; 2 Biomed. Engin. Dept.,<br />
Washington Univ., Saint Louis, MO; 3 Brain <strong>an</strong>d Cognitive Sci., Univ. of Rochester, Rochester,<br />
NY<br />
Abstract: Behavioral per<strong>for</strong>m<strong>an</strong>ce is based on population activity, <strong>an</strong>d <strong>the</strong> accuracy of<br />
population coding is constrained by correlated noise among neurons. Weak but signific<strong>an</strong>t interneuronal<br />
noise correlations have been described in m<strong>an</strong>y areas (e.g. V1, MT), suggesting that<br />
small noise correlations maybe prevalent among sensory cortical neurons. Here we examine<br />
noise correlations in multi-sensory area MSTd. More th<strong>an</strong> half of MSTd neurons are tuned to<br />
direction of self-motion based on both visual (optic flow) <strong>an</strong>d vestibular inputs <strong>an</strong>d have been<br />
implicated in multisensory integration <strong>for</strong> heading perception.<br />
Eighty-two pairs of single-units were recorded <strong>from</strong> area MSTd simult<strong>an</strong>eously using multiple<br />
electrodes. Ano<strong>the</strong>r 35 pairs were acquired by isolating a second unit off-line <strong>from</strong> data recorded
previously <strong>from</strong> a single electrode. Data were collected <strong>from</strong> <strong>an</strong>imals (n=3, ‘trained’ group)<br />
trained to per<strong>for</strong>m a fine heading discrimination task <strong>an</strong>d <strong>from</strong> untrained <strong>an</strong>imals (n=3, ‘naïve’<br />
group) per<strong>for</strong>ming a fixation task. In all cases, two stimulus conditions were presented: a<br />
‘vestibular’ condition in which <strong>an</strong>imals were tr<strong>an</strong>slated along different heading directions by a<br />
motion plat<strong>for</strong>m <strong>an</strong>d a ‘visual’ condition in which <strong>the</strong> same headings were simulated using optic<br />
flow. Each movement followed a 2s Gaussi<strong>an</strong> velocity profile <strong>an</strong>d noise correlations were<br />
computed <strong>from</strong> spike counts measured during <strong>the</strong> middle 1s of <strong>the</strong> stimulus period.<br />
ANCOVA <strong>an</strong>alysis revealed that pair-wise noise correlations depended signific<strong>an</strong>tly on dist<strong>an</strong>ce<br />
between neurons in a pair (p
Authors: *Y. GU 1 , G. C. DEANGELIS 2 , D. E. ANGELAKI 1 ;<br />
1 2<br />
Wash Univ. Sch. Med., St.Louis, MO; Dept. of Brain <strong>an</strong>d Cognitive Sciences,, Univ. of<br />
Rochester, Rochester, NY<br />
Abstract: Heading perception is a multi-sensory process that involves both visual (optic flow)<br />
<strong>an</strong>d vestibular (inertial motion) signals. We previously reported that <strong>the</strong>se two signals in<br />
extrastriate visual area MSTd are correlated, on a trial by trial basis, with monkeys’ perceptual<br />
judgments during per<strong>for</strong>m<strong>an</strong>ce of a heading discrimination task. We also used microstimulation<br />
to test <strong>for</strong> causal links between MSTd activity <strong>an</strong>d heading perception. We reported previously<br />
(SFN 2008) that microstimulation signific<strong>an</strong>tly biased monkeys’ perception based on optic flow,<br />
consistent with previous findings (Britten & v<strong>an</strong> Wezel, 1998, 2002). However, vestibular<br />
signals in MSTd were found to be weakly clustered <strong>an</strong>d microstimulation did not produce<br />
signific<strong>an</strong>t effects on vestibular heading judgments. To overcome this problem, we applied<br />
reversible inactivation to temporarily suppress neural activity in larger regions of MSTd <strong>an</strong>d<br />
examined effects on per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> heading discrimination task.<br />
Methods: Heading stimuli were defined by optic flow, inertial motion, or congruent<br />
combinations of <strong>the</strong> two cues. Heading direction was varied in fine steps around straight <strong>for</strong>ward<br />
in <strong>the</strong> horizontal pl<strong>an</strong>e. Two monkeys were trained to report whe<strong>the</strong>r <strong>the</strong>ir perceived heading was<br />
to <strong>the</strong> left or right of straight ahead. In each experiment, small amounts of muscimol (10mg/ml,<br />
1~2µl) were injected through a microinjectrode placed in area MSTd. To suppress neural activity<br />
over as large <strong>an</strong> area as possible, chemical injections were applied bilaterally at two sites within<br />
each hemisphere (typically spaced 2-3mm apart). Control <strong>an</strong>d recovery data were collected 24<br />
hours be<strong>for</strong>e <strong>an</strong>d 48 hours after each injection, respectively.<br />
Results: In <strong>the</strong> visual condition, inactivating MSTd robustly impaired monkeys’ per<strong>for</strong>m<strong>an</strong>ce<br />
(p
Program#/Poster#: 558.12/CC29<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: KAKENHI (20020025)<br />
Title: Ch<strong>an</strong>ge of spatial resolution of direction discrimination: Comparison of MT neuronal <strong>an</strong>d<br />
psychophysical per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: *H. KUMANO, T. UKA;<br />
Dept Physiol 1, Juntendo Univ., Tokyo, Jap<strong>an</strong><br />
Abstract: Observers have difficulty in identifying a target in <strong>the</strong> presence of surrounding<br />
distracters, a phenomenon known as ‘crowding’. We previously examined crowding <strong>for</strong> hum<strong>an</strong><br />
observers using a direction discrimination task in which <strong>the</strong> central target of moving r<strong>an</strong>dom dots<br />
was surrounded by a noise <strong>an</strong>nulus of varying diameter. Discrimination per<strong>for</strong>m<strong>an</strong>ce first<br />
deteriorated <strong>an</strong>d <strong>the</strong>n recovered when <strong>the</strong> noise diameter increased, suggesting <strong>an</strong> improvement<br />
in spatial resolution. To underst<strong>an</strong>d <strong>the</strong> neuronal mech<strong>an</strong>isms underlying this adaptive ch<strong>an</strong>ge of<br />
spatial resolution, we examined single neuron activities of area MT while a monkey per<strong>for</strong>med<br />
direction discrimination in a bipartite center/surround motion display.<br />
The monkey was required to choose between two opposite directions of motion in a center patch<br />
of moving r<strong>an</strong>dom dots <strong>an</strong>d to ignore <strong>the</strong> surrounding noise (0% coherence) of varying diameter.<br />
The direction, speed, <strong>an</strong>d horizontal disparity of <strong>the</strong> center patch were tailored to <strong>the</strong> selectivity<br />
of <strong>the</strong> neuron under study. The motion coherence of <strong>the</strong> center patch was chosen r<strong>an</strong>domly on<br />
each trial among 5, 10, 20, 40, or 80%. The diameter of <strong>the</strong> center patch was fixed to 30% of <strong>the</strong><br />
receptive field eccentricity. The diameter of <strong>the</strong> noise <strong>an</strong>nulus varied <strong>from</strong> trial to trial among 1,<br />
2, 4, 8, or 16 times <strong>the</strong> center diameter.<br />
The dataset consisted of 35 experiments in which we completed at least 10 trials <strong>for</strong> each<br />
stimulus condition. Of 27 experiments in which psychophysical thresholds were < 80% across all<br />
coherences, psychophysical threshold <strong>for</strong> <strong>the</strong> largest noise size was lower th<strong>an</strong> that <strong>for</strong> <strong>the</strong><br />
middle noise size in 3 experiments (bootstrap resampling, p < 0.05). Across <strong>the</strong> population,<br />
psychophysical threshold did not differ <strong>for</strong> <strong>the</strong> largest <strong>an</strong>d <strong>for</strong> <strong>the</strong> middle noise size (Wilcoxon<br />
signed-r<strong>an</strong>k test, p = 0.39). Neuronal sensitivity was qu<strong>an</strong>tified using receiver operating<br />
characteristic <strong>an</strong>alysis. Neuronal threshold <strong>for</strong> <strong>the</strong> largest noise size was lower th<strong>an</strong> that <strong>for</strong> <strong>the</strong><br />
middle noise size (p < 0.0001, N = 27; neuronal thresholds < 80% across all coherences). The<br />
increased neuronal sensitivity <strong>from</strong> <strong>the</strong> middle to <strong>the</strong> largest noise size resulted <strong>from</strong> increased<br />
differences in me<strong>an</strong> responses to <strong>the</strong> preferred <strong>an</strong>d null directions (p = 0.003, N = 35), not <strong>from</strong><br />
reduced SDs of responses (p = 0.31, N = 35).<br />
The results suggest that when presented with large distracters, MT neurons are capable of<br />
increasing <strong>the</strong>ir motion sensitivity, <strong>an</strong>d that this increased sensitivity underlies <strong>the</strong> adaptive<br />
improvement of spatial resolution of direction discrimination observed psychophysically.<br />
Disclosures: H. Kum<strong>an</strong>o, None; T. Uka, None.
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.13/CC30<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NEI EY13138<br />
DE-AC05-00OR22750<br />
Title: Examining <strong>the</strong> temporal response profiles of neurons in area MT in relation to<br />
spatiotemporal stimulus properties<br />
Authors: *P. WALLISCH 1 , A. ROSENBERG 2 , D. C. BRADLEY 3 ;<br />
1 2 3<br />
Ctr. Neural Sci., New York Univ., New York, NY; Computat. Neurosci., Psychology, Univ. of<br />
Chicago, Chicago, IL<br />
Abstract: It is commonly recognized that <strong>the</strong> temporally-varying response of MT neurons to<br />
visual motion c<strong>an</strong> be characterized by three components: An onset-tr<strong>an</strong>sient, a sustained<br />
response <strong>an</strong>d <strong>an</strong> offset-tr<strong>an</strong>sient. While this temporal structure provides a general template <strong>for</strong><br />
MT responses, <strong>the</strong> dependencies of this pattern on stimulus parameters has not been thoroughly<br />
examined. Establishing this relationship is import<strong>an</strong>t insofar as <strong>the</strong> functional signific<strong>an</strong>ce of<br />
tr<strong>an</strong>sient versus sustained response components in area MT neurons remains elusive. To examine<br />
<strong>the</strong> dependency of temporally-varying MT responses on stimulus parameters, we recorded <strong>from</strong><br />
40 neurons in area MT of two awake, behaving macaques. We presented broadb<strong>an</strong>d stimuli<br />
(r<strong>an</strong>dom-dot-kinematograms) as well as temporally enveloped <strong>an</strong>d non-enveloped sine wave<br />
gratings <strong>an</strong>d plaids. Measures of onset-tr<strong>an</strong>sience, sustained response, <strong>an</strong>d offset-tr<strong>an</strong>sience were<br />
examined across <strong>the</strong>se stimuli. In addition, <strong>the</strong> time-course of <strong>the</strong> development of pattern<br />
direction selectivity was examined across <strong>the</strong> enveloped <strong>an</strong>d non-enveloped plaid stimuli.<br />
Finally, our results are generally consistent with simulations using motion-energy detectors<br />
embedded in a normalization network.<br />
Disclosures: P. Wallisch, None; A. Rosenberg, None; D.C. Bradley, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.14/CC31<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIBIB Gr<strong>an</strong>t 5T32EB006350-02<br />
NIMH Gr<strong>an</strong>t<br />
Title: How speed-dependent timing of excitation shapes <strong>the</strong> tuning of a collision-detecting<br />
neuron<br />
Authors: *P. W. JONES, F. GABBIANI;<br />
Baylor Coll Med., Houston, TX<br />
Abstract: We have studied <strong>an</strong> identified neuron in <strong>the</strong> locust visual system, <strong>the</strong> Lobula Gi<strong>an</strong>t<br />
Movement Detector (LGMD), that is thought to be involved in detecting objects approaching on<br />
a collision course with <strong>the</strong> <strong>an</strong>imal (looming objects) <strong>an</strong>d triggering escape behaviors away <strong>from</strong><br />
<strong>the</strong>m. It receives highly convergent excitatory inputs, with synaptic inputs <strong>from</strong> >7,500 small<br />
receptive field neurons projecting to its excitatory dendritic field. In order to underst<strong>an</strong>d how <strong>the</strong><br />
total excitatory input to <strong>the</strong> cell relates to a visual stimulus, it is import<strong>an</strong>t to underst<strong>an</strong>d both <strong>the</strong><br />
relative magnitudes <strong>an</strong>d timings of individual inputs to <strong>the</strong> cell. We have <strong>the</strong>re<strong>for</strong>e characterized<br />
responses evoked by single facet visual stimuli throughout <strong>the</strong> visual pathway leading to <strong>the</strong><br />
LGMD <strong>an</strong>d how those responses vary with <strong>the</strong> velocity at which <strong>an</strong> edge sweeps across a single<br />
facet, or equivalently, <strong>the</strong> rate of lumin<strong>an</strong>ce ch<strong>an</strong>ge at that facet. We find that <strong>the</strong> rate of<br />
lumin<strong>an</strong>ce ch<strong>an</strong>ge modulates <strong>the</strong> timing <strong>an</strong>d magnitudes of single facet responses in <strong>the</strong> LGMD<br />
<strong>an</strong>d in lamina Large Monopolar Cells (LMCs), <strong>an</strong>d it modulates <strong>the</strong> slopes of photoreceptor<br />
responses.<br />
We have now built a model of <strong>the</strong> excitatory input pathway to <strong>the</strong> LGMD, guided by known<br />
physiology <strong>an</strong>d using <strong>the</strong>se single facet responses to constrain its parameters. The model consists<br />
of <strong>an</strong> array of parallel input pathways, each corresponding to <strong>the</strong> neural processing originating at<br />
a single spatial sampling point, a single facet. Each pathway has temporal filtering stages<br />
corresponding to processing per<strong>for</strong>med by <strong>the</strong> photoreceptor <strong>an</strong>d LMCs <strong>an</strong>d a leaky integrate <strong>an</strong>d<br />
fire stage corresponding to spiking medullary inputs to <strong>the</strong> LGMD. The spike output of medulla<br />
stage is <strong>the</strong>n summed <strong>an</strong>d used to generate a synaptic input to a three-compartment, active model<br />
of <strong>the</strong> LGMD. Experimental single facet responses are well fit by this model, <strong>an</strong>d we are now<br />
using it to test <strong>the</strong> effects of speed-dependent modulation of single facet input strength <strong>an</strong>d<br />
latencies on <strong>the</strong> responses of <strong>the</strong> LGMD to stimuli that activate large populations of excitatory<br />
neurons. We find that this model replicates <strong>an</strong> experimentally observed preference <strong>for</strong> pseudolooming<br />
stimuli that activate 45 facets in a looming-like, accelerating sequence over stimuli in<br />
which <strong>the</strong> same sequence was temporally shuffled. We are currently examining its responses to<br />
full size looming stimuli.<br />
In summary, we describe, through a combination of experiments <strong>an</strong>d modeling, how single facet
velocity signals are processed through <strong>the</strong> LGMD input pathway, how <strong>the</strong>se signals shape <strong>the</strong><br />
activity of <strong>the</strong> population of excitatory inputs during natural visual stimulation, <strong>an</strong>d how this<br />
helps tune <strong>the</strong> neuron to looming-like stimuli.<br />
Disclosures: P.W. Jones, None; F. Gabbi<strong>an</strong>i, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.15/CC32<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: CIHR (MOP-79352)<br />
Title: Contrast dependence of center-surround interactions in macaque visual area MT<br />
Authors: *J. M. TSUI, R. M. CIPRIANI, C. C. PACK;<br />
McGill Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Neurons throughout <strong>the</strong> primate visual system are characterized by selectivity to<br />
stimuli placed in <strong>the</strong> centers <strong>an</strong>d surrounds of <strong>the</strong>ir receptive fields. In lower level visual cortex<br />
center-surround interactions c<strong>an</strong> be quite complex depending on <strong>the</strong> contrast of <strong>the</strong> stimuli<br />
placed in each receptive field region. Because such interactions are common in <strong>the</strong> output layers<br />
of visual cortical regions, <strong>the</strong>y shape <strong>the</strong> type of in<strong>for</strong>mation that is relayed <strong>from</strong> <strong>the</strong>se structures<br />
to o<strong>the</strong>r parts of <strong>the</strong> brain. We have <strong>the</strong>re<strong>for</strong>e characterized <strong>the</strong> center-surround interactions<br />
found in area MT of <strong>the</strong> alert macaque.<br />
In one set of experiments we examined <strong>the</strong> interaction between receptive field centers <strong>an</strong>d<br />
surrounds by varying <strong>the</strong> size <strong>an</strong>d contrast of a preferred-direction motion stimulus. For each<br />
stimulus size we fit <strong>the</strong> responses at different contrasts to a Michaelis-Menton function that<br />
provided in<strong>for</strong>mation about <strong>the</strong> slope, saturation const<strong>an</strong>t, <strong>an</strong>d maximum response of each<br />
neuron. The most obvious effect of increasing stimulus size was to decrease <strong>the</strong> maximum<br />
response in m<strong>an</strong>y neurons. However, in <strong>the</strong>se surround-suppressed neurons, we also observed <strong>an</strong><br />
equally strong ch<strong>an</strong>ge in <strong>the</strong> slope of <strong>the</strong> contrast-response function, such that m<strong>an</strong>y cells were<br />
largely insensitive to contrast <strong>for</strong> large stimulus sizes. In a minority of cells (~20%) <strong>the</strong> contrast<br />
response function actually had a negative slope <strong>for</strong> large stimulus sizes, so that increasing<br />
contrast led to a steady decrease in <strong>the</strong> response.<br />
We also measured <strong>the</strong> effect of contrast on size tuning. As has been reported previously in V1<br />
<strong>an</strong>d MT, surround suppression decreased at low contrast. Surprisingly, we found that it also
decreased at <strong>the</strong> highest contrasts, with <strong>the</strong> strongest suppression being observed <strong>for</strong> intermediate<br />
(~8%) contrasts in most cells. The tuning of simult<strong>an</strong>eously-recorded local field potential signals<br />
<strong>for</strong> contrast <strong>an</strong>d size did not correlate with that obtained <strong>from</strong> <strong>the</strong> single units.<br />
In a second set of experiments we recorded <strong>from</strong> MT neurons while varying <strong>the</strong> contrast of <strong>the</strong><br />
center <strong>an</strong>d surround stimuli independently. <strong>When</strong> both stimuli were high in contrast, <strong>the</strong><br />
surround generally had <strong>the</strong> effect of reducing responses <strong>an</strong>d sharpening direction tuning curves<br />
to stimuli presented in <strong>the</strong> center. However, when <strong>the</strong> center stimulus was of low contrast <strong>an</strong>d <strong>the</strong><br />
surround of high contrast, stimulation of <strong>the</strong> surround often resulted in ch<strong>an</strong>ges in <strong>the</strong> preferred<br />
direction of motion in <strong>the</strong> center.<br />
Overall our results reveal that MT center-surround interactions depend on contrast in a m<strong>an</strong>ner<br />
similar to that observed in lower-level visual areas, suggesting a set of computations that is<br />
repeated at each level of <strong>the</strong> visual cortical hierarchy.<br />
Disclosures: J.M. Tsui, None; R.M. Cipri<strong>an</strong>i, None; C.C. Pack, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.16/CC33<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: CELEST, <strong>an</strong> NSF Science of Learning Center (SBE-0354378)<br />
SyNAPSE program of DARPA (HR001109-03-0001, HR001-09-C-0011)<br />
Title: How do object reference frames <strong>an</strong>d motion vector decomposition emerge in laminar<br />
cortical circuits?<br />
Authors: *J. LEVEILLE, S. GROSSBERG, M. VERSACE;<br />
Cognitive <strong>an</strong>d Neural Systems, Boston Univ., Boston, MA<br />
Abstract: How do spatially disjoint <strong>an</strong>d ambiguous local motion signals in multiple directions<br />
generate coherent <strong>an</strong>d unambiguous representations of object motion? Various motion percepts<br />
have been shown to obey a rule of vector decomposition, where global motion appears to be<br />
subtracted <strong>from</strong> <strong>the</strong> true motion path of localized stimulus components (Joh<strong>an</strong>sson, 1950). This<br />
results in striking percepts wherein objects <strong>an</strong>d <strong>the</strong>ir parts are seen as moving relative to a<br />
common reference frame. While vector decomposition has been amply confirmed in a variety of<br />
experiments, no neural model has explained how it may occur in neural circuits. The current
model shows how vector decomposition results <strong>from</strong> multiple-scale <strong>an</strong>d multiple-depth<br />
interactions within <strong>an</strong>d between <strong>the</strong> <strong>for</strong>m <strong>an</strong>d motion processing streams in V1-V2 <strong>an</strong>d V1-MT.<br />
These interactions include <strong>for</strong>m-to-motion interactions <strong>from</strong> V2 to MT which ensure that precise<br />
representations of object motion-in-depth c<strong>an</strong> be computed, as demonstrated by <strong>the</strong> 3D<br />
Formotion model (e.g., Grossberg, Mingolla <strong>an</strong>d Visw<strong>an</strong>ath<strong>an</strong>, 2001, Vis. Res.; Berzh<strong>an</strong>skaya,<br />
Grossberg <strong>an</strong>d Mingolla, 2007, Vis. Res.) <strong>an</strong>d supported by recent neurophysiological data of<br />
Ponce, Lomber, & Born, 2008, Nat. Neurosci.). The present work shows how <strong>the</strong>se interactions<br />
also cause vector decomposition of moving targets, notably how <strong>for</strong>m grouping, <strong>for</strong>m-to-motion<br />
capture, <strong>an</strong>d figure-ground separation mech<strong>an</strong>isms may work toge<strong>the</strong>r to simulate classical<br />
Duncker (1929) <strong>an</strong>d Joh<strong>an</strong>sson (1950) percepts of vector decomposition <strong>an</strong>d coherent object<br />
motion in a frame of reference.<br />
Disclosures: J. Leveille, None; S. Grossberg, None; M. Versace, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.17/CC34<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH NCRR 5RO1RR008688-20<br />
NIH 5T32GM008400-18<br />
Title: Higher visual processing in <strong>an</strong> insect model system: Responses in <strong>the</strong> central complex of<br />
<strong>the</strong> flesh fly, Neobellieria bullata<br />
Authors: *J. PHILLIPS PORTILLO 1 , N. J. STRAUSFELD 2 ;<br />
1 2<br />
ARL Div. of Neurobio., Univ. of Arizona|910005516|0, Tucson, AZ; Univ. of Arizona,<br />
Tucson, AZ<br />
Abstract: Outputs <strong>from</strong> retinotopic neuropils of <strong>the</strong> lobula, a cortex-like neuropil of <strong>the</strong> optic<br />
lobes, supply optic glomeruli in <strong>the</strong> lateral protocerebrum. Integrated signals are relayed <strong>from</strong><br />
optic glomeruli to higher brain regions. These include <strong>the</strong> dorsal lobes of <strong>the</strong> superior<br />
protocerebrum <strong>an</strong>d <strong>the</strong> central complex. The latter comprises a system of prominent interlinked<br />
midline neuropils that are known to receive indirect inputs carrying visual <strong>an</strong>d mech<strong>an</strong>osensory<br />
signals. The central complex is also known to have close ties to premotor descending neurons<br />
that contribute drive to motor circuits. To underst<strong>an</strong>d what aspects of <strong>the</strong> visual world are relayed
to <strong>the</strong> central complex, how this sensory in<strong>for</strong>mation is integrated, <strong>an</strong>d how it relates to<br />
downstream events, we record <strong>from</strong> <strong>an</strong>d dye fill neurons projecting to neuropils of <strong>the</strong> central<br />
complex of <strong>the</strong> flesh fly, Neobellieria bullata. A combination of intracellular recordings, tracer<br />
injections, <strong>an</strong>d immunohistochemical techniques are being used to <strong>an</strong>alyze <strong>the</strong> filter properties<br />
<strong>an</strong>d org<strong>an</strong>ization of neurons, <strong>the</strong> morphology of which is reconstructed <strong>an</strong>d related to specific<br />
synaptic areas by confocal microscopy. Neurons thus far recorded <strong>from</strong> <strong>the</strong> central complex<br />
show sensitivity to <strong>the</strong> direction of moving visual stimuli, <strong>the</strong> <strong>an</strong>gle of polarized light, <strong>an</strong>d<br />
lumin<strong>an</strong>ce. For example, neurons projecting into <strong>the</strong> protocerebral bridge <strong>from</strong> optic glomeruli in<br />
<strong>the</strong> lateral protocerebrum are weakly responsive to flashes of light, but are strongly driven by<br />
visual stimuli moving in a preferred direction. Movement in <strong>the</strong> <strong>an</strong>ti-preferred direction produces<br />
no response. There is also a major input, which responds to visual stimuli, into <strong>the</strong> f<strong>an</strong>-shaped<br />
body of <strong>the</strong> central complex. These neurons originate <strong>from</strong> <strong>the</strong> lobes of <strong>the</strong> superior<br />
protocerebrum, a region also supplied by afferents <strong>from</strong> optic glomeruli. The present studies<br />
reveal new aspects of <strong>the</strong> distributed nature of optic lobe outputs <strong>an</strong>d <strong>the</strong> representation of visual<br />
motion in a higher brain center that is ubiquitous to insects <strong>an</strong>d crustace<strong>an</strong>s.<br />
Disclosures: J. Phillips Portillo, None; N.J. Strausfeld, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.18/CC35<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH<br />
NSF<br />
Title: Recurrent excitatory connections in <strong>the</strong> larval zebrafish optic tectum<br />
Authors: A. GRAMA, *P. RAMDYA, F. ENGERT;<br />
MCB, Harvard Univ., Cambridge, MA<br />
Abstract: Recurrent inputs contribute a large proportion of <strong>the</strong> total excitatory connectivity to<br />
neurons within <strong>the</strong> visual system. Theoretical models have proposed a role <strong>for</strong> recurrent<br />
excitation in <strong>the</strong> encoding of visual features like orientation <strong>an</strong>d direction selectivity but a direct<br />
demonstration of <strong>the</strong>ir contribution to this processing is lacking. We identified recurrent<br />
connections within <strong>the</strong> larval zebrafish optic tectum using in vivo population calcium imaging
<strong>an</strong>d whole-cell recordings. Tectal neurons had excitatory spatiotemporal receptive fields<br />
composed in large part <strong>from</strong> delayed feed-back excitation. Consistent with this recurrence, we<br />
observed propagating waves of activity through <strong>the</strong> tectum in response to flashed bar <strong>an</strong>d tectal<br />
electrical stimulation. Most remarkably, we found that <strong>an</strong> asymmetry in <strong>the</strong> strength of <strong>the</strong>se<br />
delayed local connections could predict <strong>the</strong> direction selectivity of individual neurons,<br />
suggesting <strong>an</strong> import<strong>an</strong>t role <strong>for</strong> recurrent excitatory inputs in tectal visual processing.<br />
Disclosures: A. Grama, None; P. Ramdya, None; F. Engert, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.19/CC36<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: High Potential Gr<strong>an</strong>t of Utrecht University<br />
Title: The neural signature of stabilized ambiguous structure-<strong>from</strong>-motion in macaque area MT<br />
Authors: *P. C. KLINK 1 , M. J. M. LANKHEET 1 , R. J. A. VAN WEZEL 1,2 ;<br />
1 2<br />
Helmholtz Inst., Utrecht Univ., Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds; Biomed. Signals <strong>an</strong>d Systems, Univ. of<br />
Twente, Enschede, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: <strong>When</strong> ambiguous visual stimuli are presented in sequences with intermittent bl<strong>an</strong>k<br />
periods <strong>the</strong> brain chooses a perceptual interpretation at each stimulus onset. The dynamics of <strong>the</strong><br />
resulting perceptual sequence highly depends on <strong>the</strong> duration of <strong>the</strong> intermittent bl<strong>an</strong>k period.<br />
While short interruptions cause a high probability that percepts alternate on subsequent<br />
presentations, longer interruptions stabilize perception <strong>an</strong>d <strong>the</strong> same interpretation c<strong>an</strong> be<br />
perceived <strong>for</strong> minutes. Previous studies using ambiguous structure-<strong>from</strong>-motion stimuli suggest<br />
that <strong>the</strong> process of perceptual stabilization does not need <strong>an</strong> explicit <strong>for</strong>m of perceptual memory,<br />
but ra<strong>the</strong>r that it results <strong>from</strong> <strong>the</strong> low level dynamics of motion sensitive neurons. Here we<br />
present single-unit <strong>an</strong>d local field potential data, simult<strong>an</strong>eously recorded <strong>from</strong> area MT of a<br />
fixating macaque monkey while streams of ambiguous structure-<strong>from</strong>-motion stimuli were<br />
presented in <strong>the</strong> receptive field with variable intermittent bl<strong>an</strong>k durations. Our neural data<br />
support <strong>the</strong> idea that perceptual stabilization of ambiguous structure-<strong>from</strong>-motion is correlated<br />
with a low level stabilization of response variability in motion area MT. The potential role of<br />
neuronal adaptation in <strong>the</strong> stabilization process is explored by distinguishing between adaptation<br />
that takes place on ei<strong>the</strong>r short or longer timescales.
Disclosures: P.C. Klink, None; M.J.M. L<strong>an</strong>kheet, None; R.J.A. v<strong>an</strong> Wezel, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.20/CC37<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: DFG (SFB 550)<br />
EC project SEARISE<br />
Herm<strong>an</strong>n und Lilly Schilling Foundation<br />
Title: Visual encoding of goal-directed movements: A physiologically plausible neural model<br />
Authors: *M. A. GIESE, V. CAGGIANO, A. CASILE, F. FLEISCHER;<br />
Hertie Inst. For Clin. Brain Sci., Tuebingen, Germ<strong>an</strong>y<br />
Abstract: Visual responses of action-selective neurons, e.g. in premotor cortex <strong>an</strong>d <strong>the</strong> superior<br />
temporal sulcus of <strong>the</strong> macaque monkey, are characterized by a remarkable combination of<br />
selectivity <strong>an</strong>d invari<strong>an</strong>ce. On <strong>the</strong> one h<strong>an</strong>d, <strong>the</strong> responses of such neurons show high selectivity<br />
<strong>for</strong> details about <strong>the</strong> grip <strong>an</strong>d <strong>the</strong> spatial relationship between effector <strong>an</strong>d object. At <strong>the</strong> same<br />
time, <strong>the</strong>se responses show subst<strong>an</strong>tial invari<strong>an</strong>ce against <strong>the</strong> retinal stimulus position. While<br />
numerous models <strong>for</strong> <strong>the</strong> mirror neuron system have been proposed in robotics <strong>an</strong>d neuroscience,<br />
almost none of <strong>the</strong>m accounts <strong>for</strong> <strong>the</strong> visual tuning properties of action-selective neurons<br />
exploiting physiologically plausible neural mech<strong>an</strong>isms. In addition, m<strong>an</strong>y existing models<br />
assume that action encoding is based on a full reconstruction of <strong>the</strong> 3D geometry of effector <strong>an</strong>d<br />
object. This contradicts recent electrophysiological results showing view-dependence of <strong>the</strong><br />
majority of action-selective neurons, e.g. in premotor cortex.<br />
We present a neurophysiologically plausible model <strong>for</strong> <strong>the</strong> visual recognition of grasping<br />
movements <strong>from</strong> real videos. The model is based on simple well-established neural circuits.<br />
Recognition of effector <strong>an</strong>d goal object is accomplished by a hierarchical neural architecture,<br />
where scale <strong>an</strong>d position invari<strong>an</strong>ce are accomplished by nonlinear pooling along <strong>the</strong> hierarchy,<br />
consistent with m<strong>an</strong>y established models <strong>from</strong> object recognition. Effector recognition includes a<br />
simple predictive neural circuit that results in temporal sequence selectivity. Effector <strong>an</strong>d goal<br />
position are encoded within <strong>the</strong> neural hierarchy in terms of population codes, which c<strong>an</strong> be<br />
processed by a simple gain field-like mech<strong>an</strong>ism in order to compute <strong>the</strong> relative position of
effector <strong>an</strong>d object in a retinal frame of reference. Based on this signal, <strong>an</strong>d object <strong>an</strong>d effector<br />
shape, <strong>the</strong> highest hierarchy level accomplishes a distinction between functional (h<strong>an</strong>d matches<br />
object shape <strong>an</strong>d position) <strong>an</strong>d dysfunctional (no match between h<strong>an</strong>d <strong>an</strong>d object shape or<br />
position) grips, at <strong>the</strong> same time being invari<strong>an</strong>t against strong ch<strong>an</strong>ges of <strong>the</strong> stimulus position.<br />
The model was tested with several stimuli <strong>from</strong> <strong>the</strong> neurophysiological literature <strong>an</strong>d reproduces,<br />
partially even qu<strong>an</strong>titatively, results about action-selective neurons in <strong>the</strong> STS <strong>an</strong>d premotor<br />
cortex. Specifically, <strong>the</strong> model reproduces visual tuning properties <strong>an</strong>d <strong>the</strong> view-dependence of<br />
mirror neurons in premotor cortex <strong>an</strong>d makes additional predictions, which c<strong>an</strong> be easily tested<br />
in electrophysiological experiments.<br />
Disclosures: M.A. Giese, None; V. Caggi<strong>an</strong>o, None; A. Casile, None; F. Fleischer, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.21/CC38<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Title: Attention to motion: Differential cortical modulation to <strong>for</strong>ward <strong>an</strong>d pl<strong>an</strong>ar visual flow<br />
Authors: *E. FISCHER 1 , H. H. BUELTHOFF 2 , N. K. LOGOTHETIS 3,4 , A. BARTELS 3,5 ;<br />
2 Dept. Buelthoff, 3 Dept. Logo<strong>the</strong>tis, 1 MPI Biol Cybernetics, Tuebingen, Germ<strong>an</strong>y; 4 Imaging Sci.<br />
<strong>an</strong>d Biomed. Engin., Univ. of M<strong>an</strong>chester, M<strong>an</strong>chester, United Kingdom; 5 Ctr. <strong>for</strong> Integrative<br />
Neurosci., Tuebingen, Germ<strong>an</strong>y<br />
Abstract: Self- <strong>an</strong>d object-motion processing greatly relies on visual cues. There are at least two<br />
entirely independent kinds of self-induced visual motion that combine to optic flow in <strong>the</strong> visual<br />
field: exp<strong>an</strong>sion flow, such as that induced by <strong>for</strong>ward motion in depth, or pl<strong>an</strong>ar motion, such as<br />
induced by tr<strong>an</strong>slational self-motion or by pursuit eye movements across a visual scene. In real<br />
life, both signals may occur in combination, yet, only one of <strong>the</strong> cues may be of behavioral<br />
relev<strong>an</strong>ce, thus requiring to be selectively attended to. In this fMRI study we attempt to address<br />
<strong>the</strong> question whe<strong>the</strong>r differential neural substrates get modulated by selective attention to ei<strong>the</strong>r<br />
one of <strong>the</strong>se motion cues. We created a stimulus combining <strong>an</strong> exp<strong>an</strong>sion flow pattern with<br />
tr<strong>an</strong>slational motion on <strong>the</strong> same set of dots. In a feature-based detection task, subjects<br />
selectively attended ei<strong>the</strong>r to <strong>the</strong> exp<strong>an</strong>sion or to <strong>the</strong> tr<strong>an</strong>slation component of <strong>the</strong> stimulus <strong>an</strong>d<br />
reported ch<strong>an</strong>ges in <strong>the</strong> speed of <strong>the</strong> attended motion component. In control conditions that used<br />
<strong>the</strong> same stimuli subjects attended to color hue ch<strong>an</strong>ges of <strong>the</strong> fixation cross, or passively fixated<br />
<strong>the</strong> stimulus without <strong>an</strong>y attentional dem<strong>an</strong>d. In each of <strong>the</strong> three attention conditions, <strong>the</strong>
attentional load was kept const<strong>an</strong>t across conditions by a continuously updating staircase<br />
procedure.<br />
We found that attention to exp<strong>an</strong>sion modulated <strong>the</strong> separately localized areas MT/V5, MST, <strong>an</strong>d<br />
V3A signific<strong>an</strong>tly more th<strong>an</strong> attention to tr<strong>an</strong>slation. This is in line with stimulus-driven studies<br />
that showed a preference to exp<strong>an</strong>sion/contraction stimuli in <strong>the</strong>se areas (Smith et al., 2006). In<br />
contrast, V7 <strong>an</strong>d <strong>the</strong> cingulate sulcus visual area (CSv) differed <strong>from</strong> all o<strong>the</strong>r regions, in that<br />
<strong>the</strong>y did not show <strong>an</strong>y selective modulation by attention to exp<strong>an</strong>sion flow. Most interestingly,<br />
we found motion selective modulation in <strong>the</strong> foveal confluence of V1, despite a physical match<br />
between stimulus conditions. This might be due to differential attentional enh<strong>an</strong>cement within<br />
V1, or by differential feedback <strong>from</strong> higher regions such as MT/V5, MST or V3A. Our results<br />
<strong>the</strong>re<strong>for</strong>e show a differential attentional modulation within <strong>the</strong> motion-processing pathway,<br />
depending on <strong>the</strong> type of motion-component that is attended to within <strong>the</strong> same flow stimulus.<br />
Smith AT, Wall MB, Williams AL, Singh KD (2006) Sensitivity to optic flow in hum<strong>an</strong> cortical<br />
areas MT <strong>an</strong>d MST. Eur J Neurosci 23:561-569.<br />
Disclosures: E. Fischer, None; H.H. Buelthoff, None; N.K. Logo<strong>the</strong>tis, None; A. Bartels,<br />
None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.22/CC39<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: EC FACETS project<br />
Title: Mapping <strong>the</strong> horizontal synaptic association field with apparent motion in <strong>the</strong> cat visual<br />
cortex<br />
Authors: *P. V. CARELLI, M. PANANCEAU, I. KOPISOVA, Y. FREGNAC;<br />
UNIC-CNRS, Gif-sur-Yvette, Fr<strong>an</strong>ce<br />
Abstract: It has been shown using intracellular approaches that long-r<strong>an</strong>ge horizontal<br />
connections in <strong>the</strong> primary visual cortex (area 17) of <strong>the</strong> cat c<strong>an</strong> mediate propagation of visual<br />
in<strong>for</strong>mation at low speed (0.1-0.3 m/s) far beyond <strong>the</strong> classical extent of cortical receptive fields<br />
(Bringuier et al, Science, 1999). Previous psychophysical studies have shown that spatiotemporal<br />
sequential presentation of oriented Gabor Patches (GPs) c<strong>an</strong> produce different percepts<br />
of apparent motion speed depending on <strong>the</strong> relative orientations of <strong>the</strong> GPs with respect to <strong>the</strong>
motion axis (George et al, 2002). Intracellular <strong>an</strong>d modeling studies <strong>from</strong> <strong>the</strong> lab suggest that<br />
apparent motion is perceived `faster` when induced with collinear GPs th<strong>an</strong> with cross-oriented<br />
GPs, when <strong>the</strong> evoked horizontal wave of propagation travels in phase with <strong>the</strong> sequence of<br />
feed<strong>for</strong>ward inputs. In <strong>the</strong> present work we used <strong>the</strong> sequential presentation of two Gabor<br />
patches as <strong>an</strong> elementary apparent motion probe stimulus to map <strong>the</strong> spatio-temporal properties<br />
of subthreshold receptive fields in cat area 17. In order to pave uni<strong>for</strong>mly <strong>the</strong> visual field, a<br />
sparse apparent motion noise was applied on <strong>an</strong> hexagonal grid centred on <strong>the</strong> cell's minimal<br />
discharge field (MDF). Propagation of horizontal synaptic input was revealed by per<strong>for</strong>ming a<br />
tensor voting <strong>an</strong>alysis <strong>for</strong> each point of <strong>the</strong> grid, revealing a diversity of flow patterns of<br />
centripetal or centrifugal biases in synaptic drive across <strong>the</strong> `silent` surround of <strong>the</strong> MDF. The<br />
horizontal wave activation patterns were compared <strong>for</strong> collinear <strong>an</strong>d cross-oriented Gabor<br />
elements. Our results at <strong>the</strong> intracellular level are consistent with <strong>the</strong> hypo<strong>the</strong>sis of a dynamic<br />
perceptual association field. According to this hypo<strong>the</strong>sis, <strong>the</strong> horizontal propagation wave<br />
broadcasts a functional belief or expectation to <strong>the</strong> rest of <strong>the</strong> network, preparing <strong>the</strong> recruitment<br />
of o<strong>the</strong>r neurons in <strong>the</strong> <strong>for</strong>mation of dynamic functional assemblies. This putative low-level<br />
intracortical mech<strong>an</strong>ism could underlie some of <strong>the</strong> binding principles (collinearity, common<br />
fate..) of <strong>the</strong> Gestalt <strong>the</strong>ory.<br />
Disclosures: P.V. Carelli, None; M. P<strong>an</strong><strong>an</strong>ceau, None; I. Kopisova, None; Y. Fregnac, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.23/CC40<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: Supported by EU contract IST 2006, 027198 (Decisions in Motion)<br />
Swedish Foundation <strong>for</strong> strategic research<br />
Stockholm Brain Institute<br />
Wallenberg Foundation<br />
Title: Laminar firing <strong>an</strong>d subthreshold dynamics in four visual areas exposed to objects moving<br />
to occlusion
Authors: *M. A. HARVEY, S. VALENTIENE, P. E. ROLAND;<br />
Karolinska Institutet, Stockholm, Sweden<br />
Abstract: <strong>When</strong> two objects move in opposite directions one c<strong>an</strong> usually predict <strong>the</strong> moment<br />
when <strong>the</strong> objects might collide or when one object might occlude <strong>the</strong> o<strong>the</strong>r object. We measured<br />
ch<strong>an</strong>ges in <strong>the</strong> relative membr<strong>an</strong>e potentials of cells in visual areas 17,18,19, <strong>an</strong>d 21 with a<br />
voltage sensitive dye in combination with laminar recordings of action potentials <strong>from</strong> area 17 in<br />
<strong>an</strong>es<strong>the</strong>tized ferrets (mustela putorius) exposed to two bars moving to occlusion. The bars<br />
mapped moving towards each o<strong>the</strong>r along <strong>the</strong> borders between areas 17/18 <strong>an</strong>d 19/21. In <strong>the</strong><br />
cortical space representing <strong>the</strong> area between <strong>the</strong> bar representations <strong>the</strong> dye signal increased first<br />
following <strong>the</strong> 19/21 border <strong>an</strong>d <strong>the</strong>n following <strong>the</strong> 17/18 border, mapping <strong>the</strong> future course of<br />
motion over <strong>the</strong> cortex until occlusion. Ten ms prior to <strong>the</strong> time when <strong>the</strong> bars at <strong>the</strong> display<br />
abutted each o<strong>the</strong>r (at 392 ms), <strong>the</strong> derivative of <strong>the</strong> dye signal went strongly negative below<br />
baseline. At <strong>the</strong> abutting, <strong>the</strong> firing in all layers decreased rapidly to scale with <strong>the</strong> firing<br />
associated with one bar only 23 ms after <strong>the</strong> maximal occlusion on <strong>the</strong> display. This was<br />
followed by a rebound lead by <strong>the</strong> firing of neurons in <strong>the</strong> gr<strong>an</strong>ular layers. Thus <strong>the</strong> visual cortex<br />
mapped <strong>the</strong> future path on <strong>the</strong> cortex between <strong>the</strong> bars. Then cortex matched <strong>the</strong> firing to that of<br />
a single object at <strong>the</strong> occlusion.<br />
Disclosures: M.A. Harvey, None; S. Valentiene, None; P.E. Rol<strong>an</strong>d, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.24/CC41<br />
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NSF CAREER Award BCS-0748413<br />
NWO Gr<strong>an</strong>t 2006/11353/ALW<br />
Title: Selective adaptation of 3D motion mech<strong>an</strong>isms<br />
Authors: *T. B. CZUBA 1,2,3 , L. K. CORMACK 2,3,4 , A. C. HUK 2,3,4,5,6 , B. ROKERS 2,3,4,5,6 ;<br />
2 3 4 5 6<br />
Psychology, Ctr. <strong>for</strong> Perceptual Systems, Imaging Res. Ctr., Neurobio., Inst. <strong>for</strong> Neurosci.,<br />
1<br />
The Univ. Texas, Austin, TX
Abstract: Motion aftereffects (MAE) are generally considered evidence <strong>for</strong> neuronal<br />
populations tuned to specific directions of motion. Despite some early reports, <strong>the</strong>re is sc<strong>an</strong>t<br />
physiological or psychophysical evidence <strong>for</strong> neurons in visual cortex selective <strong>for</strong> <strong>the</strong> direction<br />
of motion through depth (i.e., tuned to 3D motion) (Sak<strong>an</strong>o et al., 2005). By comparing<br />
adaptation under dichoptic <strong>an</strong>d monocular conditions, we found large 3D MAEs that could not<br />
be explained by a straight<strong>for</strong>ward combination of monocular 2D MAEs.<br />
Subjects viewed r<strong>an</strong>dom dot stereograms containing corresponding dots moving in opposite<br />
horizontal directions in <strong>the</strong> two eyes, thus producing 3D motion percepts. Following adaptation<br />
to this 3D motion (100 sec, towards or away), subjects per<strong>for</strong>med a series of direction<br />
discrimination trials composed of a 4 sec top-up adaptation <strong>an</strong>d 1 sec test stimulus. On each trial,<br />
<strong>the</strong> test stimulus contained a variable proportion of signal dots moving through depth, <strong>an</strong>d <strong>the</strong><br />
remainder of <strong>the</strong> dots followed r<strong>an</strong>dom walks through depth (e.g. Hiris, Blake 1992). On each<br />
trial, subjects reported <strong>the</strong> perceived global motion direction (towards or away).<br />
Prolonged viewing of unidirectional 3D motion biased subsequent percepts of noisy 3D motion<br />
test stimuli in <strong>the</strong> direction opposite adaptation. The contribution of 2D monocular adaptation<br />
was dissociated by measuring monocular MAEs after identical 3D adaptation. Monocular MAEs<br />
were ~4x weaker th<strong>an</strong> <strong>the</strong> corresponding 3D MAE. This suggests that <strong>the</strong> effects of adapting to<br />
3D motion c<strong>an</strong>not simply be accounted <strong>for</strong> by <strong>the</strong> combination of monocular 2D aftereffects;<br />
mech<strong>an</strong>isms tuned to 3D motion must be involved. The underlying locus of adaptation was<br />
explored by extending <strong>the</strong> motion adaptation paradigm to stimuli that isolate binocular 3D<br />
motion cues (velocity-based (IOVD) cue by binocular contrast <strong>an</strong>ti-correlation; disparity-based<br />
(CD) cue by dynamic r<strong>an</strong>dom dot stereogram).<br />
Psychophysical results were supported by a matched series of hum<strong>an</strong> fMRI experiments which<br />
revealed a decreased MT+ response to test stimuli moving in <strong>the</strong> adapted direction compared to<br />
unadapted directions. Stimuli presented during event-related fMRI sessions were identical to <strong>the</strong><br />
psychophysical stimuli. Monocular contributions were evaluated by sequential presentation of<br />
<strong>the</strong> left- <strong>an</strong>d right-eye test stimuli. Attention was equated throughout sc<strong>an</strong>ning sessions by<br />
requiring subjects to detect brief dot color ch<strong>an</strong>ges that were uncorrelated with <strong>the</strong> stimulus<br />
percept.<br />
These results provide clear evidence <strong>for</strong> <strong>the</strong> existence of cell populations tuned to 3D direction,<br />
<strong>an</strong>d support a model of 3D motion computation based on interocular velocity differences.<br />
Disclosures: T.B. Czuba, None; L.K. Cormack, None; A.C. Huk, None; B. Rokers, None.<br />
Poster<br />
558. Visual Motion: Neural Mech<strong>an</strong>isms<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 558.25/CC42
Topic: D.04.m. Visual motion: Neural mech<strong>an</strong>isms<br />
Support: NIH EY18216<br />
Title: Dragging it out: Motion-sensitive areas code perceived positions in <strong>the</strong> motion-induced<br />
flash mislocalization illusion<br />
Authors: *G. MAUS 1 , J. FISCHER 1,2 , D. WHITNEY 1,2 ;<br />
1 Ctr. <strong>for</strong> Mind <strong>an</strong>d Brain, 2 Dept. of Psychology, UC Davis, Davis, CA<br />
Abstract: Visual motion c<strong>an</strong> shift <strong>the</strong> perceived position of a flash, even in dist<strong>an</strong>t regions of <strong>the</strong><br />
visual field (Whitney & Cav<strong>an</strong>agh, NN 2000). Using fMRI we investigated how <strong>the</strong> positions of<br />
mislocalized flashes in this illusion are coded in early visual cortex.<br />
Particip<strong>an</strong>ts viewed a concentric grating oscillating between inward <strong>an</strong>d outward motion at 1.25<br />
Hz. The moving grating was visible only in wedge-shaped segments along <strong>the</strong> visual field<br />
meridi<strong>an</strong>s. Peripheral flashes were presented in <strong>the</strong> empty space between <strong>the</strong> grating segments at<br />
physically const<strong>an</strong>t locations, with four different phase offsets to <strong>the</strong> oscillating motion. In each<br />
block flashes could occur at <strong>the</strong> time <strong>the</strong> direction of <strong>the</strong> oscillating grating ch<strong>an</strong>ged (<strong>from</strong><br />
inward to outward, or vice versa), or halfway through <strong>the</strong> inward or outward motion In a 4alternative<br />
<strong>for</strong>ced-choice task particip<strong>an</strong>ts judged <strong>the</strong> perceived spatial position of <strong>the</strong> flashes in<br />
relation to small comparison lines. Behavioural responses were highly variable, but exhibited a<br />
trend <strong>for</strong> <strong>the</strong> flashes to be perceptually shifted depending on <strong>the</strong> direction of motion present at<br />
<strong>the</strong> time of <strong>the</strong> flash.<br />
We <strong>an</strong>alysed <strong>the</strong> fMRI BOLD response to this task by coding conditions ei<strong>the</strong>r by <strong>the</strong> physical<br />
variation (phase offset) or by <strong>the</strong> responses in single trials (perceived positions). Using a<br />
multivariate pattern <strong>an</strong>alysis, we found that <strong>the</strong> BOLD response in motion-sensitive areas V3,<br />
V3A, <strong>an</strong>d V5/MT+ was best predicted by <strong>the</strong> perceived positions reported by particip<strong>an</strong>ts.<br />
Responses in V1/V2, on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, were similarly predicted by physical <strong>an</strong>d perceptual<br />
properties of <strong>the</strong> stimuli.<br />
We conclude that systematic shifts in <strong>the</strong> perceived position of a flash due to visual motion are<br />
represented in cortical areas involved in motion processing. A possible neural mech<strong>an</strong>ism is a<br />
shift of receptive fields in <strong>the</strong> opposite direction of motion due to feedback <strong>from</strong> higher areas.<br />
Disclosures: G. Maus, None; J. Fischer, None; D. Whitney, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.1/CC43
Topic: D.06.e. Pursuit<br />
Support: MRC<br />
Title: A neural network model of predictive behaviour <strong>an</strong>d visual-vestibular interaction during<br />
head-fixed <strong>an</strong>d head-free pursuit<br />
Authors: *R. ACKERLEY 1 , G. R. BARNES 1 , K. P. BROHAN 2 ;<br />
1 2<br />
Fac of Life Sci., Fac. of Engin. <strong>an</strong>d Physical Sci., Univ. M<strong>an</strong>chester, M<strong>an</strong>chester, United<br />
Kingdom<br />
Abstract: Prediction in pursuit overcomes delays in central processing. Evidence suggests that<br />
prediction is largely accomplished through rapid acquisition of target motion in<strong>for</strong>mation <strong>from</strong><br />
prior stimulation <strong>an</strong>d subsequent replay of stored in<strong>for</strong>mation in <strong>an</strong>ticipation of future target<br />
motion. A neural network model has now been developed that realistically represents this storage<br />
process. The network takes as input a time-base signal, which it converts into a representation of<br />
<strong>an</strong>y arbitrary target trajectory. Trajectory in<strong>for</strong>mation is derived <strong>from</strong> <strong>an</strong> internally reconstructed<br />
representation of target velocity via <strong>an</strong> efference copy mech<strong>an</strong>ism. Timing is derived <strong>from</strong> <strong>the</strong><br />
same internal signal if <strong>the</strong> stimulus is periodic or <strong>from</strong> independent timing cues signalling onset<br />
of repeated tr<strong>an</strong>sient stimuli. The network is trained at regular intervals to represent <strong>the</strong> incoming<br />
target motion trajectory <strong>an</strong>d, when repeated stimuli are recognised, c<strong>an</strong> play out a copy of <strong>the</strong><br />
trajectory to <strong>for</strong>m <strong>an</strong> <strong>an</strong>ticipatory movement by resetting <strong>the</strong> time-base signal. Network<br />
incorporation within <strong>the</strong> efference copy loop allows gain <strong>an</strong>d phase of sinusoidal pursuit to be<br />
simulated over its normal frequency r<strong>an</strong>ge (~0.1-2Hz). Additionally, <strong>the</strong> network generates<br />
realistic <strong>an</strong>ticipatory movements to repeated tr<strong>an</strong>sient stimuli. The model has now been adapted<br />
to simulate responses during head-free pursuit, in which head rotation stimulates <strong>the</strong> vestibuloocular<br />
reflex (VOR). Recent experiments have shown that a brief initial presentation (150ms) of<br />
a moving target of r<strong>an</strong>domized speed <strong>an</strong>d direction is sufficient <strong>for</strong> <strong>the</strong> subject to store<br />
in<strong>for</strong>mation about target velocity. This is revealed by <strong>the</strong> fact that when <strong>the</strong> motion stimulus is<br />
repeated but is not visible <strong>for</strong> <strong>the</strong> first 600ms, subjects make a prolonged <strong>an</strong>ticipatory smooth<br />
movement that is appropriately scaled to target velocity in <strong>the</strong> first brief presentation. In <strong>the</strong><br />
head-fixed pursuit condition this reveals <strong>the</strong> activity of internal (extraretinal) drive mech<strong>an</strong>isms.<br />
During head-free pursuit gaze (head + eye) velocity exhibits similar <strong>an</strong>ticipatory activity,<br />
implying that <strong>the</strong> internal drive has taken account of <strong>the</strong> VOR, which, during target extinction,<br />
would normally drive <strong>the</strong> eye in <strong>the</strong> opposite direction to <strong>the</strong> head. The model is able to simulate<br />
<strong>the</strong>se responses using <strong>the</strong> same network memory if it is assumed that <strong>the</strong> internal drive is used to<br />
counterm<strong>an</strong>d <strong>the</strong> VOR response <strong>an</strong>d drive <strong>the</strong> pursuit response. However, small differences<br />
between <strong>an</strong>ticipatory gaze velocity in head-free <strong>an</strong>d head-fixed conditions indicate that VOR<br />
gain is less th<strong>an</strong> unity under <strong>the</strong>se conditions, which may indicate <strong>the</strong> existence of additional<br />
non-visual VOR suppressive mech<strong>an</strong>isms.<br />
Disclosures: R. Ackerley, None; G.R. Barnes, None; K.P. Broh<strong>an</strong>, None.<br />
Poster
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.2/CC44<br />
Topic: D.06.e. Pursuit<br />
Support: MRC Gr<strong>an</strong>t G0601572<br />
Title: The effects of stimulus timing history on <strong>an</strong>ticipatory movements may account <strong>for</strong> nonlinear<br />
frequency characteristics of pseudo-r<strong>an</strong>dom pursuit<br />
Authors: *G. R. BARNES 1 , C. J. S. COLLINS 2 ;<br />
1 2<br />
Emeritus Prof of Sensorimotor Neurosci, Fac. of Life Sci., Univ. of M<strong>an</strong>chester, M<strong>an</strong>chester,<br />
United Kingdom<br />
Abstract: In responses to pseudo-r<strong>an</strong>dom target motion, smooth pursuit gain <strong>an</strong>d phase ch<strong>an</strong>ge<br />
with <strong>the</strong> r<strong>an</strong>ge of frequencies comprising <strong>the</strong> stimulus. Here, we provide evidence that this effect<br />
may be related to stimulus timing history. Hum<strong>an</strong> subjects pursued a pseudo-r<strong>an</strong>dom stimulus<br />
composed of concatenated, alternating (left/right) segments of variable duration (RD). Segments<br />
were (a) ramps: velocity ±15deg/s or (b) half-cycle sinusoids: peak velocity ±23.5deg/s. RDs<br />
were r<strong>an</strong>domized within 4 r<strong>an</strong>ges (300-720ms; 420-840ms; 600-1020ms; 840-1200ms), each<br />
r<strong>an</strong>ge comprising 8 RDs. Responses to 12 repeats of each RD were obtained <strong>from</strong> 6 subjects.<br />
Frequency <strong>an</strong>alysis of smooth pursuit responses revealed non-linearities similar to those found<br />
previously; gain of <strong>the</strong> highest frequency (associated with <strong>the</strong> shortest RD in each r<strong>an</strong>ge) was<br />
enh<strong>an</strong>ced with respect to lower frequencies, whereas phase lag was close to zero at <strong>the</strong> lowest<br />
frequency (associated with <strong>the</strong> longest RD in each r<strong>an</strong>ge). Averaging of each ramp or sine<br />
response with identical RD showed that eye velocity started to decline in <strong>an</strong>ticipation of target<br />
reversal at a similar time within each r<strong>an</strong>ge, irrespective of actual ramp duration. However, <strong>the</strong><br />
<strong>an</strong>ticipatory decline occurred progressively later as <strong>the</strong> me<strong>an</strong> of <strong>the</strong> r<strong>an</strong>ge increased. As <strong>an</strong><br />
indicator of time of <strong>an</strong>ticipatory eye velocity reversal we calculated T90, <strong>the</strong> time at which eye<br />
velocity fell to 90% of peak velocity. Within each RD r<strong>an</strong>ge, T90 was less th<strong>an</strong> RD <strong>an</strong>d<br />
relatively const<strong>an</strong>t, regardless of RD, but, as RD r<strong>an</strong>ge increased, T90 also increased; i.e. <strong>the</strong><br />
time at which smooth eye movement deceleration beg<strong>an</strong> was not influenced by current RD but<br />
was related to <strong>the</strong> overall r<strong>an</strong>ge of RDs presented within a trial, suggesting that T90 might be<br />
derived <strong>from</strong> averaging of prior stimulus timing. Multiple regression <strong>an</strong>alysis was used to<br />
examine <strong>the</strong> relationship between T90 <strong>an</strong>d up to 8 prior values of ramp duration (RD). This<br />
revealed that both <strong>the</strong> modulations within each r<strong>an</strong>ge <strong>an</strong>d differences between r<strong>an</strong>ges, could be<br />
accounted <strong>for</strong> by a single function, with coefficients progressively diminishing into <strong>the</strong> past.<br />
Timing of <strong>an</strong>ticipatory reversals was thus derived as a running, weighted average of prior<br />
stimuli. This resulted in a response that was reasonably symmetric <strong>for</strong> <strong>the</strong> highest frequency<br />
within each r<strong>an</strong>ge but highly skewed <strong>for</strong> lower frequencies as a result of <strong>an</strong>ticipation occurring<br />
too early. The non-linear gain <strong>an</strong>d phase characteristics occurred as a natural consequence of
<strong>the</strong>se ch<strong>an</strong>ges in <strong>the</strong> symmetry of <strong>the</strong> response. In effect, continual attempts to predict stimulus<br />
reversal in <strong>the</strong> r<strong>an</strong>domized wave<strong>for</strong>m favored early <strong>an</strong>ticipation <strong>an</strong>d thus accentuated <strong>the</strong> highest<br />
frequency components.<br />
Disclosures: G.R. Barnes, Medical Research Council UK, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
C.J.S. Collins, Medical Research Council, A. Employment (full or part-time).<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.3/CC45<br />
Topic: D.06.e. Pursuit<br />
Support: FNRS<br />
FRSM<br />
IAP <strong>an</strong>d PRODEX (SSTC)<br />
FSR (UCL)<br />
ARC (BELGIUM)<br />
ESA (EU)<br />
Title: Predictive oculomotor behavior in patients with frontotemporal lobar degeneration<br />
Authors: *S. COPPE 1,2 , D. YUKSEL 2,3 , A. IVANOIU 4 , P. LEFEVRE 2 ;<br />
1 Univ. catholique de Louvain, Louvain la Neuve, Belgium; 2 CESAME <strong>an</strong>d Lab. of<br />
Neurophysiol., Univ. catholique de Louvain, Brussels, Belgium; 3 Ophthalmology Dept., 4 Neurol.<br />
Dept., Cliniques Universitaires Saint-Luc, Brussels, Belgium<br />
Abstract: In normal subjects, saccades compensate <strong>for</strong> <strong>the</strong> variability of <strong>the</strong> smooth pursuit eye<br />
movements during bl<strong>an</strong>king of <strong>the</strong> target. Several studies suggest that predictive pursuit relies on<br />
frontal <strong>an</strong>d temporal lobes (Frontal <strong>an</strong>d Supplementary Eye Fields, Temporal areas). In this<br />
study, we tested whe<strong>the</strong>r <strong>an</strong> impairment of <strong>the</strong>se cortical areas would damage <strong>the</strong> predictive<br />
pursuit <strong>an</strong>d/or its saccade compensation.
Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disorder that is associated<br />
with degeneration of <strong>the</strong> frontal <strong>an</strong>d/or <strong>an</strong>terior temporal lobes with relative sparing of more<br />
posterior cortical regions. We recorded eye movements of one patient with frontotemporal<br />
dementia (FTD-patient) presenting as a behavioral/dysexecutive syndrome (frontal lobe vari<strong>an</strong>t)<br />
<strong>an</strong>d one patient with sem<strong>an</strong>tic dementia (SD-patient) presenting with sem<strong>an</strong>tic memory loss<br />
(temporal lobe vari<strong>an</strong>t). Diagnosis was confirmed by <strong>the</strong> clinical assessment, neuropsychological<br />
evaluation, magnetic reson<strong>an</strong>ce imaging <strong>an</strong>d functional imagery.<br />
Previous studies (Meyniel et al 2005, Boxer et al 2006, Garbutt et al 2008) have shown<br />
abnormalities in smooth pursuit <strong>an</strong>d in <strong>an</strong>ti but not reflexive saccades <strong>for</strong> FTD-patients. SDpatients<br />
showed normal oculomotor behavior compared to control subjects <strong>for</strong> saccades <strong>an</strong>d<br />
smooth pursuit in all <strong>the</strong> tested conditions.<br />
We tested <strong>an</strong>ticipatory smooth pursuit eye movements be<strong>for</strong>e target onset <strong>an</strong>d predictive pursuit<br />
during occlusion. In addition, <strong>the</strong> degree of impairment of ei<strong>the</strong>r <strong>the</strong> saccadic system <strong>an</strong>d/or <strong>the</strong><br />
pursuit system was compared in order to test if <strong>the</strong>re is a coupling between <strong>the</strong> two pathways <strong>for</strong><br />
compensation to <strong>the</strong> deficit. The experiment was <strong>the</strong> following; after fixation <strong>an</strong>d a gap period,<br />
<strong>the</strong> target started moving at const<strong>an</strong>t velocity be<strong>for</strong>e bl<strong>an</strong>king <strong>an</strong>d reappear<strong>an</strong>ce (r<strong>an</strong>dom<br />
direction, gap <strong>an</strong>d bl<strong>an</strong>king duration, target velocity).<br />
Anticipatory <strong>an</strong>d predictive pursuit <strong>for</strong> SD-patient showed no major impairment. In contrast,<br />
pursuit gain was slightly decreased <strong>for</strong> <strong>the</strong> FTD-patient during visually-guided tracking <strong>an</strong>d <strong>the</strong><br />
deficit was even more severe during target bl<strong>an</strong>king (especially <strong>for</strong> target velocity > 10 deg/s). In<br />
this condition, <strong>the</strong> saccadic system could not compensate <strong>for</strong> <strong>the</strong> predictive pursuit deficit.<br />
Results will be discussed in order to correlate <strong>the</strong> predictive eye movements with <strong>the</strong> lobar<br />
degeneration in order to bring insight to <strong>the</strong> role of <strong>the</strong> frontal <strong>an</strong>d temporal cortical regions in<br />
this process. Moreover, <strong>the</strong> observed abnormalities may serve as sensitive indicators to guide <strong>the</strong><br />
clinici<strong>an</strong> in <strong>the</strong> early diagnosis, <strong>an</strong>d to follow <strong>the</strong> evolution of <strong>the</strong> disease.<br />
Disclosures: S. Coppe, None; D. Yuksel, None; A. Iv<strong>an</strong>oiu, None; P. Lefevre, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.4/CC46<br />
Topic: D.06.e. Pursuit<br />
Support: Medical Research Council UK<br />
Title: Rapid acquisition of a generic predictive pursuit response to r<strong>an</strong>domized target motion in<br />
2D
Authors: *C. J. COLLINS, R. ACKERLEY, G. R. BARNES;<br />
Fac. of Life Sci., The Univ. of M<strong>an</strong>chester, M<strong>an</strong>chester, United Kingdom<br />
Abstract: Although development of predictive pursuit responses is normally associated with<br />
predictable target motion, recent experiments have shown that <strong>an</strong>ticipatory responses may be<br />
elicited even when <strong>the</strong> stimulus is r<strong>an</strong>domized. We recently showed that during horizontal<br />
pursuit of <strong>an</strong> aperiodic tri<strong>an</strong>gular wave<strong>for</strong>m <strong>an</strong>ticipatory eye deceleration occurred prior to target<br />
reversal, even though duration <strong>an</strong>d speed of successive ramp durations were r<strong>an</strong>domized. Now,<br />
we have examined <strong>the</strong> ability of subjects to follow <strong>the</strong> movement of a target in a quadrilateral<br />
pattern in which successive horizontal <strong>an</strong>d vertical ramp movements had const<strong>an</strong>t speed<br />
(15deg/s) but r<strong>an</strong>domized duration. Our aim was to determine whe<strong>the</strong>r, in addition to<br />
<strong>an</strong>ticipatory slowing, <strong>an</strong> <strong>an</strong>ticipatory acceleration would occur in <strong>the</strong> orthogonal axis in<br />
preparation <strong>for</strong> <strong>the</strong> next ch<strong>an</strong>ge of direction. Three overlapping r<strong>an</strong>ges of ramp duration (RD)<br />
were presented separately: Short (420-840ms), Medium (600-1020ms) <strong>an</strong>d Long (780-1200ms).<br />
8 RDs were presented within each r<strong>an</strong>ge; each RD was repeated 16 times in each axis. Responses<br />
were compared with controls in which each RD was held const<strong>an</strong>t <strong>for</strong> 5 cycles of <strong>the</strong> result<strong>an</strong>t<br />
square pattern motion.<br />
As found previously <strong>for</strong> single axis motion, subjects exhibited <strong>an</strong>ticipatory slowing of eye speed,<br />
both during horizontal <strong>an</strong>d vertical motion, prior to <strong>the</strong> orthogonal ch<strong>an</strong>ge of direction, even<br />
though duration was r<strong>an</strong>domized. However, in addition, <strong>the</strong>re was <strong>an</strong> accomp<strong>an</strong>ying <strong>an</strong>ticipatory<br />
acceleration of smooth eye movement in <strong>the</strong> orthogonal axis, both <strong>for</strong> vertical <strong>an</strong>d horizontal<br />
movement. Notably, <strong>the</strong> time at which <strong>an</strong>ticipatory slowing started was fairly const<strong>an</strong>t<br />
irrespective of <strong>the</strong> ramp duration. This was measured by determining <strong>the</strong> time (T90) at which eye<br />
velocity fell to 90% of its peak. T90 was const<strong>an</strong>t across each r<strong>an</strong>ge but increased as <strong>the</strong> me<strong>an</strong> of<br />
<strong>the</strong> r<strong>an</strong>ge increased. By contrast, T90 <strong>for</strong> <strong>the</strong> predictable control responses progressively<br />
increased as RD increased. Comparison of velocity profiles <strong>for</strong> each RD showed that responses<br />
to <strong>the</strong> r<strong>an</strong>domized responses lay midway between those of <strong>the</strong> predictable responses <strong>for</strong> <strong>the</strong><br />
extremes of each RD r<strong>an</strong>ge. Anticipatory acceleration was measured by calculating eye velocity<br />
(V50) 50ms after each direction ch<strong>an</strong>ge, prior to <strong>the</strong> effects of visual feedback. V50 values were<br />
greater in <strong>the</strong> horizontal th<strong>an</strong> in <strong>the</strong> vertical axis <strong>an</strong>d slightly higher in predictable (me<strong>an</strong>s:<br />
H=5.07; V= 2.05deg/s) th<strong>an</strong> r<strong>an</strong>domized conditions (H= 4.25; V=1.59deg/s). Anticipatory<br />
acceleration <strong>an</strong>d deceleration components developed within <strong>the</strong> first 2-3 ramp movements,<br />
illustrating <strong>the</strong> rapidity with which a generic prediction was developed despite stimulus<br />
r<strong>an</strong>domization.<br />
Disclosures: C.J. Collins, None; R. Ackerley, None; G.R. Barnes, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 559.5/CC47<br />
Topic: D.06.e. Pursuit<br />
Support: NSF BCS0717272<br />
Title: Predictive smooth pursuit: C<strong>an</strong> pursuit guided by motor <strong>an</strong>d/or spatial memory be<br />
distinguished?<br />
Authors: P. FU 1 , R. CHANG 1 , *W. KING 2 ;<br />
1 Col. of Literature, Sci. <strong>an</strong>d Arts, 2 Dept Otolaryngology, Univ. Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Hum<strong>an</strong>s track moving targets with two characteristic types of eye movements:<br />
saccadic pursuit <strong>an</strong>d smooth pursuit. Saccades are rapid eye movements that correct eye position<br />
errors but which may reduce visual acuity. Smooth pursuit attempts to maintain <strong>the</strong> fovea<br />
continuously on target to so as to minimize reductions in visual acuity. In general, smooth<br />
pursuit c<strong>an</strong>not be initiated without a visible moving target. However, once initiated, smooth<br />
pursuit movements c<strong>an</strong> persist if <strong>the</strong> target disappears tr<strong>an</strong>siently. Saccades c<strong>an</strong> be generated<br />
with or without a visible target.<br />
To study smooth pursuit, Rashbass (J. Physiol. Lond. 1961) used a ramp-like target movement.<br />
Later studies modified this paradigm <strong>an</strong>d occluded or “blinked” <strong>the</strong> target tr<strong>an</strong>siently to study <strong>the</strong><br />
persistence of pursuit (e.g., Mitrami & Dimitrov, Vision Res. 1978; Becker & Fuchs, Exp. Brain<br />
Res. 1985). These studies showed that smooth pursuit persisted, but at a reduced speed when <strong>the</strong><br />
target was blinked.<br />
HYPOTHESIS I: <strong>the</strong> persistence of pursuit when a target is tr<strong>an</strong>siently blinked is produced by<br />
“motor memory”, i.e., <strong>the</strong> motor comm<strong>an</strong>d or efference copy prior to <strong>the</strong> target’s disappear<strong>an</strong>ce<br />
is remembered <strong>an</strong>d re-used to continue (persevere) <strong>the</strong> pursuit along <strong>the</strong> same trajectory.<br />
Persistence alone is insufficient to account <strong>for</strong> predictive pursuit of a target that ch<strong>an</strong>ges<br />
direction or speed (e.g., Bennett & Barnes, J. Neurophysiol. 2004; Bennett & Barnes, Exp. Brain<br />
Res. 2006; Bennett et al., J. Neurophysiol. 2007).<br />
HYPOTHESIS II: predictive pursuit of a target whose trajectory ch<strong>an</strong>ges when it is tr<strong>an</strong>siently<br />
blinked is produced by <strong>an</strong> estimate of <strong>the</strong> target’s future motion <strong>an</strong>d position that is created <strong>from</strong><br />
spatial memory of <strong>the</strong> target’s prior motion <strong>an</strong>d position <strong>an</strong>d <strong>the</strong> observer’s prior experience.<br />
To examine <strong>the</strong>se hypo<strong>the</strong>ses, we introduced a modification of <strong>the</strong> Rashbass paradigm that we<br />
call a time shifted step ramp. In a shifted step ramp, <strong>the</strong> target abruptly ch<strong>an</strong>ges direction after<br />
pursuit is initiated <strong>an</strong>d during <strong>the</strong> time interval when <strong>the</strong> target is blinked. The shifted step ramp<br />
c<strong>an</strong>not be tracked by reli<strong>an</strong>ce on perseverating eye speed prior to <strong>the</strong> target’s disappear<strong>an</strong>ce<br />
(Hypo<strong>the</strong>sis I). Instead, subjects must create a new estimate of <strong>the</strong> target’s path based on prior<br />
experience <strong>an</strong>d <strong>the</strong>n correct <strong>the</strong>ir eye movements accordingly (Hypo<strong>the</strong>sis II). An individual's<br />
uncertainty about target velocity <strong>an</strong>d where it may reappear leads to a more domin<strong>an</strong>t<br />
involvement of spatial memory to drive eye movement. Thus <strong>the</strong> spatial estimate of target<br />
motion must suppress <strong>the</strong> component of pursuit guided by motor memory if <strong>the</strong> target's trajectory<br />
ch<strong>an</strong>ges during a blink.<br />
Disclosures: P. Fu, None; R. Ch<strong>an</strong>g, None; W. King, None.
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.6/CC48<br />
Topic: D.06.e. Pursuit<br />
Support: NIH Gr<strong>an</strong>t EY012212<br />
Aginsky Scholars Award<br />
Title: Superior colliculus inactivation reveals competitive interactions between motion inputs to<br />
smooth pursuit<br />
Authors: *S. U. NUMMELA 1,2 , R. J. KRAUZLIS 1 ;<br />
1 Salk Inst., La Jolla, CA; 2 Neurosciences Grad. Program, UCSD, La Jolla, CA<br />
Abstract: Smooth pursuit to <strong>the</strong> onset of two moving stimuli is a weighted average of <strong>the</strong> pursuit<br />
response to <strong>the</strong> individual stimuli (Lisberger <strong>an</strong>d Ferrera, 1997). Last year we showed that<br />
inactivation of primate superior colliculus (SC) reduced <strong>the</strong> contribution of moving stimuli<br />
located within <strong>the</strong> affected region of visual space (Nummela <strong>an</strong>d Krauzlis, SfN 2008). We now<br />
report that SC inactivation also results in a complementary increase in <strong>the</strong> contribution of <strong>the</strong><br />
moving stimulus out of <strong>the</strong> affected region of visual space.<br />
We evaluated <strong>the</strong> contribution of moving stimuli <strong>for</strong> smooth pursuit using a 'vector-averaging'<br />
task. On each trial, two identical bright spots moved orthogonally, in a step-ramp fashion<br />
towards central fixation. Once both spots reached fixation, one spot disappeared <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r<br />
continued on its trajectory; ei<strong>the</strong>r spot was equally likely to continue on as <strong>the</strong> target. The<br />
monkey was rewarded <strong>for</strong> successfully tracking <strong>the</strong> target by <strong>the</strong> end of <strong>the</strong> trial. Data were<br />
collected on <strong>the</strong> vector-averaging task be<strong>for</strong>e <strong>an</strong>d after focal inactivation of SC by injection of<br />
muscimol (0.5 µl per injection, 5 µg/µl). After SC inactivation, <strong>the</strong> affected region of visual<br />
space was determined by measuring <strong>the</strong> velocities of visually guided saccades. We qu<strong>an</strong>tified <strong>the</strong><br />
“weight” of each stimulus’ contribution by measuring <strong>the</strong> eye velocity <strong>for</strong> <strong>the</strong> first 50 ms of<br />
smooth pursuit.<br />
As previously reported, SC inactivation reduced <strong>the</strong> weight of motion inputs located in <strong>the</strong><br />
affected region of visual space (p < 0.0001, n = 14 experiments), by about half (0.56 +/- 0.12 of<br />
baseline). In addition, we now report that <strong>the</strong> weight of <strong>the</strong> orthogonal motion input, located out<br />
of <strong>the</strong> affected region, was signific<strong>an</strong>tly increased (p = 0.023, 1.12 +/- 0.12 of baseline). This<br />
increase in weight did not occur when <strong>the</strong> orthogonal stimuli were paired with a moving stimulus<br />
located in <strong>the</strong> visual hemifield opposite <strong>the</strong> affected region (p = 0.50) or when presented alone (p
= 0.37).<br />
These results indicate that <strong>the</strong> ch<strong>an</strong>ges in stimulus weight caused by SC inactivation involve<br />
interactions between <strong>the</strong> activity <strong>from</strong> each stimulus. The complementary increase in weight<br />
observed <strong>for</strong> stimuli outside of <strong>the</strong> affected region suggests that this interaction involves a<br />
normalization of motion weights <strong>for</strong> smooth pursuit at or after <strong>the</strong> stage of visuomotor<br />
processing that occurs in <strong>the</strong> SC.<br />
Disclosures: S.U. Nummela, None; R.J. Krauzlis, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.7/CC49<br />
Topic: D.06.e. Pursuit<br />
Support: MEXT 17022001<br />
MEXT 20500351<br />
NIH EY-06558 (NEI)<br />
RR-00166<br />
Title: Neuronal activity in <strong>the</strong> caudal frontal eye fields (FEF): Comparison with <strong>the</strong><br />
supplementary eye fields (SEF) <strong>an</strong>d cerebellar dorsal vermis during memory-based smooth<br />
pursuit eye movements<br />
Authors: *J. FUKUSHIMA 1 , T. AKAO 2 , N. SHICHINOHE 2 , S. KURKIN 2 , C. R. S.<br />
KANEKO 3 , K. FUKUSHIMA 2 ;<br />
1 Dept. of Hlth. Sci., 2 Dept. of Physiol., Hokkaido Univ. Sch. of Med., Sapporo, Jap<strong>an</strong>; 3 Dept<br />
Physiol & Biophys & Washington Natl. Primate Res. Ctr., Univ. Washington, Seattle, WA<br />
Abstract: Smooth pursuit eye movements maintain target images on <strong>the</strong> foveae during<br />
movement by compensating <strong>for</strong> <strong>the</strong> delays involved in processing visual motion in<strong>for</strong>mation<br />
<strong>an</strong>d/or producing eye velocity comm<strong>an</strong>ds. Memory of visual motion may be used <strong>for</strong> predictive<br />
smooth pursuit, however, definitive proof is still lacking. To distinguish neuronal activity<br />
associated with <strong>the</strong> preparation <strong>for</strong> smooth pursuit <strong>from</strong> discharge related to processing or<br />
memory of <strong>the</strong> target motion signals, we trained 2 monkeys on a memory-based smooth pursuit
task. While fixating a stationary spot, <strong>the</strong> monkeys were required to memorize <strong>the</strong> direction of<br />
r<strong>an</strong>dom dot motion (cue-1). After a delay, a second cue (cue-2) instructed monkeys to prepare<br />
ei<strong>the</strong>r <strong>for</strong> pursuit in <strong>the</strong> memorized direction (go) or to maintain fixation (no-go). After a 2nd<br />
delay, <strong>the</strong> monkeys were required to execute <strong>the</strong> correct action by selecting one of 3 spots (one<br />
fixed at <strong>the</strong> center <strong>an</strong>d two moving away in opposite directions) based on <strong>the</strong> memory of <strong>the</strong><br />
motion in cue-1 <strong>an</strong>d cue-2 instruction (color). By comparing <strong>the</strong> discharge during each period<br />
with that during fixation, we showed that <strong>the</strong> SEF contained signals that code memory <strong>an</strong>d<br />
assessment of visual motion-direction, <strong>the</strong> decision whe<strong>the</strong>r or not to pursue, <strong>an</strong>d <strong>the</strong> preparation<br />
<strong>for</strong> pursuit (Fukushima et al. NCM <strong>2009</strong> abstr). To underst<strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>se SEF signals are<br />
unique to SEF, we examined <strong>the</strong> discharge of 160 task-related neurons in <strong>the</strong> caudal FEF in <strong>the</strong><br />
same monkeys. Of <strong>the</strong>se, 82 neurons were recorded during simple pursuit; 45 were pursuitrelated<br />
<strong>an</strong>d 37 were pursuit-unrelated. Neurons that coded visual motion-memory <strong>an</strong>d no-go<br />
signals were also represented in both groups of FEF neurons, however, <strong>the</strong> percentages of such<br />
neurons was signific<strong>an</strong>tly lower compared to those in <strong>the</strong> SEF (16/160=10% vs 52/208=25% of<br />
task-related neurons <strong>for</strong> visual motion-memory, p
Title: MST activity during memory-based smooth pursuit eye movements: Comparison with<br />
supplementary eye fields (SEF)<br />
Authors: *S. KURKIN 1 , N. SHICHINOHE 1 , T. AKAO 1 , J. FUKUSHIMA 2 , K.<br />
FUKUSHIMA 1 ;<br />
1 Dept. of Physiol., 2 Dept. of Hlth. Sci., Hokkaido Univ. Sch. of Med., Sapporo, Jap<strong>an</strong><br />
Abstract: During smooth pursuit eye movements, prediction is needed to compensate <strong>for</strong><br />
response-delays <strong>an</strong>d to maintain target images on <strong>the</strong> foveae. To distinguish neuronal discharge<br />
related to memory of visual motion-direction <strong>from</strong> that related to prediction, we trained Jap<strong>an</strong>ese<br />
monkeys to per<strong>for</strong>m a memory-based smooth pursuit task. While fixating a stationary spot<br />
(control fixation), <strong>the</strong> monkeys were required to memorize <strong>the</strong> direction of r<strong>an</strong>dom dot motion<br />
(cue-1). After a delay (delay-1, 2s), a second cue (cue-2) instructed <strong>the</strong> monkeys to prepare ei<strong>the</strong>r<br />
pursuit in <strong>the</strong> memorized direction (go) or to maintain fixation (no-go). After a second delay<br />
(delay-2, 2s), <strong>the</strong> monkeys were required to execute <strong>the</strong> correct action by selecting one of 3 spots<br />
(one fixed at <strong>the</strong> center <strong>an</strong>d two moving away in opposite directions) based on <strong>the</strong> cue-1 visual<br />
motion-memory <strong>an</strong>d cue-2 instruction. By comparing <strong>the</strong> discharge during each period with that<br />
during control fixation, we reported recently that <strong>the</strong> SEF contained signals coding retinal imageslip-velocity,<br />
memory <strong>an</strong>d assessment of visual motion-direction, <strong>the</strong> decision of whe<strong>the</strong>r or not<br />
to pursue, <strong>an</strong>d <strong>the</strong> preparation <strong>for</strong> pursuit (Fukushima et al. NCM <strong>2009</strong>, abstr). Since MST plays<br />
a major role in visual motion processing <strong>an</strong>d projects to <strong>the</strong> SEF, we examined whe<strong>the</strong>r MST<br />
carries visual motion-memory using <strong>the</strong> identical task. 108 MST neurons were tested that<br />
exhibited a visual response to cue-1. Of <strong>the</strong>se, 98 neurons exhibited directional responses to cue-<br />
1, <strong>an</strong>d 10 neurons were non-directional. The majority of <strong>the</strong>m (69/108=64%) were also<br />
modulated during smooth pursuit eye movements within <strong>the</strong> action period. Only 4 of <strong>the</strong> 108<br />
(4%) exhibited maintained cue-1 discharge during delay-1 (Bonferroni corrected t-test, P
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.9/CC51<br />
Topic: D.06.e. Pursuit<br />
Support: NIH EY-12816<br />
NIH EY-18322<br />
Title: Your eyes know more th<strong>an</strong> you: Higher oculometric th<strong>an</strong> psychometric per<strong>for</strong>m<strong>an</strong>ce in a<br />
velocity discrimination task<br />
Authors: *A. TAVASSOLI, D. L. RINGACH;<br />
UCLA, Los Angeles, CA<br />
Abstract: The influential work of Goodale <strong>an</strong>d Milner (1992) suggested distinct visual pathways<br />
involved in <strong>the</strong> control of actions <strong>an</strong>d in perception. Smooth pursuit eye movements are <strong>an</strong><br />
example of visually guided motor actions. The main goal of <strong>the</strong> smooth pursuit system is to<br />
stabilize <strong>the</strong> retinal image of a moving target. Comparing <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of pursuit with that of<br />
perception in psychophysical tasks c<strong>an</strong> shed light into a possible dissociation between vision <strong>for</strong><br />
perception <strong>an</strong>d action.<br />
Previous studies have examined oculometric <strong>an</strong>d perceptual per<strong>for</strong>m<strong>an</strong>ce in speed discrimination<br />
tasks (Kowler <strong>an</strong>d McKee, 1987; Gegenfurtner et al, 2003), where thresholds were found to be<br />
similar <strong>for</strong> pursuit <strong>an</strong>d perceptual discrimination. Moreover, Gegenfurtner <strong>an</strong>d colleagues also<br />
found that motor <strong>an</strong>d perceptual errors were uncorrelated. This suggested a common signal<br />
driving both eye movements <strong>an</strong>d perception but having independent noise sources.<br />
We studied smooth pursuit eye movement <strong>an</strong>d perceptual per<strong>for</strong>m<strong>an</strong>ces in <strong>the</strong> discrimination of a<br />
brief temporal perturbation in velocity of a target o<strong>the</strong>rwise moving at a const<strong>an</strong>t speed. The<br />
velocity perturbation consisted of a single cycle of sinusoidal shape that was introduced during<br />
tracking (Churchl<strong>an</strong>d <strong>an</strong>d Lisberger, 2002; Schwartz <strong>an</strong>d Lisberger, 1994). In half of <strong>the</strong> trials,<br />
<strong>the</strong> velocity of <strong>the</strong> target (a high-contrast Gabor) initially increased during <strong>the</strong> perturbation<br />
(peak-first condition), <strong>an</strong>d in <strong>the</strong> o<strong>the</strong>r half, <strong>the</strong> velocity initially decreased (peak-last). We<br />
per<strong>for</strong>med experiments <strong>for</strong> a r<strong>an</strong>ge of temporal frequencies <strong>an</strong>d amplitudes of <strong>the</strong> perturbation<br />
cycle.<br />
Our results show a clear superiority of oculometric per<strong>for</strong>m<strong>an</strong>ce to psychometric per<strong>for</strong>m<strong>an</strong>ce,<br />
with d’ about 80% higher in some cases. In o<strong>the</strong>r words, eye movements were faithfully<br />
following <strong>the</strong> stimuli while <strong>the</strong> observer failed to discriminate among <strong>the</strong>m. We also observed<br />
that perceptual errors were uncorrelated with eye movements. These findings suggest a larger<br />
source of noise after <strong>the</strong> measurement of visual motion in <strong>the</strong> neural pathways mediating<br />
perception th<strong>an</strong> those involved in motor control.
Disclosures: A. Tavassoli, None; D.L. Ringach, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.10/CC52<br />
Topic: D.06.e. Pursuit<br />
Support: NIH Gr<strong>an</strong>t EY019266<br />
Title: Retinal error signals <strong>from</strong> <strong>the</strong> pretectal nucleus of <strong>the</strong> optic tract guide motor learning <strong>for</strong><br />
smooth pursuit<br />
Authors: *S. ONO, M. J. MUSTARI;<br />
Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: The smooth pursuit (SP) system has <strong>the</strong> capacity to adapt to ch<strong>an</strong>ges associated with<br />
development, injury <strong>an</strong>d new behavioral dem<strong>an</strong>ds. The sources of signals that support SP<br />
adaptation are incompletely understood but could involve cortical <strong>an</strong>d brainstem areas involved<br />
in retinal image motion processing <strong>an</strong>d generation of pursuit. Previous lesion <strong>an</strong>d single unit<br />
recording studies show that <strong>the</strong> macaque cerebellum plays <strong>an</strong> essential role in SP initiation,<br />
mainten<strong>an</strong>ce <strong>an</strong>d adaptation. Here, we focus on <strong>the</strong> potential role of <strong>the</strong> pretectal nucleus of <strong>the</strong><br />
optic tract (NOT) in SP adaptation. The NOT provides retinal error in<strong>for</strong>mation to <strong>the</strong><br />
cerebellum (e.g., ventral paraflocculus <strong>an</strong>d vermis), which could support adaptation.<br />
To test <strong>the</strong> role of <strong>the</strong> NOT in adaptation, we applied brief (200ms) trains of micro-electrical<br />
(100µA) stimulation (ES) in one NOT to introduce directional error signals at <strong>the</strong> point in time<br />
where a second target speed would appear in st<strong>an</strong>dard double-step paradigms. We extinguished<br />
<strong>the</strong> target coincident with onset of <strong>the</strong> ES train. We used 100 trials of SP coupled with ES to<br />
produce adaptation similar to that observed using st<strong>an</strong>dard double-step adaptation paradigm.<br />
Initial eye acceleration <strong>an</strong>d eye velocity (first 100 ms) showed signific<strong>an</strong>t increases (P< 0.001;<br />
unpaired t-test) after repetition (100 trials) of ES during ongoing pursuit in ipsiversive direction.<br />
In contrast, Initial eye acceleration <strong>an</strong>d eye velocity (first 100 ms) showed signific<strong>an</strong>t decreases<br />
(P< 0.001; unpaired t-test) after repetition (100 trials) of ES during ongoing pursuit in<br />
contraversive direction. In control testing, we delivered ES at <strong>the</strong> same interval as in our stepramp<br />
testing but without target motion or SP. In this condition, no charging of velocity-storage<br />
was found (P = 0.87; unpaired t-test). There<strong>for</strong>e, ES of <strong>the</strong> NOT paired with SP appears to<br />
simulate conditions produce when visual error signals alone guide SP adaptation. Our results
suggest that NOT serves as <strong>an</strong> import<strong>an</strong>t source of visual error signals <strong>for</strong> SP adaptation.<br />
1<br />
Disclosures: S. Ono, None; M.J. Mustari, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.11/CC53<br />
Topic: D.06.e. Pursuit<br />
Support: HHMI<br />
NIH Gr<strong>an</strong>t EY03878<br />
Title: Two loci of vector averaging <strong>for</strong> smooth pursuit eye movements<br />
Authors: *Y.-Q. NIU 1,2,3 , S. G. LISBERGER 1,2,3 ;<br />
1 UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 2 Howard Hughes Med. Inst., S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 3 W.M. Keck Fndn.<br />
Ctr. <strong>for</strong> Integrative Neurosci., S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Weighted vector averaging is one of <strong>the</strong> most import<strong>an</strong>t strategies in visual-motor<br />
processing to decode neuron population responses <strong>an</strong>d create comm<strong>an</strong>ds <strong>for</strong> movement. One<br />
piece of direct behavioral evidence <strong>for</strong> vector averaging is provided by <strong>the</strong> smooth pursuit eye<br />
movements evoked by <strong>the</strong> motion of two visual stimuli in orthogonal directions. The initial eye<br />
acceleration is towards <strong>the</strong> middle direction of <strong>the</strong> two stimuli’s directions, defined by <strong>the</strong><br />
equally-weighted vector averaging of <strong>the</strong> two target motions. To explore possible loci of vector<br />
averaging, we studied <strong>the</strong> effects on <strong>the</strong> initiation of pursuit eye movements of <strong>the</strong> relative<br />
lumin<strong>an</strong>ce <strong>an</strong>d stimulus location of two moving stimuli. We found evidence <strong>for</strong> at least two types<br />
of vector averaging. For overlapped patches of r<strong>an</strong>dom dots, <strong>the</strong> weight of vector averaging<br />
strongly favored <strong>the</strong> more luminous patch, <strong>an</strong>d rapidly devolved to winner-take-all <strong>for</strong> <strong>the</strong><br />
brighter stimulus. For r<strong>an</strong>dom dot patches at separate locations in <strong>the</strong> visual field, <strong>the</strong> weight of<br />
vector averaging ch<strong>an</strong>ged more gradually as a function of <strong>the</strong> lumin<strong>an</strong>ce ratio <strong>an</strong>d kept <strong>the</strong><br />
vector-averaging pattern <strong>for</strong> a wide r<strong>an</strong>ge of ratios. The effects of relative lumin<strong>an</strong>ce <strong>an</strong>d<br />
stimulus location were not caused by <strong>the</strong> absolute lumin<strong>an</strong>ce of <strong>the</strong> stimuli, or by differences in<br />
pursuit latency or response amplitude <strong>for</strong> <strong>the</strong> stimuli presented singly. We also observed that <strong>the</strong><br />
trial-by-trial vari<strong>an</strong>ce of eye direction was much larger <strong>for</strong> overlapped patches of equal<br />
lumin<strong>an</strong>ce th<strong>an</strong> <strong>for</strong> single patches. We suggest that <strong>the</strong>re are at least two types of vector
averaging that exist in different locations of <strong>the</strong> visual-motor circuit <strong>for</strong> pursuit. One may be<br />
located in <strong>the</strong> sensory cortex while <strong>the</strong> o<strong>the</strong>r is located in downstream areas, perhaps <strong>the</strong><br />
cerebellum.<br />
Disclosures: Y. Niu, None; S.G. Lisberger, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.12/CC54<br />
Topic: D.06.e. Pursuit<br />
Support: HHMI<br />
P50 MH77970<br />
Title: Correlations between activity in <strong>the</strong> lateral intraparietal area (LIP) <strong>an</strong>d post-saccadic<br />
smooth pursuit eye velocity<br />
Authors: *J. G. O'LEARY, S. G. LISBERGER;<br />
Univ. Cali<strong>for</strong>nia-S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: <strong>When</strong> a moving target is far <strong>from</strong> <strong>the</strong> fovea, <strong>the</strong> smooth pursuit eye movement system<br />
tracks its motion relatively poorly. If a saccade is <strong>the</strong>n made to <strong>the</strong> target, <strong>the</strong> smooth pursuit<br />
response immediately after <strong>the</strong> saccade is much more robust. This "enh<strong>an</strong>cement" of <strong>the</strong> pursuit<br />
response is linked to <strong>the</strong> occurrence of a saccade (Lisberger 1998; Gardner & Lisberger 2002),<br />
showing <strong>an</strong> import<strong>an</strong>t interaction between saccades <strong>an</strong>d pursuit. Post-saccadic enh<strong>an</strong>cement of<br />
pursuit c<strong>an</strong> be thought of as motor attention that focuses <strong>the</strong> pursuit eye velocity on <strong>the</strong> image<br />
motion at <strong>the</strong> target of <strong>the</strong> saccade. To test directly <strong>for</strong> a link <strong>from</strong> areas involved in saccades <strong>an</strong>d<br />
attention to pursuit, we recorded <strong>the</strong> activity of single units in area LIP while a monkey made<br />
saccades to <strong>an</strong>d <strong>the</strong>n pursued a moving target. We asked whe<strong>the</strong>r <strong>the</strong> trial-by-trial variation in <strong>the</strong><br />
activity of LIP neurons was correlated with <strong>the</strong> post-saccadic pursuit eye velocity. <strong>When</strong> <strong>the</strong><br />
target moved through a neuron’s receptive field, <strong>the</strong> average correlation (across units) between<br />
peri-saccadic LIP activity <strong>an</strong>d immediate post-saccadic eye velocity was signific<strong>an</strong>tly greater<br />
th<strong>an</strong> zero. <strong>When</strong> <strong>the</strong> target moved outside <strong>the</strong> receptive field, <strong>the</strong> average (across units)<br />
correlation was negative. The correlation between LIP activity <strong>an</strong>d post-saccadic smooth eye<br />
velocity could not be explained by <strong>the</strong> correlations between LIP activity <strong>an</strong>d saccade latency or
eye position at <strong>the</strong> end of <strong>the</strong> saccade. We conclude that <strong>the</strong> activity of area LIP neurons may<br />
play a role in determining <strong>the</strong> strength of <strong>the</strong> post-saccadic smooth pursuit response.<br />
Disclosures: J.G. O'Leary, None; S.G. Lisberger, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.13/CC55<br />
Topic: D.06.e. Pursuit<br />
Support: HHMI<br />
EY03878<br />
Title: Responses of MT neurons to dot textures with different amounts of directional noise<br />
Authors: *J. YANG, S. G. LISBERGER;<br />
HHMI, W.M. Keck Center, Dept Physiol., Univ. Cali<strong>for</strong>nia S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: In prior work, our laboratory has shown that <strong>the</strong> trial-by-trial vari<strong>an</strong>ce of pursuit<br />
increases when noise is added to a dot texture by having each dot follow a r<strong>an</strong>dom walk<br />
trajectory. We now explore how <strong>the</strong> same stimuli affect <strong>the</strong> response of <strong>the</strong> population of motion<br />
sensitive neurons in extrastriate visual area MT. Adding noise to <strong>the</strong> stimulus causes ch<strong>an</strong>ges in<br />
<strong>the</strong> direction tuning curves of individual neurons. On average, response amplitude decreases <strong>an</strong>d<br />
tuning b<strong>an</strong>dwidth increases as a function of <strong>the</strong> magnitude of <strong>the</strong> directional noise. The effect of<br />
noise varies widely <strong>from</strong> neuron to neuron, however, with some neurons showing no ch<strong>an</strong>ge at<br />
all <strong>an</strong>d o<strong>the</strong>rs showing <strong>an</strong> increase in tuning b<strong>an</strong>dwidth to 140% of control <strong>an</strong>d a reduction in<br />
response amplitude to 40% of control. The trial-by-trial vari<strong>an</strong>ce of individual neural spike<br />
counts was not increased uni<strong>for</strong>mly by added directional noise. Instead, spike count vari<strong>an</strong>ce<br />
retained its control relationship to spike count me<strong>an</strong>, with F<strong>an</strong>o factors close to one. We<br />
conclude that <strong>the</strong> effect of directional noise on <strong>the</strong> trial-by-trial vari<strong>an</strong>ce of pursuit must be<br />
attributed to <strong>an</strong> increase in response vari<strong>an</strong>ce <strong>an</strong>d b<strong>an</strong>dwidth across <strong>the</strong> neural population, ra<strong>the</strong>r<br />
th<strong>an</strong> to <strong>an</strong> increase in <strong>the</strong> vari<strong>an</strong>ce of <strong>the</strong> responses of individual MT neurons.<br />
Disclosures: J. Y<strong>an</strong>g, HHMI, A. Employment (full or part-time); HHMI, EY03878, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); S.G. Lisberger, HHMI, A. Employment (full or part-time); EY03878,
B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well<br />
as gr<strong>an</strong>ts already received).<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.14/CC56<br />
Topic: D.06.e. Pursuit<br />
Support: NIH Gr<strong>an</strong>t EY12212<br />
NSF Graduate Research Fellowship<br />
Title: Inactivation of <strong>the</strong> frontal pursuit area does not impair pursuit selectivity<br />
Authors: *S. P. MAHAFFY 1,2 , R. J. KRAUZLIS 1 ;<br />
1 Salk Inst., La Jolla, CA; 2 Neurosciences Grad. Program, UCSD, La Jolla, CA<br />
Abstract: Primates are adept at selectively tracking moving targets with smooth pursuit eye<br />
movements, even in <strong>the</strong> presence of competing distracter motions. The superior colliculus has<br />
been implicated recently in <strong>the</strong> spatial selection of targets <strong>for</strong> pursuit. But it is unknown how<br />
pursuit makes <strong>the</strong> directional selection required <strong>for</strong> <strong>the</strong> smooth eye velocity motor output, or<br />
which neural structures underlie this selection.<br />
There is some evidence that <strong>the</strong> frontal pursuit area (FPA) is involved in determining <strong>the</strong><br />
direction <strong>for</strong> pursuit. Neurons in <strong>the</strong> FPA are strongly tuned <strong>for</strong> pursuit direction. In addition, <strong>the</strong><br />
FPA has been shown to play a role in setting <strong>the</strong> gain of pursuit, a function which seems linked<br />
to target selection. And <strong>an</strong>alogous to areas involved in target selection <strong>for</strong> saccades, <strong>the</strong> frontal<br />
pursuit area c<strong>an</strong> be stimulated to evoke smooth movements during fixation. To test <strong>the</strong> role of <strong>the</strong><br />
FPA in selection, we have conducted several experiments using a target selection task. This task<br />
requires <strong>the</strong> monkey to generate smooth pursuit to select which of two parallel <strong>an</strong>d opposite<br />
moving targets matches <strong>the</strong> color of a remembered cue.<br />
Last year, we presented data <strong>from</strong> a recording experiment in <strong>the</strong> FPA. We found that a<br />
subst<strong>an</strong>tial minority of neurons discriminated target <strong>from</strong> distracter prior to pursuit initiation.<br />
This suggested that <strong>the</strong> FPA could play a role in target selection. However, several aspects of our<br />
data cast doubt on this conclusion. First, <strong>the</strong> majority of neurons did not become selective until<br />
after pursuit onset. Second, most of <strong>the</strong> variability in pursuit latencies was also evident in <strong>the</strong><br />
timing of FPA neurons’ selectivity, suggesting that <strong>the</strong> FPA lies downstream of <strong>the</strong> selection<br />
process.
To explicitly test <strong>the</strong> causal contribution of <strong>the</strong> FPA to target selection, we have now tr<strong>an</strong>siently<br />
inactivated <strong>the</strong> area with muscimol. If <strong>the</strong> FPA were involved in target selection, this<br />
m<strong>an</strong>ipulation should impair <strong>the</strong> monkey’s ability to select <strong>the</strong> inactivated direction. Our<br />
inactivation produced signific<strong>an</strong>t motor effects, including deficits in <strong>the</strong> initial acceleration <strong>an</strong>d<br />
maintained velocity, <strong>an</strong>d <strong>the</strong>se effects were most pronounced <strong>for</strong> ipsiversive pursuit. However, in<br />
<strong>the</strong> target selection task, we found no deficit in <strong>the</strong> monkey’s ability to select <strong>the</strong> ipsiversive<br />
target <strong>for</strong> pursuit movements. The percentage of correct responses, which was typically 70-85%<br />
be<strong>for</strong>e inactivation, remained unch<strong>an</strong>ged after each of seven FPA inactivations.<br />
These results suggest that <strong>the</strong> FPA is not necessary <strong>for</strong> selection of pursuit targets. Instead, <strong>the</strong><br />
FPA appears to be primarily related to generating motor comm<strong>an</strong>ds, <strong>an</strong>d probably lies<br />
downstream of target selection in <strong>the</strong> pathway <strong>for</strong> pursuit.<br />
Disclosures: S.P. Mahaffy, None; R.J. Krauzlis, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.15/CC57<br />
Topic: D.06.e. Pursuit<br />
Support: KAKENHI(17022019)<br />
Title: Functional subdivision of <strong>the</strong> hum<strong>an</strong> middle temporal complex based on motion reference<br />
frame: An fMRI study<br />
Authors: *T. YAMAMOTO 1 , H. YAMAMOTO 2 , H. MANO 2,3 , M. UMEDA 4 , C. TANAKA 3 ,<br />
K. KAWANO 1 ;<br />
1 Grad. Sch. of Med., 2 Grad. Sch. of Hum<strong>an</strong> <strong>an</strong>d Envrn. Studies, Kyoto Univ., Kyoto, Jap<strong>an</strong>;<br />
3 Dept. of Neurosurg., 4 Dept. of Med. In<strong>for</strong>matics, Meiji Univ. of Integrative Med., N<strong>an</strong>t<strong>an</strong>, Jap<strong>an</strong><br />
Abstract: [PURPOSE] It has been demonstrated that two motion-sensitive areas of monkeys, <strong>the</strong><br />
middle temporal area (MT) <strong>an</strong>d medial superior temporal area (MST) code distinct motion<br />
signals; MT neurons code <strong>for</strong> visual motion on <strong>the</strong> retina, whereas MST neurons code <strong>for</strong> visual<br />
motion in <strong>the</strong> external world while compensating <strong>for</strong> <strong>the</strong> counter-rotation of retinal images due to<br />
pursuit eye movements (Inaba et al., 2007). It remains, however, unclear whe<strong>the</strong>r such functional<br />
divisions also exist between hum<strong>an</strong> motion-sensitive areas MT <strong>an</strong>d MST or not. To <strong>an</strong>swer this<br />
question, <strong>the</strong> present study investigated cortical activity of hum<strong>an</strong> MT <strong>an</strong>d MST <strong>for</strong> visual<br />
motion stimuli in conjunction with smooth pursuit eye movements using functional magnetic
eson<strong>an</strong>ce imaging (fMRI).<br />
[METHOD] We first localized putative hum<strong>an</strong> MT <strong>an</strong>d MST based on retinotopy by using a new<br />
phase-encoding method, in which, instead of a st<strong>an</strong>dard large checkerboard pattern, a peripheral<br />
small motion-dot stimulus was rotated (Yamamoto et al., 2008). Next, we investigated <strong>the</strong> major<br />
source of motion signals <strong>for</strong> <strong>the</strong> putative MT <strong>an</strong>d MST. Subjects viewed a gray r<strong>an</strong>dom-dot<br />
pattern on <strong>the</strong> black background while pursuing a fixation point that moved reciprocatory. The<br />
r<strong>an</strong>dom-dot pattern was alternately between stationary <strong>an</strong>d moving with <strong>the</strong> fixation point. Thus,<br />
we compared cortical activity while <strong>the</strong> fixation point moved on <strong>the</strong> stationary r<strong>an</strong>dom-dot<br />
pattern (corresponding to retinal motion) to that while <strong>the</strong> fixation point moved with <strong>the</strong> r<strong>an</strong>domdot<br />
pattern (corresponding to external motion).<br />
[RESULTS] The putative MT showed a stronger fMRI response while <strong>the</strong> fixation point moved<br />
on <strong>the</strong> stationary r<strong>an</strong>dom-dot pattern. In contrast, <strong>the</strong> putative MST showed a stronger fMRI<br />
response while <strong>the</strong> fixation point moved with <strong>the</strong> r<strong>an</strong>dom-dot pattern. These results indicate that,<br />
like monkeys, <strong>the</strong> putative MT <strong>an</strong>d MST respond to <strong>the</strong> motion of <strong>the</strong> image on <strong>the</strong> retina <strong>an</strong>d <strong>the</strong><br />
motion of <strong>the</strong> image on <strong>the</strong> screen independent of pursuit eye movement, respectively.<br />
[CONCLUSION] Hum<strong>an</strong> motion-sensitive areas c<strong>an</strong> be also functionally subdivided into MT<br />
<strong>an</strong>d MST based on in<strong>for</strong>mation about visual motion to code; MT <strong>an</strong>d MST code <strong>for</strong> retinal <strong>an</strong>d<br />
external motion, respectively.<br />
Disclosures: T. Yamamoto, None; H. Yamamoto, None; H. M<strong>an</strong>o, None; M. Umeda,<br />
None; C. T<strong>an</strong>aka, None; K. Kaw<strong>an</strong>o, None.<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 559.16/CC58<br />
Topic: D.06.e. Pursuit<br />
Support: DoD gr<strong>an</strong>t W81XWH-08-1-0646<br />
DoD gr<strong>an</strong>t W81XWH-08-2-0177<br />
James S. McDonnell Foundation gr<strong>an</strong>t CNRC-TBI<br />
Title: Ocular pursuit per<strong>for</strong>m<strong>an</strong>ce as a metric <strong>for</strong> concussion diagnosis
Authors: *J. MARUTA 1 , M. SUH 2 , J. GHAJAR 1,3 ;<br />
1 Brain Trauma Fndn., New York, NY; 2 Dept. of Biol. Sci., Sungkyunkw<strong>an</strong> Univ., Suwon,<br />
Republic of Korea; 3 Dept. of Neurolog. Surgery, Weill Cornell Med. Col., New York, NY<br />
Abstract: We aimed to determine whe<strong>the</strong>r per<strong>for</strong>m<strong>an</strong>ce variability during predictive visual<br />
tracking c<strong>an</strong> provide a useful screening measure <strong>for</strong> mild traumatic brain injury (mTBI) <strong>an</strong>d its<br />
cognitive sequelae. Eye movements were recorded binocularly with videooculography at a<br />
sampling frequency of 500 Hz as <strong>the</strong> subject tracked a target that moved through a circular<br />
trajectory of 8.5° radius in visual <strong>an</strong>gle at 0.4 Hz <strong>for</strong> up to 12 cycles. 17 chronic mTBI <strong>an</strong>d 9<br />
control subjects were included in <strong>the</strong> study. We <strong>an</strong>alyzed <strong>the</strong> variability of eye positions by<br />
computing <strong>the</strong> st<strong>an</strong>dard deviation of gaze positional errors relative to <strong>the</strong> moving target in both<br />
<strong>the</strong> radial <strong>an</strong>d t<strong>an</strong>gential directions of <strong>the</strong> target trajectory. The two error variability parameters<br />
were closely correlated with each o<strong>the</strong>r. The me<strong>an</strong> radial error variabilities <strong>for</strong> normal <strong>an</strong>d mTBI<br />
subjects were 0.9° <strong>an</strong>d 1.4°, respectively, <strong>an</strong>d <strong>the</strong> me<strong>an</strong> t<strong>an</strong>gential error variabilities, 1.1° <strong>an</strong>d<br />
2.4°, respectively. None of <strong>the</strong> normal subjects exceeded <strong>the</strong> value of 1.5° in radial error<br />
variability while 9 of <strong>the</strong> mTBI subjects did so, <strong>an</strong>d none of <strong>the</strong> normal subjects exceeded <strong>the</strong><br />
value of 1.9° in t<strong>an</strong>gential error variability while 11 of <strong>the</strong> mTBI subjects did so. Although <strong>the</strong><br />
distributions of error variabilities of <strong>the</strong> subject groups overlapped, Student’s t-test revealed<br />
signific<strong>an</strong>t group differences <strong>for</strong> both radial <strong>an</strong>d t<strong>an</strong>gential error variabilities. We next compared<br />
<strong>the</strong> error variabilities across all subjects with <strong>the</strong> fractional <strong>an</strong>isotropy (FA) parameter of<br />
diffusion tensor imaging, <strong>an</strong> indicator of <strong>the</strong> integrity of white matter tracts. Both radial <strong>an</strong>d<br />
t<strong>an</strong>gential error variabilities were found to be signific<strong>an</strong>tly negatively correlated with <strong>the</strong> FA<br />
values of <strong>the</strong> right <strong>an</strong>terior corona radiata (ACR) <strong>an</strong>d <strong>the</strong> left superior cerebellar peduncle, tracts<br />
critical <strong>for</strong> spatial processing <strong>an</strong>d susten<strong>an</strong>ce of attention. Because <strong>the</strong> ACR was <strong>the</strong> most<br />
commonly damaged white matter tract among <strong>the</strong> mTBI subjects, <strong>the</strong> correlation implies that eye<br />
position variability during visual tracking may provide a useful diagnostic tool <strong>for</strong> mTBI. Visual<br />
tracking error variability across all subjects was also correlated with measures of cognitive<br />
functions. Specifically, both radial <strong>an</strong>d t<strong>an</strong>gential error variabilities were negatively correlated<br />
with <strong>the</strong> discriminability paramenters of <strong>the</strong> Cali<strong>for</strong>nia Verbal Learning Test II, <strong>an</strong>d positively<br />
correlated with <strong>the</strong> overall me<strong>an</strong> reaction time <strong>an</strong>d negatively correlated with <strong>the</strong> orienting effect<br />
of <strong>the</strong> Attention Network Testing. Thus, variability in visual tracking per<strong>for</strong>m<strong>an</strong>ce may reveal<br />
general working memory <strong>an</strong>d attention deficits <strong>an</strong>d indicate decreased white matter integrity in<br />
frontal white matter tracts vulnerable to mTBI.<br />
Disclosures: J. Maruta, None; M. Suh, None; J. Ghajar, Sync-Think, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
559. Pursuit Eye Movements<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 559.17/CC59<br />
Topic: D.06.c. Eye <strong>an</strong>d head control<br />
Support: Marie Curie Action, EST – „SensoPrim“<br />
Betty <strong>an</strong>d David Koetser Foundation <strong>for</strong> Brain Research<br />
Title: Direction specific eye-head coordination during target tracking in <strong>the</strong> periprimary<br />
oculomotor r<strong>an</strong>ge<br />
Authors: *J. S. THOMASSEN, B. J. M. HESS;<br />
Neurol. Department, Univ. Hosp. Zurich, Zürich, Switzerl<strong>an</strong>d<br />
Abstract: Due to <strong>the</strong> large inertia of <strong>the</strong> head, gaze control in <strong>the</strong> periprimary r<strong>an</strong>ge is usually<br />
dominated by motion of <strong>the</strong> eyes, which rapidly acquire targets of interest <strong>an</strong>d eventually track<br />
<strong>the</strong>ir motion, whereas <strong>the</strong> head movement comes in, if at all, much more sluggishly with <strong>the</strong> goal<br />
to keep <strong>the</strong> eyes close to primary position. In large gaze saccades, <strong>the</strong> target acquisition phase<br />
might require <strong>the</strong> intervention of <strong>the</strong> vestibulo-ocular reflex (VOR), whereas this reflex must be<br />
shut off during a subsequent tracking phase that also involves <strong>the</strong> head. We asked <strong>the</strong> question to<br />
what extent <strong>the</strong> head might contribute in a target tracking paradigm, which concerns only <strong>the</strong><br />
subject’s periprimary oculomotor r<strong>an</strong>ge.<br />
We trained four rhesus monkeys to per<strong>for</strong>m <strong>the</strong> following complementary paradigms: a smooth<br />
pursuit task, in which <strong>the</strong> stationary subject had to track a laser point that moved on a circular<br />
path (15° radius, 0.1Hz) projected on <strong>the</strong> inner wall of a surrounding sphere <strong>an</strong>d a fixation task,<br />
where <strong>the</strong> subject was moved in a yaw-pitch motion around a circular path relative to <strong>the</strong><br />
stationary laser point target, using a computer-controlled, motorized 4-axes motion device.<br />
Three-dimensional (3D) eye movements were measured using <strong>the</strong> magnetic search coil technique<br />
in combination with <strong>an</strong> ultrasound system <strong>for</strong> tracking 6D head movements. In both <strong>the</strong> smooth<br />
pursuit <strong>an</strong>d fixation paradigm, <strong>the</strong> movements of <strong>the</strong> head relative to <strong>the</strong> torso <strong>an</strong>d of <strong>the</strong> eye<br />
relative to <strong>the</strong> head showed a clear preference <strong>for</strong> horizontal <strong>an</strong>d vertical directions, respectively,<br />
albeit with high intra- <strong>an</strong>d inter-subjective variability regarding <strong>the</strong> head’s contribution. In both<br />
paradigms <strong>the</strong> head contribution was lagging <strong>the</strong> target. Ocular torsion resulted <strong>from</strong> Donders<br />
Law, that specifies torsional position <strong>for</strong> <strong>an</strong>y gaze direction, but depended also on <strong>the</strong> current<br />
head roll position (i.e. gravity), which generally varied <strong>from</strong> trial to trial.<br />
Our findings support <strong>the</strong> notion that <strong>the</strong> head- <strong>an</strong>d oculo-motor system share higher level gaze<br />
comm<strong>an</strong>ds. The variables underlying <strong>the</strong> particular parsing of <strong>the</strong>se comm<strong>an</strong>ds into head- <strong>an</strong>d<br />
oculo-motor are not understood.<br />
Disclosures: J.S. Thomassen, None; B.J.M. Hess, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.1/CC60<br />
Topic: D.08.bb. Opioids<br />
Support: NIDA Gr<strong>an</strong>t R01DA02328<br />
Title: BU08028: A novel buprenorphine-like lig<strong>an</strong>d, with high affinity <strong>for</strong> NOP receptors, that<br />
has long lasting <strong>an</strong>tinociceptive effects in mice<br />
Authors: *L. R. TOLL 1 , T. V. KHROYAN 1 , W. E. POLGAR 1 , G. CAMI-KOBECI 2 , S. M.<br />
HUSBANDS 2 ;<br />
1 2<br />
SRI Intl., Menlo Park, CA; Dept. of Pharm. <strong>an</strong>d Pharmacol., Univ. of Bath, Bath, United<br />
Kingdom<br />
Abstract: Buprenorphine, a high affinity mu-opioid receptor partial agonist that also binds to<br />
NOP receptors with 100 nM affinity, is a long-lasting potent <strong>an</strong>algesic. It has been postulated<br />
that buprenorphine’s inverted U shaped dose response curve <strong>for</strong> <strong>an</strong>tinociceptive activity may be<br />
due to <strong>the</strong> activation of NOP receptors <strong>an</strong>d <strong>the</strong> resulting inhibition of mu-mediated <strong>an</strong>algesia. We<br />
<strong>an</strong>d o<strong>the</strong>rs have shown that <strong>the</strong> <strong>an</strong>tinociceptive effects of buprenorphine is potentiated by NOP<br />
<strong>an</strong>tagonists <strong>an</strong>d in NOP receptor knock out mice. To better underst<strong>an</strong>d <strong>the</strong> contribution of <strong>the</strong><br />
NOP <strong>an</strong>d mu-opioid receptors in mediating buprenorphine-associated <strong>an</strong>tinociceptive behaviors<br />
we have syn<strong>the</strong>sized novel buprenorphine-like <strong>an</strong>alogs with <strong>the</strong> intent of designing in higher<br />
NOP receptor affinity <strong>an</strong>d efficacy. To this end, we have syn<strong>the</strong>sized <strong>the</strong> first truly non-selective<br />
compounds that bind with high affinity to all of <strong>the</strong> receptors in <strong>the</strong> opioid family. Our lead<br />
compound, BU08028 has Ki values of less th<strong>an</strong> 10 nM at mu, delta, kappa, <strong>an</strong>d NOP receptors.<br />
[ 35 S]GTPγS binding studies have indicated very potent partial agonist activity at mu, <strong>an</strong>d<br />
considerably higher efficacy th<strong>an</strong> buprenorphine at NOP receptors. We tested BU08028 in vivo<br />
<strong>for</strong> attenuation of acute pain, using <strong>the</strong> tail flick assay in ICR mice. Animals received <strong>an</strong><br />
injection of vehicle, morphine (10 mg/kg) or BU08028 (0.3-10 mg/kg) <strong>an</strong>d were tested <strong>for</strong> tail<br />
flick latencies 30-, 60-, 120-, 240-min, <strong>an</strong>d 24- <strong>an</strong>d 48-hr post-injection. Morphine produced a<br />
signific<strong>an</strong>t increase in tail flick latency that was evident <strong>for</strong> <strong>the</strong> first 4 hours of testing but not at<br />
24 <strong>an</strong>d 48 hours post-injection. BU08028 produced a naloxone reversible, dose-dependent<br />
increase in <strong>an</strong>tinociception. Maximal effects of BU08028 were 63%MPE <strong>an</strong>d 92% MPE at <strong>the</strong> 3<br />
<strong>an</strong>d 10 mg/kg doses, respectively. The <strong>an</strong>tinociceptive effects of <strong>the</strong>se doses were long lasting<br />
<strong>an</strong>d were present 24-hr but not 48-hr post-injection. Levels of <strong>an</strong>tinociception observed with <strong>the</strong><br />
10 mg/kg dose of BU08028 were similar to those observed with morphine, indicating that with<br />
this in vivo assay, BU08028 produced maximal <strong>an</strong>algesic effects, comparable with a high<br />
efficacy mu-opioid receptor agonist. Thus, BU08028 shows a very favorable behavioral profile<br />
that warr<strong>an</strong>ts testing using additional pain assays. Additionally, Because of <strong>the</strong> increased efficacy<br />
at NOP receptors, we will be examining whe<strong>the</strong>r BU08028 has reduced abuse liability <strong>an</strong>d may<br />
be more useful as a drug abuse medication.
Disclosures: L.R. Toll, None; T.V. Khroy<strong>an</strong>, None; W.E. Polgar, None; G. Cami-Kobeci,<br />
None; S.M. Husb<strong>an</strong>ds, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.2/CC61<br />
Topic: D.08.bb. Opioids<br />
Support: Department of Anes<strong>the</strong>siology<br />
Title: Buprenorphine-induced hyperalgesia in <strong>the</strong> rat<br />
Authors: *J. R. HOLTMAN JR, E. F. HENSON, E. P. WALA;<br />
Univ. Kentucky Col. Med., Lexington, KY<br />
Abstract: In addition to <strong>an</strong>algesia, opioids may also enh<strong>an</strong>ce pain sensitivity (opioid induced<br />
hyperalgesia, OIH). OIH has been observed in different settings characterized by <strong>the</strong> dose (high,<br />
ultra low), mode of administration (acute, chronic), <strong>an</strong>d during withdrawal. OIH is typically<br />
associated with potent mu agonists (eg. fent<strong>an</strong>yl) <strong>an</strong>d involves <strong>an</strong> NMDA receptor mech<strong>an</strong>ism.<br />
Buprenorphine (BPN), a potent opioid <strong>an</strong>algesic clinically used in treatment of opioid addiction,<br />
has partial mu agonist <strong>an</strong>d kappa <strong>an</strong>tagonist activities. BPN appears less likely to produce typical<br />
mu opioid side effects, exhibits a ceiling effect <strong>for</strong> respiratory depression, <strong>an</strong>d has less potential<br />
<strong>for</strong> abuse. Little is known regarding <strong>the</strong> potential <strong>for</strong> BPN to produce OIH. A potential lack of<br />
OIH with <strong>the</strong>se o<strong>the</strong>r characteristics could make BPN a desirable clinical choice <strong>for</strong> chronic pain<br />
m<strong>an</strong>agement. The present study was undertaken to: 1) examine responsiveness to high (HD,<br />
<strong>an</strong>tinociceptive) <strong>an</strong>d ultra low (LD, sub<strong>an</strong>algesic) doses of BPN following acute <strong>an</strong>d repeated<br />
(chronic; 10 days) IP administration in Sprague Dawley male rats; 2) determine if OIH affects<br />
<strong>the</strong> <strong>an</strong>tinociceptive potency of BPN; <strong>an</strong>d 3) investigate <strong>the</strong> possible involvement of <strong>an</strong> NMDA<br />
receptor mech<strong>an</strong>ism in BPN induced OIH. The <strong>an</strong>tinociceptive <strong>an</strong>d hyperalgesic responses were<br />
respectively assessed on high intensity (baseline 2-3 s) <strong>an</strong>d low intensity (baseline 8-10 s) tailflick<br />
assays. BPN (HD: 20-200 mcg/kg) exerted a dose-related <strong>an</strong>tinociception. However, loss of<br />
<strong>an</strong>tinociceptive efficacy (toler<strong>an</strong>ce) followed by enh<strong>an</strong>ced sensitivity to pain (OIH) occurred<br />
with repeated dosing of BPN (100 mcg/kg/2xday). This effect was delayed in <strong>the</strong> presence of <strong>the</strong><br />
NMDA <strong>an</strong>tagonist, ketamine (KET, 1.5-3 mg/kg). Recovery <strong>from</strong> toler<strong>an</strong>ce (14 days after<br />
abstinence) was greater in rats previously treated with BPN <strong>an</strong>d KET compared to BPN alone.<br />
Interestingly, LD of BPN (100 ng/kg) also resulted in OIH. KET (0.15-1.5 mg/kg) attenuated LD<br />
OIH, in a dose-related fashion. No toler<strong>an</strong>ce to LD OIH was seen with repeated (10 days)
administration of BPN (100 ng/kg/2xday). The <strong>an</strong>tinociceptive efficacy of BPN (20 mcg/kg) was<br />
diminished in rats, which previously exhibited LD OIH. The present findings demonstrated<br />
bimodal properties of BPN: pronoceptive at ultra low dose <strong>an</strong>d <strong>an</strong>tinociceptive at higher doses.<br />
Prolonged exposure to <strong>an</strong> initially <strong>an</strong>tinociceptive dose of BPN resulted in progressive activation<br />
of a pronoceptive system as evident by diminished efficacy <strong>an</strong>d OIH. The present data on BPN<br />
suggest that this opioid, like morphine, also activates simult<strong>an</strong>eously pain facilitatory <strong>an</strong>d<br />
inhibitory systems.<br />
Disclosures: J.R. Holtm<strong>an</strong> Jr, None; E.F. Henson, None; E.P. Wala, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.3/CC62<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t NS059028<br />
NIH Gr<strong>an</strong>t NS060735<br />
Title: Toler<strong>an</strong>ce <strong>an</strong>d withdrawal to opiate exposure results in different pain affect <strong>an</strong>d glial<br />
activation in <strong>the</strong> rostral ventromedial medulla (RVM)<br />
Authors: *S. C. LAGRAIZE, W. GUO, H. WANG, K. REN, R. DUBNER;<br />
Dept of Neural <strong>an</strong>d Pain Sci., Dentl Sch. & Prog. Neurosci Univ. Maryl, Baltimore, MD<br />
Abstract: Toler<strong>an</strong>ce <strong>an</strong>d withdrawal to opiates typically results in a negative affective state. It is<br />
suggested that <strong>the</strong> mech<strong>an</strong>isms of pain hypersensitivity in <strong>an</strong>imal models of injury are similar to<br />
pain hypersensitivity that is associated with morphine (MOR) toler<strong>an</strong>ce <strong>an</strong>d withdrawal. The<br />
present experiments examined negative affect that is associated with toler<strong>an</strong>ce <strong>an</strong>d withdrawal<br />
produced by opiates <strong>an</strong>d <strong>the</strong>ir correlation with pain hypersensitivity <strong>an</strong>d underlying neural<br />
mech<strong>an</strong>isms. We first examined <strong>the</strong> effect of continuous MOR treatment on hindpaw mech<strong>an</strong>ical<br />
sensitivity in <strong>an</strong>imals that were inflamed (complete Freund’s adjuv<strong>an</strong>t; CFA) or saline-treated.<br />
Twenty-four hours after CFA/saline injection, rats receiving CFA showed mech<strong>an</strong>ical<br />
hypersensitivity as compared to saline injected rats. Following MOR treatment (osmotic pump,<br />
10 mg/kg/d), <strong>the</strong>re was decreased mech<strong>an</strong>ical hypersensitivity on days 1-2 (<strong>an</strong>algesia) which<br />
returned to <strong>the</strong> pre-MOR level by day 4 (MOR toler<strong>an</strong>ce). We next examined negative affect by<br />
measuring avoid<strong>an</strong>ce behavior (CPA test). In <strong>the</strong> CPA test, <strong>an</strong>imals received suprathreshold
mech<strong>an</strong>ical stimulation (476 mN von Frey filament/15 s) of <strong>the</strong> injured paw when in <strong>the</strong><br />
preferred dark area <strong>an</strong>d <strong>the</strong> uninjured paw when in <strong>the</strong> light area of <strong>the</strong> testing chamber. Inflamed<br />
<strong>an</strong>imals spend more time in <strong>the</strong> light side of <strong>the</strong> chamber as compared to naïve rats, suggesting<br />
increased aversion to <strong>the</strong> noxious stimulus. Analgesic <strong>an</strong>imals exhibited less aversive behavior<br />
th<strong>an</strong> naïve rats <strong>an</strong>d toler<strong>an</strong>t rats showed increased aversive behavior similar to <strong>the</strong> inflamed rats<br />
without morphine. Animals in <strong>the</strong> MOR withdrawal phase showed decreased aversion,<br />
suggesting that MOR plays a role in maintaining CFA-induced aversion. It appears that <strong>the</strong> CPA<br />
test is unique in revealing a difference in affective behavior <strong>an</strong>d involves traits that are not<br />
revealed by hyperalgesia, a more sensory-discriminative measure. Western blots showed<br />
increased GFAP, CD11b, <strong>an</strong>d IL-1β immunoreactivity in <strong>the</strong> RVM of inflamed <strong>an</strong>imals that<br />
received saline treatment at 1 d post-CFA. Similar increases were observed in <strong>the</strong> toler<strong>an</strong>ce <strong>an</strong>d<br />
withdrawal phases after MOR treatment. These results show that different phases of MOR<br />
treatment lead to different responses to sensory stimuli <strong>an</strong>d negative affect associated with a<br />
noxious stimulus, <strong>an</strong>d different neurochemical ch<strong>an</strong>ges at <strong>the</strong> brainstem level.<br />
Disclosures: S.C. LaGraize, None; W. Guo, None; H. W<strong>an</strong>g, None; K. Ren, None; R.<br />
Dubner, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.4/CC63<br />
Topic: D.08.bb. Opioids<br />
Support: SRI Internal IR&D Funding W32HTA<br />
R01DA023281<br />
Title: The rewarding <strong>an</strong>d <strong>an</strong>algesic effects of morphine- Do <strong>the</strong>y ch<strong>an</strong>ge in a chronic pain state?<br />
Authors: *T. V. KHROYAN, W. POLGAR, J. ORDUNA, J. MONTENEGRO, L. TOLL;<br />
SRI Intl., Menlo Park, CA<br />
Abstract: Negative consequences following chronic opiate use, such as toler<strong>an</strong>ce <strong>an</strong>d addiction<br />
development, have resulted in a concern <strong>for</strong> <strong>the</strong>ir continued use. In rodents, <strong>the</strong> effect of chronic<br />
pain on toler<strong>an</strong>ce development is not well characterized. Moreover, <strong>the</strong> effect of chronic pain on<br />
morphine conditioned place preference (CPP) is not consistent between laboratories. In <strong>the</strong><br />
following experiments we examined whe<strong>the</strong>r chronic constriction injury (CCI) in mice could
alter morphine-induced CPP <strong>an</strong>d/or <strong>an</strong>algesic/<strong>an</strong>ti-allodynic effectiveness. Animals underwent<br />
CCI surgery <strong>an</strong>d were given one week to recover. Following baseline measures of <strong>the</strong>rmal<br />
hyperalgesia <strong>an</strong>d mech<strong>an</strong>ical allodynia, <strong>an</strong>imals underwent conditioning. To establish CPP, drug<br />
injections were paired with one of two distinctly different compartments, whereas saline<br />
injections were paired with <strong>the</strong> o<strong>the</strong>r compartment. Additionally, on drug injection days, <strong>an</strong>imals<br />
were tested <strong>for</strong> allodynia or <strong>the</strong>rmal nociception 60min following <strong>the</strong> drug injection. Following<br />
conditioning, <strong>an</strong>imals were given access to both compartments <strong>an</strong>d <strong>the</strong> amount of time spent in<br />
each compartment was recorded. CCI <strong>an</strong>imals did not exhibit a decrease in morphine-CPP, since<br />
<strong>the</strong>se <strong>an</strong>imals spent a similar amount of time in <strong>the</strong>ir drug-paired compartment relative to shams.<br />
CCI caused hyperalagesia, inducing a decrease in <strong>the</strong> paw withdrawal latency. The 3 <strong>an</strong>d 10<br />
mg/kg doses of morphine blocked <strong>the</strong> hyperalgesia <strong>an</strong>d were <strong>an</strong>algesic, since paw withdrawal<br />
latency was not only greater th<strong>an</strong> CCI controls but also greater when compared to vehicle sham<br />
controls. However, following a single morphine injection every o<strong>the</strong>r day <strong>for</strong> 8 days, toler<strong>an</strong>ce to<br />
morphine-induced <strong>an</strong>algesia <strong>an</strong>d <strong>an</strong>ti-hyperalgesia developed in CCI <strong>an</strong>imals but not in sham<br />
controls that received morphine using <strong>the</strong> same drug regimen. Mech<strong>an</strong>ical allodynia was also<br />
evident in CCI <strong>an</strong>imals relative to controls. As with <strong>the</strong>rmal hyperalgesia, morphine produced <strong>an</strong><br />
initial <strong>an</strong>ti-allodynic effect in CCI <strong>an</strong>imals, <strong>an</strong>d by <strong>the</strong> fourth injection <strong>the</strong>re was attenuation in<br />
morphine-induced <strong>an</strong>ti-allodynia. This data indicate that although <strong>the</strong> rewarding effects of<br />
morphine as measured by <strong>the</strong> CPP paradigm are unaltered in a chronic pain condition, morphine<br />
toler<strong>an</strong>ce develops to <strong>the</strong>rmal <strong>an</strong>tinociception at a more rapid pace in CCI <strong>an</strong>imals relative to<br />
<strong>an</strong>imals that are not in pain.<br />
Disclosures: T.V. Khroy<strong>an</strong>, None; W. Polgar, None; J. Orduna, None; J. Montenegro,<br />
None; L. Toll, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.5/CC64<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t RR016816<br />
UNO New Investigator Funds<br />
Title: Morphine-induced <strong>an</strong>algesia in phasic <strong>an</strong>d tonic pain models in rhes knockout mice
Authors: F. A. LEE 1 , B. BAIAMONTE 1 , *L. M. HARRISON 2 , D. SPANO 3 , R. D.<br />
SOIGNIER 1 ;<br />
1 Psychology, Univ. of New Orle<strong>an</strong>s, New Orle<strong>an</strong>s, LA; 2 LSU Hlth. Sci. Ctr., New Orle<strong>an</strong>s, LA;<br />
3 CEINGE Biotecnologie Av<strong>an</strong>zate, Naples, Italy<br />
Abstract: Rhes (Ras homolog enriched in striatum) is a striatally expressed gu<strong>an</strong>osine<br />
triphosphate-binding protein involved in dopamine signaling <strong>an</strong>d behavior. We have previously<br />
demonstrated that dopamine- denervated, supersensitive rats show decreases in its gene <strong>an</strong>d<br />
protein levels, suggesting that Rhes is normally inhibitory to dopamine signaling. Studies using a<br />
recently developed strain of mice homozygous <strong>for</strong> <strong>the</strong> Rhes null mutation (-/-) also suggest that<br />
Rhes is involved in dopamine signaling in <strong>the</strong> striatum, with Rhes (-/-) displaying increased D1<br />
agonist-induced locomotor behavior <strong>an</strong>d decreased grooming relative to Rhes (+/+) mice [Errico<br />
et al., Mol Cell Neurosci., 37(2): 335-45, 2008; Quintero et al., NeuroReport, 19(16): 1563-6,<br />
2008]. Although <strong>the</strong> exact mech<strong>an</strong>ism <strong>for</strong> <strong>the</strong>se phenomena is unknown, pull-down assays <strong>from</strong><br />
our laboratory suggest <strong>an</strong> interaction of Rhes with Gαi. Given that m<strong>an</strong>y opioid effects involve<br />
dopamine receptor signaling in <strong>the</strong> striatum <strong>an</strong>d signaling through Gαi, we sought to determine if<br />
morphine <strong>an</strong>algesia could be demonstrated in Rhes (-/-) mice. To do this we tested mice<br />
homozygous <strong>for</strong> <strong>the</strong> wild-type gene (+/+), <strong>an</strong>d heterozygous (+/-) or homozygous (-/-) <strong>for</strong> <strong>the</strong><br />
deleted Rhes gene in <strong>the</strong> tailflick <strong>an</strong>d <strong>for</strong>malin tests. Animals were habituated to <strong>the</strong> test room<br />
<strong>an</strong>d apparatus <strong>for</strong> 15 minutes prior to receiving 0.0, 1.0, 3.0 or 10.0 mg/kg morphine i.p. For <strong>the</strong><br />
tail flick test, following baseline measures, <strong>the</strong>rmal withdrawal thresholds were measured at 15minute<br />
intervals <strong>for</strong> 2 hours following morphine. For <strong>for</strong>malin testing, 30 minutes after morphine<br />
injections, <strong>the</strong> pl<strong>an</strong>tar surface of one hindpaw was injected with 40 µl of 2% <strong>for</strong>malin, <strong>an</strong>d time<br />
spent licking <strong>the</strong> injected paw was recorded every 5 minutes <strong>for</strong> 1 hour. In <strong>the</strong> tail flick assay,<br />
morphine produced long-lasting, dose-dependent <strong>an</strong>algesia in all genotypes, though <strong>the</strong>re was a<br />
trend towards decreased latency of <strong>an</strong>algesic action in (+/-) <strong>an</strong>d (-/-) mice. By contrast, we<br />
observed signific<strong>an</strong>tly increased morphine-induced <strong>an</strong>algesia in (-/+) <strong>an</strong>d (-/-) mice in <strong>the</strong><br />
<strong>for</strong>malin test. These results suggest that <strong>the</strong> Rhes protein contributes to supraspinal morphine<br />
<strong>an</strong>algesia, <strong>an</strong>d may prove a novel target <strong>for</strong> <strong>an</strong>algesics or adjunctive treatments in pain<br />
m<strong>an</strong>agement.<br />
Disclosures: F.A. Lee, None; B. Baiamonte, None; L.M. Harrison, None; D. Sp<strong>an</strong>o,<br />
None; R.D. Soignier, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.6/CC65
Topic: D.08.bb. Opioids<br />
Support: NIDCR Gr<strong>an</strong>t R01 DE16558<br />
NINDS U01-DE017018<br />
P01 NSO45685<br />
Title: The role of MOR-1K in opioid induced hyperalgesia<br />
Authors: *M. S. CONRAD, S. WENTWORTH, D. PRUSIK, J. GAUTHIER, R. SUKUMAR,<br />
W. MAIXNER, L. DIATCHENKO, A. NACKLEY;<br />
Ctr. <strong>for</strong> Neurosensory Disorders. Sch. of Dent., UNC-CH, Chapel Hill, NC<br />
Abstract: Opioids have become one of <strong>the</strong> most widely used drugs in chronic pain treatment<br />
despite m<strong>an</strong>y side effects including dependence, addiction, <strong>an</strong>d withdrawal. A subst<strong>an</strong>tial<br />
proportion of chronic opioid users also experience <strong>an</strong> increase in pain sensitivity termed opioid<br />
induced hyperalgesia (OIH). The majority of prescribed opioids, including <strong>the</strong> most broadly used<br />
opioid, morphine, mediate physiological effects through binding to <strong>the</strong> mu opioid receptor MOR-<br />
1. MOR-1 is a seven tr<strong>an</strong>smembr<strong>an</strong>e G-protein-coupled receptor whose <strong>an</strong>algesic function is<br />
facilitated through <strong>the</strong> cellular inhibition <strong>an</strong>d subsequent hyperpolarization of <strong>the</strong> cellular<br />
membr<strong>an</strong>es. Recently, our group identified a novel iso<strong>for</strong>m of MOR-1, MOR-1K, which codes<br />
<strong>for</strong> a truncated six tr<strong>an</strong>smembr<strong>an</strong>e receptor. The minor allele T of <strong>the</strong> rs563649 polymorphism<br />
carried by <strong>the</strong> MOR-1K iso<strong>for</strong>m is associated with both increased pain sensitivity <strong>an</strong>d decreased<br />
<strong>an</strong>algesic efficacy of morphine in hum<strong>an</strong>s, though it codes <strong>for</strong> higher tr<strong>an</strong>slation efficiency of <strong>the</strong><br />
receptor. Preliminary characterization of this iso<strong>for</strong>m has shown it to have <strong>an</strong> excitatory cellular<br />
function mediated by increased cAMP, Ca 2+ , <strong>an</strong>d NO production opposite to inhibitory cellular<br />
effects of <strong>the</strong> major MOR-1 iso<strong>for</strong>m. We hypo<strong>the</strong>size that MOR-1K plays a pivotal role in <strong>the</strong><br />
development of OIH through this excitatory cellular function. For this study, we utilized three<br />
strains of mice which exhibit different responses to morphine. Mice received subcut<strong>an</strong>eous<br />
injections of morphine (10-40 mg/kg) twice daily <strong>for</strong> 4 days. The C57B/6J <strong>an</strong>d 129/SVEV<br />
strains exhibited strong <strong>an</strong>algesia post-injection while <strong>the</strong> CXBK strain, which has decreased<br />
MOR-1 expression, did not show <strong>an</strong> <strong>an</strong>algesic response. Beginning with <strong>the</strong> third injection day,<br />
<strong>the</strong> C57B/6J <strong>an</strong>d CXBK strains developed OIH as evident by <strong>an</strong> increase in pain sensitivity<br />
while <strong>the</strong> 129/SVEV strain did not develop OIH. An increase in locomotor activity <strong>an</strong>d a<br />
decrease in defecation rates were seen in all three strains of mice. During <strong>the</strong> time periods in<br />
which <strong>the</strong> greatest OIH response was observed, spinal cord <strong>an</strong>d brain tissue <strong>from</strong> multiple<br />
regions was collected <strong>an</strong>d <strong>an</strong>alyzed <strong>for</strong> MOR-1 <strong>an</strong>d MOR-1K gene expression. The expression<br />
level of MOR-1 was found to be unch<strong>an</strong>ged between <strong>the</strong> vehicle <strong>an</strong>d morphine-treated mice<br />
while <strong>an</strong> increase in MOR-1K was observed in <strong>the</strong> morphine-treated mice only. Upregulation of<br />
<strong>the</strong> MOR-1K iso<strong>for</strong>m may be causing <strong>the</strong> switch <strong>from</strong> <strong>the</strong> inhibitory effect of morphine to <strong>an</strong><br />
excitatory effect at <strong>the</strong> cellular level leading to <strong>the</strong> subsequent development of OIH.<br />
Disclosures: M.S. Conrad, None; S. Wentworth, None; D. Prusik, None; J. Gauthier,<br />
None; R. Sukumar, None; W. Maixner, None; L. Diatchenko, None; A. Nackley, None.
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.7/CC66<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t DA019620<br />
Title: The direct screening of mixture-based combinatorial libraries <strong>for</strong> <strong>the</strong> discovery of novel<br />
<strong>an</strong>algesics<br />
Authors: *R. A. HOUGHTEN 1 , C. T. DOOLEY 2 , M. A. GIULIANOTTI 2 , J. P.<br />
MCLAUGHLIN 2 ;<br />
1 Torrey Pines Inst., Ft. Peirce, FL; 2 Torrey Pines Inst., Port St. Lucie, FL<br />
Abstract: Syn<strong>the</strong>tic combinatorial methods, combined with high-throughput screening, have<br />
fundamentally adv<strong>an</strong>ced <strong>the</strong> ability to syn<strong>the</strong>size <strong>an</strong>d screen large numbers of potential<br />
<strong>the</strong>rapeutics, but it is unrealistic to use discovery in vivo models to screen <strong>the</strong> hundreds of<br />
thous<strong>an</strong>ds of <strong>the</strong> individual compounds produced. A potential solution to this problem is <strong>the</strong> use<br />
of mixture-based combinatorial libraries directly <strong>for</strong> in vivo testing in <strong>the</strong> discovery phase to<br />
identify c<strong>an</strong>didates with desired biological profiles while simult<strong>an</strong>eously eliminating those<br />
compounds with poor absorption, distribution, metabolism, <strong>an</strong>d poor pharmacokinetic properties.<br />
This has been successfully used with mixture-based peptide libraries in earlier studies by this lab.<br />
We have now extended this approach to include multiple libraries prepared by in house<br />
approaches each made up of more th<strong>an</strong> 720,000 classic low MW small molecules. These<br />
mixture-based small molecule libraries are being used through direct in vivo testing to identify<br />
novel opioid compounds that act ei<strong>the</strong>r centrally or as peripherally restricted <strong>an</strong>algesic agents.<br />
Selection of <strong>the</strong>se libraries was guided by <strong>the</strong> <strong>the</strong>oretical calculation of <strong>the</strong>ir drug like properties.<br />
Library 1346, made up of mixtures varied at four positions of this scaffold had mixtures r<strong>an</strong>ging<br />
in size <strong>from</strong> 17,000 to 28,000 compounds each (120 mixtures total). These 120 mixtures were<br />
first screened in vitro by classic radio-receptor binding assays <strong>for</strong> mu, delta <strong>an</strong>d kappa opiate<br />
activity to identify those mixtures with signific<strong>an</strong>t affinity <strong>for</strong> one or more of <strong>the</strong> opioid<br />
receptors. Separately, in vivo screening of <strong>the</strong>se same mixtures was carried in <strong>the</strong> mouse 55 o C<br />
warm-water tail-withdrawal assay. This consistently resulted in a distinctly different set of active<br />
mixtures with <strong>an</strong>algesic activity that was blocked by pretreatment with <strong>the</strong> non-selective opioid<br />
<strong>an</strong>tagonist, naloxone. From <strong>the</strong>se results, syn<strong>the</strong>sis of individual compounds TPI1818-101 <strong>an</strong>d -<br />
109 were carried out that, after peripheral IP administration, resulted in a mu-(101) <strong>an</strong>d kappa-<br />
(109) opioid receptor mediated, dose-dependent in vivo <strong>an</strong>tinociception that were 3-5 times
greater th<strong>an</strong> morphine. Of note, <strong>the</strong>se 1818 series individual compounds produced little or no<br />
respiratory depression, hyperlocomotion or conditioned place preference. These results suggest<br />
that <strong>the</strong> concept of using very large, highly diverse mixture-based libraries <strong>for</strong> <strong>the</strong> identification<br />
of inherently more adv<strong>an</strong>ced individual ‘hits’ by <strong>the</strong> direct in vivo testing is both conceptually<br />
<strong>an</strong>d practically successful <strong>an</strong>d may signific<strong>an</strong>tly accelerate <strong>an</strong>d/or enh<strong>an</strong>ce <strong>the</strong> development of<br />
promising <strong>the</strong>rapeutic c<strong>an</strong>didates.<br />
Disclosures: R.A. Houghten, None; C.T. Dooley, None; M.A. Giuli<strong>an</strong>otti, None; J.P.<br />
McLaughlin, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.8/CC67<br />
Topic: D.08.bb. Opioids<br />
Support: UNO New Faculty Support<br />
Title: Toler<strong>an</strong>ce develops to morphine-induced <strong>an</strong>algesia <strong>an</strong>d cognitive deficits at different rates<br />
in rats<br />
Authors: B. A. BAIAMONTE, F. A. LEE, R. M. PEEBLES, *R. D. SOIGNIER;<br />
Psychology, Univ. New Orle<strong>an</strong>s, New Orle<strong>an</strong>s, LA<br />
Abstract: Opioid medications are <strong>the</strong> best pharmacological weapon against severe, intractable<br />
pain, but <strong>the</strong> <strong>an</strong>algesia produced by <strong>the</strong>se medications is often accomp<strong>an</strong>ied by adverse side<br />
effects, e.g. suppression of respiratory <strong>an</strong>d gastrointestinal function, <strong>an</strong>d mental clouding.<br />
Import<strong>an</strong>tly, with repeated use of opioids, toler<strong>an</strong>ce may develop to each of <strong>the</strong>se drug effects at<br />
different rates. For example, clinical studies have shown that while toler<strong>an</strong>ce develops rapidly to<br />
morphine’s cognitive effects, morphine-induced <strong>an</strong>algesia is often stable after initial dose<br />
escalations. The observation of different time-courses raises <strong>the</strong> possibility that <strong>the</strong>re are separate<br />
mech<strong>an</strong>isms responsible <strong>for</strong> toler<strong>an</strong>ce to different drug effects. In <strong>the</strong> present study we sought to<br />
dissociate <strong>the</strong> development of toler<strong>an</strong>ce to morphine induced mental clouding, akinesia <strong>an</strong>d<br />
<strong>an</strong>algesia in laboratory <strong>an</strong>imals. To do this we simult<strong>an</strong>eously examined morphine’s <strong>an</strong>algesic,<br />
locomotor <strong>an</strong>d cognitive effects in Sprague-Dawley rats over a period of 5 days. Animals trained<br />
in <strong>the</strong> 8-arm radial maze win-stay task were injected s.c. with morphine (or saline) <strong>an</strong>d maze<br />
per<strong>for</strong>m<strong>an</strong>ce was video recorded. Thermal withdrawal thresholds were assessed immediately<br />
following removal <strong>from</strong> <strong>the</strong> maze. Acutely (day 1), morphine produced a profound <strong>an</strong>algesia
accomp<strong>an</strong>ied by signific<strong>an</strong>t increase in reference <strong>an</strong>d working memory errors in <strong>the</strong> radial maze<br />
task <strong>an</strong>d pronounced locomotor slowing. Locomotor scores returned to baseline within 2 days,<br />
memory errors returned to baseline by day 5. Analgesia was reduced but still observed on <strong>the</strong><br />
final day of testing in <strong>the</strong> absence of locomotor or cognitive effects. These results raise <strong>the</strong><br />
possibility that separate mech<strong>an</strong>isms mediate toler<strong>an</strong>ce morphine-induced drug effects.<br />
Disclosures: B.A. Baiamonte, None; F.A. Lee, None; R.M. Peebles, None; R.D. Soignier,<br />
None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.9/CC68<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t R01-DA-03816<br />
NIH Gr<strong>an</strong>t K05-DA00360<br />
NIH Gr<strong>an</strong>t R01-NS43466<br />
United States Public Health Service Gr<strong>an</strong>t ES07462<br />
Title: Morphine produces <strong>an</strong>algesia through brain cytochrome P450/epoxygenase signaling<br />
Authors: J. L. CONROY 1 , C. FANG 2 , J. GU 2 , A. SNYDER-KELLER 2 , M. BEHR 2 , S. O.<br />
ZEITLIN 3 , W. YANG 2 , J. YANG 1 , M. A. VANALSTINE 1 , *J. NALWALK 1 , J. E.<br />
MAZURKIEWICZ 1 , Z. SHAN 4 , S.-Z. ZHANG 4 , M. P. WENTLAND 4 , B. I. KNAPP 5 , J. M.<br />
BIDLACK 5 , X. DING 2 , L. B. HOUGH 1 ;<br />
1 Ctr. <strong>for</strong> Neuropharmacol & Neuroscie, Alb<strong>an</strong>y Med. Col., Alb<strong>an</strong>y, NY; 2 Wadsworth Center,<br />
New York State Dept. of Health, <strong>an</strong>d Sch. of Publ. Hlth., Alb<strong>an</strong>y, NY; 3 Dept. of Neurosci., Univ.<br />
of Virginia Sch. of Med., Charlottesville, VA; 4 Dept. of Chem., Rensselaer Polytechnic Inst.,<br />
Troy, NY; 5 Dept. of Pharmacol. <strong>an</strong>d Physiol., Univ. of Rochester, Sch. of Med. <strong>an</strong>d Dent.,<br />
Rochester, NY<br />
Abstract: It is well established that morphine acts at mu opioid receptors in <strong>the</strong> brain <strong>an</strong>d spinal<br />
cord to produce <strong>an</strong>algesia, but <strong>the</strong> details of <strong>the</strong> biochemical cascade that occur after receptor<br />
activation remain unclear. Previous studies of brainstem mu opioid <strong>an</strong>algesic signaling suggest
<strong>the</strong> possible relev<strong>an</strong>ce of <strong>the</strong> arachidonic acid (AA) cascade. In order to test a role <strong>for</strong> brain P450<br />
AA epoxygenases in morphine <strong>an</strong>tinociception, we generated a tr<strong>an</strong>sgenic mouse line with a<br />
brain neuron-specific deletion of cytochrome P450 reductase (brain-Cpr-null; Null), <strong>an</strong> enzyme<br />
required <strong>for</strong> all microsomal P450 activity. This was achieved by crossing Cpr lox/lox mice with<br />
neuron-specific Cre-expressing mice (i.e. CamKIIα-Cre). Null mice showed subst<strong>an</strong>tial losses in<br />
brain Cpr <strong>an</strong>d P450 activity, as well as highly attenuated morphine <strong>an</strong>tinociception. No<br />
differences were found in gross appear<strong>an</strong>ce, levels of activity, brain mu opioid receptor<br />
characteristics, or brain morphine levels after systemic administration compared with wild-type<br />
(WT) littermates. In addition, pharmacological inhibition of P450 <strong>an</strong>d epoxygenase activity<br />
signific<strong>an</strong>tly reduced morphine <strong>an</strong>tinociception in rats. There<strong>for</strong>e, both genetic <strong>an</strong>d<br />
pharmacological evidence supports a role <strong>for</strong> brain P450/epoxygenase signaling in morphine<br />
<strong>an</strong>tinociception. An epoxygenase model of mu signaling in brainstem <strong>an</strong>algesic circuits accounts<br />
<strong>for</strong> <strong>the</strong> present observations as well as m<strong>an</strong>y literature findings. This model also suggests new<br />
approaches <strong>for</strong> <strong>the</strong> development of pain-relieving drugs.<br />
Disclosures: J.L. Conroy, None; C. F<strong>an</strong>g, None; J. Gu, None; A. Snyder-Keller, None; M.<br />
Behr, None; S.O. Zeitlin, None; W. Y<strong>an</strong>g, None; J. Y<strong>an</strong>g, None; M.A. V<strong>an</strong>Alstine, None; J.<br />
Nalwalk, None; J.E. Mazurkiewicz, None; Z. Sh<strong>an</strong>, None; S. Zh<strong>an</strong>g, None; M.P. Wentl<strong>an</strong>d,<br />
None; B.I. Knapp, None; J.M. Bidlack, None; X. Ding, None; L.B. Hough, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.10/CC69<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t DA012609<br />
Title: Leu-enkephalin does not produce <strong>an</strong>algesic toler<strong>an</strong>ce<br />
Authors: *W.-L. CHEN, J. G. MARVIZON;<br />
Dept Med., Univ. Cali<strong>for</strong>nia, Los Angeles, CA<br />
Abstract: It has been assumed that enkephalins do not produce toler<strong>an</strong>ce. However, this has<br />
been difficult to ascertain because enkephalins injected into <strong>the</strong> CNS are rapidly degraded by<br />
peptidases, leading to low <strong>an</strong>d inconsistent levels of <strong>an</strong>algesia. We found that when Leuenkephalin<br />
is injected intra<strong>the</strong>cally with three peptidase inhibitors (PI), amastatin, captopril <strong>an</strong>d<br />
phosphoramidon, it produces robust <strong>an</strong>algesia with high potency. In <strong>the</strong> present study we
investigated whe<strong>the</strong>r daily injections of Leu-enkephalin (Leu-enk, 10 nmol) with <strong>the</strong>se peptidase<br />
inhibitors (10 nmol) leads to <strong>an</strong>algesic toler<strong>an</strong>ce. As a positive control, we gave <strong>an</strong>o<strong>the</strong>r group of<br />
rats intra<strong>the</strong>cal injections of morphine (224 nmol). In addition, a third group of rats was given<br />
daily injections of endomorphin-2 (EM-2, 100 nmol), to test <strong>the</strong> hypo<strong>the</strong>sis that compounds that<br />
produce µ-opioid receptor internalization do not produce toler<strong>an</strong>ce. Analgesia was measured 15<br />
min after <strong>the</strong> intra<strong>the</strong>cal injection as paw withdrawal latencies to radi<strong>an</strong>t heat, every 5 min <strong>for</strong> 15<br />
min. The four latencies obtained during <strong>the</strong>se 15 min were averaged <strong>an</strong>d expressed as percent of<br />
maximum possible effect [%MPE = 100 x (latency-baseline) / (cuff-off - baseline)]. The baseline<br />
was measured be<strong>for</strong>e <strong>the</strong> first injection, <strong>an</strong>d cut-off time was 25 s. On day 1, all three compounds<br />
produced similar levels of <strong>an</strong>algesia (see figure). On subsequent days <strong>the</strong> effect of morphine<br />
gradually decreased to practically disappear on day 6, which was interpreted as toler<strong>an</strong>ce.<br />
Analgesia produced by EM-2 also decreased, but less pronouncedly th<strong>an</strong> that of morphine.<br />
Analgesia produced by Leu-enk remained const<strong>an</strong>t. Linear regression yielded slopes of -11.6 ±<br />
2.3 (morphine), -4.9 ± 2.6 (EM-2) <strong>an</strong>d 2.5 ± 1.7 (Leu-enk + PI). These results show that<br />
enkephalin does not produce <strong>an</strong>algesic toler<strong>an</strong>ce. The fact that EM-2 did produce toler<strong>an</strong>ce<br />
shows that <strong>the</strong> ability to induce MOR internalization is not sufficient to avoid toler<strong>an</strong>ce.<br />
Disclosures: W. Chen, None; J.G. Marvizon, None.<br />
Poster
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.11/CC70<br />
Topic: D.08.bb. Opioids<br />
Title: Dynamic mass redistribution: A tool to detect lig<strong>an</strong>d directed signaling in opioid <strong>an</strong>d<br />
c<strong>an</strong>nabinoid receptors<br />
Authors: *E. E. CODD, J. R. MABUS, B. MURRAY, C. M. FLORES;<br />
Analgesics Res., Johnson & Johnson PRD, Spring House, PA<br />
Abstract: Classically, G-protein coupled receptor activation by a lig<strong>an</strong>d has been viewed as<br />
resulting in a defined response, such as activation of a G-protein, activation or inhibition of<br />
adenylyl cyclase or alteration in calcium flux. Newer concepts of lig<strong>an</strong>d directed signaling<br />
recognize that various lig<strong>an</strong>ds may induce different down stream effects. Such signaling<br />
diversity makes difficult <strong>the</strong> selection of a screening assay, as most traditional assays focus on<br />
<strong>the</strong> lig<strong>an</strong>d’s effect on a single step in a signaling cascade. Dynamic mass redistribution (DMR), a<br />
label free technology that uses light to measure lig<strong>an</strong>d-induced ch<strong>an</strong>ges in <strong>the</strong> mass of cells<br />
proximate to <strong>the</strong> biosensor, provides <strong>an</strong> integrated cellular response that involves multiple<br />
pathways <strong>an</strong>d cellular events (F<strong>an</strong>g et al., FEBS Letters, 579:6365-6374, 2005). Fur<strong>the</strong>rmore, <strong>the</strong><br />
technology measures mass ch<strong>an</strong>ges across time, thus providing a global representation of <strong>the</strong> cell<br />
signaling event. We have used this technology to examine classic mu <strong>an</strong>d delta opioid as well as<br />
CB2 receptor pharmacology. The DMR signals induced by mu or delta opioid or CB2 agonists in<br />
cells tr<strong>an</strong>sfected with <strong>the</strong>se receptors were blocked by pharmacologically appropriate receptor<br />
<strong>an</strong>tagonists as well as by pertussis toxin, <strong>an</strong> inhibitor of Gi/o signaling. Preliminary evidence<br />
suggests that inhibitors of intermediate steps in <strong>the</strong> cell signaling cascade, such as receptor<br />
recycling inhibitors (e.g., wortm<strong>an</strong>nin) or MEK/p38 MAPK inhibitors (e.g., U0126 <strong>an</strong>d<br />
SB202190) as well as cytoskeletal disruptors (latrunculin A) alter or attenuate <strong>the</strong> lig<strong>an</strong>d-induced<br />
response. Thus, DMR appears to provide a global end point that c<strong>an</strong> be used in conjunction with<br />
inhibitors of specific steps potentially involved in lig<strong>an</strong>d-induced intracellular signaling. DMR<br />
has <strong>the</strong> potential not only to identify lig<strong>an</strong>ds that activate a given G-protein coupled receptor but<br />
also to ascertain <strong>the</strong> signaling pathways engaged by a given lig<strong>an</strong>d.<br />
Disclosures: E.E. Codd, J&J, A. Employment (full or part-time); J&J, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); J.R. mabus, J&J, A. Employment<br />
(full or part-time); J&J, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual<br />
property); B. Murray, None; C.M. Flores, J&J, A. Employment (full or part-time); J&J, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.12/CC71<br />
Topic: D.08.bb. Opioids<br />
Support: NIH gr<strong>an</strong>ts P20 DA026002<br />
NIH gr<strong>an</strong>ts DE18214<br />
NIH gr<strong>an</strong>ts DE18538<br />
Title: Enh<strong>an</strong>ced morphine conditioned place preference behavior in rats with CFA-induced<br />
hindpaw monoarthritis<br />
Authors: *G. LIM, S. WANG, J. MAO;<br />
Anes<strong>the</strong>siol. & Crit Care, Mass Gen. Hosp., Charlestown, MA<br />
Abstract: Recent reports have raised major concerns over addiction to <strong>an</strong>d abuse of opioid<br />
<strong>an</strong>algesics in <strong>the</strong> setting of chronic pain m<strong>an</strong>agement. Although <strong>the</strong> cellular mech<strong>an</strong>isms of<br />
opioid addiction might be similar regardless of whe<strong>the</strong>r opioids are prescribed <strong>for</strong> a legitimate<br />
medical use or obtained as illicit drug, addition to opioid <strong>an</strong>algesics in <strong>the</strong> setting of pain<br />
m<strong>an</strong>agement raises <strong>an</strong> intriguing <strong>an</strong>d clinically challenging issue. In <strong>the</strong> present study, we<br />
examined whe<strong>the</strong>r <strong>an</strong> experimental inflammatory pain condition would signific<strong>an</strong>tly enh<strong>an</strong>ce <strong>the</strong><br />
morphine-induced rewarding effect in a rat model of conditioned place preference (CPP). After 5<br />
days, Morphine-paired conditioning (mCPP) signific<strong>an</strong>tly increased <strong>the</strong> CPP score, which was<br />
fur<strong>the</strong>r increased in rats with Complete Freund’s Adjuv<strong>an</strong>t (CFA)-induced monoarthritis<br />
(CFA/mCPP). Fur<strong>the</strong>rmore, <strong>the</strong> mCPP paradigm induced <strong>the</strong> increase in glial activation <strong>an</strong>d<br />
enh<strong>an</strong>ced <strong>the</strong> expression of toll-like receptors (TLR) <strong>an</strong>d dopamine receptors (DA) in <strong>the</strong> nucleus<br />
accumbens (NAc). These ch<strong>an</strong>ges were enh<strong>an</strong>ced in rats with combined mCPP <strong>an</strong>d CFA-induced<br />
monoarthritis.<br />
These findings suggest that morphine-induced rewarding effect could be enh<strong>an</strong>ced by <strong>an</strong><br />
experimental inflammatory pain condition <strong>an</strong>d that activation of dopamine receptors within <strong>the</strong><br />
NAc through TLR-mediated glial activation may be a cellular mech<strong>an</strong>ism underlying <strong>the</strong>se<br />
behavioral ch<strong>an</strong>ges.<br />
Disclosures: G. Lim, Massachusetts general hospital, A. Employment (full or part-time); S.<br />
W<strong>an</strong>g, None; J. Mao, None.
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.13/CC72<br />
Topic: D.08.bb. Opioids<br />
Support: Department of Veter<strong>an</strong>s Affairs<br />
Title: Spinal µ-opiate receptor (MOR)-expressing dorsal horn neurons: Role in modulating pain<br />
<strong>an</strong>d opiate <strong>an</strong>algesia<br />
Authors: *R. H. KLINE IV 1,3 , R. G. WILEY 2,4 ;<br />
1 2 3 4<br />
Neurol., Neurol. <strong>an</strong>d Pharmacol., V<strong>an</strong>derbilt Univ., Nashville, TN; Exptl. Neurol., Neurol.<br />
Service, VA TVHS, Nashville, TN<br />
Abstract: Selective destruction of MOR-expressing interneurons in lamina II of <strong>the</strong> dorsal horn<br />
of <strong>the</strong> spinal cord increases reflex nocifensive responses to <strong>for</strong>malin <strong>an</strong>d decreases <strong>the</strong> <strong>an</strong>tinociceptive<br />
effects of morphine on <strong>the</strong> hotplate <strong>an</strong>d in <strong>the</strong> <strong>for</strong>malin test. The interpretation of<br />
<strong>the</strong>se studies is limited because reflex-based assays may not accurately reflect <strong>the</strong> cerebral<br />
component of nociception. There<strong>for</strong>e, we sought to determine <strong>the</strong> effects of selectively<br />
destroying MOR-expressing dorsal horn neurons on baseline oper<strong>an</strong>t responses to aversive<br />
<strong>the</strong>rmal <strong>an</strong>d mech<strong>an</strong>ical stimuli in a shuttle box task <strong>an</strong>d effects of systemic morphine <strong>an</strong>d<br />
naloxone in <strong>the</strong> same task. The preference apparatus consisted of a 15 X 15 X 30 cm smoked<br />
Plexiglas vented chamber placed upon two adjoining temperature-controlled smooth aluminum<br />
floor plates (<strong>the</strong>rmal preference task) or one smooth temperature-controlled floor plate adjoined<br />
to a room temperature surface covered with 40 grit s<strong>an</strong>dpaper (mech<strong>an</strong>ical preference task). For<br />
both preference tasks, response functions were obtained by pairing a 44°C plate or <strong>the</strong> s<strong>an</strong>dpaper<br />
surface with ei<strong>the</strong>r 11°, 16°, 25°, 38° or 46°C. Rats were intra<strong>the</strong>cally injected over <strong>the</strong> lumbar<br />
cord with ei<strong>the</strong>r 625ng of derm-sap (n=7) or bl<strong>an</strong>k-sap (n=6) followed by daily <strong>the</strong>rmal or<br />
mech<strong>an</strong>ical preference testing on a r<strong>an</strong>domized schedule. Derm-sap treated rats showed<br />
enh<strong>an</strong>ced avoid<strong>an</strong>ce of aversive <strong>the</strong>rmal stimuli <strong>an</strong>d <strong>the</strong> aversive mech<strong>an</strong>ical stimulus. Morphine<br />
<strong>an</strong>d naloxone signific<strong>an</strong>tly altered responses of control rats (bl<strong>an</strong>k-sap), but not derm-sap rats, in<br />
both <strong>the</strong>rmal <strong>an</strong>d mech<strong>an</strong>ical preference tasks. We interpret <strong>the</strong>se results as showing that <strong>the</strong><br />
derm-sap lesion produces hyperalgesia/allodynia, impairs <strong>the</strong> <strong>an</strong>ti-nociceptive <strong>an</strong>d <strong>an</strong>algesic<br />
effects of morphine <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e indicating that postsynaptic dorsal horn MOR-expressing<br />
neurons play a key role in modulating nociception, pain <strong>an</strong>d opiate <strong>an</strong>algesia. Dysfunction of<br />
<strong>the</strong>se neurons may also play a role in pathological pain states.<br />
Conflict of interest statement: RGW is affiliated with Adv<strong>an</strong>ced Targeting Systems, supplier of<br />
dermorphin-saporin.<br />
Support: This work was supported by <strong>the</strong> Department of Veter<strong>an</strong>s Affairs.
Disclosures: R.H. Kline IV, None; R.G. Wiley, Adv<strong>an</strong>ced Targeting Sys, F.<br />
Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.14/CC73<br />
Topic: D.08.bb. Opioids<br />
Support: DA-022599<br />
Title: Neurochemical alterations in <strong>the</strong> amygdala during opioid self-administration in nerveinjured<br />
rats<br />
Authors: *E. E. EWAN, N. L. BUECHLER, T. J. MARTIN;<br />
Wake Forest Univ., Winston-Salem, NC<br />
Abstract: The amygdala is a midbrain structure known to be involved in affective responses to<br />
noxious stimuli <strong>an</strong>d contains a dense population of mu opioid receptors (MOR’s). Previous work<br />
<strong>from</strong> our lab revealed that irreversible inhibition of MOR’s in <strong>the</strong> amygdala has a greater impact<br />
on heroin self-administration in rats with neuropathic pain compared to normal rats. This<br />
suggests that opioid pharmacology in <strong>the</strong> amygdala is altered following nerve injury, <strong>an</strong> effect<br />
that may be due to alterations in neuronal activity within this region. Microdialysis of <strong>the</strong><br />
amygdala was per<strong>for</strong>med in both normal <strong>an</strong>d nerve-injured rats during heroin self-administration<br />
<strong>an</strong>d revealed that glutamate levels were signific<strong>an</strong>tly increased only in nerve-injured rats during<br />
heroin self-administration. In rats with neuropathic pain, intra-amygdala administration of <strong>the</strong><br />
ampa/kainate <strong>an</strong>tagonist NBQX, <strong>the</strong> group II metabotropic glutamate agonist LY354740, <strong>an</strong>d <strong>the</strong><br />
group III metabotropic glutamate agonist LAP-4 failed to reverse mech<strong>an</strong>ical allodynia. These<br />
findings suggest that alterations in glutamatergic tr<strong>an</strong>smission in <strong>the</strong> amygdala may modulate <strong>the</strong><br />
rein<strong>for</strong>cing effects of opioids in <strong>the</strong> presence of neuropathic pain me<strong>an</strong>while producing no<br />
effects on hypersensitivity.<br />
Disclosures: E.E. Ew<strong>an</strong>, None; N.L. Buechler, None; T.J. Martin, None.<br />
Poster
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.15/CC74<br />
Topic: D.08.bb. Opioids<br />
Title: Nascent structure-activity relationship study of a diastereomeric series of kappa opioid<br />
receptor <strong>an</strong>tagonists derived <strong>from</strong> <strong>the</strong> peptide, CJ-15,208<br />
Authors: *A. MULLER, M. MICHAUT, B. MARTINEZ-TEIPEL, P. R. SEIDA, R. N.<br />
DEHAVEN, R. E. DOLLE;<br />
Adolor Corp, Exton, PA<br />
Abstract: Opioid <strong>an</strong>tagonists have a variety of <strong>the</strong>rapeutic uses, including <strong>the</strong> prevention <strong>an</strong>d<br />
treatment of post-operative ileus, reversal of opioid-induced constipation, treatment of<br />
alcoholism <strong>an</strong>d subst<strong>an</strong>ce abuse, <strong>an</strong>d, in ultra-low doses in combination with opioid agonists, to<br />
attenuate <strong>the</strong> risk of <strong>the</strong> development of toler<strong>an</strong>ce <strong>an</strong>d physical dependence. Saito et al (2002, J<br />
Antibiot (Tokyo) 55:847) identified a novel naturally occurring cyclic peptide, c(Phe-D-Pro-Phe-<br />
Trp; CJ-15,208), with <strong>an</strong>tagonist activities at <strong>the</strong> three opioid receptors with r<strong>an</strong>k order of<br />
potencies of kappa > mu > delta. We syn<strong>the</strong>sized this peptide <strong>an</strong>d found Ki values at κ, µ, <strong>an</strong>d δ<br />
receptors of 29 nM, 130 nM, <strong>an</strong>d 2000 nM, respectively. Its diastereomer, c(Phe-D-Pro-Phe-D-<br />
Trp), inhibited binding at κ, µ, <strong>an</strong>d δ receptors with Ki values of 3.8 nM, 30 nM, <strong>an</strong>d >1000 nM,<br />
respectively. These compounds behaved as <strong>an</strong>tagonists in receptor-mediated [ 35 S]GTPγS<br />
binding. Analogs designed to probe <strong>the</strong> effects of aromatic substituents that withdraw or donate<br />
electrons or donate or accept hydrogen bonds had Ki values at <strong>the</strong> κ receptor r<strong>an</strong>ging <strong>from</strong> 14 to<br />
220 nM <strong>an</strong>d a r<strong>an</strong>ge of Ki values at <strong>the</strong> µ receptor that resulted in µ/κ Ki ratios of 0.47 to 7.5. The<br />
<strong>an</strong>alogs were generally inactive at <strong>the</strong> δ receptor. Hydroxylation of ei<strong>the</strong>r Phe to yield Tyr or 3-<br />
OH-Phe <strong>an</strong>alogs had only modest effects on <strong>the</strong> affinities <strong>for</strong> <strong>the</strong> µ <strong>an</strong>d κ receptors, which is<br />
surprising in light of <strong>the</strong> import<strong>an</strong>ce of <strong>the</strong> Tyr hydroxyl substituent in <strong>the</strong> affinities of <strong>the</strong><br />
endogenous peptide agonists. SAR studies combined with overlays of low energy con<strong>for</strong>mations<br />
of c[Ala-D-Pro-Phe-D-Trp] provide support <strong>for</strong> <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> biological activity is<br />
principally dependent upon <strong>the</strong> contacts of <strong>the</strong> peptide’s side chains <strong>an</strong>d relatively independent<br />
of <strong>the</strong> backbone structure. Exploration of acyclic <strong>an</strong>alogs derived <strong>from</strong> CJ-15,208 <strong>an</strong>d its<br />
diastereomer demonstrated that <strong>the</strong> cyclic nature of <strong>the</strong> peptides was import<strong>an</strong>t in imparting high<br />
affinity <strong>for</strong> <strong>the</strong> µ <strong>an</strong>d κ receptors. Two acyclic <strong>an</strong>alogs, which bound with modest affinities to <strong>the</strong><br />
µ receptor, share a common motif, Phe-D-Trp-Phe, unique among opioid lig<strong>an</strong>ds.<br />
Disclosures: A. Muller, Employed by Adolor Corporation, A. Employment (full or part-time);<br />
stock options in Adolor Corporation, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); M. Michaut, None; B. Martinez-Teipel, None; P.R. Seida, Employee of<br />
Adolor Corporation, A. Employment (full or part-time); Stock option holder of Adolor<br />
Corporation, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);
R.N. DeHaven, Employee of Adolor Corporation, A. Employment (full or part-time); Stock<br />
option holder of Adolor Corporation, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); R.E. Dolle, Employee of Adolor Corporation, A. Employment (full or<br />
part-time); Stock option holder of Adolor Corporation, E. Ownership Interest (stock, stock<br />
options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.16/CC75<br />
Topic: D.08.bb. Opioids<br />
Support: CONACyT Gr<strong>an</strong>t 43604-M (SLC)<br />
CONACyT Gr<strong>an</strong>t 200325 (ASM)<br />
Fondo Miguel Alem<strong>an</strong> (ML-O)<br />
Fondo de apoyo a la investigación INPRFM (ML-O)<br />
Title: Mu opioid receptor internalization <strong>an</strong>d delay in toler<strong>an</strong>ce development with morphine <strong>an</strong>d<br />
fent<strong>an</strong>yl coadministration in rats<br />
Authors: *A. SILVA-MORENO, SR 1 , M. LEÓN-OLEA 2 , S. L. CRUZ 1 ;<br />
1 Farmacobiologia, Ctr. de Investigación y Estudios Av<strong>an</strong>zados, Mexico D.F., Mexico; 2 Lab. de<br />
Histología y Microscopia Electrónica, Inst. Nacional de Psiquiatría Ramón de la Fuente Muñiz,<br />
Mexico, Mexico<br />
Abstract: Chronic opiate administration c<strong>an</strong> be limited by <strong>the</strong> development of toler<strong>an</strong>ce <strong>an</strong>d<br />
physical dependence. These responses involve multiple adaptations including mu-opioid receptor<br />
internalization. Opiates differ in <strong>the</strong>ir ability to induce receptor internalization as well as<br />
toler<strong>an</strong>ce development. For example, morphine (MOR) induces poor internalization <strong>an</strong>d a high<br />
degree of toler<strong>an</strong>ce, while fent<strong>an</strong>yl (FEN) produces good internalization <strong>an</strong>d less toler<strong>an</strong>ce.<br />
Recent studies have examined <strong>the</strong> effects of combining opiates with different opioid receptor<br />
internalization capacities to determine whe<strong>the</strong>r toler<strong>an</strong>ce development diminishes. The objective<br />
of this work was to evaluate toler<strong>an</strong>ce development, physical dependence <strong>an</strong>d opioid receptor<br />
internalization after repeated administration of MOR alone or in combination with FEN in <strong>the</strong><br />
tail flick model. Male Wistar rats (200 ± 20 g) were i.c.v. administered twice a day (12 h apart)
with ei<strong>the</strong>r MOR (1 µg), FEN (1 ng) or <strong>the</strong>ir combination (n=6, each). Final volumen<br />
administration was 5 µl. This schedule was followed until <strong>an</strong>tinociception disappeared. After<br />
achieving complete <strong>an</strong>algesic toler<strong>an</strong>ce, 50% of <strong>the</strong> <strong>an</strong>imals were used <strong>for</strong> immunohistochemical<br />
studies to determine opioid receptor internalization. Specific <strong>an</strong>tibodies against mu opioid<br />
receptor <strong>an</strong>d beta arrestin protein were used. The brain areas <strong>an</strong>alyzed were periaqueductal gray<br />
(PAG), locus coeruleus (LC) <strong>an</strong>d ventral tegmental area (VTA). The remainig 50% of <strong>the</strong><br />
<strong>an</strong>imals were used <strong>for</strong> abstinence evaluation. In this case, <strong>an</strong>imals were challenged with a single<br />
naloxone administration (31 µg, i.c.v.). Withdrawal signs were evaluated <strong>for</strong> 20 min <strong>an</strong>d a global<br />
withdrawal score was calculated. An additional group was treated with 5.6 µg MOR using <strong>the</strong><br />
same administration schedule <strong>an</strong>d challenged with naloxone as a positive control <strong>for</strong> dependence.<br />
The results show that 1 ng FEN had no effect. A mild <strong>an</strong>tinociception was produced by 1 µg<br />
MOR, but it disappeared at <strong>the</strong> 5th administration. <strong>When</strong> FEN plus MOR were administered<br />
repeatedly, <strong>the</strong>re was <strong>an</strong> initial <strong>an</strong>tinociceptive potentiation <strong>an</strong>d a delay in toler<strong>an</strong>ce development<br />
in comparison with single treated groups. A robust abstinence response was only seen in rats<br />
treated with <strong>the</strong> high dose of morphine. In <strong>the</strong> FEN-MOR treated group opioid receptor<br />
internalization (showed as fluorescent co localization) was higher th<strong>an</strong> that observed in groups<br />
treated with FEN or MOR alone. These results suggest that repeated co-administration of FEN<br />
<strong>an</strong>d MOR produces good <strong>an</strong>tinociception along with a delay in toler<strong>an</strong>ce development. These<br />
data were correlated, at least in part, with differences in mu opioid receptor internalization.<br />
Disclosures: A. Silva-Moreno, None; M. León-Olea, None; S.L. Cruz, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.17/CC76<br />
Topic: D.08.bb. Opioids<br />
Title: The development of pain <strong>an</strong>d reward processing in rat pups: Neonatal administration of<br />
morphine activates <strong>the</strong> mesolimbic dopaminergic system<br />
Authors: *L. LOW, M. FITZGERALD;<br />
Neuroscience, Physiol. <strong>an</strong>d Pharmacol., Univ. Col. London, London, United Kingdom<br />
Abstract: Painful experiences in early life have long term effects upon <strong>the</strong> developing CNS.<br />
Work <strong>from</strong> our laboratory has shown that repeated exposure to a painful procedure during <strong>the</strong><br />
first three postnatal weeks in <strong>the</strong> rat alters behavioural responses to reward in <strong>the</strong> adult. This<br />
behavioural ch<strong>an</strong>ge is reflected in activation of <strong>the</strong> orexin system of <strong>the</strong> lateral hypothalamus,
which is closely interlinked with <strong>the</strong> mesolimbic dopamine (DA) system. We hypo<strong>the</strong>sise that <strong>an</strong><br />
overlap in pain <strong>an</strong>d reward systems is already present in early life <strong>an</strong>d that because of this,<br />
neonatal pain alters <strong>the</strong> normal maturation of <strong>the</strong> DA system.<br />
However, little is known of <strong>the</strong> postnatal functional development of reward processing <strong>an</strong>d <strong>the</strong><br />
aim of this work was to investigate whe<strong>the</strong>r <strong>the</strong> DA system is sensitive to reward in early life. To<br />
do this we have used a classic pharmacological trigger of reward pathways, acute <strong>an</strong>d chronic<br />
morphine administration.<br />
Acute morphine injection (s.c.) was given to postnatal day (P)7, P21 <strong>an</strong>d adult rats; <strong>an</strong>imals were<br />
killed <strong>an</strong>d perfused with 4% para<strong>for</strong>maldehyde after 30 minutes. Chronic morphine<br />
administration consisted of a daily morphine injection <strong>for</strong> 5 days (P3 to P7 <strong>an</strong>d P17 to P21).<br />
Dosage was 5mg/kg <strong>for</strong> <strong>the</strong> youngest <strong>an</strong>imals <strong>an</strong>d 10mg/kg <strong>for</strong> older neonates <strong>an</strong>d adults.<br />
Littermate controls received acute or chronic saline injections. N = 3-5 in each group.<br />
Activation of <strong>the</strong> dopaminergic system was qu<strong>an</strong>tified by double-immunolabelling of<br />
dopaminergic cells (tyrosine hydroxylase, TH+ve) in <strong>the</strong> ventral tegmental area (VTA) with<br />
pERK, a marker of cell activation. The VTA receives input <strong>from</strong> <strong>the</strong> lateral hypothalamus <strong>an</strong>d<br />
feeds directly into <strong>the</strong> mesolimbic dopaminergic system. Expression of pERK in <strong>the</strong> TH+ve cells<br />
of <strong>the</strong> VTA indicates activation of <strong>the</strong> DA system (Berhow et al, 1996; Ozaki et al, 2002; 2004).<br />
In P7 <strong>an</strong>d P21 <strong>an</strong>imals, acute <strong>an</strong>d chronic morphine signific<strong>an</strong>tly increased <strong>the</strong> number of TH+ve<br />
VTA cells expressing pERK compared to both saline conditions <strong>from</strong> 5-15% to 20-30%. This<br />
pattern was also seen in adults; however, <strong>the</strong> activation of TH+ve VTA cells after chronic<br />
morphine returned to control levels. General activity of <strong>the</strong> VTA (via pERK expression) was<br />
highest in <strong>the</strong> P7 <strong>an</strong>imals, decreasing with age. This developmental increase was not reflected in<br />
differences between <strong>an</strong>y experimental group.<br />
These results suggest that <strong>the</strong> mesolimbic dopamine system of <strong>the</strong> neonatal rat is functional by<br />
P3, thus providing a rationale <strong>for</strong> <strong>the</strong> hypo<strong>the</strong>sis that ch<strong>an</strong>ges in adult reward behaviour<br />
following repeated injury in early life is due to altered dopaminergic tone.<br />
Disclosures: L. Low, None; M. Fitzgerald, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.18/CC77<br />
Topic: D.08.bb. Opioids<br />
Title: The <strong>an</strong>tinociceptive potency of c<strong>an</strong>nabinoid <strong>an</strong>d opioid increased in bone c<strong>an</strong>cer model
Authors: A. DOGRUL 1 , *M. H. OSSIPOV 2 , T. KING 3 , F. PORRECA 3 ;<br />
1 Gulh<strong>an</strong>e Acad. of Med., Ankara, Turkey; 2 Pharmacol, Univ. of Arizona Col. Med., Tucson, AZ;<br />
3 Pharmacol., Univ. of Arizona, Tucson, AZ<br />
Abstract: Previous studies suggested that bone c<strong>an</strong>cer pain may have <strong>an</strong> inflammatory <strong>an</strong>d<br />
neuropathic component. The enh<strong>an</strong>cement <strong>an</strong>d decreasement of <strong>an</strong>tinociceptive action of opioid<br />
<strong>an</strong>d c<strong>an</strong>nabinoid during inflammation <strong>an</strong>d neuropathic pain, respectively, are well known. In this<br />
study, we assessed <strong>the</strong> <strong>the</strong>rmal <strong>an</strong>tinociceptive effects of WIN 55, 212-2, a mixed CB-1 <strong>an</strong>d CB-<br />
2 receptor agonist, <strong>an</strong>d morphine, a opioid receptor agonists, in bone c<strong>an</strong>cer <strong>an</strong>d control <strong>an</strong>imals.<br />
Osteoyltic sarcoma cells were injected into <strong>the</strong> intramedullar space of femur <strong>an</strong>d behavioral tests<br />
were per<strong>for</strong>med following 14 days after tumor injection. Thermal <strong>an</strong>tinociceptions were<br />
evaluated by radi<strong>an</strong>t tail-flick test in both systemic <strong>an</strong>d spinal administration of WIN 55, 212-2<br />
<strong>an</strong>d morphine. Both systemic <strong>an</strong>d spinal administration of WIN 55, 212-2 <strong>an</strong>d morphine produce<br />
dose dependent <strong>an</strong>tinociceptive effects in sham <strong>an</strong>d bone c<strong>an</strong>cer <strong>an</strong>imals. However, dose<br />
response curve <strong>for</strong> spinal <strong>an</strong>d sysstemically administered WIN 55, 212-2 <strong>an</strong>d morphine were<br />
signific<strong>an</strong>tly shifted by to <strong>the</strong> left in bone c<strong>an</strong>cer stiuation suggesting <strong>an</strong> increase in <strong>the</strong><br />
<strong>an</strong>tinociceptive potency of c<strong>an</strong>nabinoid <strong>an</strong>d opioids. These results suggest that inflammatory<br />
component play in import<strong>an</strong>t role in <strong>the</strong> mech<strong>an</strong>ism of bone c<strong>an</strong>cer pain<br />
Disclosures: A. Dogrul, None; M.H. Ossipov, None; T. King, None; F. Porreca, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.19/CC78<br />
Topic: D.08.bb. Opioids<br />
Support: DARPA Gr<strong>an</strong>t APHYP0138<br />
Title: Feedback-regulated morphine delivery: A new approach in emergency medicine<br />
Authors: *M. WALLISCH 1 , S. S. SATAV 3 , K. B. SPENCER 1 , D. A. N. SILVERMAN 1 , D. R.<br />
KOOP 1,2 , S. THAYUMANAVAN 3 , G. D. OLSEN 1 ;<br />
1 Physiol. & Pharmacol., 2 Pharmacokinetics Core, Oregon Hlth. & Sci. Univ., Portl<strong>an</strong>d, OR;<br />
3 Chem., Univ. of Massachusetts, Amherst, MA<br />
Abstract: Morphine is a powerful <strong>an</strong>algesic <strong>for</strong> <strong>the</strong> treatment of acute pain <strong>an</strong>d is widely used to<br />
alleviate pain in emergency medicine. One of <strong>the</strong> major difficulties of acute pain m<strong>an</strong>agement,
however, is adequate dosing. <strong>When</strong> morphine levels exceed <strong>the</strong>rapeutic doses, <strong>the</strong>y c<strong>an</strong> cause<br />
severe respiratory depression, apnea or death. This morphine-induced hypoventilation is<br />
characterized by <strong>an</strong> increased concentration of CO2 levels in blood plasma, which c<strong>an</strong> be used to<br />
detect toxic levels of morphine. This project evaluates <strong>the</strong> biological properties of a new<br />
polymer-based morphine delivery system that uses feedback regulation to deliver adequate<br />
<strong>an</strong>algesic doses of morphine. The delivery system uses blood plasma CO2 levels as a biomarker<br />
<strong>for</strong> drug induced toxicity <strong>an</strong>d feedback regulation. Here we report on <strong>the</strong> biological <strong>an</strong>d<br />
pharmacological properties of this delivery system. We evaluate <strong>the</strong> cytototoxic properties of<br />
<strong>the</strong>se polymers <strong>an</strong>d <strong>the</strong> pharmacokinetic profile of <strong>the</strong> morphine release. In addition, we use<br />
several <strong>an</strong>algesic tests to characterize <strong>the</strong> pharmacodynamic properties of <strong>the</strong> morphine release.<br />
Our data suggests that <strong>the</strong> new delivery system is non-toxic <strong>an</strong>d releases <strong>an</strong>algesic effective<br />
doses of morphine that are responsive to increased CO2.<br />
Disclosures: M. Wallisch, None; S.S. Satav, None; K.B. Spencer, None; D.A.N. Silverm<strong>an</strong>,<br />
None; D.R. Koop, None; S. Thayum<strong>an</strong>av<strong>an</strong>, None; G.D. Olsen, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.20/CC79<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t DA022023<br />
NIH Gr<strong>an</strong>t 5T23AG000196-17<br />
University of Florida Allumni Association Fellowship<br />
Title: Effects of age <strong>an</strong>d chronic fent<strong>an</strong>yl administration on <strong>the</strong>rmal preference in rats<br />
Authors: *J. D. MITZELFELT 1 , C. S. CARTER 2,3 , D. MORGAN 1,3 ;<br />
1 Dept Psychiatry, Col. of Med., Univ. Florida, Gainesville, FL; 2 Dept. of Aging <strong>an</strong>d Geriatric<br />
Research, Col. of Med., 3 Inst. of Aging, Univ. of Florida, Gainesville, FL<br />
Abstract: The U.S. population is getting increasingly older with people 65+ years of age<br />
projected to make up 20% of <strong>the</strong> population by 2030. The use of opioids <strong>for</strong> pain relief in nonc<strong>an</strong>cer<br />
patients <strong>an</strong>d patients with chronic pain is increasing. While traditional reflexive pain<br />
assessments have not consistently shown aging effects in pain sensitivity, <strong>the</strong>se tests may not be
sensitive enough to demonstrate aging effects. For this study, <strong>an</strong> oper<strong>an</strong>t-based <strong>the</strong>rmal<br />
preference procedure was used to test <strong>the</strong> effects of age <strong>an</strong>d chronic fent<strong>an</strong>yl administration (28<br />
days) <strong>an</strong>d withdrawal (28 days). Sensitivity to both hot <strong>an</strong>d cold <strong>the</strong>rmal stimulation was tested<br />
in both saline <strong>an</strong>d fent<strong>an</strong>yl treated rats at 16, 20, <strong>an</strong>d 24 months of age. Differences were<br />
observed between ages be<strong>for</strong>e <strong>an</strong>d during fent<strong>an</strong>yl administration. These finding suggest that<br />
<strong>the</strong>re are differences in pain sensitivity across ages <strong>an</strong>d that older individuals respond differently<br />
to fent<strong>an</strong>yl administration th<strong>an</strong> younger individuals. These findings also suggest that this <strong>the</strong>rmal<br />
preference procedure is potentially better suited <strong>for</strong> pain assessment across ages.<br />
Disclosures: J.D. Mitzelfelt, None; C.S. Carter, None; D. Morg<strong>an</strong>, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.21/CC80<br />
Topic: D.08.bb. Opioids<br />
Title: The contribution of spinal <strong>an</strong>d supraspinal loci to opioid-induced hyperalgesia<br />
Authors: A. R. WAXMAN 1 , M. CALDWELL 2 , C. A. AROUT 1 , G. ROSSI 3 , *B. KEST 4 ;<br />
1 City Univ. of New York, New York, NY; 2 Col. of Staten Isl<strong>an</strong>d, Staten Isl<strong>an</strong>d, NY; 3 Long<br />
Isl<strong>an</strong>d Univ., New York, NY; 4 Dept Psychol/Neurosci, CUNY, Staten Isl<strong>an</strong>d, NY<br />
Abstract: We have observed that acute systemic morphine injection c<strong>an</strong> increase nociception in<br />
mice. Although this opioid-induced hyperalgesia is independent of prior or concurrent opioid<br />
receptor activity, it is none<strong>the</strong>less blocked by <strong>the</strong> non-competitive NMDA receptor <strong>an</strong>tagonist<br />
MK-801. To assess whe<strong>the</strong>r spinal <strong>an</strong>d/or supraspinal CNS loci contribute to systemic morphine<br />
hyperalgesia, mice were injected with <strong>the</strong> opioid <strong>an</strong>tagonist naltrexone <strong>an</strong>d <strong>the</strong>n tested <strong>for</strong><br />
nociception on <strong>the</strong> tail-flick test be<strong>for</strong>e <strong>an</strong>d after receiving <strong>an</strong> acute morphine bolus dose via <strong>the</strong><br />
intra<strong>the</strong>cal (IT) or intracerebroventricular (ICV) route. Fur<strong>the</strong>rmore, <strong>the</strong> contribution of NMDA<br />
receptors at spinal <strong>an</strong>d supraspinal loci was also studied in naltrexone treated mice by assaying<br />
nociception be<strong>for</strong>e <strong>an</strong>d after IT <strong>an</strong>d ICV MK-801 injection in mice treated with <strong>an</strong> acute<br />
systemic morphine bolus dose. Whereas IT <strong>an</strong>d ICV morphine injection both caused signific<strong>an</strong>t<br />
hyperalgesia, IT <strong>an</strong>d ICV MK-801 blocked hyperalgesia after systemic morphine injection. The<br />
data indicate that morphine injected into spinal <strong>an</strong>d supraspinal loci c<strong>an</strong> increase nociception, but<br />
that <strong>the</strong> ensuing hyperalgesia is not mediated by opioid receptors. The data also indicate that <strong>the</strong><br />
ability of MK-801 to block opioid-induced hyperalgesia after systemic morphine injection may<br />
be mediated by NMDA receptors at ei<strong>the</strong>r spinal or supraspinal loci.
Disclosures: A.R. Waxm<strong>an</strong>, None; M. Caldwell, None; C.A. Arout, None; G. Rossi, None; B.<br />
Kest, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.22/CC81<br />
Topic: D.08.bb. Opioids<br />
Title: Do pronociceptive opioid metabolites mediate opioid-induced hyperalgesia?<br />
Authors: *C. A. AROUT 1 , A. R. WAXMAN 2 , A. DAHAN 3 , K. VAN DE WETERING 4 , B.<br />
KEST 2 ;<br />
1 Queens Col, CUNY, Flushing, NY; 2 City Univ. of New York, New York, NY; 3 Leiden Univ.<br />
Med. Ctr., Leiden, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 The Ne<strong>the</strong>rl<strong>an</strong>ds C<strong>an</strong>cer Inst., Amsterdam, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: The acute injection of opioids c<strong>an</strong> paradoxically increase nociception. Here, we<br />
assayed nociception on <strong>the</strong> tail-withdrawal test in mice concurrently treated with <strong>the</strong> general<br />
opioid <strong>an</strong>tagonist naltrexone or in opioid receptor triple knock-out (TKO) mice lacking µ, δ, <strong>an</strong>d<br />
κ opioid receptors. Hyperalgesia was m<strong>an</strong>ifest in both groups, indicating that opioid-induced<br />
hyperalgesia is independent of prior or concurrent opioid receptor activity. To assess whe<strong>the</strong>r<br />
this hyperalgesia was <strong>the</strong>re<strong>for</strong>e mediated by pronociceptive non-opioid neuroexcitatory 3conjugated<br />
glucuronide metabolites, we also tested naltrexone pelleted mice with a knock-out of<br />
Multi-drug resist<strong>an</strong>t protein 3 (Mrp3 (--/--) ), which facilitates <strong>the</strong> tr<strong>an</strong>sfer of M3G <strong>from</strong> <strong>the</strong> liver<br />
into <strong>the</strong> bloodstream. In a parallel study, we also assayed nociception after acute injection of<br />
fent<strong>an</strong>yl, which is not subject to glucuronidation <strong>an</strong>d does not yield known pronociceptive<br />
metabolites, in CD-1 mice concurrently treated with <strong>the</strong> general opioid receptor <strong>an</strong>tagonist<br />
naltrexone or in opioid receptor triple knock-out (TKO) mice lacking µ, δ, <strong>an</strong>d κ opioid<br />
receptors. The data show that morphine causes hyperalgesia in Mrp3 (--/--) mice <strong>an</strong>d fent<strong>an</strong>yl<br />
causes hyperalgesia in naltrexone pelleted <strong>an</strong>d TKO mice. These data collectively suggest that,<br />
although morphine <strong>an</strong>d fent<strong>an</strong>yl cause hyperalgesia independently of opioid receptor activity,<br />
<strong>the</strong>y do not do so via known neuroexcitatory metabolites<br />
Disclosures: C.A. Arout, None; A.R. Waxm<strong>an</strong>, None; A. Dah<strong>an</strong>, None; K. v<strong>an</strong> de Wetering,<br />
None; B. Kest, None.
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.23/CC82<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t DE019448<br />
Title: Activation of peripheral delta opioid receptors attenuates muscle hypersensitivity via<br />
ATP-sensitive potassium ch<strong>an</strong>nels (KATP) in a sex-dependent m<strong>an</strong>ner<br />
Authors: *J. Y. RO 1 , J. L. SALOMAN 2 , K. Y. NIU 1 , Y. ZHANG 1 ;<br />
1 Dept. of Neural <strong>an</strong>d Pain Sci., Univ. of Maryl<strong>an</strong>d Dent. Sch., Baltimore, MD; 2 Program in<br />
Neurosci., Univ. of Maryl<strong>an</strong>d Baltimore, Baltimore, MD<br />
Abstract: Sex-differences in opioid <strong>an</strong>algesia have received much attention in <strong>the</strong> past. Few<br />
studies have directly addressed sex-differences in peripheral opioid receptor (POR)-mediated<br />
<strong>an</strong>algesia. Since activation of peripheral opioid receptors has been shown to contribute to potent<br />
<strong>an</strong>algesic <strong>an</strong>d <strong>an</strong>ti-hyperalgesic effects in various pain models, studying <strong>the</strong> underlying<br />
mech<strong>an</strong>isms of sex-based differences in POR-mediated <strong>an</strong>algesia becomes pertinent. In this<br />
project, we examined whe<strong>the</strong>r <strong>the</strong> activation of peripheral delta opioid receptors (DORs)<br />
produces sex specific <strong>an</strong>algesic <strong>an</strong>d/or <strong>an</strong>ti-hyperalgesic responses <strong>an</strong>d potential mech<strong>an</strong>isms that<br />
underlie sex-differences in DOR effects in <strong>the</strong> context of <strong>an</strong> acute orofacial muscle pain<br />
condition. Specifically, we investigated whe<strong>the</strong>r KATP, a downstream target of OR signaling,<br />
contributes to sex-differences in DOR-mediated <strong>an</strong>algesic responses. Male <strong>an</strong>d female Sprague-<br />
Dawley rats (200-300g) were used. Estrous cycles in female rats were staged to select rats with<br />
<strong>the</strong> highest (proestrous, PER) <strong>an</strong>d <strong>the</strong> lowest (diestrous, DER) circulating estrogen levels. Local<br />
pretreatment of <strong>the</strong> masseter with a specific DOR agonist, DPDPE (1, 10, 100µg), dosedependently<br />
attenuated capsaicin-induced acute nocifensive behaviors as well as mech<strong>an</strong>ical<br />
hyperalgesia in male rats. However, <strong>the</strong> highest does of DPDPE (100µg) failed to block <strong>the</strong><br />
capsaicin responses in female rats in ei<strong>the</strong>r PER or DER. The mRNAs <strong>for</strong> <strong>the</strong> inward rectifying<br />
pore-<strong>for</strong>ming subunits, Kir6.1 <strong>an</strong>d Kir6.2, as well as <strong>the</strong> regulatory sulphonylurea subunits,<br />
SUR1 <strong>an</strong>d SUR2B, were detected in trigeminal g<strong>an</strong>glia (TG) of both male <strong>an</strong>d female rats.<br />
Immunohistochemical studies confirmed that Kir6.1 <strong>an</strong>d Kir6.2 subunits co-localize with DORs.<br />
Preliminary data <strong>from</strong> Western Blot <strong>an</strong>alysis suggest that Kir6.2 is expressed at a higher level in<br />
male TG compared to that of female rats in ei<strong>the</strong>r PER or DER. Masseteric injection of a specific<br />
KATP agonist, Pinacidil (20 µg) signific<strong>an</strong>tly attenuated <strong>the</strong> capsaicin-induced nocifensive<br />
responses <strong>an</strong>d mech<strong>an</strong>ical hyperalgesia in male rats, but not in female rats in ei<strong>the</strong>r PER or DER.<br />
Finally, pretreatment of <strong>the</strong> masseter with <strong>the</strong> KATP <strong>an</strong>tagonist glibenclamide (100µg)<br />
signific<strong>an</strong>tly blocked <strong>the</strong> effects of DPDPE in male rats suggesting that <strong>the</strong> peripheral DOR
effect is mediated by KATP . These studies revealed novel mech<strong>an</strong>isms underlying sex-differences<br />
in peripherally localized DOR-mediated <strong>an</strong>algesia in orofacial muscle pain condition, which may<br />
have import<strong>an</strong>t tr<strong>an</strong>slational implications in sexually dimorphic clinical conditions, such as<br />
temporom<strong>an</strong>dibular disorders.<br />
Disclosures: J.Y. Ro, None; J.L. Salom<strong>an</strong>, None; K.Y. Niu, None; Y. Zh<strong>an</strong>g, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.24/CC83<br />
Topic: D.08.bb. Opioids<br />
Support: NIH Gr<strong>an</strong>t DE019448<br />
Title: Sex differences in peripheral µ-opioid receptor (MOR)-mediated <strong>an</strong>algesia under a chronic<br />
orofacial myositis condition are mediated by sex-different MOR expression levels in trigeminal<br />
g<strong>an</strong>glia<br />
Authors: *K. NIU, J.-S. LEE, J. Y. RO;<br />
Dept. of Neural <strong>an</strong>d Pain Sci., Univ. of Maryl<strong>an</strong>d Dent. Sch., Baltimore, MD<br />
Abstract: Orofacial muscle pain related to temporom<strong>an</strong>dibular disorders is a prevailing <strong>an</strong>d<br />
debilitating pain condition. Women show a much greater prevalence <strong>an</strong>d severity of orofacial<br />
muscle pain compared to men. Sex differences also play <strong>an</strong> import<strong>an</strong>t role in individuals’<br />
response to treatment. There is accumulating evidence supporting <strong>the</strong> contribution of peripheral<br />
opioid receptors (POR) in attenuation of pain/hyperalgesia under various pain conditions.<br />
However, very few studies have directly studied sex differences in peripheral opioid receptor<br />
(POR)-mediated <strong>an</strong>algesia. It has been demonstrated that cytokines are one of <strong>the</strong> main<br />
regulators of OR expression in neuronal <strong>an</strong>d immune cells. This led us to propose that<br />
differential release of cytokines at <strong>the</strong> inflammatory site is one of <strong>the</strong> underlying mech<strong>an</strong>isms <strong>for</strong><br />
sex-differences in POR expressions that lead to sex-dependent <strong>an</strong>algesic responses under<br />
inflammatory conditions. The objectives of this study were to demonstrate (1) sex-differences in<br />
<strong>the</strong> effect of peripherally applied MOR agonist; (2) sex-differences in MOR expression in<br />
trigeminal g<strong>an</strong>glia (TG), <strong>an</strong>d that (3) sex-differences in POR expression correlate with sex<br />
differences in cytokine levels in <strong>the</strong> masseter muscle. Male <strong>an</strong>d female Sprague-Dawley rats<br />
(200-300g) were used. Estrous cycles in female rats were staged to select rats with <strong>the</strong> highest<br />
(proestrous, PER) <strong>an</strong>d <strong>the</strong> lowest (diestrous, DER) circulating estrogen levels. Animals were
injected with CFA in <strong>the</strong> masseter <strong>an</strong>d mech<strong>an</strong>ical hyperalgesia was examined be<strong>for</strong>e <strong>an</strong>d 3 days<br />
post CFA injection. MOR agonist (DAMGO) had no effect in naïve <strong>an</strong>imals in both males <strong>an</strong>d<br />
females. However, under inflammatory condition, DAMGO was effective in blocking <strong>the</strong><br />
masseter mech<strong>an</strong>ical hyperalgesia in males but not in females. TG <strong>an</strong>d masseter muscle were<br />
extracted <strong>from</strong> <strong>the</strong> rats to <strong>an</strong>alyze MOR expression <strong>an</strong>d TNF-α <strong>an</strong>d IL-1β levels respectively.<br />
Based on our preliminary results, MOR was upregulated after inflammation in males in a time<br />
dependent m<strong>an</strong>ner, but not in females. IL-1β in <strong>the</strong> masseter increased in males after<br />
inflammation in a similar time course as MOR upregulation, but not in females in ei<strong>the</strong>r PER or<br />
DER. There was no signific<strong>an</strong>t ch<strong>an</strong>ge of TNF-α level in masseter muscle following CFAinduced<br />
inflammation. These data suggest that sex differences in peripheral MOR-mediated<br />
<strong>an</strong>algesia under a chronic orofacial myositis condition are mediated by different MOR receptor<br />
expression levels in male <strong>an</strong>d female; <strong>an</strong>d that <strong>the</strong> differential release of cytokines at <strong>the</strong><br />
inflammatory site is one of <strong>the</strong> underlying mech<strong>an</strong>isms <strong>for</strong> sex-differences in MOR expression.<br />
Disclosures: K. Niu, None; J. Lee, None; J.Y. Ro, None.<br />
Poster<br />
560. Opioids: Mech<strong>an</strong>ism of Action<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 560.25/CC84<br />
Topic: D.08.bb. Opioids<br />
Support: NIH gr<strong>an</strong>t SC1NS063951<br />
NIH gr<strong>an</strong>t RR03032<br />
NIH gr<strong>an</strong>t U54 NS041071<br />
RISE 2R25GM59994<br />
Title: Selective activation of kappa opioid receptor reduces carrageen<strong>an</strong>-induced <strong>the</strong>rmal<br />
hyperalgesia in female rats but not tactile allodynia in male <strong>an</strong>d female rats<br />
Authors: *K. P. LAWSON, S. NAG, S. S. MOKHA;<br />
Meharry Med. Col., Nashville, TN<br />
Abstract: M<strong>an</strong>y chronic inflammatory painful conditions show a higher prevalence in women.<br />
Hyperalgesia <strong>an</strong>d allodynia are bo<strong>the</strong>rsome symptoms often associated with chronic pain.
Numerous <strong>an</strong>imal studies have reported that kappa opioid receptor (KOR) activation c<strong>an</strong><br />
attenuate <strong>the</strong>rmal or mech<strong>an</strong>ical hyperalgesia in various models of chronic pain. However,<br />
inconsistent <strong>an</strong>d conflicting results regarding sex differences have resulted <strong>from</strong> <strong>the</strong>se studies.<br />
There<strong>for</strong>e, this investigation was designed to determine whe<strong>the</strong>r <strong>the</strong> selective activation of KOR<br />
produces sex-specific modulation of nociception in <strong>an</strong> inflammation-induced model of persistent<br />
pain. The effects of U50,488H, a selective KOR agonist, were tested on carrageen<strong>an</strong>-induced<br />
<strong>the</strong>rmal hyperalgesia <strong>an</strong>d tactile allodynia in male <strong>an</strong>d OVX+E <strong>an</strong>imals. C<strong>an</strong>nulae were<br />
impl<strong>an</strong>ted in <strong>the</strong> intra<strong>the</strong>cal space of <strong>the</strong> lumbosacral spinal cord in male <strong>an</strong>d OVX female<br />
Sprague-Dawley rats. Nociceptive testing was conducted 5-7 days after surgery. The<br />
ovariectomized rats (OVX +E) were given estradiol benzoate (10 µg/ 100 µl sesame oil, s.c.) 48<br />
h be<strong>for</strong>e testing. Inflammation was induced by injection of 2% lambda carrageen<strong>an</strong> into <strong>the</strong> right<br />
hind paw 2 h prior to behavioral testing. U50,488H was intra<strong>the</strong>cally administered <strong>an</strong>d <strong>the</strong> heatevoked<br />
paw withdrawal latency (PWL) or mech<strong>an</strong>ical threshold was measured in 10-min<br />
intervals over a 90-min period. U50,488H signific<strong>an</strong>tly increased PWLs in OVX + E rats, but did<br />
not affect those of <strong>the</strong> males. Carrageen<strong>an</strong> injection signific<strong>an</strong>tly reduced PWLs in all groups.<br />
U50, 488H reversed <strong>the</strong> carrageen<strong>an</strong>-induced <strong>the</strong>rmal hyperalgesia in OVX + E rats, but did not<br />
alter PWLs in <strong>the</strong> male group. Carrageen<strong>an</strong> reduced <strong>the</strong> baseline mech<strong>an</strong>ical thresholds of both<br />
groups, but U50,488H treatment did not affect <strong>the</strong> mech<strong>an</strong>ical threshold in <strong>an</strong>imals with or<br />
without inflammation in male or OVX+E groups. Selective activation of KOR was fur<strong>the</strong>r<br />
confirmed by <strong>an</strong>tisense knockdown strategy using a nociceptive tail flick assay. Intra<strong>the</strong>cal<br />
<strong>an</strong>tisense, but not mismatch-, ODN treatment (12.5 µg/ 5 µl, 10-12 h intervals over 4 d) blocked<br />
<strong>the</strong> <strong>an</strong>tinociceptive action of U50,488H in OVX+E rats. Complete recovery of <strong>the</strong><br />
<strong>an</strong>tinociceptive effects of U50,488H was observed in this group 10 days after termination of<br />
<strong>an</strong>tisense ODN administration. We conclude that selective activation of KOR produces sexspecific,<br />
stimulus-dependent modulation of inflammatory pain in <strong>the</strong> rat. These findings could<br />
provide new insights into <strong>the</strong> design of <strong>an</strong>algesic treatment in women suffering <strong>from</strong> chronic<br />
pain.<br />
Disclosures: K.P. Lawson, None; S. Nag, None; S.S. Mokha, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.1/CC85<br />
Topic: D.08.p. Inflammatory pain: Inflammatory mediators<br />
Support: KRF- 2008-532-E00019
Title: Effect of interleukin-8 receptor inhibitor on mech<strong>an</strong>ical allodynia in a rat model of lumbar<br />
disc herniation<br />
Authors: S.-J. KIM 1 , *J. SEO 1 , S.-J. HWANG 2 , H.-W. PARK 3 , S.-M. PARK 4 , Y.-W. CHO 5 ,<br />
S.-H. JANG 5 , S.-H. AHN 5 ;<br />
1 Rehabil. medicine, Inst. of Med. Science, Youngnam Univ., Daugu, Republic of Korea; 2 Dept.<br />
of Anat. <strong>an</strong>d Cell Biology,, college of medicine, H<strong>an</strong>y<strong>an</strong>g Univ., Deagu, Republic of Korea;<br />
3 Daegu Catholic Univ. Med. Ctr., Department of Rehabilitation Medicine, Republic of Korea;<br />
4 Dept. of Rehabil. Med., Nams<strong>an</strong> hospital, Deagu, Republic of Korea; 5 Departments of Rehabil.<br />
Med., Col. of Medicine, Yeungnam Univ., Deagu, Republic of Korea<br />
Abstract: Objective: IL-8(CXCL8) has been reported to be associated with painful degenerative<br />
disc disorders <strong>an</strong>d chronic inflammatory pain states. The purpose of this study is to investigate<br />
sequential ch<strong>an</strong>ges of IL-8 expression in <strong>the</strong> spinal cord <strong>an</strong>d DRG <strong>an</strong>d <strong>the</strong> pain-related behaviors<br />
with time course <strong>an</strong>d to elucidate whe<strong>the</strong>r <strong>the</strong> IL-8 receptor inhibitor attenuates pain-related<br />
behaviors <strong>an</strong>d influences neuroglia in a rat model of lumbar disc herniation.<br />
Method: Lumbar disc herniated rat model was made by impl<strong>an</strong>tation of <strong>the</strong> autologous nucleus<br />
pulposus, harvested <strong>from</strong> <strong>the</strong> coccygeal vertebra of each tail, on <strong>the</strong> left L5 nerve root just<br />
proximal to <strong>the</strong> DRG. Rats were tested <strong>for</strong> mech<strong>an</strong>ical allodynia of <strong>the</strong> pl<strong>an</strong>tar surface of both<br />
hindpaw on 2 days be<strong>for</strong>e surgery, <strong>an</strong>d on 1, 5, 10, 20, 30 <strong>an</strong>d 60 days postoperatively.<br />
Experimental group was injected with <strong>the</strong> IL-8 receptor inhibitor (200µg/kg, i.t.) at L1 level on<br />
postoperative day 10. Mech<strong>an</strong>ical allodynia of <strong>the</strong> pl<strong>an</strong>tar surface of both hindpaw was tested on<br />
30 minutes, 1, 3hours, 1, 2, 3 <strong>an</strong>d 4 days after administration. For <strong>the</strong> staining of astrocytes <strong>an</strong>d<br />
microglia, immunohistochemical study was done 14 days after surgery.<br />
Results: Mech<strong>an</strong>ical allodynia in ipsilateral hindpaw developed 1day after surgery <strong>an</strong>d lasted<br />
until 60 days. The IL-8 receptor inhibitor attenuated <strong>the</strong> mech<strong>an</strong>ical allodynia caused by nucleus<br />
pulposus when it was administered on postoperative day 10 <strong>an</strong>d reduced microglial activation in<br />
<strong>the</strong> spinal dorsal horn.<br />
Conclusion: These data suggest that mech<strong>an</strong>ical allodynia was induced by nucleus pulposus in a<br />
rat lumbar disc herniation model. The IL-8 receptor inhibitor attenuated <strong>the</strong> mech<strong>an</strong>ical allodynia<br />
caused by nucleus pulposus <strong>an</strong>d reduced microglial activation. There<strong>for</strong>e, IL-8 might be a<br />
potential <strong>the</strong>rapeutic target in chronic radicular neuropathic pain due to disc herniation.<br />
Acknowledgements: This work was supported by <strong>the</strong> Korea Research Foundation Gr<strong>an</strong>t funded<br />
by <strong>the</strong> Kore<strong>an</strong> Government (KRF- 2008-532-E00019).<br />
Disclosures: S. Kim, None; J. Seo, None; S. Hw<strong>an</strong>g, None; H. Park, None; S. Park, None; Y.<br />
Cho, None; S. J<strong>an</strong>g, None; S. Ahn, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.2/CC86<br />
Topic: D.08.p. Inflammatory pain: Inflammatory mediators<br />
Support: KRF- 2008-532-E00019<br />
Title: Long-term attenuation of hindpaw pain response after pulsed radiofrequency application<br />
on dorsal root g<strong>an</strong>glion in lumbar disc herniated rat model<br />
Authors: *H.-W. PARK 1 , S.-J. KIM 2 , J.-M. SEO 2 , S.-J. HWANG 3 , Y.-W. CHO 4 , S.-J. DO 5 , S.-<br />
H. JANG 5 , S.-H. AHN 5 ;<br />
1 Daegu Catholic Univ. Med. Ctr., Daegu, Republic of Korea; 2 Inst. of medical science, Deagu,<br />
Republic of Korea; 3 Dept. of Anat. <strong>an</strong>d Cell Biol., Col. of Medicine, H<strong>an</strong>y<strong>an</strong>g Univ., Seoul,<br />
Republic of Korea; 4 Col. of Medicine, Yeungnam Univ., Department of Rehabilitation Medicine,<br />
Republic of Korea; 5 Dept. of Rehabil. Med., Col. of Medicine, Yeungnam Univ., Deagu,<br />
Republic of Korea<br />
Abstract: Objetive: Pulsed radiofrequency procedure has been used <strong>for</strong> treatment chronic<br />
neuropathic pain conditions without neuronal damage in clinical practice. The purpose of this<br />
study is to investigate <strong>the</strong> long-term effect of pulsed radiofrequency (PRF) on pain-related<br />
behavior in a rat model of lumbar disc herniation.<br />
Method: Lumbar disc herniated rat model was made by impl<strong>an</strong>tation of autologous nucleus<br />
pulposus on <strong>the</strong> left L5 spinal nerve root just proximal to <strong>the</strong> dorsal root g<strong>an</strong>glion (DRG). Rats<br />
were ascertained <strong>the</strong> development of mech<strong>an</strong>ical allodynia on both hindpaw until 10 days<br />
postoperatively (PO) using von Frey hair. PRF was applied on left DRG <strong>for</strong> 120 seconds with<br />
maximum temperature 42°C after 10 days PO. Mech<strong>an</strong>ical allodynia was tested on 8 hour, 1, 3,<br />
10, 20 <strong>an</strong>d 40 days PO after PRF.<br />
Results: After once PRF procedure, withdrawal threhold <strong>for</strong> mech<strong>an</strong>ical stimuli on both<br />
hindpaws was increased markedly. Immediate reduction of mech<strong>an</strong>ical allodynia was shown<br />
<strong>from</strong> 8 hours after PRF procedure <strong>an</strong>d maintained throughout <strong>the</strong> experimental period.<br />
Conclusion: Our results demonstrate that mech<strong>an</strong>ical allodynia indued by lumbar disc herniation<br />
was reversed by PRF on ipsilateral DRG immediately <strong>an</strong>d maintained <strong>for</strong> longer time. The PRF<br />
might be a simple <strong>an</strong>d effective procedure to treat radicular pain in lumbar disc herniation.<br />
Disclosures: H. Park, None; S. Kim, None; J. Seo, None; S. Hw<strong>an</strong>g, None; Y. Cho, None; S.<br />
Do, None; S. J<strong>an</strong>g, None; S. Ahn, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.3/DD1<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: Korea Research Foundation, E00460<br />
Title: Expression of corticotropin releasing factor <strong>an</strong>d its receptors in <strong>the</strong> spinal cord <strong>an</strong>d dorsal<br />
root g<strong>an</strong>glion in a rat model of neuropathic pain<br />
Authors: E. KIM 1 , D. RYU 1 , J. HAN 2 , *S. HWANG 1 ;<br />
1 Dept Anat. <strong>an</strong>d Cell Biol., H<strong>an</strong>y<strong>an</strong>g Univ., Seoul, Republic of Korea; 2 Cell <strong>an</strong>d Developmental<br />
Biol., Univ. of North Carolina, Chapel Hill, NC<br />
Abstract: Coritcotropin releasing factor (CRF) is a hormone that coordinates <strong>the</strong> endocrine<br />
response to stress through <strong>the</strong> hypothalamic-pituitary-adrenal (HPA) axis. CRF has been known<br />
to have <strong>an</strong>tinociceptive effect mediated by its receptors, CRF-R1 <strong>an</strong>d CRF-R2 at various levels<br />
of <strong>the</strong> neuraxis. However, little is known about <strong>the</strong> ch<strong>an</strong>ges in expression of CRF <strong>an</strong>d its<br />
receptors in <strong>the</strong> spinal cord <strong>an</strong>d dorsal root g<strong>an</strong>glion (DRG) in neuropathic pain states. We here<br />
used immunohistochemistry to study <strong>the</strong> expression of CRF, CRF-R1, <strong>an</strong>d CRF-R2 in <strong>the</strong> spinal<br />
dorsal horn <strong>an</strong>d DRG in a rat model of neuropathic pain, induced by spinal nerve injury (SNI). In<br />
naive rats, CRF immunoreactivity was observed in small-sized DRG neurons <strong>an</strong>d in puncta in<br />
<strong>the</strong> superficial laminae of <strong>the</strong> dorsal horn. After SNI, <strong>the</strong> number CRF-positive neurons in DRG<br />
increased tr<strong>an</strong>siently, <strong>an</strong>d decreased by day 14, toge<strong>the</strong>r with <strong>the</strong> CRF-positive puncta in <strong>the</strong><br />
dorsal horn. In naïve rats, CRF-R1 was expressed in spinal microglia, revealed by simult<strong>an</strong>eous<br />
staining with <strong>the</strong> microglia/macrophage marker Iba1, <strong>an</strong>d <strong>the</strong> number of <strong>the</strong>se cells increased in<br />
<strong>the</strong> ipsilateral dorsal horn at 14 days after SNI. Immunoreaction <strong>for</strong> CRF-R2 was found in<br />
macrophages in <strong>the</strong> ipsilateral DRG at 14 days after SNI. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest<br />
that microglia <strong>an</strong>d macrophages in <strong>the</strong> spinal cord <strong>an</strong>d DRG may be involved in <strong>the</strong> mediation of<br />
stress response to neuropathic pain.<br />
Disclosures: E. Kim, None; D. Ryu, None; J. H<strong>an</strong>, None; S. Hw<strong>an</strong>g, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.4/DD2
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NIH Gr<strong>an</strong>t DA024044<br />
NIH Gr<strong>an</strong>t DE017782<br />
NIH Gr<strong>an</strong>t DA017670<br />
Title: Constriction of <strong>the</strong> facial nerve leads to increased sensitivity to somatosensory <strong>an</strong>d<br />
auditory stimuli: Characterization of <strong>an</strong> inflammatory <strong>an</strong>d glial component<br />
Authors: *S. S. LEWIS, A. M. BENISON, S. CAMPEAU, D. S. BARTH, S. F. MAIER, L. R.<br />
WATKINS;<br />
Dept Psychol, Univ. Colorado, Boulder, CO<br />
Abstract: Neuropathic orofacial pain is a complex <strong>an</strong>d difficult to treat syndrome. M<strong>an</strong>y patients<br />
report not only increased nociception, but o<strong>the</strong>r sensory, auditory, <strong>an</strong>d general health ch<strong>an</strong>ges.<br />
We present a new model of neuropathic facial pain following facial nerve constriction, in order<br />
to study mech<strong>an</strong>isms of central sensitization across both somatosensory systems. Based on <strong>the</strong><br />
clinical condition Bell’s Palsy, our model produces nociceptive <strong>an</strong>d auditory sensitivity<br />
following constriction of <strong>the</strong> facial nerve.<br />
METHODS: A skin incision was made to expose <strong>the</strong> buccal br<strong>an</strong>ch of <strong>the</strong> facial nerve. This<br />
br<strong>an</strong>ch was constricted using 3 chromic gut ligatures loosely tied around <strong>the</strong> nerve fiber. Tactile<br />
allodynia was assessed 3, 7, 10, 14, 21, 28, 35 <strong>an</strong>d 42 days post-surgery. IL-1 receptor <strong>an</strong>tagonist<br />
(IL-1ra) was administered intracisternally to characterize <strong>the</strong> role of immune activation in pain<br />
behaviors. Pre-pulse inhibition, a measure of auditory sensitivity, was measured 35 days after<br />
surgery <strong>an</strong>d investigation of additional time points is ongoing. Immunohistochemistry will be<br />
used to define <strong>the</strong> <strong>an</strong>atomical distribution <strong>an</strong>d timecourse of microglial <strong>an</strong>d/or astrocytic<br />
activation with this model. Retrograde tracing studies are underway to determine <strong>the</strong> central<br />
nervous system origin of <strong>the</strong> nerve fibers at <strong>the</strong> site of constriction.<br />
RESULTS: Signific<strong>an</strong>t allodynia was present <strong>from</strong> 10-35 days post-constriction injury. Central<br />
(intracisternal) IL-1ra reversed established allodynia, implicating proinflammatory cytokines in<br />
this pain enh<strong>an</strong>cement. Enh<strong>an</strong>ced pre-pulse inhibition was seen 35 days after nerve injury,<br />
suggesting increased sensitivity to sound, <strong>an</strong>d fur<strong>the</strong>r characterization of this measure is ongoing.<br />
Immunohistochemical <strong>an</strong>d <strong>an</strong>atomical <strong>an</strong>alyses are ongoing.<br />
CONCLUSION: We present evidence that constriction of a motor nerve c<strong>an</strong> lead to enh<strong>an</strong>ced<br />
sensitivity to both somatosensory <strong>an</strong>d auditory stimuli. This is consistent with <strong>the</strong> complex<br />
symptomology of Bell’s Palsy <strong>an</strong>d will allow us to better investigate <strong>the</strong> mech<strong>an</strong>isms of chronic<br />
neuropathic orofacial pain, cross-modal hypersensitivity, <strong>an</strong>d <strong>the</strong> interplay between neuropathic<br />
orofacial pain <strong>an</strong>d o<strong>the</strong>r facial pain disorders.<br />
Disclosures: S.S. Lewis, None; A.M. Benison, None; S. Campeau, None; D.S. Barth,<br />
None; S.F. Maier, None; L.R. Watkins, None.
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.5/DD3<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: Wellcome trust<br />
Title: CX3CR1 receptor is required <strong>for</strong> full development of nociceptive behaviour following<br />
peripheral nerve injury<br />
Authors: *A. K. CLARK, R. WODARSKI, M. MALCANGIO;<br />
Wolfson CARD, Kings Col. London, London, United Kingdom<br />
Abstract: Underst<strong>an</strong>ding of <strong>the</strong> sequence <strong>an</strong>d nature of <strong>the</strong> events that govern neuron-microgliaastrocyte<br />
communication is vital <strong>for</strong> <strong>the</strong> discovery of new mech<strong>an</strong>isms <strong>an</strong>d targets <strong>for</strong> chronic<br />
pain treatment. Our data support <strong>the</strong> suggestion that among <strong>the</strong> m<strong>an</strong>y signaling c<strong>an</strong>didates in <strong>the</strong><br />
spinal cord <strong>the</strong> neuronal chemokine Fractalkine <strong>an</strong>d its cognate microglial receptor CX3CR1 are<br />
critical <strong>for</strong> neuron-microglial signaling during chronic pain states. There<strong>for</strong>e, we evaluated <strong>the</strong><br />
development <strong>an</strong>d mainten<strong>an</strong>ce of neuropathic pain behavior following partial ligation of <strong>the</strong><br />
sciatic nerve in CX3CR1 knockout <strong>an</strong>d wild-type mice. We <strong>the</strong>n examined <strong>the</strong> extent of spinal<br />
microglial activation in <strong>the</strong>se mice.<br />
As expected, wild-type mice developed both mech<strong>an</strong>ical (von Frey hairs) <strong>an</strong>d <strong>the</strong>rmal<br />
(Hargreaves) hypersensitivity <strong>from</strong> day 2 to day 21 post-injury, whilst sham <strong>an</strong>imals did not<br />
show signific<strong>an</strong>t ch<strong>an</strong>ges in nociceptive thresholds. In contrast, CX3CR1 knock-out mice did not<br />
develop mech<strong>an</strong>ical allodynia up to day 21 post-injury. CX3CR1 knock-out mice exhibited<br />
<strong>the</strong>rmal hyperalgesia <strong>from</strong> day 7-11, although this hyperalgesia was less severe th<strong>an</strong> in wild-type<br />
mice, <strong>an</strong>d had begun to recover by day 14 <strong>an</strong>d fully recovered by day 21 after injury.<br />
At both 14 <strong>an</strong>d 21 days following injury <strong>the</strong> number of Iba-1 positive microglial cells in <strong>the</strong><br />
ipsilateral dorsal horn of <strong>the</strong> spinal cord of CX3CR1 knock-out mice was signific<strong>an</strong>tly lower<br />
th<strong>an</strong> in wild-type mice, suggesting <strong>the</strong> occurrence of less microglial activation in mice lacking<br />
CX3CR1 receptors.<br />
These data indicate that spinal CX3CR1 receptors are critical <strong>for</strong> both <strong>the</strong> development <strong>an</strong>d<br />
mainten<strong>an</strong>ce of neuropathic mech<strong>an</strong>ical allodynia, as well as microglial activation. In addition,<br />
absence of CX3CR1 leads to a delayed development of <strong>the</strong>rmal hyperalgesia, as well as <strong>an</strong><br />
accelerated recovery following injury. We suggest that specific CX3CR1 receptor <strong>an</strong>tagonists,<br />
possibly CNS penetr<strong>an</strong>t, may be beneficial <strong>for</strong> <strong>the</strong> treatment of persistent pain.<br />
Disclosures: A.K. Clark, None; R. Wodarski, None; M. Malc<strong>an</strong>gio, None.
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.6/DD4<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: Wellcome Trust<br />
King's College London<br />
Title: Ca<strong>the</strong>psin S release by microglial cells is ATP/P2X7 receptor dependent<br />
Authors: *R. WODARSKI 1 , F. GUIDA 2 , M. MALCANGIO 1 ;<br />
1 2<br />
Wolfson CARD, King's Col. London, London, United Kingdom; Dept. of Exptl. Med., Second<br />
Univ. of Naples, Naples, Italy<br />
Abstract: Following peripheral nerve injury, ATP released <strong>from</strong> central terminals of injured<br />
afferent fibres c<strong>an</strong> activate microglial cells via P2X4 <strong>an</strong>d/or P2X7 receptors <strong>the</strong>reby contributing<br />
to <strong>the</strong> development of neuropathic pain (Inoue et al., 2007). Here we tested <strong>the</strong> hypo<strong>the</strong>sis that<br />
ATP induces <strong>the</strong> release <strong>from</strong> microglia of <strong>the</strong> lysosomal cysteine protease Ca<strong>the</strong>psin S (CatS),<br />
which we have shown to exert a pro-nociceptive effect via <strong>the</strong> shedding of neuronal fractalkine<br />
in <strong>the</strong> dorsal horn of <strong>the</strong> spinal cord (Clark et al., 2007).<br />
Mixed glial cultures were prepared <strong>from</strong> spinal cords of Wistar rat pups (P3-7) <strong>an</strong>d microglial<br />
cells were harvested by shaking after 12-14 days in culture. Cells were plated <strong>for</strong> 48 hrs,<br />
stimulated <strong>an</strong>d extracellular media as well as cell lysates were collected to measure release of<br />
enzymatically active CatS. Enzymatic activity was assessed by measuring <strong>the</strong> fluorescence<br />
produced following sample incubation with <strong>the</strong> substrate Z-Val-Val-Arg-MCA.<br />
Nei<strong>the</strong>r incubation with lipopolysaccharide (LPS; 1 µg/ml) <strong>for</strong> 3 hrs or ATP (1 mM) <strong>for</strong> 20 min<br />
alone induced release of enzymatically active CatS in extracellular media as compared to control.<br />
However, 3hrs after LPS priming, incubation with ATP (1 mM) resulted in <strong>an</strong> increase of CatS<br />
enzymatic activity by 80±20 %, which could be prevented by <strong>the</strong> irreversible CatS inhibitor<br />
morpholinurea-leucine-homophenylal<strong>an</strong>ine-vinyl sulfone-phenyl (LHVS; 10 µM). LHVS also<br />
inhibited CatS activity in cellular extracts confirming <strong>the</strong> lysosomotropism of this inhibitor.<br />
Fur<strong>the</strong>rmore, LPS+ATP induced release of enzymatically active CatS was prevented by <strong>the</strong><br />
P2X7 receptor <strong>an</strong>tagonist A438079 (10 µM).<br />
These data indicate that in <strong>the</strong> dorsal horn of <strong>the</strong> spinal cord, <strong>the</strong> release of active CatS, which<br />
plays a critical role in neuropathic pain states, is likely to be triggered by <strong>the</strong> neuro/glial<br />
tr<strong>an</strong>smitter ATP via P2X7 receptor activation.
Clark AK, Yip PK, Grist J, Gentry C, St<strong>an</strong>il<strong>an</strong>d AA, March<strong>an</strong>d F, Dehvari M, Wo<strong>the</strong>rspoon G,<br />
Winter J, Ullah J, Bev<strong>an</strong> S, & Malc<strong>an</strong>gio M (2007). Inhibition of spinal microglial ca<strong>the</strong>psin S<br />
<strong>for</strong> <strong>the</strong> reversal of neuropathic pain. Proc Natl Acad Sci U S A 104, 10655-10660.<br />
Inoue K, Tsuda M, Tozaki-Saitoh H. (2007). Modification of neuropathic pain sensation through<br />
microglial ATP receptors. Purinergic Signal. 3(4), 311-316<br />
Disclosures: R. Wodarski, None; F. Guida, None; M. Malc<strong>an</strong>gio, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.7/DD5<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NIH Gr<strong>an</strong>t K01 DA017236<br />
NIH Gr<strong>an</strong>t T32 DA07234<br />
Title: The neuropeptide precursor VGF mediates p38 activation in microglia<br />
Authors: M. RIEDL 1 , P. BRAUN 1 , K. KITTO 1 , C. FAIRBANKS 1 , *L. VULCHANOVA 2 ;<br />
1 Univ. of Minnesota, Minneapolis, MN; 2 Univ. of Minnesota, Saint Paul, MN<br />
Abstract: VGF (non-acronymic) is a neuropeptide precursor protein related to <strong>the</strong> chromogr<strong>an</strong>in<br />
family. Within <strong>the</strong> central nervous system, VGF has been implicated in neuroplasticity<br />
associated with depression, learning <strong>an</strong>d memory, <strong>an</strong>d chronic pain. The 617 amino acid<br />
sequence of VGF contains over a dozen potential proteolytic sites, suggesting that a number of<br />
signaling peptides may be generated <strong>from</strong> <strong>the</strong> precursor. The precise identities of bioactive VGFderived<br />
peptides <strong>an</strong>d <strong>the</strong>ir mech<strong>an</strong>isms of action, including putative receptors, are unknown. We<br />
have demonstrated that VGF is rapidly upregulated in models of nerve injury <strong>an</strong>d inflammation,<br />
suggesting that it may participate in processes underlying <strong>the</strong> development of persistent pain.<br />
The goal of <strong>the</strong>se experiments was to determine whe<strong>the</strong>r peptides derived <strong>from</strong> proteolytic<br />
processing of VGF (1) participate in nociceptive signaling, <strong>an</strong>d (2) are released <strong>from</strong> sensory<br />
neurons in response to depolarization. We examined <strong>the</strong> spinal effects of AQEE-30 <strong>an</strong>d LQEQ-<br />
19, potential proteolytic products previously shown to have functional effects. In <strong>the</strong> warm water<br />
tail immersion assay, each peptide evoked dose-dependent <strong>the</strong>rmal hyperalgesia that persisted <strong>for</strong><br />
up to 1.5 h <strong>an</strong>d required activation of <strong>the</strong> mitogen-activated protein kinase (MAPK) p38. In<br />
addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intra<strong>the</strong>cally
<strong>an</strong>d in <strong>the</strong> BV-2 microglial cell line when applied in vitro. The release of VGF peptides was<br />
examined in primary cultures of dorsal root g<strong>an</strong>glion neurons. Increases in <strong>the</strong> amount of VGF<br />
peptides in <strong>the</strong> culture supernat<strong>an</strong>t following stimulation with high potassium were measured<br />
using a dot blot assay. In addition, <strong>the</strong> identities of released VGF peptides were characterized by<br />
mass spectrometry. Taken toge<strong>the</strong>r, <strong>the</strong>se observations suggest that VGF peptides released <strong>from</strong><br />
sensory neurons may participate in <strong>the</strong> early response to tissue injury though activation of spinal<br />
microglia.<br />
Disclosures: M. Riedl, None; P. Braun, None; K. Kitto, None; C. Fairb<strong>an</strong>ks, None; L.<br />
Vulch<strong>an</strong>ova, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.8/DD6<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Title: Effects of spinal nerve ligation on cut<strong>an</strong>eous blood flow in rats<br />
Authors: *C. E. HAGAINS 1 , P. J. HUNTINGTON 1 , A. K. SENAPATI 1 , H. D. WILSON 1 , P.<br />
N. FUCHS 1 , H. LIU 2 , Y. B. PENG 1 ;<br />
1 Dept Psychology, 2 Dept Bioengineering, Univ. Texas-Arlington, Arlington, TX<br />
Abstract: Cut<strong>an</strong>eous blood flow (CBF) c<strong>an</strong> be affected by primary afferent fibers (PAFs) <strong>an</strong>d<br />
sympa<strong>the</strong>tic nerves. Spinal nerve ligation (SNL) alters m<strong>an</strong>y aspects of peripheral <strong>an</strong>d spinal<br />
physiology. It causes a neuroma proximal to ligation that produces ectopic firing to increase <strong>the</strong><br />
nociceptive signals into <strong>the</strong> spinal cord <strong>an</strong>d trigger dorsal root reflexes (DRR), which in turn<br />
increases peripheral CBF. The goal of this study was to determine <strong>the</strong> affects of L5 SNL on<br />
CBF. It was hypo<strong>the</strong>sized that SNL would increase CBF in both hindpaws because of increased<br />
DRRs, which lead to <strong>the</strong> release of CGRP to induce vasodilatation. Twenty-one male Sprague-<br />
Dawley rats were used in this experiment. Fifteen rats had a tight ligation in <strong>the</strong> left L5 spinal<br />
nerve. Seven days following nerve ligation, Laser Doppler Imaging was used to measure CBF in<br />
<strong>the</strong> rats’ hindpaws be<strong>for</strong>e <strong>an</strong>d after laminectomy. Then, SNL rats were split into 2 groups <strong>an</strong>d<br />
CBF measurement was conducted ei<strong>the</strong>r ipsilaterally or contralaterally <strong>for</strong> baseline, responses to<br />
GABA <strong>an</strong>d electrical stimulation applied to <strong>the</strong> left L5 dorsal root entry zone. Signific<strong>an</strong>t<br />
ch<strong>an</strong>ges were tested by using ANOVAs followed by Fisher LSD post-hoc tests. For GABA <strong>an</strong>d<br />
electrical stimulation tests, ch<strong>an</strong>ges were normalized into percentage to baseline reading. Our<br />
results showed that (1) <strong>the</strong> contralateral (non-ligated) paws had signific<strong>an</strong>tly higher CBF across
all 10 baseline images be<strong>for</strong>e laminectomy (p < 0.001); (2) after laminectomy, CBF was greater<br />
<strong>for</strong> both paws of SNL rats compared to non-SNL rats, <strong>an</strong>d <strong>the</strong> contralateral increased<br />
signific<strong>an</strong>tly more th<strong>an</strong> <strong>the</strong> ipsilateral in SNL rats (p < 0.001); (3) application of GABA<br />
signific<strong>an</strong>tly increased CBF in <strong>the</strong> contralateral paw compared to baseline (p < 0.05), but <strong>the</strong>re<br />
were no ch<strong>an</strong>ges in <strong>the</strong> ligated paw; (4) electrical stimulation elicited a signific<strong>an</strong>t increase of<br />
CBF compared to baseline (p < 0.05) in <strong>the</strong> ligated paw, whereas a signific<strong>an</strong>t increase was<br />
observed only in <strong>the</strong> image following electrical stimulation (p < 0.05) in <strong>the</strong> contralateral paw.<br />
These results support <strong>the</strong> hypo<strong>the</strong>sis that L5 SNL increases hindpaw CBF. This is probably due<br />
to a barrage of ectopic firing of <strong>the</strong> ligated nerve to <strong>the</strong> spinal cord, which in turn leads to DRRs<br />
in <strong>the</strong> spared ipsilateral nerves <strong>an</strong>d contralateral PAFs. It is concluded that spinal nerve ligation<br />
will cause long term bilateral increase of blood flow, probably due to disturbed bal<strong>an</strong>ce of DRRinduced<br />
vasodilatation <strong>an</strong>d sympa<strong>the</strong>tic vasoconstriction.<br />
Disclosures: C.E. Hagains, None; P.J. Huntington, None; A.K. Senapati, None; H.D.<br />
Wilson, None; P.N. Fuchs, None; H. Liu, None; Y.B. Peng, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.9/DD7<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: CIHR MOP-79471<br />
C<strong>an</strong>adi<strong>an</strong> Diabetes Association<br />
Title: Key role of microglial kinin B1 receptor in diabetic polyneuropathy<br />
Authors: S. TALBOT, E. CHAHMI, J. P. DIAS, *R. COUTURE;<br />
Univ. Montreal Med. Sch., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines <strong>an</strong>d<br />
hyperglycemia-induced oxidative stress. Recently, we found <strong>the</strong> pro-nociceptive B1R localized<br />
on sensory C fibres, astrocytes <strong>an</strong>d microglia in streptozotocin (STZ)-diabetic rat. The goal of<br />
this study was to define <strong>the</strong> role of microglial kinin B1R in diabetic pain polyneuropathy.<br />
Experiments were conducted in Sprague-Dawley rats made diabetic with STZ (65 mg/kg, ip)<br />
which received 4 days later, a single injection of vehicle or fluorocitrate (1 nmol, intra<strong>the</strong>cal), a<br />
specific inhibitor of microglial cells. The outcome of microglial inhibition or B1R blockade with
SSR240612 (10 mg/kg, gavage) was determined on glycemia, tactile <strong>an</strong>d cold allodynia, B1Rmediated<br />
<strong>the</strong>rmal hyperalgesia <strong>an</strong>d mRNA levels (qRT-PCR) of B1R, IL-1β, TNF-α <strong>an</strong>d TRPV1<br />
in <strong>the</strong> thoracic spinal cord. Ch<strong>an</strong>ges in <strong>the</strong> density of B1R binding sites <strong>an</strong>d microglial<br />
distribution were assessed by qu<strong>an</strong>titative autoradiography <strong>an</strong>d confocal microscopy. Results<br />
showed a marked <strong>an</strong>d reversible inhibition of STZ-induced tactile <strong>an</strong>d cold allodynia by<br />
SSR240612 <strong>an</strong>d fluorocitrate <strong>from</strong> 1 to 6h post-treatment. Fluorocitrate also abolished B1Rmediated<br />
<strong>the</strong>rmal hyperalgesia without affecting hyperglycemia in STZ-treated rats. The<br />
increased mRNA levels of B1R, IL-1β <strong>an</strong>d TNF-α in <strong>the</strong> spinal cord of STZ-diabetic rats were<br />
completely reversed by fluorocitrate which also reduced by 25 % B1R specific binding sites<br />
associated to <strong>the</strong> elimination of B1R on morphologically altered microglia. Results suggest a key<br />
role <strong>for</strong> microglia <strong>an</strong>d pro-inflammatory cytokines in <strong>the</strong> induction <strong>an</strong>d overexpression of B1R in<br />
<strong>the</strong> spinal cord of STZ-diabetic rats that contributes to pain polyneuropathy.<br />
Disclosures: S. Talbot, None; E. Chahmi, None; J.P. Dias, None; R. Couture, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.10/DD8<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NIH DK 044935<br />
NIH NS 038850<br />
DEPT of VETERANS AFFAIRS<br />
Title: Subst<strong>an</strong>ce P increases microglial mTNFα expression<br />
Authors: *Z. ZHOU 1,2 , X. PENG 1,2 , J. HAGHSHENAS 1,2 , R. INSOLERA 1,2 , M. MATA 1,2 , D.<br />
FINK 1,2 ;<br />
1 Dept Neurol, Univ. Michig<strong>an</strong>, Ann Arbor, MI; 2 VA Ann Arbor Healthcare Syst., Ann Arbor,<br />
MI<br />
Abstract: Spinal microglial activation is a feature of chronic neuropathic pain, <strong>an</strong>d tumor<br />
necrosis factor α (TNFα) plays a key role in that process. TNFα is expressed as a 26 kD type II<br />
tr<strong>an</strong>smembr<strong>an</strong>e protein (mTNFα) that is cleaved by a TNFα convertase to release <strong>the</strong> 17 kD<br />
soluble TNFα (sTNFα). We found a marked increase in mTNFα mRNA <strong>an</strong>d protein in spinal
dorsal horn in models of pain created by spinal cord injury <strong>an</strong>d <strong>for</strong>malin injection, but sTNFα<br />
was not detected. In order to study <strong>the</strong> regulation of microglial TNFα expression we exposed<br />
microglial (HAPI) cells in culuture to subst<strong>an</strong>ce P (SP) or calcitonin gene related peptide<br />
(CGRP) <strong>for</strong> 6 hrs. SP but not CGRP resulted in increased TNFα mRNA <strong>an</strong>d protein, but nei<strong>the</strong>r<br />
SP nor CGRP treatment resulted in release of sTNFα <strong>from</strong> <strong>the</strong> cells into <strong>the</strong> medium. In order to<br />
evaluate <strong>the</strong> potential role of mTNFα in microglial activation we designed a TNFα construct with<br />
a mutation in <strong>the</strong> TACE cleavage site (designated mut<strong>an</strong>t TNFα). Exposure of HAPI cells to <strong>the</strong><br />
mut<strong>an</strong>t TNFα resulted in a signific<strong>an</strong>t increase in TNFα mRNA <strong>an</strong>d protein in <strong>the</strong> cells, a<br />
subst<strong>an</strong>tial increase in monocyte chemotactic protein-1 (MCP-1) release, <strong>an</strong>d <strong>an</strong> increase in <strong>the</strong><br />
microglial marker OX42. Coculture of HAPI cells with COS7 cells tr<strong>an</strong>siently tr<strong>an</strong>sfected to<br />
overexpress mut<strong>an</strong>t TNFα resulted in increased expression of OX42 <strong>an</strong>d release of MCP-1 by <strong>the</strong><br />
HAPI cells. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that SP induces expression of mTNFα in<br />
microglial cells, <strong>an</strong>d that through cell-cell interactions full-length mTNFα may serve to fur<strong>the</strong>r<br />
activate microglial cells creating a “feed-<strong>for</strong>ward” loop of neuroimmune activation. This process<br />
may be import<strong>an</strong>t in <strong>the</strong> tr<strong>an</strong>sition <strong>from</strong> acute to chronic pain.<br />
Disclosures: Z. Zhou, None; X. Peng, None; J. Haghshenas, None; R. Insolera, None; M.<br />
Mata, None; D. Fink, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.11/DD9<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Title: Epigenetic role of <strong>the</strong> spinal IL-6-mediated signaling network in <strong>the</strong> development of a<br />
neuropathic pain-like state in mice<br />
Authors: *S. IMAI, M. NARITA, T. SHIMIZU, M. NARITA, K. NIIKURA, M. FURUYA, N.<br />
KUZUMAKI, T. SUZUKI;<br />
Dept. of Toxicology, Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Jap<strong>an</strong><br />
Abstract: The aim of <strong>the</strong> present study was to assess <strong>the</strong> role of IL-6/C-C chemokine-mediated<br />
signaling network within <strong>the</strong> spinal cord in <strong>the</strong> development of a neuropathic pain-like state<br />
induced by partial sciatic nerve ligation in mice. In <strong>the</strong> present study, we demonstrated that <strong>the</strong><br />
mRNA level of IL-6 on <strong>the</strong> ipsilateral side of <strong>the</strong> mouse DRG was markedly increased at 1 day<br />
<strong>an</strong>d 7 days after sciatic nerve ligation, whereas we did not detect IL-6 mRNA expression on <strong>the</strong><br />
ipsilateral side of <strong>the</strong> mouse spinal cord under <strong>the</strong> same conditions. Fur<strong>the</strong>rmore, we per<strong>for</strong>med
DNA microarray <strong>an</strong>alysis using <strong>the</strong> spinal cord of nerve-ligated mice at 7 days after surgery. As<br />
a result, <strong>the</strong> level of C-C chemokine mRNAs at 7 days after nerve ligation was dramatically<br />
increased among <strong>the</strong> total 39,300 genes that were detectable in <strong>the</strong> spinal cord of sham-operated<br />
<strong>an</strong>d nerve-ligated mice. The RT-PCR assay demonstrated that <strong>the</strong> expression of C-C chemokine<br />
mRNA on <strong>the</strong> ipsilateral side of <strong>the</strong> spinal cord obtained <strong>from</strong> nerve-ligated mice was<br />
signific<strong>an</strong>tly increased at 1, 3, 5 <strong>an</strong>d 7 days after surgery. The key finding of <strong>the</strong> present study<br />
was that <strong>the</strong>se robust increases in C-C chemokine mRNA expression observed in <strong>the</strong> spinal cord<br />
of nerve-ligated wild-type mice were signific<strong>an</strong>tly reduced in <strong>the</strong> spinal cord of IL-6 knockout<br />
mice at 7 days after sciatic nerve ligation. These findings suggest that IL-6 is a potent inducer of<br />
increased expression of C-C chemokines in <strong>the</strong> spinal cord of mice with nerve ligation. However,<br />
it should be noted that IL-6 signaling has no specific target on <strong>the</strong> DNA promoter region of C-C<br />
chemokines genes. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, C-C chemokine-immunoreactivity was highly co-localized<br />
with <strong>the</strong> microglial marker OX42, but not <strong>the</strong> astrocyte marker GFAP or several neuronal<br />
markers, in <strong>the</strong> spinal cord. Based on <strong>the</strong>se results, we fur<strong>the</strong>r investigated whe<strong>the</strong>r C-C<br />
chemokine-mediated signaling pathways in <strong>the</strong> spinal cord of nerve-ligated mice could directly<br />
contribute to <strong>the</strong> development of <strong>the</strong> neuropathic pain induced by nerve ligation. A single i.t.<br />
injection of a C-C chemokine caused a persistent painful state in normal mice.<br />
Taken toge<strong>the</strong>r, we proposed here that neuropathic pain increases <strong>the</strong> mRNA expression of IL-6<br />
in <strong>the</strong> DRG <strong>an</strong>d, in turn, may regulate <strong>the</strong> chromatin remodeling at promoter regions of C-C<br />
chemokine genes. These phenomena may be responsible <strong>for</strong> spinal plasticity with enh<strong>an</strong>ced <strong>the</strong><br />
neuron-microglia communication, which is associated with <strong>the</strong> development of a long-lasting<br />
neuropathic pain induced by nerve ligation in mice.<br />
Disclosures: S. Imai, None; M. Narita, None; T. Shimizu, None; M. Narita, None; K.<br />
Niikura, None; M. Furuya, None; N. Kuzumaki, None; T. Suzuki, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.12/DD10<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NIH NINDS RO1NS049136<br />
NIH/NINDS R01NS043095<br />
Title: Expression of <strong>the</strong> chemokine receptor, CCR2, in primary sensory neurons of <strong>the</strong> adult<br />
mouse following peripheral nerve tr<strong>an</strong>section <strong>an</strong>d crush injury
Authors: P. FELDMAN 1 , H. JUNG 2 , M. S. RIPSCH 3 , R. J. MILLER 2 , *F. A. WHITE 4,5 ;<br />
1 Neurosci. Grad. Program, Loyola University, Chicago, Maywood, IL; 2 Mol. Pharmacol.,<br />
Northwestern Univ., Chicago, IL; 3 Cell Biol, Neurobio & Anat, Loyola Univ., Chicago, IL;<br />
4 Loyola Univ, Chicago, Maywood, IL; 5 Anes<strong>the</strong>siol., Loyola Univ. Med. Ctr., Maywood, IL<br />
Abstract: Numerous studies in <strong>the</strong> rat indicate that monocyte chemoattract<strong>an</strong>t protein-1 (MCP1<br />
also known as CCL2) signaling through CCR2 in DRG neurons derived <strong>from</strong> peripheral nerve<br />
injured rodents is excitatory <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e pro-nociceptive. MCP1 may act at multiple sites <strong>an</strong>d<br />
<strong>the</strong> precise nature of its involvement may differ in different types of pain. To better underst<strong>an</strong>d<br />
<strong>the</strong> location of MCP1 action within <strong>the</strong> nervous system following injury, we utilized bitr<strong>an</strong>sgenic<br />
reporter mice in which CCR2 <strong>an</strong>d MCP-1 were labeled by <strong>the</strong> fluorescent proteins EGFP <strong>an</strong>d<br />
mRFP1, respectively. Nei<strong>the</strong>r MCP-1 nor CCR2 was detected in naïve spinal cord, DRG, or<br />
peripheral nerves. However, following peripheral (sciatic) nerve injury, intense CCR2-EGFP<br />
was observed in lumbar DRG neurons <strong>an</strong>d in tr<strong>an</strong>sected/crushed peripheral nerves. A timecourse<br />
study comparing expression following sciatic nerve crush <strong>an</strong>d tr<strong>an</strong>section revealed<br />
maximum CCR2 levels at day 14 but not day 7 in both models. Cell-size <strong>an</strong>d double-labeling<br />
studies revealed that CCR2 was expressed by DRG cells of all sizes <strong>an</strong>d colocalized with some<br />
classical markers of DRG subpopulations. MCP1-mRFP colocalized to some degree with CCR2<br />
positive sensory neurons. This study supports <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> site of MCP1/CCR2 action,<br />
at least in <strong>the</strong> models employed, is located in <strong>the</strong> peripheral nervous system. In summary, <strong>the</strong><br />
data reported here support <strong>the</strong> conclusion that CCR2 activation in <strong>the</strong> peripheral nervous system<br />
c<strong>an</strong> play a critical role in pain hypersensitivity in states of neuropathic pain supporting <strong>the</strong><br />
possibility that <strong>an</strong>tagonism of MCP1-CCR2 signaling may be a novel <strong>the</strong>rapeutic intervention <strong>for</strong><br />
<strong>the</strong> treatment of chronic pain syndromes.<br />
Disclosures: P. Feldm<strong>an</strong>, None; H. Jung, None; M.S. Ripsch, None; R.J. Miller, None; F.A.<br />
White, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.13/DD11<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: CIHR<br />
NIH
Title: Hum<strong>an</strong> immunodeficiency virus-1 Vpr expression in <strong>the</strong> peripheral nervous system:<br />
Evidence <strong>for</strong> neuronal injury <strong>an</strong>d neuropathic pain<br />
Authors: *S. ACHARJEE 1 , Y. ZHU 2 , F. NOORBAKHSH 2 , P. L. STEMKOWSKI 3 , C. J.<br />
OLECHOWSKI 3 , E. A. COHEN 8 , B. J. KERR 4 , K. BALLANYI 5 , P. A. SMITH 6 , C. POWER 7 ;<br />
1 Dept. of Med., 2 Med., 3 Neurosci., 4 Anes<strong>the</strong>siol. <strong>an</strong>d Pain Med., 5 Physiol., 6 Pharmacol., 7 Med.<br />
<strong>an</strong>d Psychiatry, Univ. of Alberta, Edmonton, AB, C<strong>an</strong>ada; 8 Inst. de Recherches Cliniques de<br />
Montre´al <strong>an</strong>d Dept. of Microbiology <strong>an</strong>d Immunol., Univ. of Montreal, Edmonton, AB, C<strong>an</strong>ada<br />
Abstract: INTRODUCTION: Peripheral neuropathy has become <strong>the</strong> principal neurological<br />
disorder among HIV/AIDS patients, affecting >30% patients with associated neuropathic pain.<br />
Herein, we investigated <strong>the</strong> effects of a HIV-1 accessory protein, viral protein R (Vpr) on<br />
primary sensory neurons.<br />
METHODS: Dissociated hum<strong>an</strong> <strong>an</strong>d rat dorsal root g<strong>an</strong>glia (DRG) cultures were used <strong>for</strong> this<br />
study. For intracellular calcium ([Ca]) measurements, DRG neurons were preloaded with <strong>the</strong><br />
calcium sensitive fluorescent dye, Fluo-AM <strong>an</strong>d intracellular fluorescence ch<strong>an</strong>ges indicated<br />
ch<strong>an</strong>ges in [Ca]. Neuronal membr<strong>an</strong>e responses were determined by whole-cell patch-clamp<br />
recording. Von Frey filaments were used to assess mech<strong>an</strong>ical allodynia in mice. Additionally,<br />
host immune responses in DRGs of <strong>the</strong>se mice were <strong>an</strong>alyzed by real-time PCR.<br />
RESULTS: HIV-1 infection caused hum<strong>an</strong> DRG neuronal injury, particularly, neurite retraction,<br />
soma atrophy <strong>an</strong>d neuronal loss toge<strong>the</strong>r with vpr tr<strong>an</strong>script expression. Application of Vpr<br />
caused <strong>an</strong> increase in fluorescence intensity in DRG indicating a signific<strong>an</strong>t Ca flux. Vpr also<br />
increased frequency of action potential firing <strong>an</strong>d reduced whole cell currents in both rat <strong>an</strong>d<br />
hum<strong>an</strong> DRG. RAG-1 knockout (RAG-1-/-) mice expressing tr<strong>an</strong>sgenic (Tg) HIV-1 Vpr in<br />
monocytoid cells expressed vpr in <strong>the</strong> DRGs <strong>an</strong>d displayed mech<strong>an</strong>ical allodynia compared with<br />
vpr Tg or RAG-1-/- litter mates. Fur<strong>the</strong>rmore, IL-6 tr<strong>an</strong>script abund<strong>an</strong>ce was signific<strong>an</strong>tly<br />
reduced in <strong>the</strong>se <strong>an</strong>imals’ DRG compared with control littermates.<br />
CONCLUSIONS: Vpr activated DRG neurons by inducing large rises in [Ca], hyperexcitability<br />
<strong>an</strong>d reducing whole cell currents, <strong>the</strong>reby contributing to DRG neuron injury <strong>an</strong>d neuropathic<br />
pain. In <strong>the</strong> <strong>an</strong>imal model of Vpr-induced peripheral neuropathic pain, immunosuppression<br />
appeared to be a requisite feature <strong>for</strong> development of allodynia. The absence of neurotrophic<br />
support mediated by IL-6, owing to its reduced expression in <strong>the</strong> DRGs of Vpr Tg RAG-1-/-<br />
mice might contribute neuronal injury leading to neuropathic pain.<br />
Disclosures: S. Acharjee, None; Y. Zhu, None; F. Noorbakhsh, None; P.L. Stemkowski,<br />
None; C.J. Olechowski, None; E.A. Cohen, None; B.J. Kerr, None; K. Ball<strong>an</strong>yi, None; P.A.<br />
Smith, None; C. Power, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.14/DD12<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: Gr<strong>an</strong>ts-in-Aid <strong>for</strong> Scientific Research<br />
Open Research Center Gr<strong>an</strong>t, Hyogo College of Medicine<br />
Title: Tr<strong>an</strong>sient expression of TNF alpha in <strong>the</strong> rat spinal cord following peripheral nerve injury<br />
Authors: *K. KOBAYASHI 1 , H. YAMANAKA 1 , M. OKUBO 1 , Y. DAI 1,2 , K. NOGUCHI 1 ;<br />
1 Dept. of Anat. <strong>an</strong>d Neurosci., Hyogo Col. of Med., Nishinomiya, Jap<strong>an</strong>; 2 Dept. of Pharmacy,<br />
Sch. of Pharm., Hyogo Univ. of Hlth. Sci., Kobe, Jap<strong>an</strong><br />
Abstract: Nerve injury c<strong>an</strong> lead to neuropathic pain, such as <strong>the</strong>rmal hyperalgesia <strong>an</strong>d<br />
mech<strong>an</strong>ical allodynia. Tumor necrosis factor (TNF)-alpha is one of a pro-inflammatory cytokines<br />
that may play a key role <strong>for</strong> <strong>the</strong> induction of neuropathic pain following nerve injury. However,<br />
<strong>the</strong> precise histological expression of TNF alpha <strong>an</strong>d its receptors mRNA in <strong>the</strong> spinal cord after<br />
nerve injury is not fully understood.<br />
Using spared nerve injury (SNI) model rat, we investigated <strong>the</strong> mRNA expression of TNF alpha<br />
<strong>an</strong>d its receptors in <strong>the</strong> spinal cord. Semi-qu<strong>an</strong>titative RT-PCR revealed <strong>the</strong> temporal induction<br />
of TNF alpha, TNFR1 <strong>an</strong>d TNFR2 mRNA. TNF alpha mRNA showed <strong>the</strong> tr<strong>an</strong>sient increase<br />
<strong>from</strong> 18 hour <strong>an</strong>d peaked at 24 hour following SNI. The increased TNF alpha mRNA returned to<br />
<strong>the</strong> normal level 48h after SNI. In contrast, <strong>the</strong> expression of TNFR1 <strong>an</strong>d TNFR2 mRNAs<br />
showed delayed onset <strong>an</strong>d continuous induction that peaked at day 3 after injury <strong>an</strong>d continued<br />
<strong>for</strong> at least 2 weeks. Next, we examined <strong>the</strong> histological expression pattern of TNF alpha,<br />
TNFR1 <strong>an</strong>d TNFR2 after SNI using in situ hybridization histochemistry. In <strong>the</strong> spinal cord of<br />
naive rats, TNF alpha <strong>an</strong>d TNFR2 showed a low level expression <strong>an</strong>d TNFR1 mRNA signal was<br />
detected in neurons <strong>an</strong>d glial cells in <strong>the</strong> dorsal horn. Twenty-four hours after SNI, TNF alpha<br />
mRNA signals increased in <strong>the</strong> ipsilateral glial cells. TNFR1 <strong>an</strong>d TNF R2 mRNA signals<br />
increased strikingly in ipsilateral glial cells at 3 days after SNI.<br />
Our data suggest that <strong>the</strong> release of TNF alpha <strong>from</strong> dorsal horn glial cells may be a first step in<br />
<strong>the</strong> development of neuropathic pain.<br />
Disclosures: K. Kobayashi, None; H. Yam<strong>an</strong>aka, None; M. Okubo, None; Y. Dai, None; K.<br />
Noguchi, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.15/DD13<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> Young Scientists<br />
Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research<br />
Title: Interleukin-1β converting enzyme (ICE) upregulation in spinal glial cells contributes to<br />
tactile allodynia after nerve injury<br />
Authors: *K. MIYOSHI, K. OBATA, K. NOGUCHI;<br />
Dept. Anat. & Neurosci., Hyogo Col. Med., Nishinomiya, Jap<strong>an</strong><br />
Abstract: Peripheral nerve injury leads to <strong>the</strong> activation of glia in <strong>the</strong> spinal dorsal horn. This<br />
spinal glial activation triggers <strong>the</strong> production <strong>an</strong>d secretion of pro-inflammatory cytokines, such<br />
as interleukin-1β (IL-1β), IL-6, TNF-α, <strong>an</strong>d neurotrophins. IL-1β converting enzyme (ICE) is a<br />
cysteine protease that cleaves <strong>the</strong> IL-1β <strong>an</strong>d IL-18. Recently, we have showed that <strong>the</strong> IL-18mediated<br />
microglia/astrocyte interaction enh<strong>an</strong>ces neuropathic pain processing after nerve injury.<br />
We now show that ICE induced in spinal dorsal horn is crucial <strong>for</strong> tactile allodynia after nerve<br />
injury. Peripheral nerve injury, but not inflammation, induced a striking increase in ICE<br />
expression in <strong>the</strong> ipsilateral dorsal horn, <strong>an</strong>d ICE was upregulated in hyperactive microglia, but<br />
not neurons, oligodendrocytes, or astrocytes. Functional inhibition of ICE signaling pathways<br />
suppressed nerve injury-induced tactile allodynia, <strong>an</strong>d decreased <strong>the</strong> level of IL-18 in spinal<br />
cord. Indeed, ICE-deficient mice did not develop tactile allodynia after nerve injury. Conversely,<br />
intra<strong>the</strong>cal injection of <strong>the</strong> TLR4 agonist, LPS, increased <strong>the</strong> expression of ICE in spinal cord<br />
microglia. Taken toge<strong>the</strong>r, <strong>the</strong>se results demonstrate that ICE upregulation in spinal microglia<br />
has a subst<strong>an</strong>tial role in <strong>the</strong> generation of tactile allodynia through <strong>the</strong> IL-18 pathway. Thus, <strong>the</strong><br />
ICE/IL-18 signaling in spinal glial cells might be one of attractive targets in neuropathic pain<br />
mech<strong>an</strong>isms.<br />
Disclosures: K. Miyoshi, None; K. Obata, None; K. Noguchi, None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.16/DD14
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Title: Interleukin-4 deficiency is associated with increased pain behavior<br />
Authors: *C. L. SOMMER, T. TOPUZOGLU, S. HAHNENKAMP, N. ÜCEYLER;<br />
Univ. of Wuerzburg, 97080 Wuerzburg, Germ<strong>an</strong>y<br />
Abstract: Objectives<br />
Interleukin-4 (IL-4) has <strong>an</strong>algesic effects when applied in <strong>an</strong>imal models of pain. In some patient<br />
groups with chronic pain disorders, low levels of IL-4 were found. Here we aimed at establishing<br />
a model of hyperalgesia associated with IL-4 deficiency to study <strong>the</strong> pathophysiological role of<br />
IL-4 in pain induction <strong>an</strong>d mainten<strong>an</strong>ce.<br />
Methods<br />
We used IL-4 knockout (ko) mice of BL6 background <strong>an</strong>d investigated <strong>the</strong>m in <strong>the</strong> naïve state<br />
<strong>an</strong>d in models of inflammatory pain (hindpaw injection with complete Freund’s adjuv<strong>an</strong>t, CFA,<br />
or with tumor necrosis factor-alpha, TNF, <strong>an</strong>d of neuropathic pain (chronic constrictive sciatic<br />
nerve injury, CCI)). Behavioral read-outs of pain were <strong>the</strong>rmal hyperalgesia (Hargreaves<br />
method) <strong>an</strong>d tactile allodynia (von Frey hairs) at <strong>the</strong> hindpaws, <strong>an</strong>d mech<strong>an</strong>ical hyperalgesia<br />
(R<strong>an</strong>dall Selitto method) at <strong>the</strong> gastrocnemius muscle. We per<strong>for</strong>med qu<strong>an</strong>titative RT-PCR to<br />
assess possible ch<strong>an</strong>ges in <strong>the</strong> peripheral <strong>an</strong>d central nervous as well as <strong>the</strong> cut<strong>an</strong>eous cytokine<br />
system secondary to <strong>the</strong> lack of IL-4. For this purpose gene expression of selected pro- <strong>an</strong>d <strong>an</strong>tiinflammatory<br />
cytokines was measured in peripheral <strong>an</strong>d central nervous tissue <strong>an</strong>d in paw<br />
samples of <strong>the</strong> <strong>an</strong>imals.<br />
Results<br />
IL-4 ko mice had a higher sensitivity to tactile stimuli in <strong>the</strong> naïve state, increased paw swelling<br />
after CFA, <strong>an</strong>d stronger <strong>the</strong>rmal hyperalgesia after TNF injection. Pain behavior after nerve<br />
injury (CCI) was not different between genotypes. IL-4 ko mice had higher IL-1ß gene<br />
expression in <strong>the</strong> ipsilateral DRG after CFA i.pl. (d 7), higher IL-1ß <strong>an</strong>d IL-10 gene expression<br />
in <strong>the</strong> ipsilateral footpads after TNF injection (7h), <strong>an</strong>d higher pro- <strong>an</strong>d <strong>an</strong>ti-inflammatory<br />
cytokine gene expression in sciatic nerve <strong>an</strong>d ipsilateral spinal cord after CCI (28 d).<br />
Conclusion<br />
Taken toge<strong>the</strong>r IL-4 ko mice are a useful tool to investigate pain associated with <strong>an</strong> altered<br />
cytokine profile. The model c<strong>an</strong> be used to study mech<strong>an</strong>isms of increased hyperalgesia that<br />
might also be relev<strong>an</strong>t in patients with chronic pain <strong>an</strong>d low IL-4.<br />
Disclosures: C.L. Sommer, None; T. Topuzoglu, None; S. Hahnenkamp, None; N. Üceyler,<br />
None.<br />
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.17/DD15<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NS059028<br />
NS060735<br />
DE018573<br />
T32DE007309<br />
Title: Microinjection of MCP-1 into <strong>the</strong> rostral ventromedial medulla induces microglial<br />
activation <strong>an</strong>d behavioral hyperalgesia in rats<br />
Authors: H. WANG, W. GUO, F. WEI, *R. DUBNER, K. REN;<br />
Dept Neural <strong>an</strong>d Pain Sci., Univ. Maryl<strong>an</strong>d Dent. Sch., Baltimore, MD<br />
Abstract: Our previous study showed that spinal nerve injury upregulated neuronal chemokine<br />
monocyte chemoattract<strong>an</strong>t protein-1 (MCP-1, CCL2) <strong>an</strong>d its receptor CCR2, mainly associated<br />
with microglia, in <strong>the</strong> rostral ventromedial medulla (RVM). To mimic <strong>the</strong> nerve injury-induced<br />
effect on glial activity <strong>an</strong>d nociception, we injected recombin<strong>an</strong>t rat MCP-1/CCL2 (R&D<br />
Systems) into <strong>the</strong> RVM. Injection of MCP-1 produced dose-dependent (0.03, 0.3, 3 pmol)<br />
hyperalgesia, as indicated by a reduction of paw withdrawal latency to noxious <strong>the</strong>rmal stimuli.<br />
MCP-1 also induced <strong>an</strong> increased activity of microglial cells, as indicated by <strong>an</strong> increased<br />
CD11b (microglial marker) immunostaining in RVM. The MCP-1-induced upregulation of<br />
CD11b <strong>an</strong>d <strong>the</strong>rmal hyperalgesia was prevented by pretreatment with RS-102895 (10 pmol), a<br />
CCR2b chemokine receptor <strong>an</strong>tagonist. These results are consistent with a role of MCP-1 in<br />
microglial activation <strong>an</strong>d descending facilitation of nociception <strong>an</strong>d hyperalgesia. Studies<br />
suggest that expression of MCP-1 in neurons is inducible by interleukin (IL)-1beta. We show<br />
that in rats receiving <strong>an</strong> L5 spinal nerve injury, IL-1beta was upregulated selectively in RVM<br />
reactive astrocytes, as indicated by double immunofluorescence labeling of IL-1beta with glial<br />
fibrillary acidic protein, <strong>an</strong> astrocytic marker, but not with neuN (neuronal marker) <strong>an</strong>d CD11b.<br />
Injection of IL-1beta (120 fmol) in RVM induced behavioral hyperalgesia, which was blocked<br />
by pretreatment with RS-102895 (10 pmol). However, pretreatment with <strong>an</strong> IL-1 receptor<br />
<strong>an</strong>tagonist (3 pmol) did not prevent MCP-1 (3 pmol)-induced <strong>the</strong>rmal hyperalgesia. These results<br />
suggest that <strong>the</strong> effect of MCP-1 is downstream to IL-1beta signaling. Taken toge<strong>the</strong>r, our<br />
findings suggest that <strong>the</strong> IL-1 receptor <strong>an</strong>d <strong>the</strong> MCP-1-CCR2 signaling cascade plays a role in<br />
neuron-glia-cytokine interactions <strong>an</strong>d descending facilitation of neuropathic pain.<br />
Disclosures: H. W<strong>an</strong>g, None; W. Guo, None; F. Wei, None; R. Dubner, None; K. Ren, None.
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.18/DD16<br />
Topic: D.08.t. Mech<strong>an</strong>isms of neuropathic pain: Glia <strong>an</strong>d inflammatory mediators<br />
Support: NIH Gr<strong>an</strong>t DE17794<br />
NIH Gr<strong>an</strong>t NS54932<br />
NIH Gr<strong>an</strong>t TW7180<br />
Title: Intra<strong>the</strong>cal injection of activated astrocytes induces tactile allodynia by producing MCP-1<br />
Authors: *Y.-J. GAO 1 , R.-R. JI 2 ;<br />
1 Pain Res. Center, Dept. Anes<strong>the</strong>sia, Brigham & Women's Hosp. <strong>an</strong>d Harvard Med. Sch.,<br />
Boston, MA; 2 Pain Res. Center, Dept. Anes<strong>the</strong>sia, Brigham & Women's Hosp., Boston, MA<br />
Abstract: Previous studies have shown that astrocyte activation is required <strong>for</strong> <strong>the</strong> development<br />
of neuropathic pain. But direct evidence of whe<strong>the</strong>r activation of astrocytes is sufficient to<br />
induce neuropathic pain symptoms is limited. We investigated whe<strong>the</strong>r <strong>an</strong>d how spinal injection<br />
of activated astrocytes could produce mech<strong>an</strong>ical allodynia, a cardinal feature of neuropathic<br />
pain, in naïve mice. Primary cultures of astrocytes were prepared <strong>from</strong> cerebral cortexes of<br />
neonatal mice. Two weeks after culture, astrocytes were differentiated with dibutyryl cAMP <strong>an</strong>d<br />
stimulated with TNF-α <strong>for</strong> 15 minutes. After two washes with PBS, stimulated <strong>an</strong>d nonstimulated<br />
astrocytes were intra<strong>the</strong>cally injected into naïve mice. Compared with non-stimulated<br />
astrocytes, TNF-α-stimulated astroctyes induced a signific<strong>an</strong>t decrease in paw withdrawal<br />
thresholds, indicating a development of mech<strong>an</strong>ical allodynia. This allodynia was evident after at<br />
3 hours <strong>an</strong>d maintained <strong>for</strong> more th<strong>an</strong> 24 hours. However, <strong>the</strong> allodynia was prevented when <strong>the</strong><br />
astrocytes cultures were pre-treated with a c-Jun N-terminal kinase (JNK) inhibitor, D-JNKI-1.<br />
Interestingly, a short incubation of astrocyte cultures with TNF-α (15 minutes) induces a<br />
sustained expression <strong>an</strong>d release of <strong>the</strong> chemokine CCL2/MCP-1 (monocyte chemoattract<strong>an</strong>t<br />
protein-1) <strong>for</strong> more th<strong>an</strong> 18 hours. The MCP-1 upregulation by TNF-α in astrocytes was<br />
suppressed by D-JNKI-1 pretreatment. Consistently, tactile allodynia induced by TNF-αstimulated<br />
astrocytes was reversed by a MCP-1 neutralizing <strong>an</strong>tibody. However, immunostaining<br />
showed that intra<strong>the</strong>cal injection of TNF-α-stimulated astrocytes did not increase <strong>the</strong> expression<br />
of Iba-1, a microglia marker. Taken toge<strong>the</strong>r, our results suggest that activated astrocytes are<br />
sufficient to produce tactile allodynia in naïve mice by producing <strong>the</strong> chemokine MCP-1.<br />
Disclosures: Y. Gao, None; R. Ji, None.
Poster<br />
561. Neuropathic Pain: Inflammatory Cytokine <strong>an</strong>d Protein <strong>an</strong>d Peptide Signaling<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 561.19/DD17<br />
Topic: D.08.v. Mech<strong>an</strong>isms of neuropathic pain: Signaling pathways <strong>an</strong>d models<br />
Support: SNSF Gr<strong>an</strong>t 124317<br />
NIH Gr<strong>an</strong>t DE17794<br />
NIH Gr<strong>an</strong>t NS54932<br />
IASP Collaboration RRJ <strong>an</strong>d ID<br />
Title: Activation of caspase-6 contributes to neuropathic pain-like behavior after nerve injury<br />
Authors: *T. BERTA 1 , Y.-C. LIU 1 , Y.-J. GAO 1 , I. DECOSTERD 2 , R.-R. JI 1 ;<br />
1 Pain Res. Center, Dept. of Anes<strong>the</strong>siol., Brigham <strong>an</strong>d Women Hosp. <strong>an</strong>d Harvard Med. Sch.,<br />
Boston, MA; 2 Anes<strong>the</strong>siol. Pain Res. Unit, Dept. of Anes<strong>the</strong>siol. <strong>an</strong>d Dept. of Cell Biol. <strong>an</strong>d M,<br />
Univ. Hosp. Center, Univ. of Laus<strong>an</strong>ne, Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d<br />
Abstract: Caspases are intracellular proteases that play import<strong>an</strong>t roles in neuroinflammation<br />
<strong>an</strong>d neurodegeneration. General caspase inhibitors have been shown to attenuate neuropathic<br />
pain-like behaviors in different <strong>an</strong>imal models by suppressing IL-1β syn<strong>the</strong>sis <strong>an</strong>d neuronal<br />
apoptosis. However, <strong>the</strong> role of different caspases in neuropathic pain has not been well studied.<br />
Since a recent study has implicated caspase-6 in axonal degeneration, we investigated whe<strong>the</strong>r<br />
casapase-6 plays a role in neuropathic pain after spared nerve injury (SNI). Microarray <strong>an</strong>alysis<br />
showed a signific<strong>an</strong>t up-regulation of caspase-6 mRNA levels in injured DRG neurons one week<br />
after SNI. Western blot also revealed <strong>an</strong> up-regulation of <strong>the</strong> active caspase-6 one week after<br />
SNI. Double immunofluorescence <strong>an</strong>alyses demonstrated that active caspase-6 is expressed in<br />
both small- <strong>an</strong>d large-sized DRG neurons in normal conditions, but nerve injury increased<br />
caspase-6 in small-sized DRG neurons. To determine a specific role of caspase-6 in neuropathic<br />
pain, we administrated a selective inhibitor of caspase-6, Z-VEID-FMK, via <strong>an</strong> intra<strong>the</strong>cal route<br />
that c<strong>an</strong> target both DRG <strong>an</strong>d spinal cord cells. Z-VEID-FMK largely abolished SNI-induced<br />
mech<strong>an</strong>ical allodynia. Currently, we are investigating how caspase-6 activation contributes to<br />
neuropathic pain sensitization. Taken toge<strong>the</strong>r, our results suggest that caspase-6 may be a novel<br />
target <strong>for</strong> <strong>the</strong> treatment of neuropathic pain.
Disclosures: T. Berta, None; Y. Liu, None; Y. Gao, None; I. Decosterd, None; R. Ji, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.1/DD18<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Title: The effects of moisture on fingertip skin de<strong>for</strong>mation during loading <strong>an</strong>d slipping<br />
Authors: *T. ANDRE 1 , V. LEVESQUE 2 , V. HAYWARD 3 , P. LEFEVRE 1 , J.-L.<br />
THONNARD 1 ;<br />
1 Univ. catholique de Louvain, Brussels, Belgium; 2 Ctr. <strong>for</strong> Intelligent Machines <strong>an</strong>d Dept. of<br />
Electrical <strong>an</strong>d Computer Engin., McGill Univ., Montreal, QC, C<strong>an</strong>ada; 3 Inst. des Systèmes<br />
Intelligents et de Robotique, UPMC Univ. Paris 06, Paris, Fr<strong>an</strong>ce<br />
Abstract: During tactile interaction, skin de<strong>for</strong>mation stimulates <strong>the</strong> mech<strong>an</strong>oreceptors, enabling<br />
<strong>the</strong> nervous system to become aware of <strong>the</strong> properties of <strong>the</strong> touched surface. Even on perfectly<br />
smooth, glassy surfaces, <strong>the</strong> fingertip skin de<strong>for</strong>ms in complicated ways when slip occurs. The<br />
aim of <strong>the</strong> present study is to assess <strong>the</strong> effects of moisture content <strong>an</strong>d interaction <strong>for</strong>ces on <strong>the</strong><br />
fingertip skin de<strong>for</strong>mation occurring in this condition.<br />
Twelve subjects having a large r<strong>an</strong>ge of skin moisture (<strong>from</strong> dry to wet) participated in this<br />
study. They were asked to exert a const<strong>an</strong>t normal <strong>for</strong>ce against a glass surface with <strong>the</strong> index<br />
fingertip <strong>an</strong>d to increase <strong>the</strong> t<strong>an</strong>gential <strong>for</strong>ce until slip occurs. Each subject per<strong>for</strong>med five<br />
slippages <strong>for</strong> six different levels of normal <strong>for</strong>ce (0.2, 0.5, 1, 2, 5, 10N). Forces <strong>an</strong>d moisture<br />
were measured with a <strong>for</strong>ce-torque sensor (ATI F/T Mini40) <strong>an</strong>d a moisture evaluator<br />
(Corneometer®), respectively. Skin de<strong>for</strong>mation at <strong>the</strong> contact surface was acquired with a<br />
specifically designed optical apparatus that allowed <strong>the</strong> evaluation of <strong>the</strong> relative sizes of <strong>the</strong><br />
slipping <strong>an</strong>d sticking regions.<br />
The area of <strong>the</strong> sticking region decreased when <strong>the</strong> t<strong>an</strong>gential <strong>for</strong>ce increased. It varied linearly<br />
with <strong>the</strong> ratio of net t<strong>an</strong>gential/normal <strong>for</strong>ce components. The slope of this relationship was<br />
strongly influenced by both <strong>the</strong> normal <strong>for</strong>ce exerted <strong>an</strong>d <strong>the</strong> moisture content of <strong>the</strong> skin.<br />
Finally, <strong>the</strong> static coefficient of friction could be extracted when <strong>the</strong> sticking regions v<strong>an</strong>ished<br />
<strong>an</strong>d <strong>the</strong> slipping region spread to <strong>the</strong> whole surface of contact.<br />
This study described <strong>the</strong> de<strong>for</strong>mation of <strong>the</strong> skin occurring during interaction with smooth<br />
surfaces <strong>an</strong>d evaluated <strong>the</strong> effect of interaction <strong>for</strong>ce components <strong>an</strong>d moisture on this<br />
de<strong>for</strong>mation. A skin de<strong>for</strong>mation model that takes into account <strong>the</strong>se two parameters is presently<br />
being developed.
This research was supported by a gr<strong>an</strong>t <strong>from</strong> Prodex (contract numbers 90063, 90064, 90231,<br />
90232), ESA (Europe<strong>an</strong> Space Agency), PAI, FNRS, FRSM, ARC, <strong>an</strong>d NANOBIOTACTproject<br />
(EU-FPG-NMP-033287).<br />
Disclosures: T. Andre, None; V. Levesque, None; V. Hayward, None; P. Lefevre, None; J.<br />
Thonnard, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.2/DD19<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: N<strong>an</strong>obiotact Project Gr<strong>an</strong>t EU-FP6-NMP-033287<br />
Title: Tactile discrimination of viscous lubric<strong>an</strong>ts<br />
Authors: *O. ROSENTHAL 1 , M. J. ADAMS 2 , A. M. WING 1 ;<br />
1 Sch. Psychol, 2 Sch. Chem. Engin., Univ. Birmingham, Birmingham, United Kingdom<br />
Abstract: Sc<strong>an</strong>ning a surface with <strong>the</strong> finger pad provides rich in<strong>for</strong>mation about <strong>the</strong> nature of<br />
<strong>the</strong> surface, such as its roughness, softness <strong>an</strong>d curvature. Several decades of studies indicate that<br />
much of this tactile in<strong>for</strong>mation reflects <strong>the</strong> dynamics <strong>an</strong>d spatial distribution of <strong>the</strong> friction <strong>an</strong>d<br />
normal <strong>for</strong>ces produced by <strong>the</strong> contact between <strong>the</strong> finger pad <strong>an</strong>d <strong>the</strong> surface, which are<br />
detected by cut<strong>an</strong>eous mech<strong>an</strong>oreceptors in <strong>the</strong> finger pad. <strong>When</strong> a surface is well-lubricated, <strong>the</strong><br />
contact between <strong>the</strong> surface <strong>an</strong>d <strong>the</strong> finger pad is no longer direct, but is mediated by <strong>the</strong><br />
lubric<strong>an</strong>t. Although it is well established that lubrication does not interfere with <strong>the</strong> estimation of<br />
magnitude of <strong>the</strong> properties of <strong>the</strong> underlying surface, such as <strong>the</strong> roughness, <strong>the</strong> tactile<br />
experience with a lubricated surface is considerably different <strong>from</strong> when it is dry. In <strong>the</strong> case of<br />
viscous lubric<strong>an</strong>ts, additional tactile cues may be sensed such as stickiness <strong>an</strong>d sliminess,<br />
viscosity of <strong>the</strong> lubric<strong>an</strong>t. These additional factors are captured in tribology by a Stribeck curve<br />
that delineates <strong>the</strong> various mech<strong>an</strong>isms of lubrication: boundary, mixed <strong>an</strong>d elastohydrodynamic.<br />
We will present preliminary results <strong>from</strong> a behavioural-tribological study examining <strong>the</strong><br />
discrimination of lubric<strong>an</strong>t viscosity by <strong>the</strong> finger pad under passive touch conditions, using a<br />
r<strong>an</strong>ge of hydrocarbon oil blends at varying viscosity levels as lubric<strong>an</strong>ts. The separability of<br />
friction <strong>an</strong>d viscosity in tactile sensibility will be discussed in <strong>the</strong> context of <strong>the</strong><br />
interrelationships between <strong>the</strong>se factors.<br />
This research was supported by <strong>the</strong> N<strong>an</strong>obiotact Project (EU-FP6-NMP-033287)
Disclosures: O. Rosenthal, Unilever, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies,<br />
equipment or o<strong>the</strong>r in-kind support); M.J. Adams, Unilever, A. Employment (full or parttime);<br />
A.M. Wing, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.3/DD20<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: N<strong>an</strong>obiotact project (EU-FP6-NMP-033287)<br />
<strong>the</strong> Swedish Research Council<br />
Title: Decoding hum<strong>an</strong> EEG dynamics due to tactile texture processing using independent<br />
component <strong>an</strong>alysis <strong>an</strong>d support vector machines<br />
Authors: *M. B. ABERG 1,2 , S. BECKMANN 2 , H. BACKLUND WASLING 2 , L. BECCAI 3 , C.<br />
ODDO 3 , N. VITIELLO 3 , J. WESSBERG 2 ;<br />
1 Malin Aberg, Goteborg, Sweden; 2 Physiol., Neurosci. <strong>an</strong>d physiology, Goteborg, Sweden;<br />
3 Scuola Superiore S<strong>an</strong>t'Anna, Polo S<strong>an</strong>t’Anna Valdera, ARTS Lab., Pisa, Italy<br />
Abstract: Although brain activation in relation to tactile stimulation is well studied, central<br />
processing of texture coding in hum<strong>an</strong>s is poorly understood. Here, we utilize state-of-<strong>the</strong>-art<br />
machine learning algorithms to decode <strong>an</strong>d physiologically localize subtle differences in<br />
temporal brain processing patterns related to textures perceived through <strong>the</strong> finger pad.<br />
We used a robotic setup to produce well-controlled stimuli consisting of two different textures<br />
which were moved across a subject's finger pad during electro-encephalograpy (EEG)<br />
acquisition. Two textures in <strong>the</strong> <strong>for</strong>m of gratings with spatial periods of 520 microm. <strong>an</strong>d 1920<br />
microm., respectively, were investigated. Controlled tactile stimulation of <strong>the</strong> finger pad of <strong>the</strong><br />
left middle finger was applied using a purpose-designed robotic stimulation plat<strong>for</strong>m (const<strong>an</strong>t<br />
normal <strong>for</strong>ce of 0.2 N, const<strong>an</strong>t velocity of 20 mm/s, duration of 1.0 second). The order of<br />
contact of <strong>the</strong> two surfaces was r<strong>an</strong>domized. 64-electrode EEG was recorded in one subject.<br />
After st<strong>an</strong>dard, pre-processing, independent component <strong>an</strong>alysis (ICA) was applied in two steps:<br />
first on <strong>the</strong> entire signal to remove artifact components, <strong>an</strong>d subsequently to <strong>the</strong> extracted epochs<br />
(0.5s be<strong>for</strong>e <strong>an</strong>d after stimulus onset) to obtain a final nine components with physiological<br />
relev<strong>an</strong>ce. Cortical sources were imaged using <strong>the</strong> LAURA algorithm. In order to identify<br />
components representing texture encoding, a support vector machine (SVM) classifier was
applied to differentiate between <strong>the</strong> temporal dynamics produced by <strong>the</strong> surfaces.<br />
Four components with sources in <strong>the</strong> somatosensory cortices were identified. One was found to<br />
differentiate <strong>the</strong> textures at a signific<strong>an</strong>t level (permutation test, p
staircase procedure based on a two-alternative <strong>for</strong>ced choice paradigm. In each staircase, <strong>the</strong><br />
stimulus difference was expected to track <strong>the</strong> 75% correct response threshold. Two different<br />
thresholds were measured <strong>for</strong> each particip<strong>an</strong>t, one measuring <strong>the</strong> difference between <strong>the</strong> smooth<br />
<strong>an</strong>d <strong>the</strong> reference stimuli <strong>an</strong>d one measuring <strong>the</strong> difference between <strong>the</strong> rougher <strong>an</strong>d <strong>the</strong><br />
reference stimuli.<br />
The pressure detection threshold <strong>an</strong>d <strong>the</strong> spatial resolution threshold were unworkable after <strong>the</strong><br />
ring block <strong>an</strong>es<strong>the</strong>sia of <strong>the</strong> index. In contrast, <strong>the</strong> roughness discrimination thresholds were<br />
unaffected by <strong>the</strong> <strong>an</strong>es<strong>the</strong>tic block. No signific<strong>an</strong>t differences were found (paired t-test, p ><br />
0.100) between <strong>the</strong> roughness discrimination thresholds be<strong>for</strong>e <strong>an</strong>d after <strong>an</strong>es<strong>the</strong>sia.<br />
In conclusion, digital ring block <strong>an</strong>es<strong>the</strong>sia doesn’t impair <strong>the</strong> roughness perception at <strong>the</strong><br />
fingertips.<br />
Supported by N<strong>an</strong>obiotact project (EU-FP6-NMP-033287)<br />
Disclosures: X. Libouton, None; O. Barbier, None; M. Missal, None; L. Plaghki, None; J.<br />
Thonnard, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.5/DD22<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: EPSRC Gr<strong>an</strong>t EP/D060079/1<br />
Title: Vibrotactile equivalency of real <strong>an</strong>d simulated gratings<br />
Authors: *L. R. MANFREDI, B. HENSON;<br />
Mech<strong>an</strong>ical Engin., Univ. of Leeds, Leeds, West Yorkshire, United Kingdom<br />
Abstract: A variety of stimulators have been used to investigate <strong>the</strong> perception of vibrations<br />
generated during dynamic haptic exploration, including tactile arrays, actuators <strong>an</strong>d custommade<br />
textured surfaces. In <strong>the</strong> present study, we ascertain <strong>the</strong> extent to which in<strong>for</strong>mation about<br />
textured surfaces c<strong>an</strong> be conveyed through vibratory stimulation. Specifically, we investigated<br />
whe<strong>the</strong>r in<strong>for</strong>mation about <strong>the</strong> spatial properties of a stimulus (in this case, <strong>the</strong> wavelength of a<br />
grating) is associated more closely with <strong>the</strong> frequency or amplitude of <strong>the</strong> vibrations with <strong>an</strong><br />
actuator.<br />
On each trial, <strong>the</strong> subject successively sc<strong>an</strong>ned a stationary square-wave grating <strong>the</strong>n a smooth<br />
surface mounted on a piezo electric actuator that was vibrating following a square wave<strong>for</strong>m.
The subjects’ task was to adjust <strong>the</strong> amplitude or frequency of <strong>the</strong> vibrating surface such that it<br />
yielded <strong>an</strong> equivalent sensation as <strong>the</strong> grating. The gratings, machined <strong>from</strong> Acrylonitrile<br />
Butadiene Styrene (ABS), had wavelengths of 1540, 996, 800 <strong>an</strong>d 560µm <strong>an</strong>d respective<br />
amplitudes (or surface feature depths) of 92, 98, 100 <strong>an</strong>d 64µm.<br />
Subjects were trained to maintain a const<strong>an</strong>t sc<strong>an</strong>ning velocity (50mm/s) <strong>an</strong>d <strong>an</strong>gle<br />
(perpendicular to <strong>the</strong> orientation of <strong>the</strong> grooves). The sc<strong>an</strong>ning trajectories were tracked using<br />
lasers <strong>an</strong>d found to be consistent.<br />
The default expectation was that subjects would adjust <strong>the</strong> frequency of <strong>the</strong> actuator such that it<br />
followed <strong>the</strong> following relationship:<br />
f=v/λ<br />
(1)<br />
where f is <strong>the</strong> adjusted frequency of <strong>the</strong> actuator, v is <strong>the</strong> sc<strong>an</strong>ning speed, <strong>an</strong>d λ is <strong>the</strong> wavelength<br />
of <strong>the</strong> grating. We found that <strong>the</strong> frequency adjusted to each grating was proportional to its<br />
wavelength but was fivefold higher th<strong>an</strong> expected <strong>from</strong> Equation 1. Fur<strong>the</strong>rmore, <strong>the</strong>re was no<br />
consistent relationship between adjusted actuator amplitude <strong>an</strong>d grating wavelength or<br />
amplitude.<br />
We conclude that in<strong>for</strong>mation about <strong>the</strong> wavelength of a sc<strong>an</strong>ned grating c<strong>an</strong> be conveyed by<br />
delivering vibrations at <strong>an</strong> appropriate frequency. However, <strong>the</strong> relationship between vibratory<br />
frequency <strong>an</strong>d spatial period does not follow <strong>the</strong> simple relationship described in Equation 1.<br />
Fur<strong>the</strong>rmore, we find that vibratory intensity does not convey unambiguous in<strong>for</strong>mation about<br />
stimulus wavelength or amplitude. This latter finding is likely due to <strong>the</strong> fact that subjects<br />
employ different sc<strong>an</strong>ning <strong>for</strong>ces across gratings, which may in turn affect <strong>the</strong> perceived<br />
intensity of <strong>the</strong> gratings.<br />
Disclosures: L.R. M<strong>an</strong>fredi, None; B. Henson, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.6/DD23<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: Slo<strong>an</strong> Foundation<br />
Title: Acoustical imaging of tactile exploration in freely-behaving <strong>an</strong>imals<br />
Authors: *D. H. HERMAN, T. CELIKEL;<br />
Neurosci, USC, Los Angeles, CA
Abstract: The brain needs to encode <strong>the</strong> incoming sensory in<strong>for</strong>mation to create <strong>an</strong> internal<br />
representation of <strong>the</strong> world around us. These representations are <strong>the</strong> products of <strong>the</strong><br />
spatiotemporal integration of in<strong>for</strong>mation at <strong>the</strong> periphery on individual sensory org<strong>an</strong>s, like<br />
rodent whiskers. Whiskers, like eyes, are mobile sensory org<strong>an</strong>s, which receive in<strong>for</strong>mation<br />
during quiescence periods <strong>an</strong>d in active use. Although we know <strong>the</strong> kinematics of whisker<br />
motion, in<strong>for</strong>mation flow across individual whiskers during active sensation is largely unknown.<br />
To address this issue, we developed a novel sensor designed to detect whisker contacts during<br />
active sensation in freely behaving <strong>an</strong>imals. The sensor is based on <strong>the</strong> qu<strong>an</strong>tification of<br />
capacit<strong>an</strong>ce ch<strong>an</strong>ges on a perm<strong>an</strong>ently charged conductive back-plate due to whisker contact<br />
upon a surface (Figure). We show that this construction c<strong>an</strong>, in real-time, reliably detect whisker<br />
contacts by mice at high (i.e. >10 kHz) temporal resolution with a sensitivity better th<strong>an</strong> 10 -5 N.<br />
Additionally, we show that <strong>the</strong> spatial location of individual contacts c<strong>an</strong> also be resolved by<br />
using a 2D surface array of <strong>the</strong>se sensors. Using <strong>the</strong> sensor array, in this poster, we first qu<strong>an</strong>tify<br />
<strong>the</strong> spatiotemporal pattern of multi-whisker exploration on a pl<strong>an</strong>ar surface <strong>an</strong>d subsequently<br />
qu<strong>an</strong>tify <strong>the</strong> in<strong>for</strong>mation in <strong>the</strong> patterned whisker exploration on a shape discrimination task.<br />
Figure. Real-time qu<strong>an</strong>tification of<br />
whisker touch. Top: Binary distribution of whisker touch with surface as observed in high-speed<br />
videos. Middle: Spectral distribution of touch induced ch<strong>an</strong>ge in signal recorded with <strong>the</strong> sensor<br />
described in <strong>the</strong> text. Prior to spectrum calculation <strong>the</strong> signal was processed by independent<br />
component <strong>an</strong>alysis on second-order moments. Bottom: Sum of power across frequencies.<br />
Disclosures: D.H. Herm<strong>an</strong>, None; T. Celikel, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.7/DD24<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: MCyT BFU2006-06598
Title: Rostral cuneate nucleus neurons modulate <strong>the</strong> activity of proprioceptive cuneolemniscal<br />
cells of <strong>the</strong> middle cuneate in <strong>an</strong>es<strong>the</strong>tized cats<br />
Authors: R. LEIRAS, P. VELO, F. MARTÍN-CORA, *A. CANEDO;<br />
Univ. S<strong>an</strong>tiago Compostela, S<strong>an</strong>tiago De Compostela, Spain<br />
Abstract: The great majority of cuneate proprioceptive neurons sending <strong>an</strong> axon through <strong>the</strong><br />
medial lemniscus (ML) are located in <strong>the</strong> middle of <strong>the</strong> nucleus (mCN), but <strong>the</strong>ir possible<br />
intr<strong>an</strong>uclear modulation is unknown. In this work we tested <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong><br />
proprioceptive neurons in <strong>the</strong> rostral cuneate (rCN) might modulate <strong>the</strong> activity of <strong>the</strong> mCN<br />
projection cells sensitive to primary afferent spindle activation in chloralose-<strong>an</strong>es<strong>the</strong>tized,<br />
paralyzed <strong>an</strong>d artificially ventilated cats. In a first series of experiments, extracellular recordings<br />
were separately obtained <strong>from</strong> <strong>the</strong> rCN <strong>an</strong>d <strong>the</strong> mCN while stimulating <strong>the</strong> contralateral ML, <strong>the</strong><br />
ipsilateral inferior cerebellar peduncle, <strong>the</strong> ipsilateral pedunculopontine nucleus (PPn) <strong>an</strong>d <strong>the</strong><br />
ipsilateral nucleus reticularis gig<strong>an</strong>tocellularis (NRGc). In a second series, simult<strong>an</strong>eous<br />
extracellular recordings coupled to microstimulation <strong>an</strong>d microiontophoresis were<br />
simult<strong>an</strong>eously obtained <strong>from</strong> primary spindle sensitive neurons in <strong>the</strong> rCN <strong>an</strong>d <strong>the</strong> mCN. The<br />
criteria <strong>for</strong> sampling neurons were <strong>the</strong>ir bursting activity in response to <strong>the</strong> dynamic phase of a<br />
slow muscular stretch, silenced activity to <strong>the</strong> release of such stretch; <strong>an</strong>d <strong>the</strong> capacity to follow<br />
1:1 sinusoidal vibratory stimuli at 135 Hz. Results <strong>from</strong> <strong>the</strong> first series showed that few rCN<br />
cells responded <strong>an</strong>tidromically to ML stimulation but those that did (5% of tested) also showed<br />
<strong>an</strong>tidromic responses to NRGc stimulation. About 30% of <strong>the</strong> mCN projection neurons also<br />
responded <strong>an</strong>tidromically to PPn <strong>an</strong>d to NRGc stimulation. Results <strong>from</strong> <strong>the</strong> second series<br />
demonstrated that microstimulation, applied through electrodes that also served to register rCN<br />
proprioceptive cells, activated mCN projection neurons with matched rCN receptive fields, <strong>an</strong>d<br />
inhibited, produced short-latency excitation followed by silenced firing, or did not affect <strong>the</strong><br />
mCN neurons with unmatched rCN receptive fields. Some of <strong>the</strong> excitatory effects were clearly<br />
monosynaptic since <strong>the</strong> responses could follow up to 200 Hz stimuli but did not show collision<br />
with o<strong>the</strong>r orthodromic spikes. GABA <strong>an</strong>d/or glycine ejection blocked <strong>the</strong> excitatory effects <strong>an</strong>d<br />
bicuculline <strong>an</strong>d/or strychnine reversed <strong>the</strong> inhibition of GABA <strong>an</strong>d glycine, respectively; <strong>an</strong>d<br />
removed <strong>the</strong> firing silences. The results demonstrate that <strong>the</strong> rCN proprioceptive neurons serve<br />
as interneurons modulating <strong>the</strong> activity of mCN primary spindle-sensitive projection neurons<br />
with matched <strong>an</strong>d unmatched receptive fields, <strong>an</strong>d that <strong>an</strong> appreciable percentage of <strong>the</strong>se<br />
lemniscal-projecting neurons also send axons to reticular motor-related structures.<br />
Disclosures: R. Leiras, None; P. Velo, None; F. Martín-Cora, None; A. C<strong>an</strong>edo, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 562.8/DD25<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NY Department of Health<br />
Title: Neurophysiological effects of microstimulation in rat <strong>an</strong>d monkey somatosensory<br />
thalamus<br />
Authors: *S. XU, M. SEMEWORK, A. V. ROZENBOYM, P. Y. CHHATBAR, J. T.<br />
FRANCIS, J. K. CHAPIN;<br />
Dept Physiol & Pharmacol, SUNY Downstate Med. Ctr., Brooklyn, NY<br />
Abstract: To determine whe<strong>the</strong>r microstimulation in somatosensory system c<strong>an</strong> produce<br />
“virtual” percepts that alter or mimic natural somes<strong>the</strong>sia, we have systematically assessed <strong>the</strong><br />
effects of stimulation of somatosensory thalamus (ventral posterolateral nucleus, VPL) at <strong>the</strong><br />
neurophysiological level in rats <strong>an</strong>d monkeys. Neuronal ensemble responses to discrete natural<br />
touch of <strong>the</strong> <strong>for</strong>epaw <strong>an</strong>d electrical stimulation of VPL were recorded in <strong>the</strong> primary<br />
somatosensory cortex (S1) of <strong>an</strong>es<strong>the</strong>tized <strong>an</strong>d awake rats <strong>an</strong>d in aear 1, 3b <strong>an</strong>d 2 in monkeys.<br />
Multivariate statistical <strong>an</strong>alyses were used to determine neural code <strong>for</strong> stimulation parameters<br />
such as stimulus location <strong>an</strong>d intensity. A space map was constructed using factor <strong>an</strong>alysis to<br />
represent <strong>the</strong> pattern of cortical response to each different stimulus. We found both natural touch<br />
stimuli on <strong>the</strong> peripheral <strong>an</strong>d microstimulation of VPL c<strong>an</strong> evoke complex spatiotemporal<br />
patterns <strong>from</strong> somatosensory cortical neurons. Statistical comparison showed a subset of VPL<br />
stimuli yielding cortical neural responses that were remarkably similar to those evoked by<br />
natural touch. There is a good correlation between receptive fields of thalamic nuclei <strong>an</strong>d <strong>the</strong><br />
cortical responses evoked by stimulation of <strong>the</strong> same nuclei. Responses to strong, monopolar, or<br />
non-VPL stimuli were poorly correlated to natural stimulation <strong>an</strong>d were far away on <strong>the</strong> space<br />
map. Our results indicate <strong>an</strong> optimal stimulation in VPL thalamus might generate a sensory<br />
perception close enough to <strong>the</strong> natural one <strong>for</strong> somatosensory pros<strong>the</strong>sis purpose. They might<br />
also provide <strong>an</strong> electrophysiological expl<strong>an</strong>ation <strong>for</strong> previous finding that electrical stimulation<br />
in hum<strong>an</strong> thalamus induced mostly pares<strong>the</strong>sia but occasional natural somatosensations.<br />
Disclosures: S. Xu, None; M. Semework, None; A.V. Rozenboym, None; P.Y. Chhatbar,<br />
None; J.T. Fr<strong>an</strong>cis, None; J.K. Chapin, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 562.9/DD26<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: Emmy-Noe<strong>the</strong>r Gr<strong>an</strong>t (DFG - Germ<strong>an</strong> Research Foundation)<br />
Howard Hughes Medical Institute<br />
Title: Online read-out of flutter frequency in S1 <strong>an</strong>d S2<br />
Authors: A. WOHRER 1 , R. ROMO 3 , *C. K. MACHENS 2 ;<br />
1 Dept. d'etudes cognitives, 2 Ecole Normale Superieure, Paris, Fr<strong>an</strong>ce; 3 Inst. de Fisiologia Celular<br />
- Neurosciencias, Univ. Nacional Autonoma de Mexico, Mexico, Mexico<br />
Abstract: <strong>When</strong> a vibrating stimulus is imprinted on <strong>the</strong> fingertip of a macaque, <strong>the</strong> value of <strong>the</strong><br />
stimulating frequency c<strong>an</strong> be decoded <strong>from</strong> <strong>the</strong> spiking patterns of neurons in area S1, ei<strong>the</strong>r<br />
through <strong>the</strong> periodicity of <strong>the</strong> patterns or through <strong>the</strong>ir overall spike counts. In area S2, on <strong>the</strong><br />
o<strong>the</strong>r h<strong>an</strong>d, periodic responses are much reduced <strong>an</strong>d only spike counts carry in<strong>for</strong>mation about<br />
<strong>the</strong> stimulus value [Romo <strong>an</strong>d Salinas (2003) Nat.Rev.Neurosci. 4:203-218]. The functional<br />
reason <strong>for</strong> this shift <strong>from</strong> periodic to aperiodic responses going up <strong>the</strong> somatosensory pathway is<br />
not clear yet. We hypo<strong>the</strong>size that such a shift allows neurons to <strong>for</strong>m <strong>an</strong> inst<strong>an</strong>t<strong>an</strong>eous or 'online'<br />
representation of <strong>the</strong> frequency in<strong>for</strong>mation, so that stimulus frequency is represented in <strong>the</strong><br />
*inst<strong>an</strong>t<strong>an</strong>eous* firing rate ra<strong>the</strong>r th<strong>an</strong> in overall spike counts or periodicity. To test this<br />
hypo<strong>the</strong>sis, we studied single-cell recordings of neurons in areas S1 <strong>an</strong>d S2 of monkeys. In<br />
particular, we asked to what extent <strong>the</strong> frequency in<strong>for</strong>mation c<strong>an</strong> be linearly decoded <strong>from</strong><br />
inst<strong>an</strong>t<strong>an</strong>eous estimates of <strong>the</strong> firing rates of <strong>the</strong> neural population. At each trial <strong>an</strong>d time t, <strong>the</strong><br />
spikes <strong>for</strong> each neuron are counted in a Gaussi<strong>an</strong> window centered around t, with st<strong>an</strong>dard<br />
deviation w. Considering a population of N neurons, this method produces <strong>an</strong> N-dimensional<br />
vector: The inst<strong>an</strong>t<strong>an</strong>eous population activity at time t with temporal window w. Linear<br />
prediction of <strong>the</strong> input frequency value, based on this vector at <strong>an</strong>y inst<strong>an</strong>t in time, allows to<br />
assess <strong>the</strong> amount of frequency in<strong>for</strong>mation present 'online' in <strong>the</strong> population. We find that, both<br />
in areas S1 <strong>an</strong>d S2, such <strong>an</strong> 'online neurometric decoder' c<strong>an</strong> predict <strong>the</strong> input frequency as<br />
efficiently as <strong>the</strong> monkey itself, down to temporal windows of about w=10 ms. By opposition,<br />
spike counts over <strong>the</strong> whole population <strong>an</strong>d 500ms stimulation produce predictions much more<br />
accurate th<strong>an</strong> <strong>the</strong> monkey itself, suggesting a tradeoff between <strong>the</strong> inst<strong>an</strong>t<strong>an</strong>eous counting<br />
window w <strong>an</strong>d <strong>the</strong> number of neurons N involved in <strong>the</strong> coding. Building such 'trade-off curves',<br />
we find that online coding in S1 is carried out by a very specific sub-population of cells, whereas<br />
it is much more distributed in S2. Finally, a simple neural model allows us to predict <strong>an</strong>alytically<br />
<strong>the</strong> per<strong>for</strong>m<strong>an</strong>ces of our online decoder, <strong>the</strong> effects of spike correlations, <strong>an</strong>d to suggest by what<br />
mech<strong>an</strong>isms 'online-coding' aperiodic cells could arise <strong>from</strong> more oscillatory inputs.<br />
Disclosures: A. Wohrer, None; R. Romo, None; C.K. Machens, None.<br />
Poster
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.10/DD27<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NS034086<br />
Title: The neural mech<strong>an</strong>isms underlying <strong>the</strong> spotlight of attention: A selective attention study<br />
assessing orientation <strong>an</strong>d frequency mech<strong>an</strong>isms in <strong>the</strong> somatosensory modality<br />
Authors: *M. GOMEZ-RAMIREZ, T. YOSHIOKA, S. MIHALAS, K. CHEN, E. NIEBUR,<br />
S. HSIAO;<br />
The Z<strong>an</strong>vyl Krieger Mind / Brain Inst., Johns Hopking Univ., Baltimore, MD<br />
Abstract: The aim of this study was to determine whe<strong>the</strong>r <strong>the</strong> attentional spotlight functions by<br />
enh<strong>an</strong>cing <strong>the</strong> neural responses in a local neural region or whe<strong>the</strong>r it selectively operates on<br />
subpopulation of neurons that are modality specific. Electrophysiological recordings were made<br />
in <strong>the</strong> <strong>an</strong>terior, central <strong>an</strong>d posterior h<strong>an</strong>d regions of secondary somatosensory cortex (SII) while<br />
a non-hum<strong>an</strong> primate <strong>an</strong>imal engaged in a highly-dem<strong>an</strong>ding somatosensory selective attention<br />
task. The <strong>an</strong>imal was cued on a trial-by-trial basis to per<strong>for</strong>m <strong>an</strong> orientation or frequency task<br />
<strong>an</strong>d detect a target in <strong>the</strong> relev<strong>an</strong>t sub-modality only. The imperative stimulus upon which <strong>the</strong><br />
<strong>an</strong>imal per<strong>for</strong>med <strong>the</strong> discrimination task was designed such that it contained both orientation<br />
<strong>an</strong>d frequency in<strong>for</strong>mation that selectively activated ei<strong>the</strong>r slowly adapting (SA, <strong>for</strong>m) or rapidly<br />
adaptive (RA, frequency) neurons. Fur<strong>the</strong>r, a control condition where <strong>the</strong> <strong>an</strong>imal had to disregard<br />
all tactile in<strong>for</strong>mation <strong>an</strong>d per<strong>for</strong>m a task in <strong>the</strong> visual modality was implemented. We<br />
hypo<strong>the</strong>sized that that SA neurons will show selective attentional modulations when <strong>the</strong> <strong>an</strong>imal<br />
attends to orientation but not frequency. In contrast, attention will enh<strong>an</strong>ce <strong>the</strong> RAs neuronal<br />
response when <strong>the</strong> <strong>an</strong>imal is cued to per<strong>for</strong>m <strong>the</strong> frequency task. Finally, we contended that<br />
attention to <strong>the</strong> visual modality would not enh<strong>an</strong>ce <strong>the</strong> neural responses to both <strong>the</strong> SA <strong>an</strong>d RA<br />
neurons.<br />
Disclosures: M. Gomez-Ramirez, None; T. Yoshioka, None; S. Mihalas, None; K. Chen,<br />
None; E. Niebur, None; S. Hsiao, NS034086, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.11/DD28<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NIH Gr<strong>an</strong>t NS037501<br />
Title: Nociceptor afferent drive alters SI neuron response to mech<strong>an</strong>ical stimulation of <strong>the</strong> RF<br />
Authors: *B. L. WHITSEL 1 , O. FAVOROV 2 , Y. LI 3 , J. LEE 2 , M. TOMMERDAHL 2 ;<br />
1 Univ. North Carolina Sch. Med., Chapel Hill, NC; 2 Biomed. Engin., Univ. of North Carolina at<br />
Chapel Hill, Chapel Hill, NC; 3 Slo<strong>an</strong>-Kettering C<strong>an</strong>cer Ctr., New York, NY<br />
Abstract: Extracellular recordings of <strong>the</strong> spike discharge activity of rapidly-adapting (RA)<br />
neurons in areas 3b <strong>an</strong>d 1 of primary somatosensory cortex (SI) were obtained be<strong>for</strong>e, during,<br />
<strong>an</strong>d following 25Hz sinusoidal vertical skin displacement stimulation of <strong>the</strong> receptive field center<br />
(RFC). The spike firing that accomp<strong>an</strong>ies near-threshold (0.01-0.05mm) 25Hz stimulation with a<br />
47-51 degrees Celsius probe is (i) lower in me<strong>an</strong> firing rate (MFR), <strong>an</strong>d (ii) less phase-locked<br />
(“entrained”) to <strong>the</strong> stimulus th<strong>an</strong> <strong>the</strong> activity evoked by stimulation with a 25-38 degrees<br />
Celsius probe. In contrast, when <strong>the</strong> RFC stimulus is suprathreshold (0.1-0.4mm), no signific<strong>an</strong>t<br />
suppression of RA neuron MFR <strong>an</strong>d entrainment accomp<strong>an</strong>ies 25Hz stimulation with a 47-51<br />
degrees Celsius probe.<br />
Intradermal algogen injection is shown to result in modifications of <strong>the</strong> SI RA neuron spike<br />
firing evoked by 25Hz stimulation. Both <strong>the</strong> sign (-/+) <strong>an</strong>d magnitude of <strong>the</strong> modifications are<br />
stimulus-dependent. Whereas algogen injection is followed by a subst<strong>an</strong>tial decrease of both<br />
MFR <strong>an</strong>d entrainment when <strong>the</strong> 25Hz stimulus is near-threshold (0.01-0.05mm), progressively<br />
smaller decreases in MFR <strong>an</strong>d entrainment occur as stimulus amplitude is increased. <strong>When</strong> <strong>the</strong><br />
stimulus is suprathreshold (>0.2mm), algogen injection is followed ei<strong>the</strong>r by no ch<strong>an</strong>ge or, more<br />
frequently, by <strong>an</strong> increase of MFR <strong>an</strong>d entrainment. The observations enable neuromech<strong>an</strong>istic<br />
expl<strong>an</strong>ation of <strong>the</strong> diverse <strong>an</strong>d seemingly conflicting results of studies of <strong>the</strong> effects of<br />
pain/nociceptor afferent drive on hum<strong>an</strong> (i) tactile mech<strong>an</strong>oresponsivity <strong>an</strong>d discriminative<br />
capacity, <strong>an</strong>d (ii) <strong>the</strong> SI cortical response to Abeta afferent drive.<br />
Disclosures: B.L. Whitsel, None; O. Favorov, None; Y. Li, None; J. Lee, None; M.<br />
Tommerdahl, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.12/DD29<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NIH Gr<strong>an</strong>t NS059036<br />
Title: Detection of low saliency stimuli requires synergy of tectal <strong>an</strong>d thalamic relays<br />
Authors: J. D. COHEN, *M. A. CASTRO-ALAMANCOS;<br />
Drexel Univ. Col. Med., Philadelphia, PA<br />
Abstract: Sensory in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> vibrissae enters <strong>the</strong> CNS via <strong>the</strong> trigeminal g<strong>an</strong>glion<br />
<strong>an</strong>d travels to <strong>the</strong> trigeminal complex in <strong>the</strong> brainstem. From <strong>the</strong> trigeminal complex, two main<br />
pathways ascend toward <strong>the</strong> contralateral midbrain <strong>an</strong>d <strong>for</strong>ebrain. One reaches <strong>the</strong> thalamus in<br />
<strong>the</strong> <strong>for</strong>ebrain (trigeminothalamic), <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r reaches <strong>the</strong> superior colliculus (SC) in <strong>the</strong><br />
midbrain tectum (trigeminotectal). We previously showed that trigeminothalamic <strong>an</strong>d<br />
trigeminotectal pathways c<strong>an</strong> independently detect a highly salient whisker stimulus. However,<br />
effective detection of sensory signals through <strong>the</strong>se pathways may depend on <strong>the</strong> psychophysical<br />
saliency of <strong>the</strong> stimulus. Thus, we tested if <strong>the</strong>se sensory pathways differ in <strong>the</strong>ir ability to detect<br />
low salient stimuli. Animals were trained to detect a high <strong>an</strong>d a low salience electrical whisker<br />
pad conditioned stimulus (WCS) <strong>for</strong> successful avoid<strong>an</strong>ce behavior. We found that reversible<br />
lesions of <strong>the</strong> somatosensory thalamus or superior colliculus did not impair detection of <strong>the</strong> high<br />
salience WCS. However, reversible lesions of ei<strong>the</strong>r <strong>the</strong> somatosensory thalamus or superior<br />
colliculus blocked per<strong>for</strong>m<strong>an</strong>ce on <strong>the</strong> low salience WCS task. Hence, trigeminothalamic <strong>an</strong>d<br />
trigeminotectal pathways work synergistically to detect low salient stimuli during avoid<strong>an</strong>ce<br />
behavior, but are redund<strong>an</strong>t during detection of highly salient stimuli. Fur<strong>the</strong>rmore,<br />
electrophysiological recordings in <strong>the</strong> superior colliculus <strong>an</strong>d barrel cortex of actively behaving<br />
<strong>an</strong>imals reveal signific<strong>an</strong>tly different responses between <strong>the</strong> low <strong>an</strong>d high salience WCS, which<br />
may explain <strong>the</strong> requirement <strong>for</strong> synergy during low saliency.<br />
Disclosures: J.D. Cohen, None; M.A. Castro-Alam<strong>an</strong>cos, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.13/DD30
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Title: Neural projection <strong>from</strong> area 5 to primary motor cortex in healthy adults: A TMS study<br />
Authors: *A. ZILUK, A. PREMJI, A. J. NELSON;<br />
Kinesiology, Univ. of Waterloo, Waterloo, ON, C<strong>an</strong>ada<br />
Abstract: The ability to execute precision grip <strong>an</strong>d opposable thumb movements requires a high<br />
level of h<strong>an</strong>d dexterity, <strong>an</strong> evolutionary skill acquired by hum<strong>an</strong>s <strong>an</strong>d few nonhum<strong>an</strong> primates.<br />
Such activities are dependent on <strong>the</strong> integrity of somatosensory input arising <strong>from</strong> <strong>the</strong> thumb <strong>an</strong>d<br />
digits. Brodm<strong>an</strong>n’s area 5 (BA 5) is <strong>an</strong> import<strong>an</strong>t cortical structure that appears to have evolved<br />
with <strong>the</strong> ability to per<strong>for</strong>m skilled h<strong>an</strong>d movements. In addition to processing somatosensory<br />
input, BA 5 has direct projections to primary motor cortex (M1), <strong>an</strong>d thus may influence <strong>the</strong><br />
motor output of <strong>the</strong> h<strong>an</strong>d. The goal of <strong>the</strong> present experiment was to characterize <strong>the</strong> nature<br />
(excitatory versus inhibitory) <strong>an</strong>d timing of <strong>the</strong> projection <strong>from</strong> BA 5 to M1 in healthy adults<br />
during rest <strong>an</strong>d during processing of somatosensory input using paired-pulse TMS. Eleven righth<strong>an</strong>ded<br />
subjects (4 males, 7 females, 22-36 years old) participated. TMS was per<strong>for</strong>med using<br />
BiStim-2/Magstim 200 2 stimulators with two customized 50 mm br<strong>an</strong>ding coils. The<br />
conditioning stimulus (CS) was positioned over left hemisphere BA 5 as defined by <strong>the</strong><br />
individuals MRI guided by <strong>the</strong> superior parietal lobule, <strong>an</strong>d <strong>the</strong> test stimulus coil (TS) was placed<br />
over <strong>the</strong> hotspot in left M1 to elicit a maximal motor evoked potential (MEP) in <strong>the</strong> first dorsal<br />
interosseous (FDI) muscle of <strong>the</strong> right h<strong>an</strong>d. The TS was delivered at <strong>an</strong> intensity to evoke <strong>an</strong><br />
MEP of ~ 1mV amplitude, <strong>an</strong>d <strong>the</strong> CS was delivered at 90% RMT. The interval between <strong>the</strong> CS<br />
<strong>an</strong>d TS was tested at <strong>the</strong> following interstimulus intervals; 6,8,10,12,30,40 <strong>an</strong>d 50 ms. The MEP<br />
amplitude was measured <strong>from</strong> <strong>the</strong> right FDI muscle during <strong>the</strong> conditioned state normalized to<br />
<strong>the</strong> unconditioned state (TS alone). The neural projection <strong>from</strong> BA 5 to M1 was recorded during<br />
1) rest with both h<strong>an</strong>ds completely relaxed, 2) simult<strong>an</strong>eous vibration (23 Hz) to <strong>the</strong> fingertips of<br />
digits 1 <strong>an</strong>d 2, 3) vibration (80 Hz) to <strong>the</strong> tendons of FDI <strong>an</strong>d opponens pollicis <strong>an</strong>d 4) during<br />
simult<strong>an</strong>eous vibration to <strong>the</strong> tips of digits 1 <strong>an</strong>d 2 <strong>an</strong>d <strong>the</strong>ir corresponding tendons. Preliminary<br />
<strong>an</strong>alysis suggests that during rest <strong>the</strong> output <strong>from</strong> M1 is facilitated at both early (6 through 12<br />
ms) <strong>an</strong>d late (40 ms) but not intermediate (30 ms) ISIs. Compared to rest, cut<strong>an</strong>eous vibration of<br />
digits 1 <strong>an</strong>d 2 led to <strong>an</strong> early facilitation at 6 <strong>an</strong>d 10 ms while tendon vibration elicited<br />
facilitation at 40 ms only. The combination of cut<strong>an</strong>eous <strong>an</strong>d tendon vibration led to a modest<br />
facilitation at 40 ms. Collectively <strong>the</strong>se data suggest that BA 5 influences <strong>the</strong> output <strong>from</strong> M1<br />
<strong>an</strong>d that <strong>the</strong> nature <strong>an</strong>d timing of <strong>the</strong> influence is dependent on <strong>the</strong> sub-modal type of<br />
somatosensory input being processed.<br />
Disclosures: A. Ziluk, None; A. Premji, None; A.J. Nelson, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.14/DD31<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NIH Gr<strong>an</strong>t NS36860<br />
Title: Primary motor cortical neurons show differential activity ch<strong>an</strong>ges during <strong>the</strong> reaction time<br />
period of wrist movements guided by somatosensory <strong>an</strong>d visual inputs in monkeys<br />
Authors: Y. LIU 1 , J. M. DENTON 1 , *R. J. NELSON 2 ;<br />
1 Anat. <strong>an</strong>d Neurobio., 2 Univ. Tennessee Hlth. Sci. Ctr., Memphis, TN<br />
Abstract: Neurons in primary motor cortex (MI) often show task-related activity <strong>an</strong>d evoked<br />
responses elicited by peripheral stimulation. Some MI neurons are responsive to deep (DEEP) or<br />
cut<strong>an</strong>eous stimuli while o<strong>the</strong>rs have no evoked responses (NR). We w<strong>an</strong>ted to know if <strong>the</strong><br />
activity of each neuronal type varies with motor tasks that are guided by somatosensory <strong>an</strong>d<br />
visual inputs. Two monkeys made wrist extensions <strong>an</strong>d flexions after holding steady wrist<br />
positions <strong>for</strong> 0.5-2.0 s. Movements were guided in separate paradigms by visual targets (VIS),<br />
increases in vibration to <strong>the</strong> monkey's palm (VIB), or both in combination (COM). Movement<br />
targets were 5° <strong>an</strong>d 10° <strong>from</strong> center <strong>for</strong> both extensions <strong>an</strong>d flexions. Animals were cared <strong>for</strong> in<br />
accord<strong>an</strong>ce with <strong>the</strong> NRC Guide.<br />
We <strong>an</strong>alyzed activity data <strong>from</strong> 10° wrist movement trials. Each neuron contributed two cases to<br />
<strong>the</strong> total; one each <strong>for</strong> extension <strong>an</strong>d flexion. From <strong>the</strong> 394 total cases, 370 cases showed ei<strong>the</strong>r<br />
enh<strong>an</strong>ced or suppressed activity during reaction time period when compared with <strong>the</strong> instructed<br />
delay period <strong>an</strong>d were fur<strong>the</strong>r examined. Of those, 254 cases <strong>an</strong>d 112 cases were classified as<br />
belonging to <strong>the</strong> DEEP <strong>an</strong>d NR groups, respectively. The remaining 4 cases <strong>from</strong> <strong>the</strong> cut<strong>an</strong>eous<br />
group were excluded due to small sample size. For <strong>the</strong> DEEP cases, earlier onsets of both<br />
enh<strong>an</strong>ced <strong>an</strong>d suppressed activity were observed during VIB <strong>an</strong>d COM trials when compared<br />
with VIS trials. For <strong>the</strong> NR cases, <strong>the</strong> onsets of enh<strong>an</strong>ced activity were earlier during VIS <strong>an</strong>d<br />
COM trials th<strong>an</strong> VIB trials. The onsets of suppressed activity, however, occurred earlier during<br />
VIB trials th<strong>an</strong> VIS <strong>an</strong>d COM trials. In addition, enh<strong>an</strong>ced activities in both DEEP <strong>an</strong>d NR<br />
groups were lower during VIB trials. Despite this, reaction times were faster <strong>an</strong>d initial<br />
movement speeds were slower <strong>for</strong> VIB <strong>an</strong>d COM trials when compared with VIS trials.<br />
Studies have suggested that MI neuronal activity during <strong>the</strong> reaction time period is involved in<br />
<strong>the</strong> preparation <strong>an</strong>d execution of voluntary movement. In MI, DEEP <strong>an</strong>d NR neurons may play<br />
different roles in motor control. Interestingly, <strong>the</strong> patterns of activity ch<strong>an</strong>ges of DEEP cases<br />
during reaction time periods are similar to some neurons in primary somatosensory cortex (SI)<br />
that we previously observed. Those SI neurons were thought to be interneurons. Most had no<br />
clear receptive fields (NCRF). DEEP neurons in MI <strong>an</strong>d NCRF neurons in SI may receive inputs<br />
with similar functional features <strong>an</strong>d may be related to corollary discharge or efference copy of<br />
motor comm<strong>an</strong>ds. NR neurons may have local or intercortical connections <strong>an</strong>d modulate <strong>the</strong><br />
activity of cortical input <strong>an</strong>d/or output neurons.<br />
Disclosures: Y. Liu, None; J.M. Denton, None; R.J. Nelson, None.
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.15/DD32<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NIH RO1 DC003311<br />
NIH P30 HD02528<br />
NIH P30 DC005803<br />
Title: MEG registration of short-term cortical adaptation to TAC-Cell inputs to hum<strong>an</strong> h<strong>an</strong>d <strong>an</strong>d<br />
face<br />
Authors: *L. VENKATESAN 1 , S. M. BARLOW 2,1,3 , M. POPESCU 4 , A. POPESCU 4 ;<br />
1 Neurosci., 2 Spch-L<strong>an</strong>g-Hrg, 3 Hum<strong>an</strong> Biol., Univ. of K<strong>an</strong>sas, Lawrence, KS; 4 Hoglund Brain<br />
Imaging Ctr., KU Med. Ctr., K<strong>an</strong>sas City, KS<br />
Abstract: OBJECTIVE: To use magnetoencephalography (MEG) to characterize short-term<br />
spatiotemporal adaptation of <strong>the</strong> primary somatosensory cortex (S1) in response to tactile inputs<br />
to <strong>the</strong> hum<strong>an</strong> h<strong>an</strong>d <strong>an</strong>d lips using a custom built device called TAC-Cell.<br />
METHODS: The TAC-Cell is custom, small-bore pneumatic actuator based on a 5 ml<br />
polyethylene vial with a 0.005” silicone sheet placed between <strong>the</strong> lip of <strong>the</strong> vial <strong>an</strong>d a specially<br />
machined retaining ring. A whole-head MEG system with 151 axial-gradiometer sensors was<br />
used to record <strong>the</strong> cortical response to a pneumatic tactile stimulus that consisted of a repeating<br />
6-pulse train (50-ms pulse width, intertrain interval = 5 s, 125 reps/train rate, train rates [2, 4, &<br />
8 Hz]). A double-adhesive tape collar secured <strong>the</strong> TAC-Cell at two locations, including <strong>the</strong> 1)<br />
glabrous surface of <strong>the</strong> right h<strong>an</strong>d (index/middle finger), <strong>an</strong>d 2) midline of <strong>the</strong> upper <strong>an</strong>d lower<br />
lip vermillion. The active surface of <strong>the</strong> TAC-Cell (19.3 mm dia) generated a punctate input to<br />
<strong>the</strong> skin (4.25 mm displacement, 25 ms rise/fall time). Peak latencies <strong>an</strong>d amplitudes were<br />
estimated on <strong>the</strong> me<strong>an</strong> global field (MGF). Source reconstruction was per<strong>for</strong>med using CURRY<br />
software.<br />
RESULTS: The best-fit dipole corresponding to <strong>the</strong> domin<strong>an</strong>t peak of <strong>the</strong> evoked responses was<br />
localized to <strong>the</strong> h<strong>an</strong>d representation of <strong>the</strong> left S1 following stimulation of <strong>the</strong> right h<strong>an</strong>d. The<br />
same stimulus applied to <strong>the</strong> lip vermilion activated <strong>the</strong> face representation in S1 bilaterally. The<br />
evoked S1 response amplitude <strong>an</strong>d latency were dependent on pulse index <strong>an</strong>d pulse train rate.<br />
Evoked S1 activity in response to lip stimulation occurred at a signific<strong>an</strong>tly shorter latency when
compared to <strong>the</strong> evoked response to h<strong>an</strong>d stimulation at 2 Hz (F[1,14] = 9.88, p=.007) , 4 Hz<br />
(F[1,14] = 14.54, p=.002), <strong>an</strong>d 8 Hz (F[1,14] = 13.24, p=.003). Peak MGF potentials were<br />
observed at 60-90 ms <strong>for</strong> lip, <strong>an</strong>d 100-125 ms <strong>for</strong> h<strong>an</strong>d inputs. The magnitude of adaptation<br />
reflected in <strong>the</strong> attenuation of <strong>the</strong> MGF between <strong>the</strong> 1 st <strong>an</strong>d 2 nd evoked response was greater <strong>for</strong><br />
h<strong>an</strong>d (24.2%) stimulation compared to <strong>the</strong> lip (15.8%), regardless of pulse train frequency. This<br />
may be explained in part by differences in mech<strong>an</strong>oreceptor representation <strong>for</strong> <strong>the</strong>se two<br />
sensorimotor control systems.<br />
CONCLUSIONS: The localization <strong>an</strong>d pattern of short-term adaptation evident in <strong>the</strong> evoked<br />
cortical S1 MGF response pattern is signific<strong>an</strong>tly different between trigeminal (lip) <strong>an</strong>d medi<strong>an</strong><br />
nerve (h<strong>an</strong>d) using TAC-Cell inputs. The spatiotemporal org<strong>an</strong>ization of <strong>the</strong> MGF profile<br />
m<strong>an</strong>ifests signific<strong>an</strong>t modulation <strong>an</strong>d adaptation as a function of stimulus frequency.<br />
Disclosures: L. Venkates<strong>an</strong>, None; S.M. Barlow, None; M. Popescu, None; A. Popescu,<br />
None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.16/DD33<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: CNRS<br />
ANR (NATACS)<br />
EC contract FP6-015879 (FACETS)<br />
Title: Neuronal representation of tactile apparent motion in <strong>the</strong> somatosensory thalamus<br />
Authors: J. LE CAM 1 , V. EGO-STENGEL 1 , *D. E. SHULZ 2 ;<br />
1 Unite de Neurosciences Integratives et Computationnelles, CNRS, Gif sur Yvette, Fr<strong>an</strong>ce;<br />
2 CNRS, Gif Sur Yvette, Fr<strong>an</strong>ce<br />
Abstract: Rats use <strong>the</strong> facial whiskers to explore <strong>the</strong>ir environment by making multiple contacts<br />
with objects. Our goal is to underst<strong>an</strong>d how <strong>the</strong> somatosensory system processes this<br />
multiwhisker in<strong>for</strong>mation. Using a 24-whisker stimulator in <strong>an</strong>es<strong>the</strong>tized (ureth<strong>an</strong>e) rats, we have<br />
previously shown that cortical responses c<strong>an</strong> be modulated by <strong>the</strong> sensory context. Cortical<br />
neurons exhibited global direction selectivity to <strong>the</strong> apparent motion of a multivibrissal stimulus,
uncorrelated to <strong>the</strong>ir local direction selectivity (Jacob et al. 2008, Neuron).<br />
Since a certain level of multiwhisker integration has been reported in <strong>the</strong> ventro-posterior medial<br />
nucleus (VPM) of <strong>the</strong> thalamus (Simons <strong>an</strong>d Carvell, 1989), we explored whe<strong>the</strong>r emergent<br />
properties of multiwhisker stimulations are already coded by VPM neurons. Using <strong>the</strong> same<br />
protocol as <strong>for</strong> our previous cortical study, we characterized receptive fields (RFs) <strong>an</strong>d responses<br />
to multivibrissal stimuli in <strong>the</strong> thalamus. We tested sequences of locally invari<strong>an</strong>t rostro-caudal<br />
deflections that generated <strong>an</strong> apparent motion in 8 different directions across <strong>the</strong> mystacial pad,<br />
while recording unitary electrophysiological activity <strong>from</strong> <strong>the</strong> VPM. This was combined to<br />
cytochrome oxydase histochemistry to verify <strong>the</strong> location of <strong>the</strong> recording sites.<br />
The RFs of VPM cells were monovibrissal (78%). This spatial extent was more restricted th<strong>an</strong> in<br />
<strong>the</strong> cortex, which could suggest that VPM neurons were not able to extract <strong>the</strong> global<br />
in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> apparent motion stimulus.<br />
Preliminary results (VPM neurons n=33) showed different responses to <strong>the</strong> apparent motion:<br />
some cells (n=15) were strongly inhibited by <strong>the</strong> stimuli, preventing <strong>the</strong> qu<strong>an</strong>tification of<br />
direction tuning curves. On cells responsive to <strong>the</strong> multivibrissal stimuli (n=18), we calculated a<br />
direction selectivity index <strong>from</strong> <strong>the</strong> responses to <strong>the</strong> 8 directions. 50% of <strong>the</strong>se responsive cells<br />
were signific<strong>an</strong>tly selective. In our previous study, we observed that most cortical neurons (69%)<br />
were selective to <strong>the</strong> direction of <strong>the</strong> global stimulus.<br />
These results suggest two interpretations: a) <strong>the</strong> selectivity is generated at <strong>the</strong> VPM level <strong>an</strong>d is<br />
tr<strong>an</strong>sferred to <strong>an</strong>d amplified in <strong>the</strong> cortex; b) <strong>the</strong> complex tactile stimuli are integrated at <strong>the</strong><br />
cortical level <strong>an</strong>d <strong>the</strong> selectivity is tr<strong>an</strong>sferred back to <strong>the</strong> VPM through cortico-thalamic<br />
connections. Indeed, layer VI of <strong>the</strong> barrel cortex sends cortico-thalamic projections back to<br />
VPM. To test <strong>the</strong>se hypo<strong>the</strong>ses, <strong>the</strong> same experiment will be done during cortical inactivation.<br />
Disclosures: J. Le Cam, None; V. Ego-Stengel, None; D.E. Shulz, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.17/DD34<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: This study was supported by a gr<strong>an</strong>t of <strong>the</strong> Oriental Medicine R&D Project, Ministry<br />
of Healthy, Welfare <strong>an</strong>d Family Affairs, Republic of Korea (B080049).<br />
Title: Consecutive acupuncture stimulations affected fMRI signals in <strong>the</strong> latter acupuncture<br />
stimulation
Authors: *S. YEO 1,2 , I.-H. CHOE 3,4 , K.-H. RHEU 3 , Y.-G. CHOI 2,3 , Y.-M. HONG 1,2 , S.<br />
LIM 1,2,3 ;<br />
1 Meridi<strong>an</strong> <strong>an</strong>d Acupoint, Grad. Sch. of Basic Eastern Med. Science, Kyung Hee Univ., Seoul,<br />
Republic of Korea; 2 Acupuncture <strong>an</strong>d Meridi<strong>an</strong>, WHO Collaborating Ctr. <strong>for</strong> Traditional<br />
Medicine, East-West Med. Res. Institute, Kyung Hee Univ., Seoul, Republic of Korea; 3 Applied<br />
Meridi<strong>an</strong> Med., Grad. Sch. of Applied Eastern Medicine, Kyung Hee Univ., Seoul, Republic of<br />
Korea; 4 Ctr. <strong>for</strong> Neural Science, Korea Inst. of Sci. <strong>an</strong>d Technol., Seoul, Republic of Korea<br />
Abstract: Objective: Previous studies have used block design paradigms in fMRI <strong>an</strong>d<br />
acupuncture researches. Most studies were designed with consecutive acupuncture stimulations.<br />
So we thought that consecutive acupuncture stimulations could affect fMRI signals of <strong>the</strong> latter<br />
acupuncture stimulation. This study aims to investigate fMRI signal ch<strong>an</strong>ges evoked by<br />
consecutive acupuncture stimulations.<br />
Method: 15 subjects were measured 2 times each by 2 Physici<strong>an</strong>s in <strong>the</strong> same sc<strong>an</strong>ning session.<br />
We compared <strong>the</strong> first acupuncture stimulus with <strong>the</strong> second one. This study employed a block<br />
design <strong>for</strong> mapping brain activity <strong>an</strong>d acupuncture was per<strong>for</strong>med on BL62 on right foot. To<br />
reduce vari<strong>an</strong>ces between <strong>the</strong> physici<strong>an</strong>s, we set up <strong>the</strong> depth of insertion <strong>an</strong>d <strong>the</strong> number of<br />
rotation.<br />
Result: Consecutive acupuncture stimulations increased whole brain fMRI signals in <strong>the</strong> latter<br />
acupuncture stimulation. The signals of ROI, limbic lobe, <strong>an</strong>terior cingulate, frontal lobe, frontal<br />
gyrus, <strong>an</strong>d medial frontal gyrus, which were defined by group <strong>an</strong>alysis, also increased.<br />
Conclusion: We proved that consecutive acupuncture stimulations affect fMRI signals of <strong>the</strong><br />
latter acupuncture stimulation. There<strong>for</strong>e we need to concern about consecutive acupuncture<br />
stimulations when we design <strong>the</strong> experiment pl<strong>an</strong>s on <strong>the</strong> fMRI <strong>an</strong>d acupuncture researches.<br />
Disclosures: S. Yeo, None; I. Choe, None; K. Rheu, None; Y. Choi, None; Y. Hong, None; S.<br />
Lim, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 562.18/DD35<br />
Topic: D.09.e. Stimulus feature representation <strong>an</strong>d coding<br />
Support: NIH Gr<strong>an</strong>t GM060826<br />
Rutgers University Fund
Title: Women’s clitoris, vagina <strong>an</strong>d cervix mapped on <strong>the</strong> sensory cortex, using fMRI<br />
Authors: *B. R. KOMISARUK 1,2 , N. WISE 1 , E. FRANGOS 1 , W.-C. LIU 2 ;<br />
1 Psychology, Rutgers Univ., Newark, NJ; 2 Radiology, New Jersey Med. Sch., Newark, NJ<br />
Abstract: The sensory somatotopic (homuncular) representation of <strong>the</strong> body surface was<br />
originally mapped by Penfield <strong>an</strong>d Rasmussen (1950) as located in <strong>the</strong> postcentral gyrus, based<br />
on electrical stimulation exclusively in men. They also reported that <strong>the</strong> foot <strong>an</strong>d <strong>the</strong> genital<br />
sensory representations are situated in <strong>the</strong> medial cortex, i.e., in <strong>the</strong> “paracentral lobule,” which<br />
is just superior to <strong>the</strong> cingulate gyrus. To our knowledge, women’s genital regions have not been<br />
mapped on <strong>the</strong> sensory cortex. In <strong>the</strong> present study, we mapped <strong>the</strong> somatosensory cortical<br />
representation of <strong>the</strong> genital structures of 11 healthy women using fMRI in response to<br />
mech<strong>an</strong>ical self-stimulation. Data were obtained using a Siemens Allegra 3T sc<strong>an</strong>ner with a<br />
"boxcar" stimulation paradigm (30sec-on, 30sec-off <strong>for</strong> 5min), processed <strong>an</strong>d <strong>an</strong>alyzed using<br />
SPM 99 <strong>an</strong>d MRIcro. We hypo<strong>the</strong>sized that stimulation of <strong>the</strong> specific genital structures would<br />
differentially activate <strong>the</strong> general genital region of <strong>the</strong> sensory cortex. This is based on evidence<br />
that <strong>the</strong> clitoris, vagina <strong>an</strong>d cervix receive differential afferent innervation via <strong>the</strong> pudendal,<br />
pelvic, hypogastric <strong>an</strong>d vagus nerves. To establish control points of reference, we also mapped<br />
<strong>the</strong> regions of <strong>the</strong> somatosensory cortex activated by finger <strong>an</strong>d toe stimulation <strong>an</strong>d nipple selfstimulation.<br />
Results: We found that all <strong>the</strong> genital sensory projection regions are located in <strong>the</strong><br />
paracentral lobule, in <strong>the</strong> region just superior to <strong>the</strong> cingulate gyrus. The genital sensory regions<br />
are all located inferior to <strong>the</strong> region activated by toe stimulation, which we used as a reference<br />
point. The site activated by vaginal self-stimulation was superior <strong>an</strong>d posterior to those activated<br />
by clitoral <strong>an</strong>d cervical self-stimulation. The cervical self-stimulation resulted in <strong>the</strong> strongest<br />
activation among <strong>the</strong> genital regions <strong>an</strong>d was located deepest (i.e., far<strong>the</strong>st <strong>from</strong> <strong>the</strong> midline<br />
along <strong>the</strong> perpendicular to <strong>the</strong> cortical surface). Surprisingly, self-stimulation of <strong>the</strong> nipple<br />
resulted not only in activation of <strong>the</strong> thoracic sensory region, but also of <strong>the</strong> paracentral lobule<br />
within <strong>the</strong> genital sensory region. A general caveat of <strong>the</strong> interpretation of fMRI data is that <strong>the</strong><br />
extent of activation <strong>an</strong>d <strong>the</strong> degree of overlap among brain regions depends on <strong>the</strong> level of<br />
statistical signific<strong>an</strong>ce of <strong>the</strong> threshold that is selected. Taking this caveat into account, we<br />
conclude, on <strong>the</strong> basis of <strong>the</strong> present findings, that <strong>the</strong> sensory projections of women’s genitalia<br />
are clustered, yet differentiable, in <strong>the</strong> same general paracentral lobule region as indicated by<br />
Penfield <strong>an</strong>d Rasmussen in men.<br />
Disclosures: B.R. Komisaruk, None; N. Wise, None; E. Fr<strong>an</strong>gos, None; W. Liu, None.<br />
Poster<br />
562. Tactile Stimulation <strong>an</strong>d Processing<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 562.19/DD36<br />
Topic: D.09.f. Functional studies<br />
Support: FP7 ICT-215910 (BIOTACT)<br />
Italy<br />
Centro Servizi Polivalenti di Ateneo Animal Facility, University of Trieste, Trieste,<br />
Title: Vibrissal movement in <strong>the</strong> marsupial Monodelphis Domestica: A clue to <strong>the</strong> evolution of<br />
mammali<strong>an</strong> sensorimotor cortex?<br />
Authors: *T. J. PRESCOTT, R. A. GRANT, B. MITCHINSON;<br />
Psychology, Univ. Sheffield, Sheffield, United Kingdom<br />
Abstract: The gray, short-tailed (Brazili<strong>an</strong>) opossum, M. Domestica, is considered to be a good<br />
model <strong>an</strong>imal in which to trace <strong>the</strong> evolution of <strong>the</strong> mammali<strong>an</strong> brain. Fossil evidence suggests<br />
<strong>the</strong> presence of <strong>an</strong>imals similar to <strong>the</strong> Didelphid opossums as early as <strong>the</strong> Jurassic period<br />
(100mya), hence <strong>the</strong>se <strong>an</strong>imals may have m<strong>an</strong>y of <strong>the</strong> traits possessed by a common <strong>an</strong>cestor of<br />
both marsupial <strong>an</strong>d placental mammals. M. Domestica has relatively few corticospinal neurons,<br />
<strong>an</strong>d lacks a distinct motor cortex. Frost et al. (J. Comp. Neuro, 421, 29-51, 2000) demonstrated<br />
<strong>the</strong> presence of two somatosensory maps (S1, S2) <strong>an</strong>d showed that movements of <strong>the</strong> vibrissae<br />
could be induced by stimulation of S1 sites with receptive fields <strong>for</strong> <strong>the</strong> vibrissae, lower jaw, or<br />
snout. These authors found no evidence of movement elsewhere in <strong>the</strong> body by stimulation of<br />
non-facial S1. This data suggests that control of vibrissal movement was <strong>an</strong> early milestone in<br />
<strong>the</strong> evolution of sensorimotor cortex, possibly preceding <strong>the</strong> evolution of a motor cortex <strong>an</strong>d of<br />
cortical control of limbs <strong>an</strong>d digits. An examination of vibrissal movement in awake,<br />
unrestrained M. Domestica should <strong>the</strong>re<strong>for</strong>e serve as a useful next step in characterising <strong>the</strong> role<br />
of vibrissae, <strong>an</strong>d of cortical control of movement, in <strong>the</strong> life of early mammals.<br />
Adult opossums were observed in a custom-built arena, illuminated <strong>from</strong> below using a bright,<br />
infrared light source, <strong>an</strong>d filmed <strong>from</strong> above using a monochrome high-speed camera as <strong>the</strong>y<br />
emerged <strong>from</strong> a cardboard tube (used to tr<strong>an</strong>sfer <strong>the</strong>m <strong>from</strong> <strong>the</strong>ir home cage) <strong>an</strong>d explored <strong>the</strong><br />
floor, walls, <strong>an</strong>d o<strong>the</strong>r objects. All <strong>an</strong>imals exhibited bouts of ‘whisking’ (repeated <strong>for</strong>wardbackward<br />
sweeps) at rates of 5-8Hz, both whilst traversing <strong>the</strong> floor <strong>an</strong>d while exploring or<br />
searching <strong>for</strong> objects or surfaces when stationary. Whisking bouts were interspersed with periods<br />
when <strong>the</strong> whiskers were relatively motionless. Whisking was strongly synchronous between <strong>the</strong><br />
two sides of <strong>the</strong> snout but often asymmetric (i.e. <strong>the</strong> two whisker fields moved with different<br />
amplitudes) in a m<strong>an</strong>ner that appeared contact-dependent, <strong>an</strong>d as previously observed in rats<br />
(Mitchinson et al., Proc. Biol. Sci., 274, 1035-41, 2007).<br />
The similarities between whisking in murid rodents <strong>an</strong>d in M. Domestica suggest that active<br />
control of vibrissal movement, may have evolved in a common <strong>an</strong>cestor of marsupial <strong>an</strong>d<br />
placental mammals, <strong>an</strong>d that <strong>the</strong> requirement to control <strong>the</strong> positioning of <strong>the</strong> whiskers, during<br />
active touch, may have been a driving <strong>for</strong>ce in <strong>the</strong> evolution of cortical control of <strong>the</strong> facial<br />
musculature.<br />
Disclosures: T.J. Prescott, None; R.A. Gr<strong>an</strong>t, None; B. Mitchinson, None.
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.1/DD37<br />
Topic: D.10.a. Plasticity<br />
Support: Craig H. Neilsen Foundation<br />
Title: Effects of repetitive acute intermittent hypoxia on lumbosacral motor function in hum<strong>an</strong><br />
SCI<br />
Authors: *R. D. TRUMBOWER 1 , A. JAYARAMAN 2 , B. D. SCHMIT 3 , T. G. HORNBY 4 , G.<br />
S. MITCHELL 5 , W. Z. RYMER 2 ;<br />
1 Rehabil Inst. Chicago, Chicago, IL; 2 Rehabil. Inst. of Chicago, Chicago, IL; 3 Marquette Univ.,<br />
Milwaukee, WI; 4 Univ. of Illinois at Chicago, Chicago, IL; 5 Univ. of Wisconsin, Madison, WI<br />
Abstract: Neural plasticity is a signific<strong>an</strong>t contributor to motor recovery following spinal cord<br />
injury (SCI). Repetitive exposure to acute intermittent hypoxia (AIH) induces spinal plasticity,<br />
streng<strong>the</strong>ning synapses onto respiratory motor neurons. It has been suggested that similar<br />
potentiation occurs in non-respiratory pathways of spinally injured rats, but no studies have<br />
addressed this possibility in hum<strong>an</strong>s with SCI. The purpose of this study was to determine if a<br />
ch<strong>an</strong>ge in neuromotor excitability is present in <strong>the</strong> lower limb of persons with incomplete SCI in<br />
response to repetitive exposure to AIH. We hypo<strong>the</strong>sized that repetitive AIH-induced facilitation<br />
of lumbosacral motor output would increase <strong>the</strong> capacity <strong>for</strong> <strong>an</strong>kle torque generation in persons<br />
with incomplete SCI.<br />
Four chronic incomplete SCI subjects (ASIA C <strong>an</strong>d D; injury onset 15±8 years) participated in 4<br />
weeks of repetitive AIH training. During training, subjects were secured to a semi-reclined seat<br />
with domin<strong>an</strong>t leg positioned with <strong>the</strong> knee constrained in approximately 120 degrees of flexion<br />
<strong>an</strong>d <strong>the</strong> neutral <strong>an</strong>kle attached to a single degree-of-freedom isokinetic machine. Repetitive AIH<br />
training consisted of 3 AIH sessions per week; each training session included fifteen, 90-second<br />
bouts of hypoxia (FIO2=0.09) with 180-second intervals of normoxia. Biomech<strong>an</strong>ical<br />
measurements of isometric <strong>an</strong>kle pl<strong>an</strong>tar flexion torque, as well as, electromyograms (EMG)<br />
were recorded <strong>from</strong> 3 muscles representing <strong>the</strong> major muscles of <strong>an</strong>kle torque production.<br />
Repeated measurements were taken at baseline, at <strong>the</strong> end of each training week, <strong>an</strong>d one week<br />
post-training. Blood oxygen saturation, blood pressure <strong>an</strong>d heart rate were continuously<br />
monitored.<br />
Enh<strong>an</strong>ced pl<strong>an</strong>tar flexion torque generation was observed in 3 of 4 subjects during repetitive AIH<br />
exposure. Average pl<strong>an</strong>tar flexion torque increased 38±37% (+/- S.E.) relative to baseline after
week 1, 36±38% after week 2, 55±32% after week 3, <strong>an</strong>d 24±16% after week 4. Although <strong>the</strong><br />
increase in <strong>an</strong>kle torque generation was evident during AIH training, <strong>the</strong> increase was not<br />
sustained one-week post-training. Results <strong>from</strong> this work offer <strong>the</strong> first evidence <strong>for</strong> enh<strong>an</strong>ced<br />
lumbosacral motor function induced by repetitive exposure to AIH, which is consistent with our<br />
hypo<strong>the</strong>sis <strong>an</strong>d compliments previous findings of facilitation in respiratory motor excitability.<br />
Disclosures: R.D. Trumbower, None; A. Jayaram<strong>an</strong>, None; B.D. Schmit, None; T.G.<br />
Hornby, None; G.S. Mitchell, None; W.Z. Rymer, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.2/DD38<br />
Topic: D.10.a. Plasticity<br />
Support: NIH Gr<strong>an</strong>t NS09881<br />
NIH Gr<strong>an</strong>t NS 54883<br />
Veterens Administration<br />
International Spinal Research Trust<br />
The Bernard <strong>an</strong>d Anne Spitzer Charitable Trust<br />
Dr. Miriam <strong>an</strong>d Sheldon G. Adelson Medical Research Foundation<br />
Title: Enh<strong>an</strong>ced axonal tr<strong>an</strong>sport as a novel <strong>for</strong>m of tr<strong>an</strong>sient “plasticity” after primate spinal<br />
cord injury<br />
Authors: *J. H. BROCK 1 , E. S. ROSENZWEIG 1 , H. YANG 1 , V. R. EDGERTON 2 , M. H.<br />
TUSZYNSKI 1,3 ;<br />
1 2 3<br />
Dept Neurosci, UCSD, La Jolla, CA; Physiological Sci., UCLA, Los Angeles, CA; VA S<strong>an</strong><br />
Diego, La Jolla, CA<br />
Abstract: To examine axonal plasticity <strong>an</strong>d regeneration in <strong>the</strong> primate spinal cord, five<br />
adult rhesus monkeys underwent unilateral tr<strong>an</strong>sections of <strong>the</strong> right dorsolateral corticospinal<br />
tract (CST) at T8. In <strong>the</strong> same surgical session, CST axons were <strong>an</strong>terogradely traced by
injecting biotinylated dextr<strong>an</strong> amine (BDA) into <strong>the</strong> right primary motor cortex. Qu<strong>an</strong>tification<br />
of axonal number 6 months after <strong>the</strong> original injury demonstrated complete or near-complete<br />
tr<strong>an</strong>section of all CST axons in <strong>the</strong> left dorsolateral CST below <strong>the</strong> lesion, <strong>an</strong>d a signific<strong>an</strong>t 3fold<br />
increase in <strong>the</strong> number of BDA-labeled ventral CST axons in <strong>the</strong> right ventral CST<br />
(projecting ipsilateral to <strong>the</strong> cortex of origin) compared to intact <strong>an</strong>imals (p
Morton Cure Paralysis Fund<br />
Title: A distinct subset of tr<strong>an</strong>scripts that encode <strong>for</strong> synaptic vesicle proteins increases in <strong>the</strong><br />
lamprey brain during functional recovery <strong>from</strong> spinal cord injury<br />
Authors: *B. Y. LAU, N. ALIEVA, D. J. BUSCH, J. R. MORGAN;<br />
Section of Mol. Cell <strong>an</strong>d Developmental Biol., Univ. of Texas at Austin, Austin, TX<br />
Abstract: The mech<strong>an</strong>isms by which synapse function is restored after spinal cord injury (SCI)<br />
remain unclear, because synapse regeneration is inefficient in m<strong>an</strong>y commonly used vertebrate<br />
models. However, after SCI in lamprey (Petromyzon marinus), <strong>the</strong> reticulospinal (RS) axons<br />
regenerate across <strong>the</strong> lesion <strong>an</strong>d <strong>for</strong>m functional synapses onto <strong>the</strong>ir targets, providing a unique<br />
opportunity to begin underst<strong>an</strong>ding <strong>the</strong> molecular mech<strong>an</strong>isms <strong>for</strong> restoring synapse function. To<br />
do so, we first generated cDNA libraries <strong>from</strong> microdissected brain regions of control lampreys<br />
<strong>an</strong>d spinal-lesioned lampreys at 1 week, 3 weeks, <strong>an</strong>d 11-12 weeks post-tr<strong>an</strong>section, time points<br />
that correspond to axon degeneration, early axon regeneration toward <strong>the</strong> lesion, <strong>an</strong>d complete<br />
regeneration across <strong>the</strong> lesion, respectively. Next, semi-qu<strong>an</strong>titative reverse tr<strong>an</strong>scriptase-PCR<br />
was employed to determine <strong>the</strong> relative expression levels of select tr<strong>an</strong>scripts encoding <strong>for</strong><br />
synaptic vesicle-associated proteins at <strong>the</strong>se time points. Three patterns of gene expression<br />
ch<strong>an</strong>ges were identified: 1) genes that were unch<strong>an</strong>ged (e.g. protein kinase C, synaptophysin IIa<br />
<strong>an</strong>d γ-synuclein-like-1), 2) genes that were downregulated by >50% at 3 weeks <strong>an</strong>d that<br />
rebounded to control levels by 11-12 weeks (e.g. γ-synuclein-like 2), <strong>an</strong>d 3) genes that were<br />
upregulated >150% at 3 weeks <strong>an</strong>d <strong>the</strong>reafter [e.g. synapsin I <strong>an</strong>d synaptic vesicle glycoprotein 2<br />
(SV2)]. Thus, synaptic vesicle-associated genes are differentially regulated after SCI, implying a<br />
complex compensatory reaction. That two positive regulators of release probability, synapsin I<br />
<strong>an</strong>d SV2, continue to be upregulated after <strong>the</strong> RS synapses have regenerated suggests <strong>an</strong><br />
enh<strong>an</strong>cement of synaptic tr<strong>an</strong>smission as one mech<strong>an</strong>ism <strong>for</strong> restoring synapse function after<br />
SCI.<br />
Disclosures: B.Y. Lau, None; N. Alieva, None; D.J. Busch, None; J.R. Morg<strong>an</strong>, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.4/DD40<br />
Topic: D.10.a. Plasticity<br />
Support: NS51667
Title: Use of sodium ch<strong>an</strong>nels<br />
Authors: *J. LEE 1 , S. JERGOVA 2 , R. YEZIERSKI 3 , J. SAGEN 2 ;<br />
1 Biol., William Paterson Univ., Wayne, NJ; 2 MiamiProject to Cure Paralysis, Miami, FL; 3 Dept.<br />
of Orthodontics, Univ. of Florida, Gainesville, FL<br />
Abstract: Excitotoxic spinal cord injury (SCI) induced by intraspinal injection of quisqualic acid<br />
(QUIS), <strong>an</strong> AMPA receptor agonist, produces mech<strong>an</strong>ical <strong>an</strong>d cold allodynia in rats. In addition,<br />
<strong>the</strong>se <strong>an</strong>imals develop self-directed excessive grooming behavior. We have shown previously of<br />
relationship between grooming <strong>an</strong>d decreased number of endogenous dorsal horn GABAergic<br />
neurons with decreased density of PGP9.5+ free nerve endings in <strong>the</strong> grooming dermatome.<br />
Voltage-gated sodium ch<strong>an</strong>nels are involved in <strong>the</strong> tr<strong>an</strong>smission of nociception <strong>an</strong>d neuropathic<br />
pain, e.g. <strong>the</strong> absence of NaV1.7 ch<strong>an</strong>nels lead to insensitivity to pain whereas abnormal<br />
expression causes <strong>the</strong>rmal hypersensitivity. The present study examined whe<strong>the</strong>r QUIS SCI, a<br />
CNS lesion, would brings about ch<strong>an</strong>ges in <strong>the</strong> expression of voltage gated sodium ch<strong>an</strong>nels in<br />
<strong>the</strong> peripheral nerve <strong>an</strong>d differential protein expression in DRG <strong>an</strong>d spinal dorsal horn.<br />
QUIS (125mM) was injected unilaterally in rostro-caudal direction (3x0.4ul, 0.5mm apart).<br />
Controls received intraspinal PBS injections. Rats were perfused at days 3, 7, or 14 post-lesion in<br />
order to examine <strong>the</strong> time course of ch<strong>an</strong>ges in <strong>the</strong> expression of TTX-sensitive Nav 1.7 <strong>an</strong>d<br />
TTX-resist<strong>an</strong>t Nav 1.8 in <strong>the</strong> primary afferents <strong>an</strong>d at-level DRGs. Peripheral nerve injury<br />
marker ATF-3 (at-level DRG) as well as primary afferent fiber markers IB-4 <strong>an</strong>d subst<strong>an</strong>ce P<br />
(SP) in <strong>the</strong> lumbar dorsal horn were also examined.<br />
In <strong>the</strong> at-level dorsal horn, QUIS induced rapid bilateral upregulation of Nav1.7+ fibers in <strong>the</strong><br />
superficial dorsal horn laminae with higher Nav1.7+ density on <strong>the</strong> ipsilateral side <strong>from</strong> 7D <strong>an</strong>d<br />
14D. The fiber densities <strong>for</strong> IB4 <strong>an</strong>d SP in <strong>the</strong> dorsal horn remained unch<strong>an</strong>ged compared to<br />
control. In <strong>the</strong> at-level DRG, <strong>the</strong> number of Nav1.8+ DRG neurons remained unch<strong>an</strong>ged in<br />
QUIS rats compared to control. QUIS injury, however, induced bilateral expression of ATF3 in<br />
medium <strong>an</strong>d small neurons in <strong>the</strong> at-level DRG.<br />
These results suggests that excitotoxic CNS injury may bring about ch<strong>an</strong>ges in <strong>the</strong> at-level DRG<br />
<strong>an</strong>d upregulation of voltage-gated sodium ch<strong>an</strong>nels 1.7, which may play a role in mediating<br />
neuropathic pain symptoms following spinal cord injury.<br />
Disclosures: J. Lee, None; S. Jergova, None; R. Yezierski, None; J. Sagen, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.5/DD41
Topic: D.10.a. Plasticity<br />
Support: NIH NS055976<br />
Shriners Hospital <strong>for</strong> Children<br />
Title: Ch<strong>an</strong>ges in gene expression are maintained over short <strong>an</strong>d long periods of cycling exercise<br />
(Ex) after spinal cord injury (SCI)<br />
Authors: *B. E. KEELER 1 , R. N. SIEGFRIED 2 , G. LIU 2 , K. N. MILLER 2 , L. SANTI 2 , J. D.<br />
HOULE 2 ;<br />
1 Drexel Univ., Philadelphia, PA; 2 Neurobio. <strong>an</strong>d Anat., Drexel Univ. Col. of Med., Philadelphia,<br />
PA<br />
Abstract: We have previously demonstrated <strong>the</strong> effects of short term (1wk) passive cycling after<br />
spinal tr<strong>an</strong>section on mRNA expression in <strong>the</strong> whole spinal cord <strong>an</strong>d in specific cell types<br />
(motoneurons <strong>an</strong>d dorsal root g<strong>an</strong>glion [DRG] neurons). In <strong>the</strong> present study, we investigated<br />
whe<strong>the</strong>r <strong>the</strong> effects of long term (4wks) exercise on gene expression remain stable as long as<br />
exercise is continued or whe<strong>the</strong>r cells eventually become insensitive to <strong>the</strong> stimulation. We used<br />
qu<strong>an</strong>titative PCR to measure ch<strong>an</strong>ges in expression of mRNA <strong>for</strong> neurotrophins, neurotrophin<br />
receptors, <strong>an</strong>d markers of apoptosis <strong>an</strong>d cellular response to trauma; obtaining samples <strong>from</strong><br />
whole spinal cord (lumbar 4-6) <strong>an</strong>d <strong>from</strong> laser micro dissected motoneurons, intermediate grey<br />
matter, <strong>an</strong>d large DRG neurons. So far we have determined that <strong>an</strong> exercise-induced increase in<br />
BDNF, GDNF, <strong>an</strong>d NT-4 mRNA in whole cord remains at a signific<strong>an</strong>tly high <strong>an</strong>d stable level<br />
as long as exercise is continued, but injury-induced increases in GFRα1 <strong>an</strong>d NT-3 at 1 wk recede<br />
by 4 wks, although exercise was able to block this increase in <strong>the</strong> short-term. Exercise also<br />
attenuated <strong>the</strong> acute injury-induced increase in caspase-9 mRNA, but levels decreased naturally<br />
over <strong>the</strong> longer post-injury period <strong>an</strong>d exercise did not fur<strong>the</strong>r reduce <strong>the</strong> baseline level. In<br />
motoneurons, BDNF <strong>an</strong>d GDNF mRNA expression increased with short-term exercise <strong>an</strong>d<br />
remained high after 4wks of exercise, while NT-4 mRNA expression increased somewhat with<br />
short term exercise, but much more with <strong>the</strong> longer exercise period. These results indicate<br />
possible cellular <strong>an</strong>d molecular responses underlying activity dependent plasticity that is<br />
observed with <strong>an</strong>atomical <strong>an</strong>d physiological measures. Experiments investigating ch<strong>an</strong>ges in<br />
mRNA expression in intermediate grey matter <strong>an</strong>d large DRG neurons are ongoing as well as<br />
protein <strong>an</strong>alysis of whole spinal cord tissue to verify mRNA tr<strong>an</strong>slation to protein.<br />
Disclosures: B.E. Keeler, None; R.N. Siegfried, None; G. Liu, None; K.N. Miller, None; L.<br />
S<strong>an</strong>ti, None; J.D. Houle, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.6/DD42<br />
Topic: D.10.a. Plasticity<br />
Support: NIH Gr<strong>an</strong>t NS055911-01<br />
Title: Activity-dependent expression of BDNF <strong>an</strong>d TrkB in neurons <strong>an</strong>d glial cells in <strong>the</strong> lumbar<br />
spinal cord of spinal rats<br />
Authors: C. CHOW 1 , P. A. SEE 2 , J. LUU 2 , *R. DE LEON 3,2 ;<br />
1 Dept. of Biol. Sci., 2 Sch. of Kinesiology <strong>an</strong>d Nutritional Sci., Cali<strong>for</strong>nia State Univ. Los<br />
Angeles, Los Angeles, CA; 3 Cali<strong>for</strong>nia State Univ, LA, Los Angeles, CA<br />
Abstract: Previous studies have shown that brain-derived neurotrophic factors (BDNF) is upregulated<br />
in <strong>the</strong> lumbar spinal cord of spinal cord injured rats by exercise. Which spinal cells<br />
may be expressing BDNF in response to exercise after spinal cord injury is unknown. In this<br />
study, we examined <strong>the</strong> effects of imposing treadmill training on <strong>the</strong> cellular expression of<br />
BDNF <strong>an</strong>d TrkB in rats that received a complete spinal cord tr<strong>an</strong>section (ST) at 5 days of age.<br />
Thirty days after tr<strong>an</strong>section, <strong>the</strong> rats were r<strong>an</strong>domly assigned to three experimental groups based<br />
on <strong>the</strong> number of steps imposed during daily treadmill training: 0, 100 <strong>an</strong>d 1000 steps/training<br />
session (n=5/group). After 8 weeks, <strong>the</strong> rats were killed <strong>an</strong>d <strong>the</strong> spinal cords were processed <strong>for</strong><br />
BDNF <strong>an</strong>d TrkB immunohistochemical experiments. Neurons <strong>an</strong>d oligodendrocytes were<br />
identified by double-labeling with NeuN <strong>an</strong>d RIP <strong>an</strong>tibodies respectively. A signific<strong>an</strong>t<br />
difference in BDNF expression was found between <strong>the</strong> groups (p100>0). No signific<strong>an</strong>t difference in TrkB expression between <strong>the</strong> groups was found<br />
(P
Program#/Poster#: 563.7/DD43<br />
Topic: D.10.a. Plasticity<br />
Support: The University of Hong Kong Seed Funding Program <strong>for</strong> Basic Research (10206818)<br />
Regional Gr<strong>an</strong>t Council General Research Fund (HKU 780508M)<br />
Title: Differentiation plasticity of neural stem cells in <strong>the</strong> injured adult mouse spinal cord is<br />
regulated by bone morphogenetic proteins (BMPs)<br />
Authors: *Q. XIAO, Y. DU, W. WU, H. K. YIP;<br />
Dept. of Anatomy, The Univ. of Hong Kong, Hong Kong, China<br />
Abstract: Bone morphogenetic proteins (BMPs) <strong>an</strong>d <strong>the</strong>ir <strong>an</strong>tagonist noggin take part in<br />
regulating pattern <strong>for</strong>mation <strong>an</strong>d cell fate determination during central nervous system (CNS)<br />
development. Our previous findings have shown that BMP-2/4/7 <strong>an</strong>d downstream signaling<br />
molecules pSmad-1/5/8 expressions were up-regulated after spinal cord injury, <strong>an</strong>d <strong>the</strong> increased<br />
expressions of BMPs <strong>an</strong>d pSmads were detected in a wide variety of neural cells (neural stem<br />
cells, neurons <strong>an</strong>d glial cells). The results suggested that BMP signaling pathway may play a role<br />
in regulating cellular composition in spinal cord remodeling after injury. As quiescent neural<br />
stem cells (NSCs) in <strong>the</strong> normal adult spinal cord will proliferate <strong>an</strong>d differentiate extensively<br />
into neural cells after traumatic injury, we hypo<strong>the</strong>sized that BMP effect on <strong>the</strong> cellular<br />
components in <strong>the</strong> injured spinal cord was probably mediated by reulating NSC differentiation.<br />
In neurosphere culture <strong>from</strong> normal adult mouse spinal cord, we found addition of BMP-4<br />
promoted astrocytes differentiation <strong>from</strong> NSCs while suppressed production of neurons <strong>an</strong>d<br />
oligodendrocytes. Fur<strong>the</strong>r, BMP-4 not only restricted neuronal differentiation, but also delayed<br />
neuronal maturation. Blocking of BMP signaling by noggin be<strong>for</strong>e BMP-4 treatment results in<br />
<strong>the</strong> inhibition of astrocyte differentiation while promotes neuron production. In fusion of noggin<br />
to <strong>the</strong> contused spinal cord through <strong>an</strong> osmotic pump signific<strong>an</strong>tly decreased expression of<br />
pSmads, suggesting that BMP signaling pathway may be involved in <strong>the</strong> cellular responses in<br />
spinal cord injury. However, noggin had no signific<strong>an</strong>t effect on <strong>the</strong> down-regulation of GFAP<br />
<strong>an</strong>d NG2 expression, indicating that suppressing BMP pathway alone may not be effective to<br />
decrease glial scar <strong>for</strong>mation in <strong>the</strong> injured spinal cord.<br />
Disclosures: Q. Xiao, None; Y. Du, None; W. Wu, None; H.K. Yip, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 563.8/DD44<br />
Topic: D.10.a. Plasticity<br />
Support: ANR<br />
IRME<br />
Christopher <strong>an</strong>d D<strong>an</strong>a Reeve Foundation (CRF #VB1-0502-2)<br />
Title: Contribution of <strong>the</strong> BDNF-induced down-regulation of cation-chloride cotr<strong>an</strong>sporters<br />
KCC2 to <strong>the</strong> development of hyperreflexia after SCI. Possible involvement in spasticity<br />
Authors: P. BOULENGUEZ 1 , S. LIABEUF 1 , R. BOS 1 , H. BRAS 1 , C. JEAN-XAVIER 1 , C.<br />
BROCARD 1 , A. STIL 1 , D. CATTAERT 2 , E. DELPIRE 3 , M. MARSALA 4 , *L. R. VINAY 1 ;<br />
1 2 3<br />
CNRS, Marseille, Fr<strong>an</strong>ce; CNRS, Bordeaux, Fr<strong>an</strong>ce; V<strong>an</strong>derbilt Univ., Nashville, TN;<br />
4<br />
UCSD, S<strong>an</strong> Diego, CA<br />
Abstract: Spasticity which is commonly observed after spinal cord injury (SCI) is mainly<br />
characterized by hyperreflexia <strong>an</strong>d is associated with decreased inhibitory processes in <strong>the</strong><br />
sublesional spinal cord.<br />
The opening of GABAA <strong>an</strong>d glycine receptor-gated chloride ch<strong>an</strong>nels inhibits neurons as a result<br />
of low intracellular chloride concentration <strong>an</strong>d hyperpolarized Cl - equilibrium potential. Low [Cl -<br />
]i is maintained by <strong>the</strong> Cl - -extruding K + -Cl - cotr<strong>an</strong>sporter KCC2. However, in immature neurons<br />
<strong>an</strong>d in some pathological conditions, GABA <strong>an</strong>d glycine have depolarizing <strong>an</strong>d even excitatory<br />
effects as a result of higher [Cl - ]i , <strong>an</strong>d this has been associated with lower KCC2 expression.<br />
We presently report that KCC2 expression is down-regulated in <strong>the</strong> lumbar spinal cord after<br />
thoracic tr<strong>an</strong>section or contusive SCI in both <strong>the</strong> acute <strong>an</strong>d chronic phase. Western blot <strong>an</strong>d<br />
immunohistochemical <strong>an</strong>alyses suggest that SCI alters <strong>the</strong> targeting of KCC2 to motoneuron<br />
membr<strong>an</strong>es. The observed 10-20% down-regulation of KCC2 was associated with a 10 mV<br />
depolarizing shift of EIPSPs, above VREST, after SCI. Such a 10 mV positive shift of EIPSP reduces<br />
<strong>the</strong> strength of inhibitory inputs.<br />
The H reflex is commonly used to assess <strong>the</strong> I-a afferents-mediated motoneuronal excitability.<br />
The magnitude of <strong>the</strong> H wave is normally attenuated by repeated activation at frequencies higher<br />
th<strong>an</strong> 0.1 Hz (rate dependent depression, RDD). After SCI, a progressive reduction of <strong>the</strong> RDD is<br />
associated with <strong>the</strong> development of hyperreflexia <strong>an</strong>d spasticity. In <strong>the</strong> present study, <strong>the</strong><br />
blockade of KCC2 with 30 µM [(dihydroindenyl)oxy]alk<strong>an</strong>oic acid (DIOA) reduced <strong>the</strong> RDD of<br />
<strong>the</strong> monosynaptic dorsal root-evoked response in vitro. In vivo, intra<strong>the</strong>cal (it) lumbar injection<br />
of 20 µg DIOA in adult intact rats was shown to reduce <strong>the</strong> RDD of <strong>the</strong> H reflex. RDD was also<br />
reduced in KCC2-depleted tr<strong>an</strong>sgenic mice. We also observed a 10% down-regulation of KCC2<br />
in rats presenting a reduced RDD 4-5 months after a spinal contusion.<br />
KCC2 expression is modulated by BDNF. We now show that it injection of 10 µg BDNF in<br />
intact rats reduced both KCC2 expression <strong>an</strong>d RDD of <strong>the</strong> H reflex 24h later. Conversely, when<br />
BDNF effects were blocked by it injection of a TrkB chimera be<strong>for</strong>e spinal tr<strong>an</strong>section, KCC2<br />
expression increased instead of decreasing after <strong>the</strong> lesion.<br />
These results argue in favour of a critical role of a down-regulation of KCC2 in <strong>the</strong> development
of hyperreflexia after SCI <strong>an</strong>d open a new field of investigations <strong>for</strong> <strong>the</strong> development of drugs<br />
aimed at preventing or better alleviating spasticity.<br />
Disclosures: P. Boulenguez, None; S. Liabeuf, None; R. Bos, None; H. Bras, None; C. Je<strong>an</strong>-<br />
Xavier, None; C. Brocard, None; A. Stil, None; D. Cattaert, None; E. Delpire, None; M.<br />
marsala, None; L.R. Vinay, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.9/DD45<br />
Topic: D.10.a. Plasticity<br />
Support: NIH Gr<strong>an</strong>t NS057399 To HQ<br />
NIH Gr<strong>an</strong>t NS16446 To JHK<br />
Christopher <strong>an</strong>d D<strong>an</strong>a Reeve Foundation To JHK<br />
NIH Gr<strong>an</strong>t EY014680 To ABB<br />
Title: Spinal cord injury <strong>an</strong>d multiple approaches of evaluation: Behavioral consequences,<br />
neuronal response properties in deprived somatosensory cortex, <strong>an</strong>d immunocytochemical<br />
ch<strong>an</strong>ges in <strong>the</strong> brainstem <strong>an</strong>d spinal cord<br />
Authors: *H. QI 1 , J. L. REED 2 , O. A. GHARBAWIE 1 , M. J. BURISH 2 , Z. ZHOU 3 , M. R.<br />
BERNARD 3 , A. B. BONDS 3,4 , J. H. KAAS 1 ;<br />
1 Dept Psychol, 2 Neurosci. Grad. Program, 3 Dept Biomed. Engin., 4 Dept Electrical Engin. <strong>an</strong>d<br />
Computer Sci., V<strong>an</strong>derbilt Univ., Nashville, TN<br />
Abstract: Lesions at <strong>the</strong> level <strong>the</strong> cervical dorsal spinal cord (SC) produce a perm<strong>an</strong>ent but<br />
topographically restricted loss of primary sensory afferents. This has served as <strong>an</strong> ideal model to<br />
study <strong>the</strong> effects of SC injury in primates. Import<strong>an</strong>t progress has been made in recent years;<br />
however, much remains to be learned be<strong>for</strong>e we fully comprehend <strong>the</strong> relationship between<br />
neuronal plasticity, recovery, <strong>an</strong>d behavioral per<strong>for</strong>m<strong>an</strong>ce. We hypo<strong>the</strong>size that <strong>the</strong> reactivated<br />
cortical neurons have response properties beneficial to behavioral recovery, regenerative <strong>an</strong>d<br />
recovery-promoting histological ch<strong>an</strong>ges in brainstem <strong>an</strong>d SC are import<strong>an</strong>t <strong>an</strong>atomical<br />
substrates <strong>for</strong> recovery at higher levels of <strong>the</strong> processing network. To test this hypo<strong>the</strong>sis,
incomplete dorsal column (DC) sections at <strong>the</strong> C4-7 level of <strong>the</strong> SC were made in five New<br />
World owl <strong>an</strong>d squirrel monkeys. Sensorimotor functions of h<strong>an</strong>d use be<strong>for</strong>e <strong>an</strong>d after DC<br />
section were evaluated in a reaching <strong>an</strong>d retrieving task. A 100-electrode array was used to<br />
record <strong>from</strong> large numbers of neurons in deprived somatosensory cortex during tactile h<strong>an</strong>d<br />
stimulation in <strong>an</strong>es<strong>the</strong>tized monkeys. We characterized response properties of neurons in<br />
deprived somatosensory cortex to tactile stimulation by computing <strong>the</strong> peak firing rates <strong>an</strong>d onset<br />
latencies with MatLab software. The extent of <strong>the</strong> SC lesion <strong>an</strong>d consequences of morphological<br />
ch<strong>an</strong>ges in brainstem <strong>an</strong>d SC were investigated with neuro<strong>an</strong>atomical tracing <strong>an</strong>d<br />
immunocytochemical approaches. The main findings are: (1) h<strong>an</strong>d use was initially impaired but<br />
recovered 4-6 weeks post operation; <strong>the</strong> degree of <strong>the</strong> behavioral impairment of h<strong>an</strong>d in a food<br />
retrieval task after a DC lesion was correlated with <strong>the</strong> extent of <strong>the</strong> lesion, such that <strong>the</strong> lesser<br />
<strong>the</strong> damage to <strong>the</strong> DC <strong>an</strong>d/or <strong>the</strong> lower <strong>the</strong> cervical SC level of <strong>the</strong> lesion (e.g., C6-7), <strong>the</strong> lesser<br />
<strong>the</strong> impairment on <strong>the</strong> behavioral task; (2) <strong>the</strong> preserved inputs <strong>from</strong> <strong>the</strong> h<strong>an</strong>d activated larger<br />
cortical territories th<strong>an</strong> normal, <strong>an</strong>d <strong>the</strong> somatotopic org<strong>an</strong>izations of h<strong>an</strong>d representations were<br />
abnormal; (3) <strong>the</strong> reactivated neuronal population had larger response fields <strong>an</strong>d slightly longer<br />
me<strong>an</strong> latencies th<strong>an</strong> those of normal monkeys; <strong>an</strong>d (4) <strong>the</strong> immunoreactivity of brain derived<br />
neurotrophic factors dramatically increased in <strong>the</strong> white matter of <strong>the</strong> SC <strong>an</strong>d brainstem on <strong>the</strong><br />
lesioned side following behavioral recovery. The results suggested that endogenous<br />
neurotrophins play import<strong>an</strong>t roles in axon repair, that may prevent fur<strong>the</strong>r axon degeneration,<br />
<strong>an</strong>d ultimately promote new growth after injury. The results are consistent with <strong>the</strong> hypo<strong>the</strong>sis<br />
that reactivated neurons responsive to spared DC inputs play a useful sensory role in <strong>the</strong><br />
recovery of h<strong>an</strong>d use.<br />
Disclosures: H. Qi, None; J.L. Reed, None; O.A. Gharbawie, None; M.J. Burish, None; Z.<br />
Zhou, None; M.R. Bernard, None; A.B. Bonds, None; J.H. Kaas, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.10/DD46<br />
Topic: D.10.a. Plasticity<br />
Support: NIH Gr<strong>an</strong>t NS043246<br />
ISRT Gr<strong>an</strong>t ISRT-STR100<br />
Title: Alterations in chondroitin sulfate proteoglyc<strong>an</strong> expression both at <strong>an</strong>d distal to <strong>the</strong> site of<br />
spinal cord injury in rats
Authors: *E. M. ANDREWS 1,2 , Q. F. FENG YIN 1,2 , M. S. VIAPIANO 3 , L. B. JAKEMAN 1,2 ;<br />
1 2 3<br />
Physiol & Cell Biol, Ctr. <strong>for</strong> Brain <strong>an</strong>d Spinal Cord Repair, Ctr. <strong>for</strong> Mol. Neurobio., Ohio<br />
State Univ., Columbus, OH<br />
Abstract: The limited recovery of function after incomplete spinal cord injury (SCI) is due, in<br />
part, to <strong>the</strong> restricted capacity of <strong>the</strong> remaining circuitry of adult spinal cord to respond to<br />
physiological activation. The composition <strong>an</strong>d distribution of extracellular matrix molecules<br />
including chondroitin sulfate proteoglyc<strong>an</strong>s (CSPGs) contribute to limited plasticity in <strong>the</strong> adult<br />
nervous system. While m<strong>an</strong>y current approaches are directed at enh<strong>an</strong>cing regeneration <strong>an</strong>d<br />
sprouting through modification of CSPGs at <strong>the</strong> injury site, we propose that ch<strong>an</strong>ges in <strong>the</strong> CSPG<br />
expression <strong>an</strong>d composition resulting <strong>from</strong> subsequent denervation of dist<strong>an</strong>t neuropil regions<br />
also contribute to limited recovery. In <strong>the</strong> present study, we investigated CSPG expression in <strong>the</strong><br />
lesion epicenter, <strong>the</strong> cervical enlargement <strong>an</strong>d <strong>the</strong> lumbar enlargement at 3, 7, 14 <strong>an</strong>d 28 days<br />
after severe spinal contusion injury in <strong>the</strong> adult female rat. The levels of total CSPGs were not<br />
ch<strong>an</strong>ged signific<strong>an</strong>tly one week after injury in <strong>an</strong>y regions as seen with Western blots using 6S,<br />
4S <strong>an</strong>d 0S stub <strong>an</strong>tibodies. However, ch<strong>an</strong>ges in key core CSPG proteins were found at all levels.<br />
Neuroc<strong>an</strong> is expressed both by neurons <strong>an</strong>d reactive astrocytes after injury. As expected, full<br />
length neuroc<strong>an</strong> (275 kDa), was increased at <strong>the</strong> lesion epicenter 7 days post injury (dpi), <strong>an</strong>d<br />
remained elevated throughout <strong>the</strong> 28 day time point. In addition this species was elevated in both<br />
<strong>the</strong> cervical <strong>an</strong>d lumbar enlargements as early as 3 dpi <strong>an</strong>d also remained elevated throughout <strong>the</strong><br />
course of <strong>the</strong> study. The prominent cleavage product of neuroc<strong>an</strong> (150 kDa b<strong>an</strong>d) followed <strong>the</strong><br />
same pattern, with <strong>the</strong> highest expression at 7 dpi <strong>an</strong>d elevated expression through 28 dpi in all<br />
three regions. Both full length brevic<strong>an</strong> (produced by astrocytes, oligodendrocytes <strong>an</strong>d neurons)<br />
<strong>an</strong>d <strong>the</strong> prominent brevic<strong>an</strong> cleavage product decreased in <strong>the</strong> lesion epicenter 3 dpi as compared<br />
to laminectomy controls, while expression of brevic<strong>an</strong> in <strong>the</strong> cervical <strong>an</strong>d lumbar enlargements<br />
showed tr<strong>an</strong>sient ch<strong>an</strong>ges. Finally, aggrec<strong>an</strong> was dramatically decreased at <strong>the</strong> lesion epicenter at<br />
all time points. Aggrec<strong>an</strong> expression showed small decreases in <strong>the</strong> cervical spinal cord <strong>an</strong>d<br />
increased expression in <strong>the</strong> lumbar spinal cord at 14 <strong>an</strong>d 28 dpi. Histological studies also indicate<br />
ch<strong>an</strong>ges in <strong>the</strong> expression pattern <strong>an</strong>d location of CSPG ch<strong>an</strong>ges after injury. These results<br />
demonstrate that CSPG ch<strong>an</strong>ges are widespread after spinal contusion injury <strong>an</strong>d that <strong>the</strong>rapies<br />
targeted at m<strong>an</strong>ipulating <strong>the</strong> extracellular matrix both at <strong>an</strong>d far away <strong>from</strong> <strong>the</strong> lesion site may<br />
improve <strong>the</strong> prospects of recovery after SCI.<br />
Disclosures: E.M. Andrews, None; Q.F. Feng Yin, None; M.S. Viapi<strong>an</strong>o, None; L.B.<br />
Jakem<strong>an</strong>, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 563.11/DD47<br />
Topic: D.10.a. Plasticity<br />
Support: Swiss National Science Foundation<br />
NCCR "Neural plasticity <strong>an</strong>d Repair"<br />
Spinal Cord Consortium of <strong>the</strong> Christopher <strong>an</strong>d D<strong>an</strong>a Reeve Foundation<br />
EU-NeuroNe Network of Excellence<br />
Title: Anatomical plasticity in <strong>the</strong> brain stem <strong>an</strong>d propriospinal networks accomp<strong>an</strong>y locomotor<br />
recovery after incomplete spinal cord injury in rats<br />
Authors: *S. KAPITZA 1 , B. ZÖRNER 2 , L. FILLI 2 , R. GONZENBACH 2 , M.<br />
RÖTHLISBERGER 2 , M. SCHWAB 2 ;<br />
1 Zurich, Switzerl<strong>an</strong>d; 2 Univ. of Zurich, Zurich, Switzerl<strong>an</strong>d<br />
Abstract: Following incomplete spinal cord injury (SCI) <strong>an</strong>d in <strong>the</strong> absence of long-dist<strong>an</strong>ce<br />
axonal regeneration, compensatory sprouting of uninjured corticospinal (CST) fibers is a<br />
fundamental mech<strong>an</strong>ism of spont<strong>an</strong>eous functional recovery. In addition, lesioned CST fibers<br />
c<strong>an</strong> establish new contacts with propriospinal interneurons resulting in <strong>the</strong> <strong>for</strong>mation of detour<br />
pathways bridging <strong>the</strong> lesion site. We hypo<strong>the</strong>sized that <strong>an</strong>atomical plastic ch<strong>an</strong>ges are also<br />
present in descending brain stem systems contributing, in combination with rearr<strong>an</strong>gements of<br />
intraspinal networks, to functional recovery after incomplete SCI. Adult rats received a cervical<br />
(C4) unilateral hemisection of <strong>the</strong> spinal cord. Whereas basic hindlimb locomotor functions<br />
crucial <strong>for</strong> walking <strong>an</strong>d swimming improved signific<strong>an</strong>tly within <strong>the</strong> first weeks after injury <strong>the</strong>re<br />
was no spont<strong>an</strong>eous recovery of fine motor control of <strong>the</strong> <strong>for</strong>e- <strong>an</strong>d hindlimbs. Antero- <strong>an</strong>d<br />
retrograde tracing indicated <strong>an</strong>atomical ch<strong>an</strong>ges in descending brain stem <strong>an</strong>d propriospinal<br />
projections in response to <strong>the</strong> injury. Rostral to <strong>the</strong> injury, massive sprouting of lesioned<br />
reticulospinal fibers was observed. In addition, <strong>the</strong> number of spared reticulospinal fibers<br />
crossing <strong>the</strong> midline below <strong>the</strong> lesion site was signific<strong>an</strong>tly increased. Sprouting of propriospinal<br />
interneurons suggested <strong>the</strong> <strong>for</strong>mation of novel intraspinal pathways capable of conveying<br />
supraspinal input around <strong>the</strong> injury site to <strong>the</strong> denervated spinal networks below. This <strong>an</strong>atomical<br />
plasticity might contribute to <strong>the</strong> subst<strong>an</strong>tial hindlimb locomotor recovery seen after incomplete<br />
SCI in rats.<br />
Disclosures: S. Kapitza, None; B. Zörner, None; L. Filli, None; R. Gonzenbach, None; M.<br />
Röthlisberger, None; M. Schwab, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.12/DD48<br />
Topic: D.10.a. Plasticity<br />
Support: NIH R01 NS050699<br />
Dept. of Veter<strong>an</strong>s Affairs<br />
VA RR&D Center<br />
Title: Plasticity of propriospinal <strong>an</strong>d rubrospinal neurons following thoracic hemisection<br />
Authors: *A. E. BLUM 1,2,3 , S. C. JEFFERSON 1,2,3 , S. E. MONDELLO 1,2,3 , D. R.<br />
HOWLAND 1,2,3 ;<br />
1 Brain Rehabil. Res. Center, Malcom R<strong>an</strong>dall VAMC, Gainesville, FL; 2 Neurosci., Univ. of<br />
Florida, Gainesville, FL; 3 McKnight Brain Inst., Gainesville, FL<br />
Abstract: Due to <strong>the</strong> role of long descending supraspinal systems in voluntary movements,<br />
including goal-directed locomotion, m<strong>an</strong>y studies have focused on promoting regeneration<br />
<strong>an</strong>d/or collateral sprouting of <strong>the</strong>se pathways around or through a lesion site. Recent evidence,<br />
however, suggests that functional recovery following incomplete spinal cord injuries (SCI) in<br />
rodents may be supported by propriospinal neurons (PSNs) <strong>for</strong>ming bypass circuitry around <strong>the</strong><br />
lesions. The present study assesses <strong>the</strong> potential plasticity of intraspinal pathways, long <strong>an</strong>d short<br />
PSNs, as well as a descending supraspinal pathway, <strong>the</strong> rubrospinal tract (RST), following SCI<br />
in <strong>the</strong> cat. Three groups of locomotor trained cats are studied: normal controls, acute injuries, <strong>an</strong>d<br />
chronic injuries. All cats, independent of group, receive Flouro-Gold (FG) injections at spinal<br />
T11/12. Normal cats do not receive spinal hemisections. Cats with acute injuries receive left<br />
spinal T10 hemisections followed by FG injections during <strong>the</strong> same surgical procedures. Cats<br />
with chronic injuries receive FG injections ~16 weeks post-hemisection. All tissue is harvested<br />
13 days after FG injections. Retrogradely labeled FG neurons are counted in tissue sections <strong>from</strong><br />
C5, T6, T8, <strong>an</strong>d <strong>the</strong> level of <strong>the</strong> red nucleus (RN). Acutely, following injury, both short <strong>an</strong>d long<br />
PSNs with axons below <strong>the</strong> level of <strong>the</strong> hemisection show signific<strong>an</strong>t decreases bilaterally<br />
compared to normal controls. By 16 weeks, signific<strong>an</strong>t increases in <strong>the</strong> numbers of short PSNs<br />
are seen bilaterally at both T6 <strong>an</strong>d T8. These increases are greatest at T8 <strong>an</strong>d on <strong>the</strong> contralateral<br />
side <strong>for</strong> both segments. A signific<strong>an</strong>t decrease in C5 PSNs is seen acutely, but <strong>an</strong> increase is not<br />
detected between acute <strong>an</strong>d chronic time points. Because <strong>the</strong> rubrospinal tract crosses at <strong>the</strong> level<br />
of <strong>the</strong> RN <strong>an</strong>d <strong>the</strong>se fibers remain lateralized, <strong>the</strong> left RN is used as a control <strong>an</strong>d <strong>the</strong> number of<br />
right (experimental) RN neurons are assessed as a percentage of <strong>the</strong> left RN (uninjured). Acutely,<br />
<strong>the</strong> right RN shows a signific<strong>an</strong>t decrease in <strong>the</strong> percent of retrogradely labeled neurons to
hemisection <strong>an</strong>d resulting axotomy. Specifically, of <strong>the</strong> systems studied, <strong>the</strong> short PSNs in close<br />
proximity to <strong>the</strong> injury show <strong>the</strong> greatest potential <strong>for</strong> <strong>for</strong>ming novel bypass circuits past <strong>the</strong><br />
lesion site following incomplete spinal cord injuries.<br />
Disclosures: A.E. Blum, None; S.C. Jefferson, None; S.E. Mondello, None; D.R. Howl<strong>an</strong>d,<br />
None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.13/DD49<br />
Topic: D.10.a. Plasticity<br />
Support: Rom<strong>an</strong> Reed Spinal Cord Injury Fund of Cali<strong>for</strong>nia, Gr<strong>an</strong>t RR 07-199<br />
NIH Gr<strong>an</strong>t R56 NS048425-05<br />
Title: Adult neurogenesis in rat sensorimotor cortex following a cervical dorsal root injury<br />
Authors: *M. VESSAL 1 , J. SUN 2 , J. FALCONE 2 , A. LILAK 1 , C. DARIAN-SMITH 1 ;<br />
1 Dept Comparative Med., 2 Hum<strong>an</strong> Biol. Program, St<strong>an</strong><strong>for</strong>d Univ. Sch. Med., St<strong>an</strong><strong>for</strong>d, CA<br />
Abstract: Adult neurogenesis occurs under normal conditions in two neurogenic areas in <strong>the</strong><br />
brain: <strong>the</strong> subgr<strong>an</strong>ular zone (SGZ) of <strong>the</strong> dentate gyrus <strong>an</strong>d in <strong>the</strong> subventriular zone (SVZ) of<br />
<strong>the</strong> lateral ventricles. It also occurs in adult rats following certain pathological conditions such as<br />
epilepsy, ischemia <strong>an</strong>d neurodegenerative disorders, <strong>an</strong>d in primates following a cervical dorsal<br />
rhizotomy (Vessal et al., 2007. Eur J. Neurosci 26:2777-2794). We recently found that<br />
neurogenesis is induced in <strong>the</strong> primate somatosensory <strong>an</strong>d motor cortex following a cervical<br />
dorsal rhizotomy (Vessal <strong>an</strong>d Dari<strong>an</strong>-Smith, unpublished). Here our goal was to determine<br />
whe<strong>the</strong>r this <strong>for</strong>m of cortical neurogenesis also occurs in <strong>the</strong> adult rat sensorimotor cortex<br />
following <strong>the</strong> same lesion. Ten rats were used (n = 6, lesion; n = 4, control). Animals in <strong>the</strong><br />
lesion group received a unilateral dorsal rhizotomy (Left) at cervical level C6-C8 which<br />
deafferented <strong>the</strong>ir left <strong>for</strong>epaw. Rats were injected (i.p.) with bromodeoxyuridine (BrdU) 1 week<br />
post-lesion to label dividing cells. At 4 weeks post-BrdU injections (5 weeks post-lesion)<br />
<strong>an</strong>imals were given <strong>an</strong> overdose of sodium pentobarbital <strong>an</strong>d upon cessation of breathing, were<br />
perfused tr<strong>an</strong>scardially. Sections were cut (40µm) through <strong>the</strong> entire rostrocaudal extent of <strong>the</strong><br />
primary somatosensory <strong>an</strong>d motor cortex. Section series (every 240, or 480µm) were<br />
immunoprocessed with <strong>an</strong>tibodies against (BrdU), <strong>an</strong>d one of <strong>the</strong> following neuronal markers:
NeuN, calbindin (CB), neuron specific enolase (NSE), GABA <strong>an</strong>d glutamic acid decarboxylase<br />
67 (GAD67). BrdU/NeuN, <strong>an</strong>d BrdU/CB colabeled cells were qu<strong>an</strong>tified through <strong>the</strong><br />
sensorimotor cortex, <strong>an</strong>d total numbers estimated using <strong>the</strong> fractionator method. Comparisons<br />
were made bilaterally as well as with <strong>the</strong> control group. A signific<strong>an</strong>t number (P
movements recovered within a few months after lesion of <strong>the</strong> corticospinal tract (CST) by <strong>the</strong><br />
compensatory ch<strong>an</strong>ges in activation patterns of motor-related cortical areas, especially <strong>the</strong><br />
bilateral primary motor area (M1) at early recovery stage <strong>an</strong>d more extensive region of <strong>the</strong><br />
contralesional M1 <strong>an</strong>d bilateral ventral premotor area (PMv) at late recovery stage (Nishimura et<br />
al., 2007). To assess <strong>the</strong> molecular basis <strong>for</strong> this functional compensation, we investigated<br />
comprehensive gene expression profiles of M1 <strong>an</strong>d dorsal <strong>an</strong>d ventral part of PMs in CSTlesioned<br />
macaque monkeys during recovery (2 weeks after lesion <strong>for</strong> <strong>the</strong> early recovery stage; 3<br />
months after lesion <strong>for</strong> <strong>the</strong> late recovery stage) <strong>an</strong>d non-lesioned monkeys (<strong>for</strong> <strong>the</strong> pre-recovery<br />
stage) with DNA microarray method. We observed that about 300-500 genes were differentially<br />
expressed among recovery stages in each motor-related area. Fur<strong>the</strong>rmore, <strong>the</strong> number of genes<br />
that differentially expressed among motor-related areas at early recovery stage was markedly<br />
smaller th<strong>an</strong> those at <strong>the</strong> pre- <strong>an</strong>d late recovery stages. The dynamic ch<strong>an</strong>ges in gene expression<br />
patterns during recovery may represent <strong>the</strong> molecular mech<strong>an</strong>isms of functional compensation in<br />
each motor-related area after CST lesion.<br />
Disclosures: A. Sato, None; Y. Nishimura, None; T. Oishi, None; N. Higo, None; Y. Murata,<br />
None; H. Onoe, None; K. Yoshino-Saito, None; F. Tsuboi, None; M. Takahashi, None; T.<br />
Isa, None; T. Kojima, None.<br />
Poster<br />
563. Spinal Cord Injury: Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 563.15/DD51<br />
Topic: D.10.b. Injury <strong>an</strong>d recovery<br />
Title: Expression of cell fate determin<strong>an</strong>ts <strong>an</strong>d plastic ch<strong>an</strong>ges after neurotoxic lesion of adult<br />
mice spinal cord by cholera toxin-B saporin<br />
Authors: R. GULINO, *M. GULISANO;<br />
Lab. Mol Biol / Dept Physiol Sci., Univ. Cat<strong>an</strong>ia, Cat<strong>an</strong>ia, Italy<br />
Abstract: Recent studies have attempted to achieve recovery after spinal cord (SC) injury or<br />
disease by ei<strong>the</strong>r increase neurogenesis or stimulate neuroplasticity. Sonic hedgehog (Shh)<br />
Notch-1 <strong>an</strong>d Numb are involved in <strong>the</strong> regulation of stem cell function. Additionally, Notch-1<br />
has a role as modulator of synaptic plasticity. Little is known about <strong>the</strong> role of <strong>the</strong>se proteins in<br />
<strong>the</strong> adult SC after selective removal of motoneurons. We injected Cholera toxin-B saporin into<br />
<strong>the</strong> gastrocnemius muscle to induce a selective depletion of motoneurons within lumbar mice SC<br />
<strong>an</strong>d <strong>an</strong>alysed <strong>the</strong> expression levels of Shh, Notch-1, Numb, Choline acetyltr<strong>an</strong>sferase (ChAT)
<strong>an</strong>d Synapsin-I proteins. The functional outcome of <strong>the</strong> lesion was monitored by grid walk test<br />
<strong>an</strong>d rotarod. The neurotoxin lesion determined a motoneuron depletion <strong>an</strong>d a decrease of ChAT<br />
<strong>an</strong>d Synapsin-I protein levels in <strong>the</strong> lumbar SC. ChAT <strong>an</strong>d Synapsin-I appeared correlated each<br />
o<strong>the</strong>r <strong>an</strong>d with <strong>the</strong> motor per<strong>for</strong>m<strong>an</strong>ce, suggesting that <strong>the</strong> recovery of locomotion could depend<br />
<strong>from</strong> synaptic plasticity. Moreover, we observed a number of proliferating cells within <strong>the</strong><br />
depleted SC, which were identified as active astrocytes. Shh <strong>an</strong>d Notch-1 appeared reduced in<br />
<strong>the</strong> lesioned tissue <strong>an</strong>d correlated with ChAT <strong>an</strong>d Synapsin-I levels, suggesting a role in<br />
modulating synaptic plasticity. Numb expression was also reduced after lesion <strong>an</strong>d appeared<br />
correlated with motor per<strong>for</strong>m<strong>an</strong>ce There<strong>for</strong>e, given <strong>the</strong> role of <strong>the</strong>se proteins in adult<br />
neurogenesis, we presume <strong>the</strong>ir involvement also in <strong>the</strong> observed glial reaction. The in vivo<br />
m<strong>an</strong>ipulation of Shh, Notch-1 <strong>an</strong>d Numb signalling after lesion could be a way to reduce glial<br />
reaction <strong>an</strong>d improve functional recovery.<br />
Disclosures: R. Gulino, None; M. Gulis<strong>an</strong>o, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.1/DD52<br />
Topic: D.11.d. Afferent control<br />
Support: NIH gr<strong>an</strong>t HL68007<br />
Title: Respiratory network targets of central chemosensory modulation are identified in <strong>the</strong><br />
neonate rat tilted sagittal slab preparation<br />
Authors: *N. M. MELLEN;<br />
Kosair Children's Hosp Res. Inst., Univ. Louisville, Louisville, KY<br />
Abstract: Respiration is strongly modulated by central chemosensory inputs. The retrotrapezoid<br />
nucleus (RTN), which lies ventral <strong>an</strong>d slightly medial to <strong>the</strong> facial nucleus (VIIn), contains<br />
neurons that fire briskly during focal acidosis, <strong>an</strong>d <strong>the</strong>ir firing is associated with increases in<br />
inspiratory amplitude <strong>an</strong>d respiratory frequency in vivo. Little is known about <strong>the</strong> effects of RTN<br />
modulation on respiratory neurons, characterized here via optical recording. The effect of focal<br />
acidosis on respiratory rhythm, pattern, <strong>an</strong>d network constituents was characterized in <strong>the</strong> tilted<br />
sagittal slab preparation (n=6), isolated <strong>from</strong> neonate (P0-P3) Sprague-Dawley rats, tr<strong>an</strong>sected<br />
first rostral to <strong>the</strong> pontine nuclei <strong>an</strong>d <strong>the</strong>n at <strong>the</strong> rostral margin of <strong>the</strong> VIIn. Preparations were<br />
incubated 1 h with <strong>the</strong> low-affinity Ca 2+ indicator fluo-8L AM (KD=1.86 µM). Optical recordings
(3 Hz) of respiratory network activity were made over ~ 700 µm extending caudally <strong>from</strong> <strong>the</strong><br />
caudal pole of VIIn, <strong>an</strong>d inspiratory bursts were recorded <strong>from</strong> ventral root C2. Recordings were<br />
made be<strong>for</strong>e <strong>an</strong>d after ponto-medullary tr<strong>an</strong>saction; each recording epoch lasted 360 s, with<br />
acidotic puff (6.9 pH) applied at <strong>the</strong> level of VIIn <strong>for</strong> 20 s mid-way through <strong>the</strong> recording. In<br />
preparations retaining pontine structures, acidotic puff led to a biphasic response: respiratory<br />
frequency tr<strong>an</strong>siently increased, <strong>the</strong>n slowed relative to control. In medullary preparations,<br />
acidotic puff produced <strong>an</strong> increase in respiratory frequency (158±22%). Respiratory neurons in<br />
pons-attached (n=125) <strong>an</strong>d medullary (n=94) preparations were recorded. Puff-induced ch<strong>an</strong>ges<br />
in firing pattern included tr<strong>an</strong>sient inhibition, excitation <strong>an</strong>d recruitment of respiratory neurons.<br />
In pons-attached preparations, larger numbers of neurons were excited or inhibited, while in<br />
medullary preparations, larger numbers were recruited. By comparing acidosis-induced ch<strong>an</strong>ges<br />
in firing pattern be<strong>for</strong>e <strong>an</strong>d after pontomedullary tr<strong>an</strong>section, <strong>the</strong> role of respiratory network<br />
constituents in <strong>the</strong> tr<strong>an</strong>smission of chemosensory drive originating <strong>from</strong> RTN is inferred.<br />
Disclosures: N.M. Mellen, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.2/DD53<br />
Topic: D.11.d. Afferent control<br />
Support: Russi<strong>an</strong> Foundation <strong>for</strong> Basic Research <strong>an</strong>d U.S. Civili<strong>an</strong> Research <strong>an</strong>d Development<br />
Foundation Gr<strong>an</strong>t RUB1-2872-ST-07<br />
NIH Gr<strong>an</strong>t NS 06<strong>2009</strong>-01A1<br />
Title: Novel access to <strong>the</strong> cat <strong>an</strong>d hum<strong>an</strong> locomotor circuitry<br />
Authors: *Y. P. GERASIMENKO 1,2 , R. GORODNICHEV 5 , P. MUSIENKO 1 , R. R. ROY 2,3 ,<br />
V. R. EDGERTON 2,3,4 ;<br />
1 Pavlov Inst. of Physiol, St Petersburg, Russi<strong>an</strong> Federation; 2 Dept. of Physiological Sci., 3 Brain<br />
Res. Inst., 4 Dept. of Neurobio., Univ. of Cali<strong>for</strong>nia, Los Angeles, CA; 5 Velikie Luky State Acad.<br />
of Physical Educ. <strong>an</strong>d Sport, Velikie Luky, Russi<strong>an</strong> Federation<br />
Abstract: The mammali<strong>an</strong> lumbar spinal cord has neuronal circuits that c<strong>an</strong> generate locomotor<br />
patterns in response to pharmacological or electrical stimulation of <strong>the</strong> dorsum of <strong>the</strong> spinal cord.<br />
Here we present evidence that locomotor-like activity c<strong>an</strong> be induced in non-injured hum<strong>an</strong>s as
well as in decerebrated cats by spinal electromagnetic stimulation (SEMS). SEMS (Magstim-<br />
Rapid2) at 3 Hz <strong>an</strong>d <strong>an</strong> intensity of ~2 Tesla applied continuously over <strong>the</strong> T11-T12 vertebrae<br />
induced involuntary locomotor-like stepping movements in subjects with <strong>the</strong>ir legs in a gravityindependent<br />
position. The kinematics of <strong>the</strong>se movements was similar to that of voluntary air<br />
stepping, although <strong>the</strong> amplitude of <strong>the</strong> knee joint excursion during SEMS was less th<strong>an</strong> during<br />
voluntary air stepping. The bursts of flexor <strong>an</strong>d extensor EMG activity were not tightly timelinked<br />
to <strong>the</strong> individual stimuli of <strong>the</strong> SEMS. Averaged responses within <strong>the</strong> EMG bursts to<br />
single stimuli did not induce reflex responses, suggesting that SEMS more directly activates <strong>the</strong><br />
locomotor-generating circuits as opposed to more indirect activation via afferents. During SEMS<br />
<strong>the</strong> latency of <strong>the</strong> activation of flexor locomotor circuits r<strong>an</strong>ged <strong>from</strong> 85-95 ms, whereas <strong>the</strong><br />
latency of extensor circuits r<strong>an</strong>ged <strong>from</strong> 130-190 ms. Vibration of <strong>the</strong> leg muscles fur<strong>the</strong>r<br />
facilitated <strong>the</strong> involuntary stepping movements induced by SEMS. In decerebrated cats SEMS of<br />
<strong>the</strong> lumbar spinal cord at 3-5 Hz was more effective th<strong>an</strong> epidural spinal cord stimulation at L5<br />
in inducing well-coordinated hindlimb stepping on <strong>the</strong> treadmill. SEMS over C4 at 3-5 Hz at<br />
intensity of ~0.4 Tesla induced robust quadrupedal locomotion on <strong>the</strong> moving treadmill.<br />
Quadrupedal locomotion continued <strong>for</strong> ~30 sec after <strong>the</strong> termination of SEMS. Combined, <strong>the</strong>se<br />
data demonstrate <strong>the</strong> potential of a non-invasive me<strong>an</strong>s of stimulating <strong>the</strong> spinal cord (SEMS)<br />
that c<strong>an</strong> be used to directly activate locomotor circuits, thus providing a new tool <strong>for</strong> identifying<br />
spinal locomotor circuits <strong>an</strong>d facilitating locomotion after a spinal cord injury.<br />
Disclosures: Y.P. Gerasimenko, None; R. Gorodnichev, None; P. Musienko, None; R.R.<br />
Roy, None; V.R. Edgerton, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.3/DD54<br />
Topic: D.11.d. Afferent control<br />
Support: NINDS/NIH gr<strong>an</strong>t R01 NS048844<br />
Title: Role of afferent feedback in <strong>the</strong> recovery of locomotor activity after SCI: Insights <strong>from</strong> a<br />
simple neuromech<strong>an</strong>ical model<br />
Authors: *S. N. MARKIN 1 , A. N. KLISHKO 2 , N. A. SHEVTSOVA 1 , M. LEMAY 1 , K.<br />
OLLIVIER-LANVIN 1 , B. I. PRILUTSKY 2 , I. A. RYBAK 1 ;<br />
1 Drexel Univ. Col. of Med., Philadelphia, PA; 2 Sch. of Applied Physiology, Ctr. <strong>for</strong> Hum<strong>an</strong><br />
Movement Studies, Georgia Inst. of Technol., Atl<strong>an</strong>ta, GA
Abstract: It is commonly accepted that restoration of locomotor function after spinal cord injury<br />
(SCI), e.g. by locomotor training, relies at least in part on <strong>the</strong> amplification of afferent inputs to<br />
<strong>the</strong> locomotor central pattern generator (CPG). However, <strong>the</strong> feasibility of sustaining <strong>the</strong><br />
locomotor pattern with this mech<strong>an</strong>ism has never been <strong>the</strong>oretically investigated. We have<br />
developed a simple neuro-mech<strong>an</strong>ical model that describes a locomotor CPG controlling<br />
movement of a simple biomech<strong>an</strong>ical system. The two-level CPG consists of a half-center<br />
rhythm generator (RG) <strong>an</strong>d pattern <strong>for</strong>mation (PF) circuits <strong>an</strong>d generates a basic “locomotor”<br />
rhythm while receiving tonic “supra-spinal” drive (<strong>an</strong>d is normally unable to generate<br />
oscillations without this drive). The controlled biomech<strong>an</strong>ical system represents a simplified<br />
model of a one-joint limb described as a pendulum that is <strong>for</strong>ced to oscillate by <strong>the</strong> <strong>for</strong>ces of two<br />
<strong>an</strong>tagonistic muscles, flexor <strong>an</strong>d extensor, <strong>the</strong> gravitational <strong>for</strong>ce, <strong>an</strong>d <strong>the</strong> ground reaction <strong>for</strong>ce;<br />
<strong>the</strong> latter acts during <strong>the</strong> “st<strong>an</strong>ce” phase, when <strong>the</strong> pendulum moves counterclockwise (<strong>an</strong>gular<br />
velocity is positive). Muscles are described by a Zajac-type model featuring <strong>for</strong>ce-length <strong>an</strong>d<br />
<strong>for</strong>ce-velocity properties. They are activated by <strong>the</strong> corresponding motoneurons, which in turn<br />
receive corresponding phasic excitation <strong>from</strong> <strong>the</strong> CPG (PF level). The muscles also provide<br />
length-dependent (Ia <strong>an</strong>d II types) <strong>an</strong>d <strong>for</strong>ce-dependent (Ib <strong>from</strong> <strong>the</strong> extensor) feedback to <strong>the</strong><br />
CPG, controlling both <strong>the</strong> timing of phase tr<strong>an</strong>sitions (flexion-extension <strong>an</strong>d extension-flexion)<br />
in <strong>the</strong> CPG (at <strong>the</strong> RG level) <strong>an</strong>d <strong>the</strong> activity of motoneurons via <strong>the</strong> PF circuits <strong>an</strong>d disynaptic<br />
excitation of <strong>the</strong> extensor motoneuron during extension. We show that <strong>the</strong> afferent feedback<br />
org<strong>an</strong>ization proposed in <strong>the</strong> model adjusts CPG operation to kinematics <strong>an</strong>d dynamics of <strong>the</strong><br />
controlled system providing stable “locomotion” within a wide r<strong>an</strong>ge of initial conditions <strong>an</strong>d<br />
during mech<strong>an</strong>ical perturbations. Elevation of supra-spinal drive to <strong>the</strong> CPG increases <strong>the</strong> speed<br />
of locomotion by reducing <strong>the</strong> duration of st<strong>an</strong>ce phase with a relatively const<strong>an</strong>t duration of<br />
swing. <strong>When</strong> <strong>the</strong> supra-spinal drive to <strong>the</strong> CPG is removed (to imitate SCI), <strong>the</strong> system fails to<br />
“locomote”. However, a subsequent increase in <strong>the</strong> weights of afferent feedback to <strong>the</strong> CPG<br />
(simulating <strong>the</strong> hypo<strong>the</strong>sized results of locomotor training) restores “locomotor capabilities” of<br />
<strong>the</strong> model. In <strong>the</strong> new operation regime, <strong>the</strong> afferent feedback produced by passive limb<br />
movement entrains <strong>the</strong> CPG activity which in turn provides stable locomotion. The role of<br />
different afferent inputs in <strong>the</strong> CPG entrainment is considered.<br />
Disclosures: S.N. Markin, None; A.N. Klishko, None; N.A. Shevtsova, None; M. Lemay,<br />
None; K. Ollivier-L<strong>an</strong>vin, None; B.I. Prilutsky, None; I.A. Rybak, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.4/DD55<br />
Topic: D.11.d. Afferent control
Support: C<strong>an</strong>adi<strong>an</strong> Institute of Health Research to J-P. Gossard<br />
Christopher <strong>an</strong>d D<strong>an</strong>a Reeve Foundation to A. Frigon<br />
Title: Different rhythm generators have specialized control mech<strong>an</strong>isms<br />
Authors: *A. FRIGON, J. SIROIS, J.-P. GOSSARD;<br />
Dept Physiol., Univ. Montreal, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Different rhythmic motor behaviors, are generated by shared <strong>an</strong>d specialized<br />
circuitry 1,2 but control mech<strong>an</strong>isms of different rhythm generators are unclear. We recorded 56<br />
episodes of fictive locomotion (n = 35) <strong>an</strong>d scratching (n = 21) in 39 adult cats to determine how<br />
different rhythm generators control cycle period (i.e. <strong>the</strong> time between two successive bursts in a<br />
given motor nerve). Cycle period (CP) c<strong>an</strong> spont<strong>an</strong>eously vary during <strong>an</strong> episode by ch<strong>an</strong>ging<br />
<strong>the</strong> duration of extension (extensor-dominated) or flexion (flexor-dominated), or both (covarying).<br />
Following pre-collicular/pre-mammillary decerebration, spont<strong>an</strong>eous fictive<br />
locomotion c<strong>an</strong> occur. Fictive scratching was evoked by applying a small cotton ball soaked in dtubocurarine<br />
topically at upper cervical levels <strong>an</strong>d by stimulating <strong>the</strong> pinna. The majority of<br />
fictive locomotor episodes were extensor-dominated (24/35 episodes; CP = 1212ms), whereas a<br />
smaller number covaried (6/35; CP = 909ms) or were flexor-dominated (3/35 episodes; CP =<br />
973ms). 2 episodes did not vary. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, fictive scratching was primarily flexordominated<br />
(16/21 episodes; CP = 321ms). Remaining episodes did not vary (5/21 episodes; CP =<br />
320ms).<br />
In 3 cats, we applied <strong>an</strong>kle dorsiflexion (DF) during episodes of fictive locomotion (1 cat, 4<br />
episodes) or scratching (2 cats, 6 episodes). During locomotion, DF increased CP <strong>from</strong> 803 to<br />
1206ms. Be<strong>for</strong>e dorsiflexion, all 4 locomotor episodes were extensor-dominated with a slope of<br />
extensor phase relative to CP (rext) of 0.66. During DF, all episodes remained extensordominated;<br />
rext increased to 1.05 <strong>an</strong>d extensor burst duration increased <strong>from</strong> 417 to 901ms.<br />
During scratching, CP was 282ms <strong>an</strong>d 285ms be<strong>for</strong>e <strong>an</strong>d during DF, respectively. Be<strong>for</strong>e DF, 5/6<br />
scratching episodes were flexor-dominated <strong>an</strong>d 1 was invari<strong>an</strong>t. During DF, 2 episodes remained<br />
flexor-dominated, 1 remained invari<strong>an</strong>t, 2 became invari<strong>an</strong>t, <strong>an</strong>d 1 became extensor-dominated.<br />
For <strong>the</strong> group, rext ch<strong>an</strong>ged <strong>from</strong> 0.18 be<strong>for</strong>e DF to 0.41 during DF. The slope of <strong>the</strong> flexor phase<br />
relative to CP decreased <strong>from</strong> 0.88 to 0.48 during DF.<br />
There<strong>for</strong>e, <strong>the</strong> locomotor rhythm generator alters CP by varying extension, whereas <strong>the</strong> scratch<br />
rhythm generator varies flexion. Preliminary results suggest that sensory inputs <strong>from</strong> stretchrelated<br />
receptors in <strong>an</strong>kle extensors streng<strong>the</strong>n extensor-domin<strong>an</strong>ce of <strong>the</strong> locomotor rhythm<br />
generator. Conversely, <strong>the</strong> same inputs appear to shift <strong>the</strong> bias in <strong>the</strong> control of CP by <strong>the</strong> spinal<br />
scratch rhythm generator. Thus, distinct rhythm generators have specialized control mech<strong>an</strong>isms.<br />
1. Berkowitz, A. (2008). J. Neurophysiol. 99, 2887-2901.<br />
2. Frigon, A. (<strong>2009</strong>). J. Neurophysiol. 101, 2201-2203.<br />
Disclosures: A. Frigon, None; J. Sirois, None; J. Gossard, None.<br />
Poster
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.5/DD56<br />
Topic: D.11.d. Afferent control<br />
Support: Idaho State University FRC Gr<strong>an</strong>t 68153720<br />
Title: Sensory feedback modulates quipazine-induced stepping in <strong>the</strong> neonatal rat<br />
Authors: *M. R. BRUMLEY, M. E. ROBERTO, M. M. STRAIN;<br />
Idaho State Univ., Pocatello, ID<br />
Abstract: Research has shown that locomotor behavior is modulated by sensory feedback in<br />
adult <strong>an</strong>imals. However, little is known about <strong>the</strong> role of sensory feedback in <strong>the</strong> modulation,<br />
development, <strong>an</strong>d neural mech<strong>an</strong>isms of locomotion during <strong>the</strong> perinatal period. In this study, we<br />
examined if locomotor activity in newborn rats is sensitive to cut<strong>an</strong>eous <strong>an</strong>d proprioceptive<br />
feedback. One-day old rats were treated with <strong>the</strong> serotonergic agonist quipazine, which<br />
previously has been shown to induce air-stepping behavior. During stepping, a substrate was<br />
placed beneath <strong>the</strong>ir limbs so that <strong>the</strong> feet could make pl<strong>an</strong>tar surface contact with a substrate.<br />
The entire test session was 50-min: after a 5-min baseline, subjects received a 0.05 ml i.p.<br />
injection of quipazine (3.0 mg/kg) or saline (vehicle control) <strong>an</strong>d a substrate (elastic, hard, or<br />
none) was placed beneath <strong>the</strong>ir limbs. Subjects treated with quipazine showed signific<strong>an</strong>tly more<br />
alternated steps compared to subjects treated with saline. Motor activity was modified by <strong>the</strong><br />
presence <strong>an</strong>d type of substrate. For example, although <strong>the</strong>re was no overall difference in <strong>the</strong><br />
number of hindlimb steps among quipazine-treated subjects in <strong>the</strong> different substrate conditions,<br />
quipazine-treated subjects made more steps in contact with <strong>the</strong> elastic substrate th<strong>an</strong> with <strong>the</strong><br />
hard substrate. Thus, limb trajectories during hindlimb stepping were altered by characteristics of<br />
<strong>the</strong> substrate. Quipazine-treated subjects also showed <strong>an</strong> overall lower percentage of movements<br />
made in contact with <strong>the</strong> hard substrate, suggesting that <strong>the</strong>y may have been avoiding contact in<br />
this substrate condition. The types of movements made on <strong>the</strong> substrates (paw pushes, taps,<br />
swipes, <strong>an</strong>d st<strong>an</strong>ce) were also qualitatively characterized. Findings suggest that sensory feedback<br />
modulates locomotor mech<strong>an</strong>isms <strong>an</strong>d behavior during <strong>the</strong> period of development in which <strong>the</strong><br />
neural mech<strong>an</strong>isms of locomotion are just beginning to <strong>for</strong>m. It remains to be determined how<br />
sensorimotor experiences during <strong>the</strong> perinatal period shape subsequent coordinated behavior,<br />
including adult locomotion.<br />
Disclosures: M.R. Brumley, None; M.E. Roberto, None; M.M. Strain, None.<br />
Poster
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.6/DD57<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t HD33862 to SRR<br />
NIH Gr<strong>an</strong>t NIGMS58939 to Iowa Biosciences Adv<strong>an</strong>tage<br />
Title: Perinatal Darwinism: Variation <strong>an</strong>d selection of coordinative patterns during limb activity<br />
support motor learning in <strong>the</strong> neonatal rat<br />
Authors: *S. R. ROBINSON, V. MENDEZ-GALLARDO, A. L. EINFELDT, M. C.<br />
SCHREIBER;<br />
Psychology, Univ. of Iowa, Iowa City, IA<br />
Abstract: Sporns <strong>an</strong>d Edelm<strong>an</strong> (Child Dev., 64, 960-998, 1993) proposed <strong>the</strong> dual processes of<br />
variation <strong>an</strong>d selection as mech<strong>an</strong>isms responsible <strong>for</strong> tr<strong>an</strong>s<strong>for</strong>ming <strong>the</strong> primary movement<br />
repertoire of <strong>the</strong> newborn inf<strong>an</strong>t into a more sophisticated array of motor synergies <strong>an</strong>d adaptive<br />
action patterns. More recently, our laboratory has applied a simple motor learning paradigm to<br />
confirm that fetal <strong>an</strong>d neonatal rats c<strong>an</strong> adaptively modify patterns of interlimb motor<br />
coordination in response to a biomech<strong>an</strong>ical challenge. To effect motor learning, perinatal rats<br />
experience <strong>an</strong> interlimb yoke that creates a physical linkage between two hindlimbs. After 30min<br />
of yoke training, subjects express conjugate limb movements—simult<strong>an</strong>eous limb<br />
movements with parallel trajectories—which persist after removal of <strong>the</strong> yoke. Conversely,<br />
en<strong>for</strong>cement of alternated limb movement promotes expression of <strong>an</strong>tiphase coordination after<br />
<strong>the</strong> limbs are released <strong>from</strong> constraint. The ability of <strong>the</strong> perinatal rat to modify motor<br />
coordination coincides with dramatic ch<strong>an</strong>ges in body size <strong>an</strong>d allometry, which create <strong>the</strong> need<br />
<strong>for</strong> recalibration of motor comm<strong>an</strong>ds by <strong>the</strong> developing motor system. The demonstration that<br />
fetal motor learning c<strong>an</strong> be supported by spinal circuitry alone implies that improved<br />
coordination after interlimb yoke training may result <strong>from</strong> relatively simple adaptive processes,<br />
such as Darwini<strong>an</strong> selection within a variable motor system. Meta-<strong>an</strong>alysis of previous<br />
experiments <strong>an</strong>d kinematic <strong>an</strong>alysis of interlimb coordination during yoke training provide<br />
convergent evidence that motor learning involves enh<strong>an</strong>ced variation early in training <strong>an</strong>d<br />
selective elimination of movement variation as training proceeds. By this selectionist hypo<strong>the</strong>sis,<br />
experimental enh<strong>an</strong>cement of motor activity should result in greater movement variation <strong>an</strong>d<br />
<strong>the</strong>reby increase <strong>the</strong> rate of motor learning. We evaluated this prediction in three experiments in<br />
which 1-day-old rat pups received sensory stimulation at repeated intervals during yoke training.<br />
Among pups receiving yoke training, <strong>an</strong>ogenital brush strokes <strong>an</strong>d tail pinch promoted general<br />
increases in <strong>the</strong> variability of hindlimb movement, but perioral cold stimulation did not.<br />
Anogenital <strong>an</strong>d tail stimulation also resulted in more rapid improvement in interlimb
coordination during training. These findings are consistent with a selectionist mech<strong>an</strong>ism,<br />
suggesting that <strong>the</strong> Darwini<strong>an</strong> processes of movement variation <strong>an</strong>d selection may be responsible<br />
<strong>for</strong> adaptive ch<strong>an</strong>ges in motor coordination during perinatal motor learning.<br />
Disclosures: S.R. Robinson, None; V. Mendez-Gallardo, None; A.L. Einfeldt, None; M.C.<br />
Schreiber, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.7/DD58<br />
Topic: D.11.d. Afferent control<br />
Support: BSF 2005020<br />
ISF 1930/08<br />
ISF 1591/08<br />
Title: Proprioneurons with multifunicular projections are activated by sacrocaudal afferents to<br />
turn on <strong>the</strong> pattern generating circuitry in <strong>the</strong> absence of supraspinal control<br />
Authors: A. ETLIN 1 , D. BLIVIS 2 , M. BEN ZWI 1 , *A. A. LEV-TOV 1 ;<br />
1 Hebrew Univ. Sch. of Med., Jerusalem, Israel; 2 NINDS, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: Sacrocaudal afferent (SCA) stimulation in isolated spinal cords of neonatal rats is<br />
used to study <strong>the</strong> neural pathways involved in sensory-activation of <strong>the</strong> thoracolumbar central<br />
pattern generators (CPGs). Surgical m<strong>an</strong>ipulations of <strong>the</strong> white matter revealed that <strong>the</strong> SCAinduced<br />
rhythm involves activation of segmental <strong>an</strong>d non-segmental sacral relay neurons<br />
projecting through <strong>the</strong> ventral, ventrolateral, <strong>an</strong>d dorsolateral funiculi (VF, VLF, <strong>an</strong>d DLF), that<br />
<strong>the</strong> contribution of VF projections is prominent, <strong>an</strong>d that activation of proprioneurons with short<br />
r<strong>an</strong>ge projections is sufficient to turn on <strong>the</strong> CPGs on SCA stimulation. Confocal imaging<br />
following fluorescent backfilling of funicular axon bundles revealed that <strong>the</strong> sacral relay neurons<br />
projecting via <strong>the</strong> VF <strong>an</strong>d VLF were distributed within laminae V, <strong>an</strong>d VII-X; but those<br />
projecting through <strong>the</strong> DLF were located in <strong>the</strong> ipsilateral laminae II-V, VII, <strong>an</strong>d <strong>the</strong> DLF itself.<br />
The VF projections were mainly crossed, while those of <strong>the</strong> VLF were predomin<strong>an</strong>tly uncrossed.<br />
Most of <strong>the</strong> crossed VLF axons ascended through <strong>the</strong> VF be<strong>for</strong>e joining <strong>the</strong> VLF. There<strong>for</strong>e,<br />
<strong>the</strong>se axons are interrupted following VF cuts, but are spared after VLF lesions. This complex
org<strong>an</strong>ization <strong>for</strong>ms a potent <strong>an</strong>d durable me<strong>an</strong>s <strong>for</strong> activation of <strong>the</strong> CPGs in <strong>the</strong> absence of<br />
descending control of <strong>the</strong> brain <strong>an</strong>d <strong>the</strong>reby providing a possible basis of alternative <strong>the</strong>rapeutic<br />
approaches <strong>for</strong> <strong>the</strong> rescue of lost motor functions after spinal cord injury.<br />
Disclosures: A. Etlin, None; D. Blivis, None; M. Ben Zwi, None; A.A. Lev-Tov, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.8/DD59<br />
Topic: D.11.d. Afferent control<br />
Support: BSF 2005020<br />
NINDS Intramural Program<br />
Title: Studies of sacral neurons involved in activation of <strong>the</strong> lumbar central pattern generator <strong>for</strong><br />
locomotion in <strong>the</strong> neonatal rodent spinal cord<br />
Authors: *D. BLIVIS 1 , G. Z. MENTIS 1 , M. J. O'DONOVAN 1 , A. A. LEV-TOV 2 ;<br />
1 NIH/NINDS, Be<strong>the</strong>sda, MD; 2 Hebrew Univ. Sch. of Med., Jerusalem, Israel<br />
Abstract: The central pattern generator (CPG) <strong>for</strong> locomotion in <strong>the</strong> isolated spinal cord of<br />
neonatal rodents c<strong>an</strong> be activated by electrical stimulation of sacrocaudal afferents (SCAs). This<br />
effect is mediated by a group of sacral neurons whose axons project rostrally through <strong>the</strong> white<br />
matter funiculi. Selective application of <strong>the</strong> µ-opioid receptor agonist DAMGO to <strong>the</strong> sacral<br />
segments abolishes <strong>the</strong> SCA-evoked rhythm (Blivis et al., 2007). We have identified <strong>the</strong>se sacral<br />
relay neurons by retrogradely labeling <strong>the</strong>m with fluorescent dyes applied to <strong>the</strong> cut ventral<br />
funiculus (VF) at <strong>the</strong> lumbosacral junction. The labeled cells comprised a heterogeneous<br />
population with somatic diameters r<strong>an</strong>ging <strong>from</strong> 15 to 45µm. The majority of labeled neurons<br />
were found contralateral to <strong>the</strong> dye application. A subset of <strong>the</strong> labeled neurons received<br />
<strong>an</strong>terogradely labeled primary afferent boutons immunopositive <strong>for</strong> <strong>the</strong> vesicular glutamate<br />
tr<strong>an</strong>sporter 1. Electrical recordings <strong>from</strong> <strong>the</strong> cut VF <strong>an</strong>d optical recordings <strong>from</strong> VF sacral<br />
neurons labeled with a calcium-sensitive dye showed that <strong>the</strong>y were tonically active during SCA<br />
stimulus trains. This tonic activity was depressed in <strong>the</strong> presence of DAMGO <strong>an</strong>d restored by <strong>the</strong><br />
addition of Naloxone. Consistent with <strong>the</strong>se results, immunocytochemistry revealed <strong>the</strong> presence<br />
of <strong>the</strong> µ-opioid receptor MOR-1 on VF interneurons retrogradely labeled with fluorescein<br />
dextr<strong>an</strong> or cascade blue. Collectively, <strong>the</strong>se findings suggest that a class of sacral neurons
delivers a tonic drive rostrally to activate <strong>the</strong> locomotor CPG. They also suggest that <strong>the</strong><br />
suppressive effects of DAMGO on <strong>the</strong> SCA induced rhythm may involve a direct action of <strong>the</strong><br />
drug on <strong>the</strong>se tr<strong>an</strong>smission neurons. Identification of <strong>the</strong> rostral targets of <strong>the</strong>se neurons will be<br />
necessary to identify how <strong>the</strong>ir activation excites <strong>the</strong> rhythmogenic CPG.<br />
Disclosures: D. Blivis, None; G.Z. Mentis, None; M.J. O'Donov<strong>an</strong>, None; A.A. Lev-Tov,<br />
None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.9/DD60<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t EB006179<br />
NSF DGE – 0333411<br />
NSF GRFP (JLT)<br />
Title: Sacral dorsal column stimulation induced locomotor-like activity is enh<strong>an</strong>ced via<br />
tubocurarine <strong>an</strong>d output-based positive feedback<br />
Authors: *J. L. TODD 1 , S. P. DEWEERTH 1 , S. HOCHMAN 2 ;<br />
1 2<br />
Dept Biomed Eng, Georgia Inst. Tech/Emory Univ., Atl<strong>an</strong>ta, GA; Dept Physiol, Emory Univ.<br />
Sch. of Med., Atl<strong>an</strong>ta, GA<br />
Abstract: Stimulation of primary afferent fibers c<strong>an</strong> activate <strong>the</strong> spinal locomotor central pattern<br />
generator (CPG) <strong>an</strong>d modify ongoing activity. We have demonstrated that electrical stimulation<br />
of <strong>the</strong> sacral (S2-S4) dorsal columns initiates bouts of locomotor-like activity (LLA) recorded at<br />
<strong>the</strong> lumbar ventral roots. Here we present data which fur<strong>the</strong>r characterizes <strong>the</strong> pathway(s)<br />
involved <strong>an</strong>d implements feedback-controlled sacral dorsal column (sDC) stimulation as a me<strong>an</strong>s<br />
to rein<strong>for</strong>ce ongoing motor patterns. Experiments were undertaken in <strong>the</strong> isolated neonatal rat<br />
spinal cord with stimulation trains of 2 Hz pulses used to activate <strong>the</strong> CPG.<br />
We previously described adrenoceptor involvement in sDC stimulation-evoked LLA as αadrenoceptor<br />
<strong>an</strong>tagonists abolish <strong>the</strong> induced LLA (3/3). Using a split bath preparation<br />
(separated at L4 or L6), we now report that adrenoceptor <strong>an</strong>tagonist actions are restricted to <strong>the</strong><br />
caudal bath, attenuating alternating activity 67% of <strong>the</strong> time (8/12). Application of <strong>the</strong> same
<strong>an</strong>tagonists to <strong>the</strong> rostral bath had no effect (5/5). In addition, we previously reported sDC<br />
stimulation evoked LLA is attenuated by cholinergic receptor <strong>an</strong>tagonists (atropine, DHbE, <strong>an</strong>d<br />
mecamylamine) in <strong>the</strong> rostral bath. We now report that <strong>the</strong> nicotinic receptor <strong>an</strong>tagonist,<br />
tubocurarine (5-10 µM), enh<strong>an</strong>ces <strong>the</strong> duration of stimulus-evoked LLA (3/4). We will test <strong>the</strong><br />
hypo<strong>the</strong>sis that tubocurarine reduces presynaptic inhibition in activated primary afferents.<br />
We next examined <strong>the</strong> effect of sDC stimulation on 5HT/NMDA induced LLA. Single pulses<br />
produced resetting phase adv<strong>an</strong>ces of <strong>the</strong> locomotor pattern <strong>an</strong>d revealed that sDC stimulation<br />
caused <strong>an</strong> overall enh<strong>an</strong>cement of <strong>the</strong> CPG output (3/3). We <strong>the</strong>n devised a simple closed-loop<br />
feedback system that autonomously triggered stimulation of <strong>the</strong> sDC following threshold-based<br />
motor burst detection. Weak, irregular LLA was neurochemically induced; after implementing<br />
closed-loop stimuli, we observed less variable motor patterns in all ventral roots with phase<br />
relations consistent with LLA (2/2).<br />
Overall, we pl<strong>an</strong> to merge pharmacological insight of <strong>the</strong> neural circuitry generating sDC<br />
stimulation-induced LLA with closed-loop m<strong>an</strong>ipulation of <strong>the</strong> motor pattern to optimize<br />
locomotor rhythmogenesis. Results obtained may tr<strong>an</strong>slate into novel rehabilitative <strong>the</strong>rapies <strong>for</strong><br />
those with spinal cord injury.<br />
Disclosures: J.L. Todd, None; S.P. DeWeerth, None; S. Hochm<strong>an</strong>, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.10/DD61<br />
Topic: D.11.d. Afferent control<br />
Support: NSF GRFP to H.B.H.<br />
PEO Scholar Award to H.B.H.<br />
NSF Gr<strong>an</strong>t 0745164 to S.H.<br />
Paralyzed Veter<strong>an</strong>s of America Gr<strong>an</strong>t to S.H.<br />
Title: Modulation of sensory input <strong>an</strong>d interneuronal activity during non-fictive locomotion in<br />
<strong>the</strong> in vitro spinal cord - Hindlimb rat preparation
Authors: *H. B. HAYES 1 , Y.-H. CHANG 2,1 , S. HOCHMAN 3 ;<br />
1 2 3<br />
Biomed. Engin., Applied Physiol., Georgia Inst. of Technol., Atl<strong>an</strong>ta, GA; Physiol., Emory<br />
Univ. Sch. of Med., Atl<strong>an</strong>ta, GA<br />
Abstract: During locomotion, <strong>the</strong> spinal cord receives sensory input that must be processed <strong>an</strong>d<br />
used to refine <strong>an</strong>d rein<strong>for</strong>ce locomotor output. To study this sensory input <strong>an</strong>d processing, we<br />
developed a neonatal rat spinal cord-hindlimb preparation (SCHP) consisting of <strong>an</strong> isolated<br />
neonatal rat spinal cord with hindlimbs intact <strong>an</strong>d oriented dorsal-up to locomote on a surface.<br />
The SCHP combines <strong>the</strong> neural accessibility of in vitro preparations with sensory-modulated,<br />
non-fictive locomotion. Fur<strong>the</strong>r, <strong>the</strong> spinal cord c<strong>an</strong> be mech<strong>an</strong>ically stabilized to allow<br />
intracellular <strong>an</strong>d extracellular recordings in a controlled solute environment. Using <strong>the</strong><br />
adv<strong>an</strong>tages of <strong>the</strong> SCHP, we studied <strong>the</strong> modulation of sensory activity in dorsal roots (DRs) <strong>an</strong>d<br />
interneurons during non-fictive monoamine-induced locomotion <strong>an</strong>d in response to mech<strong>an</strong>ical<br />
perturbations.<br />
During 5HT, DA, <strong>an</strong>d NA locomotion, DRs often exhibited rhythmic bursting. Their rhythmicity<br />
was modulated with locomotor strength <strong>an</strong>d streng<strong>the</strong>ned by ground contact. Assistive<br />
locomotor-like limb movements could also initiate locomotion <strong>an</strong>d DR rhythmicity in sublocomotor-threshold<br />
5HT or DA, or increase ongoing DR rhythmicity. Toe <strong>an</strong>d tail pinches<br />
could reset ongoing 5HT <strong>an</strong>d DA locomotion, or initiate brief locomotor bouts with DR<br />
rhythmicity in <strong>the</strong> absence of drugs. Thus, afferents in <strong>the</strong> SCHP have strong access to <strong>the</strong> spinal<br />
locomotor network, allowing <strong>the</strong>m to alter or induce locomotion.<br />
We also observed locomotor-related rhythmic dorsal root potentials (DRPs), representing<br />
presynaptic inhibition of afferent input. L2 DRPs tended to be in-phase with flexor activity,<br />
while L5 DRPs tended to be out-of-phase. DRP amplitude was most pronounced during robust<br />
locomotion <strong>an</strong>d was clearly facilitated during limb loading <strong>an</strong>d ground contact, suggesting that<br />
<strong>the</strong> extent of presynaptic inhibition varies with locomotor conditions. Part of <strong>the</strong> locomotorrelated<br />
DRPs was GABAA receptor independent as <strong>the</strong>y persisted in bicuculline. Large rhythmic<br />
DRPs were also evoked or enh<strong>an</strong>ced by assistive limb movements <strong>an</strong>d toe or tail pinches with or<br />
without drugs present.<br />
Finally, we per<strong>for</strong>med <strong>the</strong> first whole-cell patch recordings <strong>from</strong> mammali<strong>an</strong> dorsal horn<br />
interneurons during unrestrained locomotion. We identified dorsal horn interneurons that are<br />
nearly quiescent at rest, but receive rhythmic drive potentials <strong>an</strong>d fire repetitively during<br />
locomotion at a rate proportional to locomotor strength. In response to toe pinch, <strong>the</strong>se neurons<br />
received strong inhibition followed by rebound firing. Although preliminary, intracellular<br />
recordings during locomotion are <strong>an</strong> import<strong>an</strong>t step in <strong>the</strong> study of spinal sensory processing.<br />
Disclosures: H.B. Hayes, None; Y. Ch<strong>an</strong>g, None; S. Hochm<strong>an</strong>, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.11/DD62<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t F32NS054367<br />
NIH Gr<strong>an</strong>t R01NS054274<br />
Title: Reson<strong>an</strong>ce entrainment in <strong>the</strong> lamprey central pattern generator <strong>for</strong> locomotion<br />
Authors: *E. D. TYTELL, A. H. COHEN;<br />
Univ. Maryl<strong>an</strong>d, College Park, MD<br />
Abstract: The lamprey central pattern generator (CPG) <strong>for</strong> locomotion is a spinal circuit that c<strong>an</strong><br />
generate <strong>the</strong> basic rhythmic pattern <strong>for</strong> swimming in <strong>the</strong> absence of sensory feedback or<br />
descending control. Sensory input is not necessary <strong>for</strong> <strong>the</strong> rhythm, but never<strong>the</strong>less such input,<br />
<strong>an</strong>d particularly proprioceptive input, c<strong>an</strong> ch<strong>an</strong>ge <strong>the</strong> CPG frequency or reset it entirely. M<strong>an</strong>y<br />
laboratory studies of <strong>the</strong> CPG have examined its response to sensory input in <strong>an</strong> “open loop”<br />
fashion: a defined stimulus is given <strong>an</strong>d <strong>the</strong> response of <strong>the</strong> CPG is measured. However, when a<br />
lamprey swims in vivo, <strong>the</strong> CPG functions in a closed-loop system: <strong>the</strong> CPG generates a rhythm,<br />
activating motor neurons, which cause <strong>the</strong> axial musculature to contract <strong>an</strong>d stimulate<br />
proprioceptors that, in turn, feed back to modulate <strong>the</strong> CPG rhythm. Simulation results have<br />
suggested that when a simple model of a CPG is coupled in a closed loop to a mech<strong>an</strong>ical system<br />
with a reson<strong>an</strong>t frequency, <strong>the</strong> result<strong>an</strong>t oscillation frequency tends to converge to <strong>the</strong><br />
mech<strong>an</strong>ical reson<strong>an</strong>t frequency. This “reson<strong>an</strong>ce entrainment” might be import<strong>an</strong>t biologically<br />
because <strong>the</strong> mech<strong>an</strong>ical efficiency of body movements would be highest at <strong>the</strong> body’s reson<strong>an</strong>t<br />
frequency. However, relatively little is known about existence of reson<strong>an</strong>ce entrainment in vivo,<br />
or <strong>the</strong> mech<strong>an</strong>isms that might underlie it. To this end, a closed-loop stimulation protocol was<br />
developed <strong>for</strong> <strong>the</strong> isolated lamprey spinal cord preparation. Motor neuron output was recorded<br />
extracellularly <strong>from</strong> <strong>the</strong> ventral roots <strong>an</strong>d filtered in real time using a simulation of <strong>the</strong> contractile<br />
properties of muscle <strong>an</strong>d <strong>the</strong> elastic properties of <strong>the</strong> lamprey body. The simulation results,<br />
which define <strong>the</strong> estimated effect of a given motor burst on <strong>the</strong> body, were <strong>the</strong>n used to provide a<br />
bending stimulus. Preliminary results indicate that <strong>the</strong> lamprey CPG c<strong>an</strong> be entrained to a<br />
simulated mech<strong>an</strong>ical reson<strong>an</strong>t frequency. Multiple trials with different simulated reson<strong>an</strong>t<br />
frequencies indicated that <strong>the</strong> CPG could only entrain to reson<strong>an</strong>t frequencies above its<br />
unstimulated frequency, unlike previous results <strong>from</strong> open-loop stimulation protocols, in which<br />
<strong>the</strong> CPG could entrain to frequencies above <strong>an</strong>d below its unstimulated frequency. However, <strong>the</strong><br />
r<strong>an</strong>ge of <strong>the</strong>se reson<strong>an</strong>t frequencies was subst<strong>an</strong>tially larger th<strong>an</strong> <strong>the</strong> typical r<strong>an</strong>ge of entrainment<br />
frequencies in open-loop procedures. Finally, closed-loop stimulation appeared to regularize<br />
bursting, decreasing <strong>the</strong> vari<strong>an</strong>ce in <strong>the</strong> burst period below <strong>the</strong> vari<strong>an</strong>ce when unstimulated or<br />
during open-loop stimulation.<br />
Disclosures: E.D. Tytell, None; A.H. Cohen, None.
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.12/DD63<br />
Topic: D.11.d. Afferent control<br />
Support: DFG Gr<strong>an</strong>t CR58/11-1<br />
Title: Sensory control of backward walking in stick insects: Intrasegmental influences<br />
Authors: *D. DUESTERHUS, J. SCHMITZ;<br />
Univ. of Bielefeld, Bielefeld, Germ<strong>an</strong>y<br />
Abstract: The motor pattern in <strong>for</strong>ward walking stick insects is generated by <strong>the</strong> interplay of<br />
central rhythm generators (CRG) <strong>an</strong>d sensory feedback <strong>from</strong> <strong>the</strong> legs. This sensory feedback<br />
enables <strong>the</strong> insect to adapt its walking <strong>an</strong>d climbing movements to variations in substrate<br />
conditions on a step by step basis. In stick insects different walking directions c<strong>an</strong> be elicited by<br />
specific tactile stimulations. Touching <strong>the</strong> abdomen induces <strong>for</strong>ward walks whereas backward<br />
walks c<strong>an</strong> be induced by gently pulling <strong>the</strong> <strong>an</strong>tennae. It has to be mentioned that continuous<br />
backward walking - a behavior not expressed spont<strong>an</strong>eously by <strong>the</strong> <strong>an</strong>imal - appears to be less<br />
regular th<strong>an</strong> <strong>for</strong>ward walking. Thus, backward walking might be <strong>an</strong> emergency program that<br />
might be produced predomin<strong>an</strong>tly by <strong>the</strong> actions of CRGs <strong>an</strong>d less precisely controlled by<br />
sensory feedback.<br />
Here we examined a swing phase reflex well-known <strong>from</strong> <strong>for</strong>ward walking <strong>an</strong>imals. If during<br />
swing a leg hits <strong>an</strong> obstacle with <strong>the</strong> <strong>an</strong>terior part of <strong>the</strong> femur or <strong>the</strong> tibia, <strong>the</strong> swing movement<br />
is immediately stopped due to inhibition of <strong>the</strong> swing (protractor coxae) <strong>an</strong>d excitation of <strong>the</strong><br />
st<strong>an</strong>ce (retractor coxae) motoneurons. This reflex conduces to avoid <strong>an</strong>d get over obstacles<br />
during swing by me<strong>an</strong>s of repetitive lifting movements of <strong>the</strong> coxa-troch<strong>an</strong>ter joint until<br />
eventually <strong>the</strong> obstruction c<strong>an</strong> be surmounted <strong>an</strong>d <strong>the</strong> swing movement c<strong>an</strong> be continued.<br />
In backward walking <strong>the</strong> functional swing <strong>an</strong>d st<strong>an</strong>ce muscles are inverted. In this situation <strong>the</strong><br />
retractor coxae is active during swing <strong>an</strong>d <strong>the</strong> protractor coxae is active during st<strong>an</strong>ce.<br />
Obstruction of a backward swing movement now inhibits <strong>the</strong> retractor coxae motoneurons <strong>an</strong>d<br />
activates <strong>the</strong> protractor coxae toge<strong>the</strong>r with <strong>the</strong> levator troch<strong>an</strong>teris motoneurons. Hence, <strong>the</strong><br />
reflex is functional <strong>an</strong>d obviously <strong>the</strong> sensors on both sides of <strong>the</strong> leg are connected<br />
appropriately to <strong>the</strong> respective motoneuron pools.<br />
We examined whe<strong>the</strong>r <strong>the</strong>re are two distinct tactile-sensible regions, <strong>an</strong> <strong>an</strong>terior <strong>an</strong>d a posterior<br />
region or whe<strong>the</strong>r it is <strong>the</strong> obstruction of <strong>the</strong> indented movement measured by <strong>the</strong> load sensors.<br />
Experiments were per<strong>for</strong>med with shaved femoral tactile hairs <strong>an</strong>d ablated camp<strong>an</strong>i<strong>for</strong>m
sensillae (CS). Two types of reactions in such impaired <strong>an</strong>imals were found. Still, <strong>the</strong> reflex<br />
sometimes could be observed as in <strong>the</strong> intact <strong>an</strong>imal but often obstructions led to a prolonged<br />
activity in <strong>the</strong> respective swing muscle. In <strong>the</strong> latter case <strong>the</strong>re was no switch between <strong>the</strong><br />
<strong>an</strong>tagonistic muscles. In experiments where only <strong>the</strong> tactile hairs were shaved we found no<br />
observable difference to <strong>the</strong> intact <strong>an</strong>imal, concluding that <strong>the</strong> CS c<strong>an</strong> compensate <strong>for</strong> <strong>the</strong> lack of<br />
tactile in<strong>for</strong>mation.<br />
Disclosures: D. Duesterhus, None; J. Schmitz, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.13/DD64<br />
Topic: D.11.d. Afferent control<br />
Support: DFG Gr<strong>an</strong>t CR58/11-1<br />
Title: Sensory control of backward walking in stick insects: Intersegmental influences<br />
Authors: *J. SCHMITZ, D. DUESTERHUS;<br />
Dept. Biol. Cybernetics, Univ. of Bielefeld, Bielefeld, Germ<strong>an</strong>y<br />
Abstract: The motor pattern <strong>for</strong> each leg in <strong>for</strong>ward walking stick insects is generated by <strong>the</strong><br />
interplay of central rhythm generators (CRG) <strong>an</strong>d sensory feedback. Coordination of <strong>the</strong> legs is<br />
achieved by state-dependent influences of <strong>the</strong> adjacent neighboring legs. In stick insects different<br />
walking directions c<strong>an</strong> be elicited by specific tactile stimulations. Touching <strong>the</strong> abdomen induces<br />
<strong>for</strong>ward walks whereas backward walks c<strong>an</strong> be induced by gently pulling <strong>the</strong> <strong>an</strong>tennae. It has to<br />
be mentioned that continuous backward walking - a behavior not expressed spont<strong>an</strong>eously by <strong>the</strong><br />
<strong>an</strong>imal - appears to be less regular th<strong>an</strong> <strong>for</strong>ward walking. Thus, coordination of <strong>the</strong> legs might be<br />
less stringent or even absent during backward walking.<br />
Here we investigated <strong>the</strong> activities of <strong>the</strong> swing <strong>an</strong>d st<strong>an</strong>ce motoneurons of a leg in prolonged<br />
st<strong>an</strong>ce phase. The leg under inspection was placed on a fixed plat<strong>for</strong>m while <strong>the</strong> remaining five<br />
legs were allowed to walk <strong>for</strong>ward or backward on a treadwheel. The leg on <strong>the</strong> plat<strong>for</strong>m will<br />
only lift off if it is moved passively to <strong>the</strong> respective st<strong>an</strong>ce-swing tr<strong>an</strong>sition point. This shows<br />
that also during backward walking <strong>the</strong> sensory feedback <strong>from</strong> <strong>the</strong> leg is import<strong>an</strong>t. As long as <strong>the</strong><br />
leg is held in a position far enough <strong>from</strong> this tr<strong>an</strong>sition point <strong>the</strong> leg will remain st<strong>an</strong>ding while it<br />
shows tonic activity in <strong>the</strong> respective st<strong>an</strong>ce muscle, <strong>the</strong> retractor coxae in <strong>for</strong>ward <strong>an</strong>d <strong>the</strong><br />
protractor coxae in backward walking. This tonic activity is modulated in <strong>the</strong> walking rhythm of
its neighboring legs. In <strong>the</strong> <strong>for</strong>ward walking <strong>an</strong>imal, <strong>the</strong> middle leg retractor activity is minimal<br />
<strong>an</strong>d often accomp<strong>an</strong>ied by a suprathreshold activation of <strong>the</strong> protractor motoneurons when <strong>the</strong><br />
swing phase of <strong>the</strong> ipsilateral hind leg has finished <strong>an</strong>d <strong>the</strong> hind leg starts its st<strong>an</strong>ce phase. This is<br />
interpreted as <strong>the</strong> impact of <strong>the</strong> coordination rule 2 (Cruse, TINS, 1990) which is active <strong>from</strong><br />
hind to middle <strong>an</strong>d <strong>from</strong> middle to front legs. Also during backward walking minimal activity of<br />
<strong>the</strong> st<strong>an</strong>ce <strong>an</strong>d suprathreshold activity of <strong>the</strong> swing motoneurons is correlated with <strong>the</strong> swingst<strong>an</strong>ce<br />
tr<strong>an</strong>sition of <strong>the</strong> hindleg. However, <strong>the</strong> functional swing <strong>an</strong>d st<strong>an</strong>ce motoneurons are<br />
inverted in this case. The direction of this intersegmental influence is not reversed.<br />
Ano<strong>the</strong>r coordination influence (rule 1) exerts <strong>an</strong> inhibitory effect on <strong>an</strong>terior legs during<br />
<strong>for</strong>ward walking. A swing movement of a middle leg suppresses lift-off of <strong>the</strong> <strong>an</strong>terior<br />
neighboring leg, <strong>the</strong> front leg. During backward walking, a prolonged swing phase of <strong>the</strong> middle<br />
leg blocks <strong>the</strong> lift-off of <strong>the</strong> hind leg. In this case, <strong>the</strong> direction of <strong>the</strong> influence is reversed.<br />
Fur<strong>the</strong>r studies should elucidate whe<strong>the</strong>r in backward walks <strong>an</strong>atomical hind legs act as<br />
functional front legs.<br />
Disclosures: J. Schmitz, None; D. Duesterhus, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.14/DD65<br />
Topic: D.11.d. Afferent control<br />
Support: DFG gr<strong>an</strong>t Bu 857/10<br />
Title: Rein<strong>for</strong>cement of movement in local joint control: Its role in single leg stepping <strong>an</strong>d <strong>the</strong><br />
influence of neighboring stepping legs <strong>an</strong>d walking direction<br />
Authors: *A. BUSCHGES, G. VON UCKERMANN, K. HELLEKES, E. BLINCOW;<br />
Zoology/Animal Physiol, Univ. Koln, Koln D-50923, Germ<strong>an</strong>y<br />
Abstract: Rein<strong>for</strong>cement of movement by sensory feedback is thought to play <strong>an</strong> import<strong>an</strong>t role<br />
in walking pattern generation, even though most of <strong>the</strong> supporting evidence arises <strong>from</strong> reduced<br />
or pharmacologically activated preparations. We studied <strong>the</strong> potential role of this control<br />
mech<strong>an</strong>ism in <strong>the</strong> stick insect (Carausius morosus, Br.) by intracellularly recording <strong>the</strong> activity<br />
of identified premotor nonspiking interneurons (NSI) during stepping in <strong>the</strong> semi-intact single<br />
leg preparation. In reduced, active preparations, NSIs contribute to rein<strong>for</strong>cement of movementlike<br />
motor output, when <strong>the</strong> “active reaction” (AR) is generated in tibial motoneurons upon
flexion signals <strong>from</strong> <strong>the</strong> tibia (Bässler, 1976, Biol. Cybern. 24:42; Dries<strong>an</strong>g & Büschges, 1993,<br />
JCP A173: 445). We detected clear similarities between <strong>the</strong> activity pattern of NSIs during single<br />
leg stepping <strong>an</strong>d <strong>the</strong>ir responses to stimulation of <strong>the</strong> femoral chordotonal org<strong>an</strong> (fCO) during<br />
generation of <strong>the</strong> AR, strongly supporting <strong>the</strong> notion that <strong>the</strong> motor output generated during <strong>the</strong><br />
AR reflects part of <strong>the</strong> control regime <strong>for</strong> stepping.<br />
In addition, we studied <strong>the</strong> influence of flexion <strong>an</strong>d extension signals <strong>from</strong> <strong>the</strong> FTi-joint elicited<br />
through stimulation of a middle leg fCO while recording <strong>the</strong> activity of <strong>the</strong> corresponding tibial<br />
motoneurons <strong>an</strong>d muscles of that leg while one or more of <strong>the</strong> neighboring legs were stepping on<br />
a slippery surface (Gruhn et al. 2006; J. Neurosci. Methods 158: 195). We found that<br />
rein<strong>for</strong>cement of flexor activity in tibial motoneurons in response to flexion signals <strong>from</strong> <strong>the</strong> fCO<br />
was generated only, when ipsilateral neighboring legs were present <strong>an</strong>d stepping. It was not<br />
generated when contralateral legs were stepping. Fur<strong>the</strong>rmore, rein<strong>for</strong>cement of movement upon<br />
fCO flexion signals was found to be dependent on walking direction. It occurred exclusively,<br />
when <strong>the</strong> <strong>an</strong>imals were walking <strong>for</strong>wards, but not backwards. These findings indicate: (i)<br />
Rein<strong>for</strong>cement of movement does indeed contribute to walking pattern generation in vivo. (ii)<br />
The behavioral state of <strong>the</strong> experimental <strong>an</strong>imal does not alone determine <strong>the</strong> properties of local<br />
sensorimotor processing. (iii) Our results show task dependency of processing of movement<br />
related feedback in motor pattern generation <strong>for</strong> locomotion. (iv) The results suggest that neural<br />
control <strong>for</strong> individual legs differs between walking tasks. Supp. by DFG gr<strong>an</strong>t Bu857/10<br />
Disclosures: A. Buschges, None; G. von Uckerm<strong>an</strong>n, None; K. Hellekes, None; E. Blincow,<br />
None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.15/DD66<br />
Topic: D.11.d. Afferent control<br />
Support: DFG Gr<strong>an</strong>t Bu857/10<br />
Title: Characterization of intersegmental sensory influences in <strong>the</strong> stick insect walking system<br />
Authors: *A. BORGMANN 1,2 , K. HELLEKES 2 , A. BUESCHGES 2 ;<br />
1 CSNS, NIH/NINDS, Be<strong>the</strong>sda, MD; 2 Inst. of Zoology, Univ. of Cologne, Cologne, Germ<strong>an</strong>y<br />
Abstract: Walking movements result <strong>from</strong> a complex interplay of central pattern generating<br />
networks (CPG), local sensory feedback about movements <strong>an</strong>d <strong>for</strong>ces generated in <strong>the</strong> legs <strong>an</strong>d
coordinating signals <strong>from</strong> neighboring limbs. Studies on <strong>the</strong> behavioral level have shown that<br />
coordination <strong>the</strong> between legs relies on neural signals between neighboring segments as well as<br />
on mech<strong>an</strong>ical coupling between legs, summarized in various rules <strong>for</strong> coordination between <strong>the</strong><br />
legs [1]. As yet, <strong>the</strong> neural basis <strong>for</strong> intersegmental interactions in coordination is barely<br />
unraveled.<br />
We used a semi-intact stick insect preparation with single legs stepping [2, 3] <strong>an</strong>d/or stimulation<br />
of specific leg sensors (i.e. camp<strong>an</strong>i<strong>for</strong>m sensilla (CS), femoral chordotonal org<strong>an</strong> (fCO)) in<br />
neighboring segments. Locomotor activity was induced by tactile stimulation or by activation of<br />
individual g<strong>an</strong>glia with pilocarpine. We focused on intersegmental influences <strong>from</strong> <strong>the</strong> front leg<br />
on middle leg thoraco-coxal (TC-) joint CPG.<br />
Our study yielded <strong>the</strong> following results:<br />
1. Front leg stepping entrains <strong>the</strong> active middle leg TC-joint CPG, most probably due to sensory<br />
signals em<strong>an</strong>ating <strong>from</strong> <strong>the</strong> front leg [3, 4]. The front leg retraction coincides with middle leg<br />
retractor activity. We characterized <strong>the</strong> influence of front leg sensory signals on <strong>the</strong> active<br />
middle leg TC-joint CPG by phase response curves (PRCs). The PRC <strong>for</strong> front leg steps reveals<br />
a strong influence on middle leg TC-joint CPG. The PRCs <strong>for</strong> front leg CS <strong>an</strong>d fCO stimulation<br />
on <strong>the</strong> active middle leg TC-joint CPG show a weaker but similar course as <strong>the</strong> PRC <strong>for</strong> intact<br />
front leg steps. This indicates that signals <strong>from</strong> both front leg CS <strong>an</strong>d fCO contribute in part to<br />
<strong>the</strong> observed entrainment of <strong>the</strong> middle leg TC-joint CPG by front leg steps.<br />
2. We investigated <strong>the</strong> interplay of intersegmental signals <strong>from</strong> front leg stepping <strong>an</strong>d local load<br />
signals on <strong>the</strong> middle leg TC-joint [4]. Middle leg CS were stimulated during front leg stepping<br />
sequences while middle leg protractor <strong>an</strong>d retractor MN activity was monitored. Protractor <strong>an</strong>d<br />
retractor MN activity was influenced both by front leg stepping <strong>an</strong>d middle leg CS stimulation.<br />
Front leg stepping induced alternating activity in protractor <strong>an</strong>d retractor MNs. Stimulation of<br />
middle leg CS during front leg steps mimicking load increase increased retractor MN activity,<br />
while protractor MN activity was decreased. Alternating protractor <strong>an</strong>d retractor MN activity<br />
could be entrained by CS signals, when <strong>the</strong> stimulation frequency was similar to front leg<br />
stepping frequency.<br />
[1] Cruse 1990 Trends Neurosci 13:15.<br />
[2] Bässler 1993 Biol Cybernetics 69:305.<br />
[3] Ludwar et al. 2005 J Neurophysiol 93:1255.<br />
[4] Borgm<strong>an</strong>n et al. <strong>2009</strong> J Neurosci 29:2972.<br />
Disclosures: A. Borgm<strong>an</strong>n, None; K. Hellekes, None; A. Bueschges, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.16/DD67
Topic: D.11.d. Afferent control<br />
Support: MH-51393<br />
Title: Characteristics of peripherally applied stimuli determine spike propagation in <strong>the</strong> Aplysia<br />
mech<strong>an</strong>oafferent B21<br />
Authors: C. G. EVANS, B. C. LUDWAR, T. KANG, *E. C. CROPPER;<br />
Dept Neurosci, Mt Sinai Sch. Med., New York, NY<br />
Abstract: Spikes do not actively propagate throughout <strong>the</strong> central processes of <strong>the</strong> Aplysia<br />
mech<strong>an</strong>oafferent B21 if it is peripherally activated at its resting membr<strong>an</strong>e potential. Spikes fail<br />
to propagate to <strong>the</strong> lateral process, which is <strong>the</strong> primary point of contact with <strong>the</strong> B8 motor<br />
neurons, which receive excitatory chemical synaptic input. The spike propagation failure is<br />
relieved if B21 is centrally depolarized prior to <strong>an</strong>d during peripheral activation. Previous<br />
publications that considered this phenomenon focused on <strong>the</strong> physiological signific<strong>an</strong>ce of input<br />
<strong>from</strong> electrically coupled feeding interneurons. These neurons generate 10-15 mV<br />
depolarizations during motor programs that are sufficient to modify spike propagation in B21.<br />
This input is confined to <strong>the</strong> radula retraction phase of motor programs. Consequently it was<br />
postulated that it served as a mech<strong>an</strong>ism <strong>for</strong> phasic control of B21 to B8 mech<strong>an</strong>oafferent<br />
tr<strong>an</strong>smission. The current study was motivated by recent data that indicate that spike propagation<br />
in B21 c<strong>an</strong> also be modified by central depolarizations that are less th<strong>an</strong> those observed during a<br />
motor program. We sought to determine whe<strong>the</strong>r spikes would propagate to <strong>the</strong> lateral process<br />
with <strong>the</strong> application of stimuli that stretch <strong>the</strong> subradula tissue (SRT). Stretch stimuli generate a<br />
burst of spikes in B21 <strong>an</strong>d coactivate o<strong>the</strong>r cells of <strong>the</strong> radula mech<strong>an</strong>oafferent cluster. This<br />
induces a small, relatively brief central depolarization in B21. Under physiological conditions<br />
previous data indicated that B21 will be activated in this m<strong>an</strong>ner during <strong>the</strong> radula protraction<br />
phase of motor programs (when input to B8 would be dysfunctional). We find that active spiking<br />
c<strong>an</strong> be observed in <strong>the</strong> lateral process when <strong>the</strong> SRT is stretched, <strong>an</strong>d identify parametric features<br />
of stimuli that promote spike propagation. Thus, our results indicate that motor program induced<br />
depolarizations are not essential <strong>for</strong> spike propagation to <strong>the</strong> lateral process. To determine <strong>the</strong><br />
potential physiological signific<strong>an</strong>ce of this finding <strong>for</strong> phasic control of B21-B8 mech<strong>an</strong>oafferent<br />
tr<strong>an</strong>smission, we stretched <strong>the</strong> SRT <strong>an</strong>d recorded B8 responses. We found that B21 induced PSPs<br />
were virtually absent. In this <strong>an</strong>d a comp<strong>an</strong>ion study we describe characteristics of B21-B8<br />
sensorimotor tr<strong>an</strong>smission that account <strong>for</strong> this. Finally, we consider <strong>the</strong> potential import<strong>an</strong>ce of<br />
protraction phase activation of B21 <strong>for</strong> sensorimotor tr<strong>an</strong>smission to o<strong>the</strong>r follower neurons.<br />
Disclosures: C.G. Ev<strong>an</strong>s, None; B.C. Ludwar, None; T. K<strong>an</strong>g, None; E.C. Cropper, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.17/DD68<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t MH-51393<br />
Title: Single spike induced alterations of intracellular calcium in a sensory neuron<br />
Authors: *B. C. LUDWAR 1 , C. G. EVANS 1,2 , E. C. CROPPER 1 ;<br />
1 Neurosci, Mount Sinai Sch. Med., New York, NY; 2 Phase Five Communications, New York,<br />
NY<br />
Abstract: We study sensorimotor tr<strong>an</strong>smission <strong>an</strong>d have described two distinct regulatory<br />
mech<strong>an</strong>isms in our model system, which consists of <strong>the</strong> Aplysia mech<strong>an</strong>oafferent B21 <strong>an</strong>d a<br />
follower motorneuron B8. The two mech<strong>an</strong>isms potentiate synaptic tr<strong>an</strong>smission, <strong>an</strong>d are<br />
induced by increases in <strong>the</strong> presynaptic membr<strong>an</strong>e potential. We recently showed that one<br />
mech<strong>an</strong>ism is calcium dependent. <strong>When</strong> a dihydropyridine sensitive calcium current is<br />
pharmacologically blocked, B21 induced PSPs are virtually eliminated in B8. This<br />
dihydropyridine sensitive calcium current is activated when B21 is held at depolarized potentials<br />
below <strong>the</strong> spike threshold <strong>an</strong>d its induction results in a widespread increase in <strong>the</strong> intracellular<br />
free calcium concentration. The current study focuses on ch<strong>an</strong>ges in calcium concentration<br />
induced by spiking.<br />
To determine whe<strong>the</strong>r ch<strong>an</strong>ges in <strong>the</strong> intracellular calcium concentration c<strong>an</strong> be detected during<br />
individual spikes, neuron B21 was injected with <strong>the</strong> calcium indicator dye Calcium Or<strong>an</strong>ge <strong>an</strong>d<br />
spikes were triggered by brief mech<strong>an</strong>ical stimulation of <strong>the</strong> subradula tissue. Spike induced<br />
increases in calcium fluoresence were observed in <strong>the</strong> medial, somatic, <strong>an</strong>d lateral region of B21.<br />
These increases were larger at more depolarized potentials, <strong>an</strong>d <strong>the</strong> effects of membr<strong>an</strong>e<br />
potential were graded. This suggests that potentiating effects of depolarization on B21-B8<br />
synaptic tr<strong>an</strong>smission may in part be mediated by effects of holding potential on calcium influx<br />
that occurs during spiking.<br />
Interestingly, spike induced increases in calcium fluoresence had a relatively long relaxation rate,<br />
which suggested that <strong>the</strong>y would summate if multiple spikes were triggered. To study<br />
summation, we peripherally activated B21 with a physiologically relev<strong>an</strong>t stimulus that induces<br />
burst of spikes (stretch of <strong>the</strong> subradula tissue). Summation was observed when bursts of spikes<br />
were repeatedly generated at inter-cycle intervals typical <strong>for</strong> feeding motor programs. This type<br />
of summation could <strong>the</strong>re<strong>for</strong>e constitute a mech<strong>an</strong>ism <strong>for</strong> progressive enh<strong>an</strong>cement of<br />
sensorimotor tr<strong>an</strong>smission as B21 is repeatedly activated during <strong>an</strong> ongoing motor program.<br />
Disclosures: B.C. Ludwar, None; C.G. Ev<strong>an</strong>s, None; E.C. Cropper, MH-51393, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received).
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.18/DD69<br />
Topic: D.11.d. Afferent control<br />
Support: NS42813 (MPN)<br />
Title: Two distinct gastric mill rhythms respond differently to <strong>the</strong> same sensory feedback<br />
Authors: *R. S. WHITE, M. P. NUSBAUM;<br />
Dept Neurosci, Univ. Penn. Sch. of Med., Philadelphia, PA<br />
Abstract: Sensorimotor integration occurs at <strong>the</strong> level of <strong>the</strong> motor circuit <strong>an</strong>d/or its projection<br />
neuron inputs. Here we are studying how <strong>the</strong> same proprioceptor, <strong>the</strong> gastropyloric receptor<br />
neuron (GPR), influences two separate versions of <strong>the</strong> gastric mill (chewing) rhythm (GMR) in<br />
<strong>the</strong> isolated crab stomatogastric nervous system. These distinct rhythms are triggered by<br />
stimulating ei<strong>the</strong>r of two extrinsic pathways, <strong>the</strong> POC- or VCN-neurons, which activate <strong>the</strong><br />
projection neurons MCN1 <strong>an</strong>d CPN2 to burst with a POC- or VCN-specific pattern <strong>an</strong>d <strong>the</strong>reby<br />
drive <strong>the</strong> GMR in <strong>the</strong> stomatogastric g<strong>an</strong>glion (STG) (Beenhakker & Nusbaum, 2004; Blitz et al,<br />
2008).<br />
GPR is a muscle stretch receptor that is likely activated during <strong>the</strong> gastric mill retraction phase<br />
(Katz et al, 1989). Hence, to determine <strong>the</strong> GPR actions during a triggered GMR, GPR is<br />
stimulated during <strong>the</strong> retractor phase. In <strong>the</strong> absence of a GMR, GPR excites MCN1 <strong>an</strong>d CPN2<br />
in <strong>the</strong> commissural g<strong>an</strong>glia (CoGs) (Blitz et al. 2004), but <strong>the</strong>se actions are gated out during <strong>the</strong><br />
VCN-GMR (Beenhakker et al, 2007). During this GMR, GPR stimulation selectively alters<br />
(prolongs) <strong>the</strong> retractor phase, via its synaptic actions in <strong>the</strong> STG.<br />
Here, we are assessing <strong>the</strong> GPR influence on <strong>the</strong> POC-GMR. GPR stimulation prolongs <strong>the</strong><br />
POC-retractor phase (Ctl: 6.8±0.7 s, GPR: 10.5±1.4 s, n=10, p
GMR, <strong>the</strong> GPR influence on CPN2 is selectively gated out. We aim to continue testing <strong>the</strong><br />
hypo<strong>the</strong>sis that <strong>the</strong> same sensory input c<strong>an</strong> have a distinct effect on different versions of a motor<br />
pattern, due to differential gating at <strong>the</strong> level of projection neurons, <strong>an</strong>d to determine <strong>the</strong><br />
mech<strong>an</strong>isms underlying this distinct GPR action on <strong>the</strong> POC-GMR.<br />
Disclosures: R.S. White, None; M.P. Nusbaum, None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.19/DD70<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t NS29436<br />
Title: Two peptide hormones configure similar network states that respond differently to sensory<br />
feedback<br />
Authors: *J. C. RODRIGUEZ, M. S. KIRBY, D. M. BLITZ, M. P. NUSBAUM;<br />
Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Multiple modulators converging on <strong>the</strong> same central pattern generator (CPG) circuit<br />
c<strong>an</strong> elicit similar motor output. However, little is known as to whe<strong>the</strong>r <strong>the</strong>se motor patterns are<br />
similarly sensitive to o<strong>the</strong>r inputs. We are using <strong>the</strong> gastric mill (chewing) CPG in <strong>the</strong> isolated<br />
crab (C<strong>an</strong>cer borealis) stomatogastric g<strong>an</strong>glion (STG) to determine how motor patterns<br />
configured by two peptide hormones respond to <strong>the</strong> same sensory input. The projection neuron<br />
MCN1 drives <strong>the</strong> biphasic gastric mill rhythm (GMR) via slow peptidergic <strong>an</strong>d fast GABAergic<br />
excitation to <strong>the</strong> reciprocally inhibitory CPG neurons LG (protraction) <strong>an</strong>d Int1 (retraction),<br />
respectively. Stimulating <strong>the</strong> proprioceptor GPR neuron selectively prolongs <strong>the</strong> MCN1-GMR<br />
retraction phase, by presynaptically inhibiting <strong>the</strong> STG terminals of MCN1 <strong>an</strong>d <strong>the</strong>reby<br />
decreasing activation of <strong>the</strong> voltage-dependent modulator-activated inward current (IMIC) in LG<br />
(Beenhakker et al, 2005; DeLong et al, in prep.).<br />
Bath-application of <strong>the</strong> peptide hormone CCAP (>10 -11 M) selectively prolongs <strong>the</strong> MCN1-GMR<br />
protraction phase (Kirby & Nusbaum, 2007), by activating IMIC in LG (DeLong et al, in prep.).<br />
CCAP also weakens <strong>the</strong> ability of GPR to prolong <strong>the</strong> retraction phase, by its compensatory<br />
activation of IMIC in LG (DeLong & Nusbaum, SFN Abstr 2007).<br />
Here we show that bath-applying <strong>the</strong> peptide hormone CabPK (>10 -11 M) shares with CCAP <strong>the</strong><br />
ability to prolong <strong>the</strong> MCN1 protraction phase (Saline: 5.25±0.6s, CabPK: 6.55±0.7s, n=9,
p0.05). To assess <strong>the</strong> CabPK influence on GPR feedback under conditions comparable to<br />
CCAP application (selective prolongation of protraction), we suppressed DG activity by<br />
hyperpolarization. CabPK (10 -9 M) differed <strong>from</strong> CCAP by not altering <strong>the</strong> GPR prolongation of<br />
retraction (ratio GPR/Control Retraction Duration: Saline, 1.88±0.1; CabPK: 2.04±0.6, n=4,<br />
p=0.78). Also in contrast to CCAP only activating IMIC, CabPK modulates multiple currents in<br />
LG (n=2). One such current identified in a pilot voltage-clamp experiment is a voltage-dependent<br />
current that is distinct <strong>from</strong> IMIC. We aim to continue testing <strong>the</strong> hypo<strong>the</strong>sis that CCAP <strong>an</strong>d<br />
CabPK activate distinct currents in LG but none<strong>the</strong>less share <strong>the</strong> ability to prolong GMRprotraction,<br />
while diverging in <strong>the</strong>ir ability to regulate sensory feedback.<br />
Disclosures: J.C. Rodriguez, None; M.S. Kirby, None; D.M. Blitz, None; M.P. Nusbaum,<br />
None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 564.20/DD71<br />
Topic: D.11.d. Afferent control<br />
Support: NIH Gr<strong>an</strong>t GM08224<br />
NSF Gr<strong>an</strong>t DBI-0115825<br />
NIH Gr<strong>an</strong>t RR03051<br />
NIH Gr<strong>an</strong>t NS058017<br />
NIH Gr<strong>an</strong>t GM61838<br />
Title: Exploring <strong>the</strong> role of feedback in a simple central pattern generator-effector network: Deefferentation<br />
studies in <strong>the</strong> crustace<strong>an</strong> cardiac system<br />
Authors: K. GARCÍA-CRESCIONI 1 , E. STERN 2 , V. BREZINA 2 , *M. W. MILLER 3 ;<br />
1 Inst. of Neurobio. <strong>an</strong>d Dept. of Anat. & Neurobio., Univ. of Puerto Rico Med. Sci. Campus, S<strong>an</strong>
Ju<strong>an</strong>, Puerto Rico; 2 Dept. of Neurosci., Mount Sinai Sch. of Med., New York, NY; 3 Anat. Dept<br />
Inst. of Neur, S<strong>an</strong> Ju<strong>an</strong>, Puerto Rico<br />
Abstract: Although feedback mech<strong>an</strong>isms are thought to play import<strong>an</strong>t roles in <strong>the</strong> operation of<br />
central pattern generator (CPG) circuits, details of such regulation are difficult to assess in<br />
complex motor systems. This study examined feedback in a simple CPG-effector system, <strong>the</strong><br />
heart of <strong>the</strong> blue crab, Callinectes sapidus. The neurogenic heartbeat of <strong>the</strong> crab is driven by<br />
rhythmic burst activity of <strong>the</strong> cardiac g<strong>an</strong>glion (CG), a simple CPG (four premotor interneurons<br />
<strong>an</strong>d five motor neurons) that is embedded within <strong>the</strong> cardiac musculature. Anatomical studies<br />
have shown that, in addition to <strong>the</strong>ir axonal projections to <strong>the</strong> myocardium, <strong>the</strong> CG motor<br />
neurons (MNs) possess dendritic projections that ramify within <strong>the</strong> muscle fiber bundles adjacent<br />
to <strong>the</strong> g<strong>an</strong>glion (Alex<strong>an</strong>drowicz 1932; Tazaki <strong>an</strong>d Cooke 1979). We have now used several<br />
approaches to explore <strong>the</strong> participation of <strong>the</strong>se local collaterals in <strong>the</strong> tr<strong>an</strong>sduction of feedback<br />
<strong>from</strong> <strong>the</strong> myocardium back to <strong>the</strong> CG (García-Crescioni et al., 2008 Neuroscience Abstracts,<br />
375.24). In this study, we took adv<strong>an</strong>tage of <strong>the</strong> topographical separation of <strong>the</strong> MN axonal <strong>an</strong>d<br />
dendritic projections to fur<strong>the</strong>r test <strong>the</strong> hypo<strong>the</strong>sis that CG motor patterns c<strong>an</strong> be directly<br />
influenced by <strong>the</strong> contractions <strong>the</strong>y produce. The myocardium was systematically de-efferented<br />
by sequentially cutting <strong>the</strong> four connectives that carry <strong>the</strong> MN axons to <strong>the</strong> myocardium, leaving<br />
<strong>the</strong> dendritic collaterals intact. As contractions were progressively reduced by <strong>the</strong> deefferentation,<br />
<strong>the</strong> CG burst rate was concomit<strong>an</strong>tly accelerated <strong>an</strong>d burst parameters (impulses<br />
per burst, burst duration) were decreased. To control <strong>for</strong> possible injury effects of <strong>the</strong> axotomy,<br />
identical connective tr<strong>an</strong>sections were per<strong>for</strong>med on <strong>the</strong> isolated CG <strong>from</strong> which <strong>the</strong><br />
myocardium had been removed. In that case <strong>the</strong> axotomy had no effect on <strong>the</strong> burst rate, burst<br />
parameters, or MN membr<strong>an</strong>e potential, suggesting that <strong>the</strong> effects of de-efferentation in <strong>the</strong><br />
complete system could, in fact, be attributed to altered feedback influence of <strong>the</strong> diminished<br />
contractions. These observations are consistent with our previous findings in which deafferentation,<br />
cutting or ligation of <strong>the</strong> dendrites, likewise increased <strong>the</strong> CG burst rate (García-<br />
Crescioni et al. 2008), as well as with findings that stretches of <strong>the</strong> myocardium alter <strong>the</strong> CG<br />
motor pattern (Stern et al., <strong>the</strong>se <strong>abstract</strong>s). Toge<strong>the</strong>r, <strong>the</strong>se observations support <strong>the</strong> proposal<br />
that feedback mech<strong>an</strong>isms, in part probably mediated by <strong>the</strong> MN dendrites, operate in <strong>the</strong><br />
Callinectes cardiac system to regulate <strong>the</strong> CG motor pattern <strong>an</strong>d so, in turn, <strong>the</strong> rate <strong>an</strong>d strength<br />
of <strong>the</strong> heartbeat.<br />
Disclosures: K. García-Crescioni, None; E. Stern, None; V. Brezina, None; M.W. Miller,<br />
None.<br />
Poster<br />
564. Rhythmic Movements: Afferent Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 564.21/DD72<br />
Topic: D.11.d. Afferent control<br />
Title: A soft, biologically inspired worm-like robot called Softworm<br />
Authors: *A. S. BOXERBAUM 1 , R. D. QUINN 2 , H. CHIEL 3 ;<br />
2 Mech<strong>an</strong>ical Engin., 3 Biol., 1 Case Western Res. Univ., Clevel<strong>an</strong>d Heights, OH<br />
Abstract: We have developed several innovative designs <strong>for</strong> a new kind of robot that uses<br />
peristalsis, <strong>the</strong> same method of locomotion earthworms use, <strong>an</strong>d are currently building <strong>the</strong> first<br />
prototypes. This method of locomotion is particularly effective in constrained spaces, <strong>an</strong>d<br />
although <strong>the</strong> motion has been understood <strong>for</strong> some time, it has never been effectively or<br />
accurately implemented in a robotic plat<strong>for</strong>m. We address <strong>the</strong> reasons <strong>for</strong> this, including some<br />
common misconceptions within <strong>the</strong> field, <strong>an</strong>d present a technique of using a braided mesh<br />
exterior to produce fluid waves of motion along <strong>the</strong> body of <strong>the</strong> robot. The concept is highly<br />
scalable, <strong>an</strong>d we present methods of construction at two different scales. We also present a<br />
concept <strong>for</strong> a robot specifically designed <strong>for</strong> pipe crawling, where it c<strong>an</strong> take adv<strong>an</strong>tage of<br />
flowing fluid to locomote very efficiently, even upstream.<br />
Disclosures: A.S. Boxerbaum, Authors have patent pending, E. Ownership Interest (stock,<br />
stock options, patent or o<strong>the</strong>r intellectual property); R.D. Quinn, Authors have patent pending,<br />
E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); H. Chiel,<br />
Authors have patent pending, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property).<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.1/DD73<br />
Topic: D.11.f. Models<br />
Support: DFG STE 937/5-1 <strong>an</strong>d 5-2<br />
Title: Simple or complex? How to provide virtual sensory feedback in <strong>an</strong> isolated nervous<br />
system
Authors: *N. DAUR, W. STEIN;<br />
Univ. Ulm, Ulm, Germ<strong>an</strong>y<br />
Abstract: Proprioceptors provide <strong>the</strong> nervous system with in<strong>for</strong>mation about <strong>the</strong> state of <strong>the</strong><br />
body <strong>an</strong>d are thus vital <strong>for</strong> all <strong>an</strong>imals. To test <strong>the</strong> response of <strong>the</strong> nervous system to<br />
proprioceptive feedback on <strong>the</strong> cellular level it is often inevitable to revert to reduced<br />
preparations. Yet, in <strong>the</strong>se preparations it is difficult to provide adequate sensory feedback.<br />
Traditionally, predefined stimuli are used or time/phase-correct stimulations are applied. Both<br />
conditions do not reflect <strong>the</strong> activation of <strong>the</strong> proprioceptors in <strong>the</strong> <strong>an</strong>imal <strong>an</strong>d may not be<br />
adequate to study <strong>the</strong> effects of sensory feedback. We are addressing this issue by investigating<br />
<strong>the</strong> muscle tendon org<strong>an</strong> AGR (<strong>an</strong>terior gastric receptor; Smar<strong>an</strong>dache & Stein 2007, JEB 210)<br />
in <strong>the</strong> stomatogastric nervous system of <strong>the</strong> crab. AGR measures <strong>the</strong> tension of a muscle<br />
involved in <strong>the</strong> chewing of food, which is driven by <strong>the</strong> rhythmic activity of <strong>the</strong> gastric mill<br />
central pattern generator.<br />
AGR’s soma possesses intrinsic properties such as sag potential <strong>an</strong>d spike frequency adaptation<br />
<strong>an</strong>d spikes <strong>from</strong> <strong>the</strong> periphery have to pass <strong>the</strong> soma to reach <strong>the</strong>ir target circuits. AGR is<br />
typically activated phasically during teeth protraction <strong>an</strong>d its firing frequency depends on <strong>the</strong><br />
prevailing motor activity. Here, we test whe<strong>the</strong>r a simple sensory feedback is sufficient to elicit<br />
<strong>the</strong> adequate motor response or if a more complex feedback is required. For this, we used a<br />
combination of <strong>the</strong> isolated biological nervous system <strong>an</strong>d real-time sensory feedback provided<br />
by a computer. We <strong>the</strong>n compared <strong>the</strong> response of <strong>the</strong> gastric mill motor system in two<br />
conditions: (I) We activated AGR in a phase-correct way, but with const<strong>an</strong>t frequency or (II)<br />
AGR activity was calculated by a model based on physiological data, which reproduced <strong>the</strong><br />
known response of AGR. In <strong>the</strong> latter case, <strong>the</strong> activity of motor neurons was tr<strong>an</strong>s<strong>for</strong>med into<br />
muscle tension, which <strong>the</strong>n was used <strong>for</strong> current injection into <strong>the</strong> AGR model. The model<br />
output, in turn, drove <strong>the</strong> biological AGR.<br />
We found that <strong>the</strong> resulting motor patterns showed great differences, <strong>for</strong> example in <strong>the</strong>ir period<br />
<strong>an</strong>d phase relationships. On average, <strong>the</strong> cycle periods of rhythms with <strong>the</strong> AGR model were<br />
faster (about 10 s) th<strong>an</strong> those of rhythms with const<strong>an</strong>t AGR frequency (about 14 s), although <strong>the</strong><br />
control period prior to AGR activation was similar. Additionally, <strong>the</strong> protraction phase of <strong>the</strong><br />
rhythm was signific<strong>an</strong>tly shorter when AGR was activated with const<strong>an</strong>t frequency, while <strong>the</strong><br />
retraction phase was prolonged.<br />
Our results thus indicate that to provide adequate proprioceptive feedback it is necessary to<br />
consider <strong>the</strong> dynamics of <strong>the</strong> sensory response instead of just relying on predefined parameters.<br />
Disclosures: N. Daur, None; W. Stein, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 565.2/DD74<br />
Topic: D.11.e. Descending control<br />
Support: DFG STE 937/2-1<br />
DFG STE 937/2-2<br />
Title: The activation of one projection neuron c<strong>an</strong> tip <strong>the</strong> scales: Motor pattern selection by<br />
different sensory pathways<br />
Authors: *U. B. HEDRICH 1 , C. R. SMARANDACHE 3 , W. STEIN 2 ;<br />
1 Dept Neurol. <strong>an</strong>d Applied Physiol., 2 Inst. of Neurobio., Ulm Univ., Ulm, Germ<strong>an</strong>y; 3 Dept. of<br />
Neurobiology, Physiology, <strong>an</strong>d Behavior, Univ. of Cali<strong>for</strong>nia, Davis CA, Davis, CA<br />
Abstract: We are using <strong>the</strong> stomatogastric nervous system (STNS) of <strong>the</strong> crab, C<strong>an</strong>cer pagurus,<br />
to study <strong>the</strong> effects of different sensory systems on projection neurons <strong>an</strong>d <strong>the</strong> resulting selection<br />
of motor patterns <strong>from</strong> multifunctional circuits. Within <strong>the</strong> stomatogastric g<strong>an</strong>glion (STG) two<br />
central pattern generators produce <strong>the</strong> spont<strong>an</strong>eously active, rapid pyloric rhythm (filtering of<br />
food) <strong>an</strong>d <strong>the</strong> slow gastric mill rhythm (chewing food within <strong>the</strong> <strong>for</strong>egut). These central pattern<br />
generators are modulated by <strong>the</strong> activities of several modulatory projection neurons in <strong>the</strong><br />
commissural g<strong>an</strong>glia (CoG). Three of <strong>the</strong>se projection neurons are identified namely <strong>the</strong><br />
modulatory commissural neuron 1 (MCN1) <strong>an</strong>d 5 (MCN5) as well as <strong>the</strong> commissural projection<br />
neuron 2 (CPN2). These projection neurons descend <strong>from</strong> <strong>the</strong> paired CoGs via <strong>the</strong> superior<br />
oesophageal nerve (son) or <strong>the</strong> inferior ventricular nerve (ion) <strong>an</strong>d <strong>the</strong> stomatogastric nerve (stn)<br />
to <strong>the</strong> stomatogastric g<strong>an</strong>glion (STG) where <strong>the</strong>ir modulatory effects appear to be <strong>the</strong> main<br />
reason <strong>for</strong> a broad spectrum of observed rhythms. The activities of <strong>the</strong> modulatory projection<br />
neurons are in turn driven by sensory pathways which typically activate more th<strong>an</strong> one projection<br />
neuron. Here we study <strong>the</strong> actions of <strong>the</strong> proprioceptive <strong>an</strong>terior gastric receptor (AGR) <strong>an</strong>d <strong>the</strong><br />
exteroceptive inferior ventricular (IV) neurons on <strong>the</strong> modulatory projection neurons. AGR is a<br />
muscle tendon org<strong>an</strong> in <strong>the</strong> STG (Smar<strong>an</strong>dache & Stein, J Exp Biol, 2007) whereas <strong>the</strong> IV<br />
neurons are located in <strong>the</strong> brain <strong>an</strong>d relay exteroceptive chemosensory in<strong>for</strong>mation <strong>from</strong> <strong>the</strong><br />
<strong>an</strong>tennae to <strong>the</strong> STNS (Hedrich & Stein, J Exp Biol, 2008). A stimulation of AGR <strong>an</strong>d <strong>the</strong> IV<br />
neurons affects all three projection neurons, but elicit distinct gastric mill rhythms. We found<br />
that both sensory pathways excite CPN2 <strong>an</strong>d MCN5 but differ in <strong>the</strong>ir type of excitation<br />
(electrical vs. chemical). On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>the</strong> proprioceptor AGR directly inhibits MCN1<br />
whereas it is polysynaptically excited by <strong>the</strong> exteroceptive IV neurons. By systematically<br />
eliminating <strong>the</strong> effects of <strong>the</strong> different projection neurons we found that MCN1 is essential <strong>for</strong><br />
eli<strong>citing</strong> a gastric mill rhythm during IV neuron stimulation but is less import<strong>an</strong>t during AGR<br />
stimulation. Adding IV neuron like MCN1 excitation during AGR stimulation made <strong>the</strong> gastric<br />
mill rhythm more similar to <strong>the</strong> IV neuron elicited rhythm.<br />
Our results thus support <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> combination of different projection neurons<br />
activated by sensory pathways contributes to <strong>the</strong> selection of distinct motor patterns <strong>an</strong>d that <strong>the</strong><br />
activation of one additional projection neuron c<strong>an</strong> tip <strong>the</strong> scales <strong>an</strong>d generate a signific<strong>an</strong>tly<br />
different motor pattern.
Disclosures: U.B. Hedrich, None; C.R. Smar<strong>an</strong>dache, None; W. Stein, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.3/EE1<br />
Topic: D.11.e. Descending control<br />
Support: DFG gr<strong>an</strong>t Bu 857/8,10<br />
Title: Walking speed control in <strong>the</strong> stick insect: Elucidating mech<strong>an</strong>isms<br />
Authors: *M. GRUHN, A. BORGMANN, G. VON UCKERMANN, S. WESTMARK, A.<br />
WOSNITZA, A. BÜSCHGES;<br />
Univ. Koeln, Koeln, Germ<strong>an</strong>y<br />
Abstract: It is still a largely unresolved question how walking speed <strong>an</strong>d <strong>the</strong> corresponding<br />
ch<strong>an</strong>ges in coordination of <strong>the</strong> six legs are controlled in insects. It has been shown in <strong>the</strong> single<br />
leg stick insect preparation, that mech<strong>an</strong>isms to control stepping velocity become effective only<br />
during <strong>an</strong> already ongoing st<strong>an</strong>ce phase motor output (1,2). Using electrophysiological <strong>an</strong>d<br />
behavioral experiments in reduced preparations <strong>an</strong>d intact stick insects (Carausius morosus Br.)<br />
we investigated mech<strong>an</strong>isms underlying <strong>the</strong> control of walking speed on different levels of <strong>the</strong><br />
nervous system.<br />
In <strong>the</strong> single stick insect leg, stepping velocity has already been shown to arise <strong>from</strong> modification<br />
in flexor MN activity (2). We found no additional correlation between spike frequency of <strong>the</strong> fast<br />
extensor tibiae motor neuron (FETi MN), which is active during swing, <strong>an</strong>d stepping velocity. A<br />
correlation with stepping velocity was only found <strong>for</strong> <strong>the</strong> pause between <strong>the</strong> motoneuronal st<strong>an</strong>ce<br />
<strong>an</strong>d swing activity at <strong>the</strong> tr<strong>an</strong>sition <strong>from</strong> st<strong>an</strong>ce to swing. Intracellular recordings <strong>from</strong> nonspiking<br />
premotor interneurons (NSI), which play <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> control of posture <strong>an</strong>d<br />
movement, supported <strong>the</strong>se findings. In <strong>the</strong>se neurons alterations in stepping velocity were<br />
exclusivley correlated with modifications in <strong>the</strong> activity of NSI involved in st<strong>an</strong>ce generation.<br />
This corroborates previous findings that stepping velocity in <strong>the</strong> single leg is solely dependent on<br />
st<strong>an</strong>ce phase MN <strong>an</strong>d not on swing phase MN activity. By me<strong>an</strong>s of extra- <strong>an</strong>d intracellular<br />
recordings in reduced stick insect preparations, we <strong>the</strong>n tested whe<strong>the</strong>r <strong>the</strong> stepping speed in one<br />
leg has <strong>an</strong> influence on <strong>the</strong> general excitation in o<strong>the</strong>r legs. On <strong>the</strong> inter-leg level, no systematic<br />
linear relationship was found between <strong>the</strong> velocity of a stepping front leg <strong>an</strong>d <strong>the</strong> motoneuronal<br />
activity in contralateral mesothoracic protractor <strong>an</strong>d retractor, or flexor MNs. The lack of<br />
coordination of stepping velocity between legs under normal walking conditions was confirmed
in behavioral experiments with intact stick insects te<strong>the</strong>red above a slippery surface, <strong>the</strong>reby<br />
effectively removing mech<strong>an</strong>ical coupling through <strong>the</strong> ground. In this situation, <strong>the</strong>re were again<br />
no systematic correlations between <strong>the</strong> stepping velocities of different legs. However, when <strong>the</strong><br />
te<strong>the</strong>red <strong>an</strong>imal increased walking speed due to a short tactile stimulus, stepping velocities of<br />
ipsilateral legs were found to be correlated <strong>for</strong> several steps.<br />
These results show that neuronal influences exist which coordinate stepping velocities between<br />
legs under certain circumst<strong>an</strong>ces, but that <strong>the</strong>se are usually weak in <strong>the</strong> slowly stepping <strong>an</strong>imal.<br />
1) Gabriel et al., 2003<br />
2) Gabriel & Büschges, 2007<br />
Disclosures: M. Gruhn, None; A. Borgm<strong>an</strong>n, None; G. von Uckerm<strong>an</strong>n, None; S.<br />
Westmark, None; A. Wosnitza, None; A. Büschges, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.4/EE2<br />
Topic: D.11.e. Descending control<br />
Support: NSF IBN-0615631 NSF<br />
Title: Modulation of leech swim duration by caudal g<strong>an</strong>glia<br />
Authors: *O. J. MULLINS 1 , J. T. HACKETT 2 , W. O. FRIESEN 3 ;<br />
1 2 3<br />
Neurosci. Grad. Program, Mol. Physiol. <strong>an</strong>d Biol. Physics, Biol., Univ. of Virginia,<br />
Charlottesville, VA<br />
Abstract: Using <strong>the</strong> leech swim system, we examine how a tr<strong>an</strong>sient stimulus c<strong>an</strong> elicit a<br />
prolonged behavioral response. While <strong>the</strong> mech<strong>an</strong>isms underlying swim mainten<strong>an</strong>ce are largely<br />
unidentified, caudal nerve cord g<strong>an</strong>glia are known to influence swim duration. Functional <strong>an</strong>d<br />
physical removal of caudal g<strong>an</strong>glia decreases swim duration about 55% while local excitation of<br />
<strong>the</strong> most caudal g<strong>an</strong>glion, or ‘tail brain’, elicits swim episodes 2-8 fold longer th<strong>an</strong> control<br />
swims. A model <strong>for</strong> <strong>the</strong>se swim-prolonging effects comprises continual mutual excitation<br />
between neurons in <strong>the</strong> caudal g<strong>an</strong>glia <strong>an</strong>d remaining nerve cord. To test <strong>the</strong> model, caudal<br />
g<strong>an</strong>glia were functionally connected or disconnected <strong>from</strong> <strong>an</strong> isolated leech nerve cord during<br />
on-going swim episodes. A stream of sodium-free isotonic sucrose, a ‘sucrose knife’, passing<br />
over a connective blocks impulses, allows fast, functional <strong>an</strong>d reversible disconnection of<br />
g<strong>an</strong>glia. Caudal g<strong>an</strong>glia were 1) connected, C, 2) disconnected, D, 3) connected <strong>the</strong>n
disconnected, C/D, or 4) disconnected <strong>the</strong>n connected, D/C, <strong>from</strong> <strong>the</strong> remainder of <strong>the</strong> nerve<br />
cord. For C <strong>an</strong>d D, preparations were in one state <strong>for</strong> <strong>the</strong> entire swim episode. For C/D <strong>an</strong>d D/C,<br />
a ‘state-switch’ followed <strong>an</strong> initiating stimulus (0.1-20 s delay). Swim durations in condition C<br />
averaged 50.3 bursts per episode. Condition D/C swim durations were not signific<strong>an</strong>tly different<br />
th<strong>an</strong> C swims, but nearly doubled in vari<strong>an</strong>ce. Conditions D <strong>an</strong>d C/D were signific<strong>an</strong>tly shorter<br />
th<strong>an</strong> C, with swim durations decreased by 41% <strong>an</strong>d 61%, respectively. Although C/D swims<br />
tended to be shorter th<strong>an</strong> D swims, <strong>the</strong> difference was not signific<strong>an</strong>t. Most C/D swims displayed<br />
one of two features: 1) very short swim episodes or 2) swim lengths comparable to D swims, but<br />
with increased cycle periods or a swim interruption several seconds following block initiation.<br />
On <strong>the</strong> cellular level, gating cell 204 is a likely particip<strong>an</strong>t in <strong>the</strong> mutual excitation between <strong>the</strong><br />
caudal <strong>an</strong>d o<strong>the</strong>r g<strong>an</strong>glia. Prolonged depolarization of cell 204 drives swimming, while<br />
hyperpolarizing two cell 204s shortens swim duration. There<strong>for</strong>e, we examine ch<strong>an</strong>ges in cell<br />
204 depolarization <strong>an</strong>d firing frequency following state-switches during swim episodes.<br />
Additionally, <strong>the</strong> effect of state-switches on cell 204 ‘sustained excitation’ following stimulation<br />
in shortened non-swimming preparations is examined.<br />
The results of our experiments indicate that <strong>the</strong> presence of caudal g<strong>an</strong>glia is necessary<br />
throughout a swim episode to achieve swim prolongation. Mutual excitation between caudal<br />
g<strong>an</strong>glion neurons <strong>an</strong>d cell 204 is a likely mech<strong>an</strong>ism that sustains <strong>the</strong> swim response following a<br />
brief initiating stimulus.<br />
Disclosures: O.J. Mullins, None; J.T. Hackett, None; W.O. Friesen, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.5/EE3<br />
Topic: D.11.e. Descending control<br />
Support: NIH F31NS060332-01<br />
NSF Gr<strong>an</strong>t IOB 0523959<br />
Title: Keeping it toge<strong>the</strong>r: Choreographing <strong>the</strong> motor pattern <strong>for</strong> crawling in <strong>the</strong> medicinal leech<br />
Authors: *J. G. PUHL 1 , K. A. MESCE 2 ;<br />
1 Grad Prgm Neurosci & Dept Entomol, Univ. Minnesota, St. Paul, MN; 2 Depts of Entomology<br />
<strong>an</strong>d Neurosci., Univ. of Minnesota, St. Paul, MN
Abstract: A fundamental problem of locomotion in metameric <strong>an</strong>imals is <strong>the</strong> overall activation<br />
<strong>an</strong>d coordination of segmental oscillators. In some <strong>an</strong>imal systems, segmental central pattern<br />
generators possess inherent intersegmental delays, whereas o<strong>the</strong>rs have segmental oscillators<br />
whose activities are less interdependent. The medicinal leech exhibits two primary <strong>for</strong>ms of<br />
locomotion, crawling <strong>an</strong>d swimming, <strong>an</strong>d we have determined that crawling serves as a model<br />
whereby independent unit burst generators (UBGs) are loosely coupled <strong>an</strong>d depend on a<br />
hierarchical org<strong>an</strong>ization of control. Because each of <strong>the</strong> 21 segmental g<strong>an</strong>glia of <strong>the</strong> leech CNS<br />
contains a complete UBG <strong>for</strong> crawling, which c<strong>an</strong> be independently activated by dopamine (DA)<br />
(Puhl <strong>an</strong>d Mesce, 2008, J. Neurosci., 28:4192-4200), we have been able to study UBG<br />
interdependence <strong>an</strong>d <strong>the</strong> global activation of <strong>the</strong> crawl network. We first determined <strong>the</strong> role of<br />
<strong>the</strong> cephalic g<strong>an</strong>glion in choreographing overt crawling by reversibly blocking descending<br />
signals. We used a sucrose block placed at <strong>the</strong> connectives of a nearly intact leech preparation<br />
wherein <strong>the</strong> <strong>an</strong>terior 20% of <strong>the</strong> leech was dissected <strong>an</strong>d <strong>the</strong> remaining 80% of <strong>the</strong> body was<br />
intact <strong>an</strong>d free to exhibit locomotion. Prior to <strong>the</strong> block, we observed spont<strong>an</strong>eous crawling (8/8<br />
leeches) <strong>an</strong>d could easily elicit crawling via gentle body stimulation (8/8 leeches). During <strong>the</strong><br />
sucrose block, we saw no crawling, ei<strong>the</strong>r spont<strong>an</strong>eous or elicited (8/8 leeches). After removal of<br />
<strong>the</strong> blockade, spont<strong>an</strong>eous crawling was restored (6/8 leeches) <strong>an</strong>d we could again elicit crawling<br />
(8/8 leeches). Next, we tested whe<strong>the</strong>r UBG-to-UBG (i.e., local) signals were sufficient to<br />
generate normal intersegmental delays. We monitored fictive crawling activated by DA <strong>an</strong>d<br />
observed that crawling stopped when descending control was blocked (3/12). However, under<br />
<strong>the</strong> bias of DA <strong>the</strong> majority of preparations continued to crawl, but with altered intersegmental<br />
delays <strong>an</strong>d phase relationships (9/12). To test <strong>the</strong> sufficiency of local signals to produce normal<br />
delays, we activated a subset of g<strong>an</strong>glia (within chains of 4-6 g<strong>an</strong>glia) that were removed <strong>from</strong><br />
<strong>the</strong> cephalic g<strong>an</strong>glion. Although we did observe rhythmic crawl activity in chains, intersegmental<br />
coordination was always absent. Fur<strong>the</strong>r experiments demonstrated that a single crawl oscillator,<br />
activated by DA, could provide drive to <strong>an</strong>terior (N=12) <strong>an</strong>d posterior (N=14) adjacent g<strong>an</strong>glia,<br />
but intersegmental (i.e., phase) delays were never normal. We conclude that, in sharp contrast to<br />
swimming, <strong>the</strong> cephalic g<strong>an</strong>glion is necessary <strong>for</strong> <strong>the</strong> global activation of <strong>the</strong> crawl oscillators<br />
<strong>an</strong>d <strong>the</strong> intersegmental coordination among <strong>the</strong>m.<br />
Disclosures: J.G. Puhl, None; K.A. Mesce, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.6/EE4<br />
Topic: D.11.e. Descending control
Support: NIH Gr<strong>an</strong>t NS40755<br />
Title: Synaptic output patterns of uniquely identified reticulospinal neurons to different<br />
populations of spinal motoneurons in <strong>the</strong> lamprey<br />
Authors: *J. T. BUCHANAN, J. F. EINUM;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: Uniquely identifiable reticulospinal neurons in <strong>the</strong> lamprey (Müller <strong>an</strong>d Mauthner<br />
cells) were shown previously to have specific patterns of output to ipsilateral motoneurons <strong>an</strong>d<br />
interneurons in <strong>the</strong> spinal cord. In <strong>the</strong> present study, we extended <strong>the</strong>se findings by examining<br />
<strong>the</strong> synaptic output patterns of identified reticulospinal neurons to bilateral subpopulations of<br />
motoneurons: those innervating dorsal body muscles versus those innervating ventral body<br />
muscles. Differential activation of <strong>the</strong>se motoneuron subpopulations is import<strong>an</strong>t in motor<br />
control in <strong>the</strong> lamprey, a lower vertebrate fish which lacks lateral fins. An in vitro spinal cord<br />
preparation with body muscles left attached was used to stimulate individual motoneurons with<br />
<strong>an</strong> intracellular microelectrode <strong>an</strong>d visually determine <strong>the</strong> dorsal/ventral location of each<br />
motoneuron’s muscle fibers. The synaptic input <strong>from</strong> <strong>the</strong> I1 reticulospinal cell axon was <strong>the</strong>n<br />
tested using paired intracellular recording of <strong>the</strong> ipsilateral I1 axon <strong>an</strong>d <strong>the</strong> postsynaptic<br />
motoneuron. Ventral motoneurons received monosynaptic dual electrical/chemical excitatory<br />
synaptic potentials (epsps), while dorsal motoneurons received disynaptic glycinergic ipsps.<br />
Motoneurons innervating middle muscle fibers had mixed results <strong>for</strong> I1 input: ei<strong>the</strong>r no input or<br />
a combined epsp/ipsp. To test outputs of all Müller <strong>an</strong>d Mauthner cells, <strong>an</strong> isolated<br />
brainstem/spinal cord preparation was used, <strong>an</strong>d spinal motoneurons were identified as<br />
innervating dorsal or ventral body muscles based on <strong>the</strong> synaptic input <strong>from</strong> <strong>the</strong> ipsilateral I1 cell<br />
body: those with ipsps were classified as dorsal motoneurons <strong>an</strong>d those with epsps were<br />
classified as ventral motoneurons. Each bilateral pair of <strong>the</strong> Müller <strong>an</strong>d Mauthner cells was <strong>the</strong>n<br />
tested with <strong>an</strong> intracellular microelectrode <strong>for</strong> synaptic outputs to <strong>the</strong> motoneuron. For each<br />
reticulospinal cell, <strong>the</strong>re was a consistent output pattern to <strong>the</strong> motoneurons of <strong>the</strong> four muscle<br />
quadr<strong>an</strong>ts <strong>from</strong> <strong>an</strong>imal to <strong>an</strong>imal. For example, B2 - B4 Müller cells produced excitation of<br />
ipsilateral dorsal <strong>an</strong>d ventral motoneurons with little effect on contralateral motoneurons. The<br />
Mauthner cell excited motoneurons to all four quadr<strong>an</strong>ts, while <strong>the</strong> B1 Müller cell inhibited<br />
contralateral dorsal <strong>an</strong>d ventral motoneurons with weaker ipsilateral ipsps. The M1 - M3 Müller<br />
cells each had a unique pattern of excitation <strong>an</strong>d inhibition of different quadr<strong>an</strong>ts. In conclusion,<br />
descending motor control in <strong>the</strong> lamprey is accomplished in part by specific patterns of synaptic<br />
output <strong>from</strong> reticulospinal neurons to motoneurons innervating muscle fibers in <strong>the</strong> different<br />
body quadr<strong>an</strong>ts to produce cell-specific body movement vectors.<br />
Disclosures: J.T. Buch<strong>an</strong><strong>an</strong>, None; J.F. Einum, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.7/EE5<br />
Topic: D.11.e. Descending control<br />
Support: NIH RO-1 EY014429-01A2<br />
Title: Activation of motor patterns <strong>an</strong>d reticulospinal neurons by electrical stimulation in larval<br />
zebrafish brainstem<br />
Authors: *K. E. SEVERI 1 , D. O'MALLEY 1 , F. ENGERT 2 ;<br />
1 Dept Biol, Nor<strong>the</strong>astern Univ., Boston, MA; 2 Dept of MCB, Harvard Univ., Cambridge, MA<br />
Abstract: The brainstem neurons of <strong>the</strong> larval zebrafish are <strong>the</strong> primary me<strong>an</strong>s of sending<br />
descending motor comm<strong>an</strong>ds to <strong>the</strong> spinal cord. How <strong>the</strong>se cells mediate <strong>the</strong> initiation <strong>an</strong>d<br />
control of spinal circuits involved in larval swimming behaviors remains unclear. One nucleus in<br />
particular, <strong>the</strong> Nucleus of <strong>the</strong> Medial Longitudinal Fasiculus (NucMLF) lies rostral to <strong>the</strong><br />
midbrain-hindbrain border, <strong>an</strong>d has been shown to be active during <strong>the</strong> optomotor response<br />
(Orger et al., 2008), which elicits <strong>for</strong>ward swimming. In <strong>an</strong> ef<strong>for</strong>t to better underst<strong>an</strong>d how <strong>the</strong><br />
NucMLF contributes to <strong>the</strong> initiation <strong>an</strong>d control of locomotion we have combined electrical<br />
stimulation <strong>an</strong>d high-speed video to activate this nucleus <strong>an</strong>d observe <strong>the</strong> resulting locomotor<br />
output in larvae 5-10 days post fertilization. In partially restrained larval zebrafish, electrical<br />
stimulation c<strong>an</strong> elicit rhythmic bending patterns, whose amplitude, direction <strong>an</strong>d tail-beat<br />
frequency correspond to locomotor behaviors. These same stimulus protocols are also able to<br />
elicit calcium responses in fluorescently labeled neurons in <strong>the</strong> same larvae. Using <strong>the</strong>se<br />
techniques, we hope to reveal functional <strong>an</strong>d <strong>an</strong>atomical connections between reticulospinal cells<br />
<strong>an</strong>d <strong>the</strong> downstream spinal targets active during locomotion.<br />
Disclosures: K.E. Severi , None; D. O'Malley, None; F. Engert, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.8/EE6<br />
Topic: D.11.b. Connectivity
Support: Lampetra project FP7-ICT-2007-1-216100<br />
MENRT<br />
Title: Dynamics of <strong>the</strong> axial locomotor network in <strong>the</strong> isolated spinal cord of <strong>the</strong> salam<strong>an</strong>der<br />
Authors: *D. RYCZKO 1 , S. LAMARQUE 2 , H. DIDIER 2 , J.-M. CABELGUEN 2 ;<br />
1 Univ. De Montreal, Montreal, QC, C<strong>an</strong>ada; 2 INSERM U 862, Bordeaux, Fr<strong>an</strong>ce<br />
Abstract: Salam<strong>an</strong>ders are amphibi<strong>an</strong>s that display a diversity of locomotor modes both in water<br />
<strong>an</strong>d on l<strong>an</strong>d. During locomotion, <strong>the</strong> rhythmic <strong>an</strong>d coordinated activations of axial <strong>an</strong>d limb<br />
muscles are generated by neural networks, referred as Central Pattern Generators (CPGs), that<br />
are located in <strong>the</strong> spinal cord. A prerequisite in <strong>the</strong> underst<strong>an</strong>ding of <strong>the</strong> neural control <strong>for</strong><br />
locomotion in <strong>the</strong> salam<strong>an</strong>der is <strong>the</strong> detailed knowledge of <strong>the</strong> dynamics of <strong>the</strong> spinal CPG (<strong>for</strong><br />
inst<strong>an</strong>ce, <strong>the</strong> relations between <strong>the</strong> locomotor cycle duration, <strong>the</strong> intersegmental phase lag, <strong>an</strong>d<br />
<strong>the</strong> duty cycle).<br />
In <strong>the</strong> present study we investigated <strong>the</strong> different patterns of axial motor activity produced by <strong>the</strong><br />
in vitro isolated spinal cord of salam<strong>an</strong>ders superfused with NMDA (20 µM) <strong>an</strong>d D-serine<br />
(10µM). We used three different preparations: <strong>the</strong> isolated whole spinal cord, <strong>the</strong> mid-trunk<br />
spinal cord (i.e., disconnected <strong>from</strong> <strong>the</strong> limb oscillators), <strong>an</strong>d <strong>the</strong> mid-trunk hemicord (i.e.,<br />
longitudinally split).<br />
Three patterns of longitudinal intersegmental coordination were observed in <strong>the</strong>se three different<br />
preparations: a rostrocaudal propagated activation of <strong>the</strong> ipsilateral ventral roots (VRs), a<br />
synchronous activation of <strong>the</strong> ipsilateral VRs, <strong>an</strong>d a caudorostral propagated activation of <strong>the</strong><br />
ipsilateral VRs.<br />
We previously showed that each hemisegment of <strong>the</strong> mid-trunk spinal cord is able to generate a<br />
rhythmic activity. Altoge<strong>the</strong>r <strong>the</strong>se results suggest that <strong>the</strong> axial network is constituted by a<br />
double chain of oscillators. Notably, it me<strong>an</strong>s that <strong>the</strong> mech<strong>an</strong>isms underlying <strong>the</strong> coordination<br />
of <strong>the</strong> hemisegmental oscillators are distributed on left <strong>an</strong>d right sides. Our results provide<br />
evidence <strong>for</strong> a large flexibility of <strong>the</strong> in vitro axial network in relation with <strong>the</strong> diversity of <strong>the</strong><br />
locomotor patterns observed in intact salam<strong>an</strong>ders.<br />
Disclosures: D. Ryczko, None; S. Lamarque, None; H. Didier, None; J. Cabelguen, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.9/EE7
Topic: D.11.b. Connectivity<br />
Support: NSERC 217435<br />
CIHR 15129 <strong>an</strong>d 84765<br />
FRSQ 5249 <strong>an</strong>d 12770<br />
Title: Connectivity within <strong>the</strong> brainstem respiratory network in lampreys<br />
Authors: *J.-F. GARIEPY 1 , K. MISSAGHI 2 , S. CHARTRÉ 2 , M. ROBERT 2 , F. AUCLAIR 1 , J.<br />
P. LUND 3,1 , R. DUBUC 2,1 ;<br />
1 Dept. of Physiol., Univ. Montreal, Montreal, QC, C<strong>an</strong>ada; 2 Dept. of Kinesiology, Univ. du<br />
Québec à Montréal, Montreal, QC, C<strong>an</strong>ada; 3 Fac. of Dent., McGill Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Respiration is generated by a neuronal network located in <strong>the</strong> brainstem. The detailed<br />
connectivity of <strong>the</strong> respiratory network is not fully understood. We are using <strong>the</strong> lamprey, a<br />
simple vertebrate model, to examine <strong>the</strong> connectivity within <strong>the</strong> respiratory network in <strong>the</strong><br />
brainstem. In this <strong>an</strong>imal, <strong>the</strong> respiratory rhythm is generated by neurons located in <strong>the</strong> rostrolateral<br />
hindbrain. This site is referred to as <strong>the</strong> paratrigeminal respiratory group (pTRG). This<br />
study aimed at characterizing <strong>the</strong> projections between <strong>the</strong> respiratory generators on both sides of<br />
<strong>the</strong> brainstem <strong>an</strong>d <strong>the</strong>ir projections to respiratory motoneurons. Fluorescent tracers were injected<br />
into <strong>the</strong> respiratory motoneuronal pools <strong>an</strong>d/or <strong>the</strong> pTRG. Injections in <strong>the</strong> pTRG labelled cell<br />
bodies in <strong>the</strong> contralateral pTRG <strong>an</strong>d descending fibres in close proximity to <strong>the</strong> respiratory<br />
motoneurons. Injections in <strong>the</strong> motoneuronal pools of <strong>the</strong> VII, IX <strong>an</strong>d X motor nuclei labelled<br />
cells in <strong>the</strong> pTRG on both sides. Double-tracing experiments were also carried out with one<br />
tracer injection into <strong>the</strong> motoneuronal pool <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r into <strong>the</strong> pTRG. In <strong>the</strong>se cases, we found<br />
double-labelled neurons in <strong>the</strong> non-injected pTRG area. These experiments show that <strong>the</strong> pTRG<br />
on one side of <strong>the</strong> brainstem projects to <strong>the</strong> contralateral pTRG as well as to ipsi- <strong>an</strong>d<br />
contralateral respiratory motoneuronal pools. Physiological experiments were per<strong>for</strong>med to<br />
fur<strong>the</strong>r characterize <strong>the</strong> descending projections <strong>from</strong> <strong>the</strong> pTRG to <strong>the</strong> motoneuronal pools.<br />
Respiratory motoneurons were recorded using sharp intracellular electrodes. The ipsi- or<br />
contralateral pTRG was electrically stimulated. Stimulation of <strong>the</strong> pTRG induced large EPSPs of<br />
similar sizes in ipsi- <strong>an</strong>d contralateral respiratory motoneurons (p > 0.05). The latency was<br />
longer <strong>for</strong> contralateral motoneurons (10.2 ± 3.8 vs 7.4 ± 4.0 ms; p < 0.05). Blockade of<br />
glutamatergic ionotropic receptors by locally injecting 1 mM CNQX <strong>an</strong>d 500 µM AP-5 on <strong>the</strong><br />
recorded respiratory motoneuron completely abolished <strong>the</strong> EPSPs. This study allowed us to<br />
identify projections between <strong>the</strong> respiratory generators on both sides of <strong>the</strong> brainstem. We also<br />
show that pTRG neurons excite both <strong>the</strong> ipsi- <strong>an</strong>d contralateral motoneurons through<br />
glutamatergic synapses. The bilateral connections between <strong>the</strong> generators <strong>an</strong>d <strong>the</strong>ir bilateral<br />
descending projections may play a salient role in synchronizing <strong>the</strong> respiratory activity. Funded<br />
by NSERC, CIHR <strong>an</strong>d FRSQ.<br />
Disclosures: J. Gariepy, None; K. Missaghi, None; S. Chartré, None; M. Robert, None; F.<br />
Auclair, None; J.P. Lund, None; R. Dubuc, None.
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.10/EE8<br />
Topic: D.11.e. Descending control<br />
Support: CIHR Gr<strong>an</strong>t 15129<br />
FRSQ Gr<strong>an</strong>t 5249<br />
Title: Patterns of activity of reticulospinal neurons during locomotion in lampreys<br />
Authors: *L. JUVIN 1 , R. DUBUC 2 ;<br />
1 2<br />
Univ. Montreal, Montreal, QC, C<strong>an</strong>ada; Dept. of Kinesiology, Univ. du Québec à Montreal,<br />
Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Locomotion is controlled by specific supraspinal centers, one of which is <strong>the</strong><br />
Mesencephalic Locomotor Region (MLR). The MLR initiate <strong>an</strong>d control <strong>the</strong> locomotor through<br />
projections to reticulospinal (RS) neurons. This org<strong>an</strong>ization seems to be conserved in all<br />
vertebrates. We have used <strong>the</strong> lamprey as <strong>an</strong> experimental model to characterize <strong>the</strong> detailed<br />
cellular <strong>an</strong>d synaptic mech<strong>an</strong>isms underlying MLR-induced locomotion. We have previously<br />
described a differential recruitment of RS cells located in two reticular nuclei: <strong>the</strong> middle <strong>an</strong>d<br />
posterior rhombencephalic reticular nuclei (MRRN <strong>an</strong>d PRRN). RS cells in <strong>the</strong> MRRN show<br />
spiking activity at lower MLR stimulation intensities th<strong>an</strong> RS cells in <strong>the</strong> PRRN. Because <strong>the</strong>se<br />
results were obtained using intracellular recordings, <strong>the</strong> sampling of RS cells remains small. The<br />
present study was undertaken to examine <strong>the</strong> recruitment pattern in larger populations of RS cells<br />
using calcium imaging. We observed three distinct patterns of activity in <strong>the</strong>se cells during MLR<br />
stimulation. Large RS cells mostly located in <strong>the</strong> rostral <strong>an</strong>d lateral part of <strong>the</strong> MRRN showed a<br />
sustained calcium response maintained throughout <strong>the</strong> stimulation period. O<strong>the</strong>r RS cells<br />
displayed a calcium response that progressively decayed during <strong>the</strong> stimulation period. This<br />
response pattern was seen in RS cells distributed throughout <strong>the</strong> MRRN as well as in <strong>the</strong> PRRN.<br />
A third pattern consisted of a calcium response that occurred tr<strong>an</strong>siently both at <strong>the</strong> onset <strong>an</strong>d at<br />
<strong>the</strong> end of <strong>the</strong> MLR stimulation period. This pattern was more predomin<strong>an</strong>tly observed in<br />
smaller RS cells located in <strong>the</strong> caudal MRRN. The activation threshold of <strong>the</strong> calcium responses<br />
was similar <strong>for</strong> RS cells in <strong>the</strong> MRRN <strong>an</strong>d <strong>the</strong> PRRN. Intracellular recordings were <strong>the</strong>n<br />
combined to calcium imaging <strong>an</strong>d <strong>the</strong> membr<strong>an</strong>e depolarizations elicited by MLR stimulation<br />
did show a similar pattern to <strong>the</strong> calcium responses. Intracellular recordings of RS cells in a<br />
semi-intact preparation also showed that <strong>the</strong> me<strong>an</strong> firing frequency of RS cells displaying a
sustained activity pattern was correlated to <strong>the</strong> swimming frequency. In addition, some of <strong>the</strong> RS<br />
cells showed a tr<strong>an</strong>sient firing frequency increase at <strong>the</strong> end of <strong>the</strong> swimming bout. The link<br />
between <strong>the</strong>se patterns of activity <strong>an</strong>d locomotor behavior is under study using intracellular<br />
recordings combined to kinematic <strong>an</strong>alysis.<br />
Disclosures: L. Juvin, None; R. Dubuc, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.11/EE9<br />
Topic: D.11.e. Descending control<br />
Support: CDRF gr<strong>an</strong>t GB2-0205-2<br />
NFR gr<strong>an</strong>t<br />
Title: Functional org<strong>an</strong>ization of medullary reticulospinal inputs to lumbar comissural<br />
interneurons in <strong>the</strong> neonatal mouse<br />
Authors: *K. SZOKOL, J. C. GLOVER, M.-C. PERREAULT;<br />
Dep Physiol., Univ. Oslo Inst. Basic Med. Sci., Oslo, Norway<br />
Abstract: To better underst<strong>an</strong>d how <strong>the</strong> medullary reticular <strong>for</strong>mation (MRF) coordinates <strong>an</strong>d<br />
controls spinal motor networks we use calcium (Ca 2+ ) imaging in <strong>the</strong> brainstem-spinal cord<br />
preparation of <strong>the</strong> neonatal mouse. Recently, we have described a mediolateral org<strong>an</strong>ization<br />
within <strong>the</strong> ventral MRF which may provide <strong>the</strong> <strong>an</strong>atomical base <strong>for</strong> flexible, differential control<br />
of trunk <strong>an</strong>d hindlimb muscles (Szokol et al. 2008, Szokol <strong>an</strong>d Perreault <strong>2009</strong>). The goal of <strong>the</strong><br />
present study is to investigate whe<strong>the</strong>r L2 commissural interneurons with descending axonal<br />
projections (dCINs) could participate in this control.<br />
We stimulated two regions of <strong>the</strong> ventral MRF that exert differential effects on motoneurons of<br />
<strong>the</strong> medial <strong>an</strong>d lateral motor columns (MMC <strong>an</strong>d LMC) <strong>an</strong>d which we refer to as ‘medial MRF’<br />
<strong>an</strong>d ‘lateral MRF’ (Szokol et al. 2008). To investigate <strong>the</strong> Ca 2+ responses evoked by stimulation<br />
of <strong>the</strong>se regions in individual dCINs we have developed a modified obliquely cut, face up<br />
brainstem-spinal cord preparation (Szokol <strong>an</strong>d Perreault <strong>2009</strong>). We recorded 330 dCINs in 22<br />
preparations. We found that stimulation of <strong>the</strong> ipsi- or <strong>the</strong> contralateral medial or lateral MRF<br />
elicited Ca2+ responses only in subpopulations of <strong>the</strong> recorded dCINs. On <strong>the</strong> ipsilateral side,<br />
stimulation of <strong>the</strong> medial MRF activated about 50% of <strong>the</strong> 178 recorded dCINs whereas
stimulation of <strong>the</strong> lateral MRF activated about 70% of <strong>the</strong> 180 recorded dCINs. M<strong>an</strong>y of <strong>the</strong><br />
responsive dCINs (about 60%) were activated by both MRF regions. These dCINs <strong>an</strong>d those that<br />
were activated by stimulation of <strong>the</strong> medial or lateral MRF were intermingled. On <strong>the</strong><br />
contralateral side, stimulation of <strong>the</strong> medial MRF activated about 40% of <strong>the</strong> 102 recorded<br />
dCINs while stimulation of <strong>the</strong> lateral MRF activated only about 30% of <strong>the</strong> 69 recorded dCINs.<br />
The proportion of responsive dCINs that were activated by both MRF regions was smaller th<strong>an</strong><br />
on <strong>the</strong> ipsilateral side (about 40%).<br />
Our data indicate that already at birth functional connections between medullary reticulospinal<br />
neurons <strong>an</strong>d lumbar dCINs exist. Specifically, <strong>the</strong>y suggest <strong>the</strong> presence of two dCIN-mediated<br />
double-crossed pathways between respectively medial <strong>an</strong>d lateral medullary reticulospinal<br />
neurons <strong>an</strong>d lumbar motoneurons. These evidently operate in addition to a dCIN-mediated<br />
double-crossed pathway <strong>from</strong> pontine reticulospinal neurons to lumbar motoneurons previously<br />
described in adult mammal (J<strong>an</strong>kowska et al., 2003; 2006).<br />
Disclosures: K. Szokol, None; J.C. Glover, None; M. Perreault, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.12/EE10<br />
Topic: D.11.e. Descending control<br />
Support: CDRF gr<strong>an</strong>t GB2-0205-2<br />
NFR gr<strong>an</strong>t<br />
Title: Vestibular contribution to control of axial <strong>an</strong>d limb motoneurons in <strong>the</strong> neonatal mouse<br />
Authors: *M.-C. PERREAULT, J. GLOVER, N. KASUMACIC;<br />
Univ. Oslo Domus Medica, Oslo N-0317, Norway<br />
Abstract: Descending pathways <strong>from</strong> <strong>the</strong> vestibular nuclei to <strong>the</strong> spinal cord are essential <strong>for</strong><br />
control of bal<strong>an</strong>ce <strong>an</strong>d posture. During early embryonic development, axons of vestibulospinal<br />
neurons are among <strong>the</strong> first to reach <strong>the</strong> spinal cord extending along three main descending<br />
tracts. Two of <strong>the</strong>se, <strong>the</strong> ipsi- <strong>an</strong>d <strong>the</strong> contralateral medial vestibulospinal tracts (iMVST <strong>an</strong>d<br />
cMVST), run medially in <strong>the</strong> medial longitudinal fascicle (MLF). The third runs laterally as <strong>the</strong><br />
lateral vestibulospinal tract (LVST). Whe<strong>the</strong>r <strong>the</strong>se axons have already at birth established<br />
functional connections to spinal motoneurons (MNs) is unknown. The aim of this study was to
examine <strong>the</strong> org<strong>an</strong>ization of <strong>the</strong> vestibulospinal drive to spinal MNs controlling axial <strong>an</strong>d limb<br />
muscles in <strong>the</strong> neonatal mouse.<br />
Using calcium (Ca2+) imaging, we recorded responses evoked by electrical stimulation of <strong>the</strong><br />
vestibular nerve in MNs of <strong>the</strong> medial <strong>an</strong>d lateral motor columns (MMC <strong>an</strong>d LMC) throughout<br />
<strong>the</strong> spinal cord (segments C2, C6, T7, L2 <strong>an</strong>d L5). Unilateral lesion of <strong>the</strong> MLF allowed <strong>for</strong><br />
discrimination of iMVST- <strong>an</strong>d cMVST-mediated responses whereas bilateral lesion of <strong>the</strong> MLF<br />
interrupted both MVSTs <strong>an</strong>d allowed <strong>for</strong> discrimination of MVST- <strong>an</strong>d LVST-mediated<br />
responses.<br />
The MVST-mediated responses were detected in <strong>the</strong> cervical segments both on <strong>the</strong> ipsi- <strong>an</strong>d<br />
contralateral sides relative to <strong>the</strong> stimulated nerve. Responses in <strong>the</strong> iMMC of C2 (<strong>the</strong>re is no<br />
LMC in C2) were mediated by <strong>the</strong> iMVST alone whereas responses in <strong>the</strong> iMMC of C6 derived<br />
<strong>from</strong> both <strong>the</strong> iMVST <strong>an</strong>d <strong>the</strong> LVST. Responses in <strong>the</strong> iLMC of C6 were mediated by <strong>the</strong> LVST<br />
alone. All contralateral cervical responses were mediated by <strong>the</strong> cMVST alone. All responses<br />
detected in <strong>the</strong> thoracic <strong>an</strong>d lumbar segments derived <strong>from</strong> <strong>the</strong> LVST alone. In T7, responses<br />
were evoked in both ipsi- <strong>an</strong>d contralateral axial <strong>an</strong>d hypaxial MNs. In both L2 <strong>an</strong>d L5,<br />
responses were evoked in <strong>the</strong> iLMC, cLMC <strong>an</strong>d cMMC but not <strong>the</strong> iMMC.<br />
Our results indicate that already at birth <strong>the</strong> vestibulospinal system is functional <strong>an</strong>d org<strong>an</strong>ized to<br />
differentially regulate activity in axial (neck <strong>an</strong>d trunk) <strong>an</strong>d limb MNs throughout <strong>the</strong> spinal cord<br />
in a m<strong>an</strong>ner similar to that seen in adult mammals. We are currently investigating <strong>the</strong> possibility<br />
that some of <strong>the</strong> responses evoked by vestibular nerve stimulation may be mediated via <strong>the</strong><br />
medullary reticular <strong>for</strong>mation.<br />
Disclosures: M. Perreault, None; J. Glover, None; N. Kasumacic, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.13/EE11<br />
Topic: D.11.e. Descending control<br />
Support: CDRF gr<strong>an</strong>t GB2-0205-2<br />
University of Oslo Ph D stipend<br />
Title: Vestibular influence on somatic <strong>an</strong>d autonomic thoracic motoneurons in <strong>the</strong> neonatal<br />
mouse
Authors: *N. KASUMACIC, J. C. GLOVER, M.-C. PERREAULT;<br />
Dept Physiol, Univ. Oslo, Oslo, Norway<br />
Abstract: The vestibulospinal system is essential <strong>for</strong> <strong>the</strong> control of bal<strong>an</strong>ce <strong>an</strong>d posture. While<br />
m<strong>an</strong>y studies have provided in<strong>for</strong>mation about connections between vestibulospinal neurons <strong>an</strong>d<br />
neck <strong>an</strong>d limb-innervating motoneurons (MNs) in adult mammals, little is known about<br />
vestibular regulation of <strong>the</strong> trunk <strong>an</strong>d preg<strong>an</strong>glionic autonomic motoneurons. The aim of <strong>the</strong><br />
current study was to identify motoneuron groups in <strong>the</strong> thoracic cord that receive vestibular<br />
inputs, <strong>an</strong>d to characterize <strong>the</strong> origin of those inputs.<br />
We investigated vestibular inputs to <strong>the</strong> thoracic motoneurons (segments T7 <strong>an</strong>d T10) by<br />
recording calcium responses induced by electrical stimulation of <strong>the</strong> vestibular nerve in<br />
brainstem-spinal cord preparations of <strong>the</strong> neonatal mouse (P1-P4). The individual MNs were<br />
visualized ei<strong>the</strong>r through <strong>the</strong> ventral white matter, or in <strong>the</strong> tr<strong>an</strong>sverse pl<strong>an</strong>e of <strong>an</strong> obliquely cut,<br />
face up preparation (Szokol <strong>an</strong>d Perreault <strong>2009</strong>).<br />
All MN populations recorded (lateral <strong>an</strong>d medial somatic, <strong>an</strong>d intermediate autonomic)<br />
responded to ipsi- as well as contralateral vestibular nerve stimulation, but with different<br />
magnitudes. The lateral somatic MNs exhibited <strong>the</strong> largest calcium response, on average 3 times<br />
larger th<strong>an</strong> <strong>the</strong> medial somatic MNs. The responses in <strong>the</strong> autonomic MNs were on average<br />
about 15 times smaller th<strong>an</strong> that of <strong>the</strong> lateral somatic MNs. Unilateral or bilateral lesions of <strong>the</strong><br />
MLF that interrupted tr<strong>an</strong>smission along <strong>the</strong> ipsi- <strong>an</strong>d/or <strong>the</strong> contralateral medial vestibulospinal<br />
tracts did not signific<strong>an</strong>tly ch<strong>an</strong>ge <strong>the</strong> responses in <strong>the</strong> somatic MNs, suggesting that <strong>the</strong>se<br />
responses are mediated by <strong>the</strong> lateral vestibulospinal tract. Lesion of <strong>the</strong> midline at T7 or T10<br />
completely eliminated <strong>the</strong> responses of somatic MNs to contralateral vestibular nerve<br />
stimulation, suggesting that <strong>the</strong>se are mediated by a crossed pathway involving commissural<br />
vestibulospinal axon collaterals <strong>an</strong>d/or commissural interneurons.<br />
Our data suggest that already at birth vestibulospinal neurons have established functional<br />
connections with somatic <strong>an</strong>d preg<strong>an</strong>glionic autonomous MNs in <strong>the</strong> thoracic cord. The inputs to<br />
somatic motoneurons appeared to be mediated mainly, if not solely, by vestibulospinal neurons<br />
with axons running in <strong>the</strong> lateral vestibulospinal tract.<br />
Disclosures: N. Kasumacic, None; J.C. Glover, None; M. Perreault, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.14/EE12<br />
Topic: D.11.e. Descending control
Support: CIHR 79416<br />
Will-to-Win Scholar Fund<br />
Title: Enh<strong>an</strong>ced excitation of propriospinal neurons facilitates bulbospinal tr<strong>an</strong>smission of <strong>the</strong><br />
locomotor comm<strong>an</strong>d signal in <strong>the</strong> in vitro neonatal rat brain stem-spinal cord preparation<br />
Authors: K. C. COWLEY, E. ZAPOROZHETS, *B. J. SCHMIDT;<br />
Physiol., Univ. of M<strong>an</strong>itoba, Winnipeg, MB, C<strong>an</strong>ada<br />
Abstract: It is widely accepted that long direct bulbospinal projections contribute to spinal<br />
locomotor network activation. Using <strong>an</strong> in vitro neonatal rat brainstem-spinal cord preparation,<br />
we showed that a propriospinal relay system of neurons also contributes to tr<strong>an</strong>smission of <strong>the</strong><br />
descending locomotor signal, <strong>an</strong>d c<strong>an</strong> be sufficient in this role (Cowley et al., J. Physiol. 586:<br />
2008). Thus, electrical stimulation of <strong>the</strong> brainstem evoked hindlimb locomotor-like activity in<br />
27% of preparations in which all long direct descending bulbospinal projections were disrupted<br />
by staggered contralateral hemisections. Given that 73% of <strong>the</strong> staggered hemisection<br />
preparations did not allow sufficient propagation of <strong>the</strong> bulbospinal signal to elicit locomotor<br />
activity, we are now focusing on this preparation as a model of partial spinal cord injury. We<br />
hypo<strong>the</strong>size that artificial excitation of propriospinal neurons may facilitate propagation of <strong>the</strong><br />
locomotor comm<strong>an</strong>d signal <strong>an</strong>d produce hindlimb locomotor-like activity in preparations that<br />
o<strong>the</strong>rwise fail to respond to brainstem stimulation alone. In <strong>the</strong> present work, hemisections were<br />
made in <strong>the</strong> rostral (right T1) <strong>an</strong>d contralateral caudal (left T11) thoracic regions. Bath barriers<br />
were placed such that thoracic segments (T2 through T10 inclusive) were selectively exposed to<br />
neurochemicals. These were applied at concentrations subthreshold <strong>for</strong> evoking locomotor-like<br />
activity in <strong>the</strong> absence of brainstem stimulation <strong>an</strong>d included: 5-HT (10 µM) with NMDA (2<br />
µM), acetylcholine (10-15 µM) with <strong>the</strong> acetylcholinesterase inhibitor edrophonium (50-60 µM),<br />
<strong>an</strong>d <strong>the</strong> alpha adrenergic receptor agonist clonidine (5-60 µM). The effect of increasing neuronal<br />
excitability through elevation of bath potassium concentration (up to 7 mM) or lowering<br />
magnesium ion concentration was also examined. Brainstem electrical stimulation alone failed to<br />
evoke hindlimb locomotor-like activity in 26/39 preparations. In two of three such preparations,<br />
application of 5-HT <strong>an</strong>d NMDA to <strong>the</strong> thoracic region promoted locomotor-like activity in<br />
response to brainstem stimulation. This facilitatory effect was reversed by drug wash-out. On <strong>the</strong><br />
o<strong>the</strong>r h<strong>an</strong>d, acetylcholine / edrophonium (n=0/7), clonidine (n=0/5), increased bath potassium<br />
(n=0/5), <strong>an</strong>d reduced magnesium (n=0/9) failed to facilitate <strong>the</strong> effect of brainstem stimulation.<br />
The results thus far suggest that at least some of <strong>the</strong> thoracic propriospinal neurons involved in<br />
propagation of <strong>the</strong> descending locomotor comm<strong>an</strong>d signal are 5-HT <strong>an</strong>d glutamate sensitive.<br />
These neurons may be suitable targets <strong>for</strong> <strong>the</strong>rapeutic interventions to restore locomotor function<br />
after injury.<br />
Disclosures: K.C. Cowley, None; E. Zaporozhets, None; B.J. Schmidt, None.<br />
Poster
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.15/EE13<br />
Topic: D.11.e. Descending control<br />
Support: NIH Gr<strong>an</strong>t NS046404<br />
Title: Spinal monoamine release in freely-moving <strong>an</strong>imals<br />
Authors: S. XIE, F. J. SANCHEZ, M. M. CARBALLOSA-GONZALEZ, A. D. BLYTHE, I. D.<br />
HENTALL, *B. R. NOGA;<br />
Miami Proj To Cure Paralysis, Univ. Miami Sch. Med., Miami, FL<br />
Abstract: Monoaminergic pathways (raphespinal <strong>an</strong>d ceruleospinal) play signific<strong>an</strong>t roles in<br />
modulating locomotion. To provide a tool to assess <strong>the</strong>ir functionality, we developed a protocol<br />
<strong>for</strong> real-time monitoring of monoamine release in <strong>the</strong> spinal cord of freely-moving <strong>an</strong>imals.<br />
Based upon fast cyclic voltammetry (FCV), miniature, “floating” carbon fiber microelectrodes<br />
(fCFMEs) with 33 micron carbon fibers were constructed <strong>an</strong>d impl<strong>an</strong>ted within <strong>the</strong> lumbar cord<br />
of Sprague-Dawley rats to electrochemically detect monoamine release during spont<strong>an</strong>eous or<br />
mesencephalic locomotor region (MLR) evoked locomotion. To stimulate <strong>the</strong> MLR, 60-µA<br />
electrical pulses with 1 ms duration were continually generated by a battery-powered stimulator<br />
<strong>an</strong>d applied to <strong>an</strong> integral, protruding microelectrode (Hentall et al. 2007) stereotaxically<br />
positioned into <strong>the</strong> MLR. Stimulation frequency was varied between 8 <strong>an</strong>d 16 Hz. At low<br />
frequencies of MLR stimulation (8 Hz), <strong>an</strong>imals became more alert <strong>an</strong>d occasionally stepped.<br />
Increasing <strong>the</strong> stimulation frequency to 16 Hz induced rapid locomotion. The <strong>an</strong>imals stopped<br />
walking, almost immediately upon <strong>the</strong> termination of electrical pulses. A similar pattern was also<br />
present <strong>for</strong> <strong>the</strong> monoamine level detected within <strong>the</strong> lumbar cord, which was greatly enh<strong>an</strong>ced<br />
during MLR stimulation <strong>an</strong>d, in particular, at higher stimulation frequencies. However, probably<br />
due to <strong>the</strong> reuptake mech<strong>an</strong>isms, monoamine levels gradually decreased toward a steady state<br />
concentration when pulse frequency was kept const<strong>an</strong>t <strong>for</strong> prolonged periods of time.<br />
Interestingly, during this period, locomotor activity would usually be maintained at a relatively<br />
stable frequency. Hence, monoamine level in <strong>the</strong> lumbar cord appears to be tightly coupled with<br />
<strong>the</strong> locomotion. To our knowledge, <strong>the</strong>se are <strong>the</strong> first voltammetric sensors allowing direct<br />
assessment of monoamine release in <strong>the</strong> spinal cord of freely-moving <strong>an</strong>imals. This technique<br />
c<strong>an</strong> potentially be used to assess monoaminergic function following incomplete spinal cord<br />
injury.<br />
Disclosures: S. Xie, None; F.J. S<strong>an</strong>chez, None; M.M. Carballosa-Gonzalez, None; A.D.<br />
Bly<strong>the</strong>, None; I.D. Hentall, None; B.R. Noga, None.
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.16/EE14<br />
Topic: D.11.e. Descending control<br />
Support: Supported by NIH gr<strong>an</strong>t NS-058659 to INB<br />
Title: Do pyramidal tract neurons (PTNs) with different somatosensory receptive fields have<br />
distinct firing properties during locomotion?<br />
Authors: *E. E. STOUT, I. N. BELOOZEROVA;<br />
Barrow Neurol Inst., Phoenix, AZ<br />
Abstract: The vast majority of motor cortical neurons contributing axons to <strong>the</strong> pyramidal tract<br />
(PTNs) during locomotion discharge in close relation to steps (Beloozerova & Sirota 1993;<br />
Armstrong & Drew 1993). What effect do <strong>the</strong>se discharges have on spinal locomotion networks?<br />
To <strong>an</strong>swer this question, in this study, we took <strong>an</strong> adv<strong>an</strong>tage of <strong>the</strong> fact that microstimulation of<br />
motor cortex evokes movements of <strong>the</strong> same parts of <strong>the</strong> body <strong>from</strong> which somatosensory<br />
in<strong>for</strong>mation is received (As<strong>an</strong>uma 1989). Thus, we used <strong>the</strong> somatosensory receptive field of a<br />
PTN as a rough indicator of which part of <strong>the</strong> body <strong>the</strong> neuron might influence <strong>the</strong> movement of.<br />
In 5 cats, <strong>the</strong> activity of 107 motor cortex PTNs located rostral to <strong>the</strong> cruciate sulcus that had<br />
single well-defined <strong>for</strong>elimb somatosensory receptive fields were recorded during simple<br />
locomotion on a flat surface <strong>an</strong>d complex locomotion on a horizontal ladder. Cells were grouped<br />
according to <strong>the</strong>ir reactivity to passive joint movements: 46 PTNs responded to shoulder<br />
movements, 31 to elbow, <strong>an</strong>d 30 to wrist. During simple locomotion, <strong>the</strong> activity of 95% of<br />
PTNs <strong>from</strong> all groups was modulated with <strong>the</strong> step cycle <strong>an</strong>d <strong>the</strong>re were only minor differences<br />
between <strong>the</strong> groups. Upon tr<strong>an</strong>sition <strong>from</strong> simple to complex locomotion PTNs <strong>from</strong> all groups<br />
showed ch<strong>an</strong>ges to <strong>the</strong>ir activity. However, only wrist-related PTNs showed concerted ch<strong>an</strong>ges<br />
to <strong>the</strong>ir activity while shoulder <strong>an</strong>d elbow PTNs activities ch<strong>an</strong>ged in diverse m<strong>an</strong>ners. The<br />
duration of period of elevated firing decreased in 50% of wrist PTNs, most often by 20% of step<br />
cycle or more. The preferred phases of 25% of wrist PTNs shifted into swing, resulting in a<br />
strong activity increase of <strong>the</strong> whole wrist group during swing. Wrist-related PTNs had <strong>the</strong><br />
largest average increase in <strong>the</strong> coefficient of frequency modulation upon <strong>the</strong> tr<strong>an</strong>sition.<br />
We suggest that <strong>the</strong> decrease in <strong>the</strong> duration of period of elevated firing in wrist-related PTNs<br />
sharpens <strong>the</strong>ir contribution to spinal locomotion networks, resulting in a smaller variability of<br />
step lengths during <strong>the</strong> complex task. An increase of wrist PTN activity during swing provides a<br />
possible mech<strong>an</strong>ism <strong>for</strong> one of <strong>the</strong> prominent kinematic differences between simple <strong>an</strong>d complex<br />
locomotion: increased wrist pl<strong>an</strong>tar flexion during <strong>the</strong> complex task.
Disclosures: E.E. Stout, None; I.N. Beloozerova, None.<br />
Poster<br />
565. Rhythmic Motor Networks: Descending Control<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 565.17/EE15<br />
Topic: D.11.e. Descending control<br />
Support: Brain Research Institute, University of Chicago<br />
Title: MIo neuron activity modulates to phases of <strong>the</strong> gape cycle during feeding in macaque<br />
monkeys<br />
Authors: *C. F. ROSS 1 , N. HASTOPOULOS 1 , M. KONECKI 2 , J. IRIARTE-DIAZ 1 , K.<br />
TAKAHASHI 1 ;<br />
1 Org<strong>an</strong>ismal Biol. <strong>an</strong>d Anat., 2 Pritzker Sch. of Med., Univ. of Chicago, Chicago, IL<br />
Abstract: The MI orofacial area of cerebral cortex (MIo) is involved in control of voluntary,<br />
semiautomatic <strong>an</strong>d rhythmic movements of <strong>the</strong> tongue <strong>an</strong>d jaw during ingestion, chewing <strong>an</strong>d<br />
swallowing behaviors. MIo neuronal activity is known to be modulated according to <strong>the</strong> type of<br />
task being per<strong>for</strong>med (e.g., chewing, tongue protrusion, swallowing) <strong>an</strong>d to <strong>the</strong> stages of <strong>the</strong><br />
feeding sequence (ingestion, chewing <strong>an</strong>d post-chewing). However, <strong>the</strong> functional role of MIo<br />
neurons during feeding is poorly understood because of a lack of precise data on <strong>the</strong> timing of<br />
<strong>the</strong>ir activity relative to <strong>the</strong> phases of <strong>the</strong> gape cycle (fast close, slow close, fast open <strong>an</strong>d slow<br />
open). We hypo<strong>the</strong>sized that MIo neurons would modulate <strong>the</strong>ir activity according to <strong>the</strong> phase<br />
of <strong>the</strong> gape cycle: specifically, we hypo<strong>the</strong>sized that MIo neurons would ch<strong>an</strong>ge firing rates<br />
around <strong>the</strong> time that <strong>the</strong> tongue ch<strong>an</strong>ges directions <strong>from</strong> <strong>an</strong>terior to posterior movement. Activity<br />
in MIo neurons was recorded <strong>from</strong> two macaque subjects using Utah microarrays impl<strong>an</strong>ted in<br />
<strong>the</strong> MIo region while <strong>the</strong> tongue <strong>an</strong>d jaw kinematics were measured using videofluorscopy <strong>an</strong>d<br />
3-d jaw tracking. Ch<strong>an</strong>ges in frequency of spiking of cortical neurons were matched with <strong>the</strong><br />
timing of <strong>the</strong> chewing phases while <strong>the</strong> <strong>an</strong>imals ate different kinds of food. Tongue <strong>an</strong>d jaw<br />
kinematic data indicate that <strong>the</strong> tongue reverses direction <strong>from</strong> <strong>an</strong>terior to posterior movement <strong>an</strong><br />
average of 0.0401 seconds after <strong>the</strong> slow open-fast open tr<strong>an</strong>sition, suggesting that <strong>the</strong> ch<strong>an</strong>ge in<br />
rate of jaw movement may be import<strong>an</strong>t in signaling reversal in tongue movement direction.<br />
During both potato <strong>an</strong>d grape chewing 25% of 45 neurons were identified as modulating <strong>the</strong>ir<br />
firing rate with a phase of <strong>the</strong> gape cycle, with half of those exhibiting maximum firing rate<br />
around <strong>the</strong> slow open-fast open tr<strong>an</strong>sition, some neurons prior to maximum gape <strong>an</strong>d some<br />
around fast close-slow close tr<strong>an</strong>sition. The data presented here are <strong>the</strong> first to precisely
document modulation of MIo neuron activity to <strong>the</strong> phases of <strong>the</strong> gape cycle during natural<br />
mastication. They suggest that although MIo may not be import<strong>an</strong>t in initiating rhythmic jaw<br />
movements, MIo does monitor <strong>the</strong>se movements, or play <strong>an</strong> active role in coordinating jaw <strong>an</strong>d<br />
tongue movement.<br />
Disclosures: C.F. Ross, University of Chicago, A. Employment (full or part-time); N.<br />
Hastopoulos, None; M. Konecki, None; J. Iriarte-Diaz, None; K. Takahashi, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.1/EE16<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NS 047452 (ARW)<br />
Parkinson's Disease Foundation<br />
NARSAD young investigator award (ARW)<br />
Title: Dopamine D2 receptor-dependent modulation of nitric oxide synthase activity in <strong>the</strong> rat<br />
striatal complex<br />
Authors: *K. E. HOQUE, S. SAMMUT, A. R. WEST;<br />
Rosalind Fr<strong>an</strong>klin Univ., North Chicago, IL<br />
Abstract: he gaseous neurotr<strong>an</strong>smitter nitric oxide (NO) is syn<strong>the</strong>sized throughout <strong>the</strong> striatal<br />
complex by aspiny interneurons containing neuronal NO synthase (nNOS). Striatal nNOS<br />
activity is readily assayed using nicotinamide adenine dinucleotide phosphate diaphorase<br />
(NADPH-d) histochemical staining <strong>an</strong>d is regulated by dopamine (DA) D1- <strong>an</strong>d D2 receptor<br />
activation. However, <strong>the</strong> mech<strong>an</strong>isms underlying dopaminergic modulation of nNOS activity in<br />
identified subterritories of <strong>the</strong> striatal complex remain unexplored. This study examined <strong>the</strong><br />
impact of pharmacological m<strong>an</strong>ipulations of D2 <strong>an</strong>d NMDA receptors on nNOS activity using<br />
optical density measures of NADPH-d staining pre<strong>for</strong>med in <strong>the</strong> dorsal striatum <strong>an</strong>d in multiple<br />
subdivisions (core, medial shell, intermediate shell <strong>an</strong>d lateral shell) of <strong>the</strong> nucleus accumbens<br />
(NAc). Awake behaving rats received systemic administration of saline vehicle (sal) <strong>an</strong>d/or <strong>the</strong><br />
following drugs: <strong>the</strong> nNOS inhibitor N G -propyl-L-arginine (NPA, 20 mg/kg, i.p.), <strong>the</strong> D2<br />
receptor agonist quinpirole (QNP, 100µg/kg, i.p.), <strong>the</strong> D2 receptor <strong>an</strong>tagonist eticlopride (ETI,
100µg/kg, i.p.), or <strong>the</strong> NMDA receptor <strong>an</strong>tagonist CPP (1 mg/kg, i.p.). All rats were returned to<br />
<strong>the</strong>ir home cage immediately after <strong>the</strong> first sal or drug treatment. Exactly 20 min later, rats were<br />
given a second injection of sal or drug <strong>an</strong>d returned to <strong>the</strong>ir home cage. All brains were harvested<br />
25 min after <strong>the</strong> second treatment. In sal/sal treated <strong>an</strong>imals a distinct medial-lateral<br />
histochemical gradient of NADPH-d activity was observed which was characterized by moderate<br />
staining in <strong>the</strong> core <strong>an</strong>d medial shell <strong>an</strong>d more robust staining in <strong>the</strong> intermediate <strong>an</strong>d lateral shell<br />
<strong>an</strong>d dorsal striatum. Administration of NPA/sal <strong>an</strong>d CPP/sal attenuated staining in <strong>the</strong> dorsal<br />
striatum as well as <strong>the</strong> intermediate <strong>an</strong>d lateral shell of <strong>the</strong> NAc as compared to sal/sal treated<br />
controls. Additionally, administration of QNP/sal attenuated staining in <strong>the</strong> intermediate <strong>an</strong>d<br />
lateral shell of <strong>the</strong> NAc. Conversely, sal/ETI administration robustly increased staining in <strong>the</strong><br />
dorsal striatum <strong>an</strong>d <strong>the</strong> medial <strong>an</strong>d lateral shell. Pretreatment with QNP, NPA or CPP attenuated<br />
<strong>the</strong> increase in NADPH-d staining induced by ETI <strong>an</strong>d decreased staining in more lateral regions<br />
of <strong>the</strong> NAc as compared to sal/sal treated controls. These observations demonstrate that nNOS<br />
activity measured in distinct subregions of <strong>the</strong> striatal complex is differentially modulated by DA<br />
D2 receptor activation. Moreover, <strong>the</strong>se findings demonstrate <strong>for</strong> <strong>the</strong> first time that tonic D2<br />
receptor activation opposes <strong>the</strong> facilitatory influence of glutamatergic tr<strong>an</strong>smission on striatal<br />
nNOS activity via suppression of NMDA receptor function.<br />
Disclosures: K.E. Hoque, None; S. Sammut, None; A.R. West, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.2/EE17<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NIH Gr<strong>an</strong>t NS41302<br />
NIH Gr<strong>an</strong>t DA07234<br />
Title: β-adrenergic receptor activation induces CREB phosphorylation in striatal neurons<br />
Authors: *J. MEITZEN, J. I. LUOMA, P. G. MERMELSTEIN;<br />
Neurosci., Univ. of Minnesota, Minneapolis, MN<br />
Abstract: Drugs of abuse induce long lasting ch<strong>an</strong>ges in striatal neurotr<strong>an</strong>smission <strong>an</strong>d <strong>the</strong><br />
cellular properties of striatal neurons. While attention has focused upon dopamine signaling as<br />
<strong>the</strong> principal mech<strong>an</strong>ism by which this neuroplasticity occurs, amphetamine <strong>an</strong>d o<strong>the</strong>r drugs of
abuse also trigger <strong>the</strong> release of <strong>the</strong> catecholamine norepinephrine. Norepinephrine is commonly<br />
thought of as a stress-related neurotr<strong>an</strong>smitter with fast-acting effects. However, norepinephrine<br />
activation of beta-adrenergic receptors increase intracellular cAMP levels: a signaling<br />
mech<strong>an</strong>ism by which tr<strong>an</strong>scription factors c<strong>an</strong> also be activated. To test this hypo<strong>the</strong>sis, we<br />
exposed cultured rat striatal neurons to <strong>the</strong> beta-adrenergic receptor agonist isoproterenol, <strong>an</strong>d<br />
measured <strong>the</strong> activation (i.e. serine-133 phosphorylation) of <strong>the</strong> tr<strong>an</strong>scription factor cAMP<br />
response element binding protein (CREB). We found that isoproterenol exposure increased<br />
CREB phosphorylation. This increase was blocked by <strong>the</strong> beta-adrenergic receptor <strong>an</strong>tagonist<br />
propr<strong>an</strong>olol, <strong>an</strong>d was mimicked by <strong>the</strong> adenylyl cyclase activator <strong>for</strong>skolin. As expected,<br />
isoproterenol exposure also increased cAMP concentrations, <strong>an</strong>d this increase was blocked by<br />
propr<strong>an</strong>olol. These findings demonstrate a mech<strong>an</strong>ism by which norepinephrine <strong>an</strong>d o<strong>the</strong>r betaadrenergic<br />
receptor agonists could act to influence gene tr<strong>an</strong>scription in striatal neurons, possibly<br />
contributing to <strong>the</strong> long-lasting ch<strong>an</strong>ges following exposure to drugs of abuse or chronic stress.<br />
Disclosures: J. Meitzen, None; J.I. Luoma, None; P.G. Mermelstein, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.3/EE18<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NINDS gr<strong>an</strong>t R01 NS047452<br />
NARSAD<br />
Title: Dopaminergic modulation of nitric oxide synthase activity elicited in <strong>the</strong> nucleus<br />
accumbens via stimulation of hippocampal afferents<br />
Authors: K. E. HOQUE, *A. R. WEST;<br />
Rosalind Fr<strong>an</strong>klin Univ. Med. Sci., North Chicago, IL<br />
Abstract: Dopaminergic modulation of in<strong>for</strong>mation integration in <strong>the</strong> nucleus accumbens (NAc)<br />
is thought to play a central role in motivated behaviors associated with <strong>the</strong> rein<strong>for</strong>cing effects of<br />
natural rewards <strong>an</strong>d drugs of abuse. Several lines of evidence indicate that abnormal stimulation<br />
of <strong>the</strong> gaseous neuromodulator nitric oxide (NO) may contribute to adaptations <strong>an</strong>d synaptic<br />
remodeling occurring in <strong>the</strong> NAc following exposure to psychostimul<strong>an</strong>ts. Un<strong>for</strong>tunately, <strong>the</strong><br />
role of glutamatergic <strong>an</strong>d dopaminergic afferents in <strong>the</strong> regulation of NO syn<strong>the</strong>sis in <strong>the</strong> NAc
emains poorly understood. This study examined <strong>the</strong> impact of electrical stimulation of <strong>the</strong><br />
hippocampal fimbria on neuronal NO synthase (nNOS) activity in <strong>the</strong> NAc using regional<br />
histochemical measures of NADPH-diaphorase (NADPH-d) staining. The potential modulatory<br />
impact of dopamine (DA) receptor activation on <strong>the</strong>se measures was also assessed using<br />
systemic administration of D1 receptor agonist <strong>an</strong>d D2 receptor <strong>an</strong>tagonist. Electrical stimulation<br />
of <strong>the</strong> fimbria at low frequencies (0.5 Hz, 750µA, 0.5 ms, 2 s inter-train interval, trains delivered<br />
during a single 20 min trial) increased NADPH-d staining in <strong>the</strong> medial shell, but had no effect<br />
on measures taken in <strong>the</strong> core, intermediate shell, <strong>an</strong>d lateral shell of <strong>the</strong> NAc as compared to<br />
sham-stimulated controls. High frequency simulation (50 Hz) delivered in a similar m<strong>an</strong>ner<br />
induced a robust increase in staining in all regions of <strong>the</strong> NAc shell without affecting similar<br />
measures in <strong>the</strong> core. This frequency-dependent increase in staining was selectively attenuated in<br />
<strong>the</strong> medial shell following systemic administration of <strong>the</strong> nNOS inhibitor N G -propyl-L-arginine<br />
(20 mg/kg, i.p.). Fur<strong>the</strong>rmore, systemic administration of ei<strong>the</strong>r <strong>the</strong> D1 receptor agonist SKF<br />
81297 (500µg/kg, i.p.) or <strong>the</strong> D2 receptor <strong>an</strong>tagonist eticlopride (100µg/kg, i.p.) potentiated<br />
staining in all subregions of <strong>the</strong> NAc shell as compared to saline treated controls. Pretreatment<br />
with eticlopride also potentiated staining observed in <strong>the</strong> NAc core. These observations<br />
demonstrate <strong>for</strong> <strong>the</strong> first time that NO syn<strong>the</strong>sis in <strong>the</strong> NAc is facilitated by robust activation of<br />
hippocampal afferents in a m<strong>an</strong>ner that is at least partially dependent on activation of nNOS <strong>an</strong>d<br />
potently modulated by D1 <strong>an</strong>d D2 receptor activation. We <strong>an</strong>ticipate that fur<strong>the</strong>r characterization<br />
of <strong>the</strong> regulation of nNOS activity in <strong>the</strong> NAc in <strong>an</strong>imal models of psychiatric disorders will<br />
provide valuable in<strong>for</strong>mation as to how NO signaling may be dysfunctional in hum<strong>an</strong> disorders<br />
of cognition <strong>an</strong>d motivation.<br />
Disclosures: K.E. Hoque, None; A.R. West , None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.4/EE19<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: Work supported by NIDA/IRP funds.<br />
Title: Adenosine A2A receptors expression <strong>an</strong>d function are increased by Iron deficiency: Effects<br />
on behavior <strong>an</strong>d cortico-striatal neurotr<strong>an</strong>smission<br />
Authors: *C. R. QUIROZ 1 , V. PEARSON 2 , S. GULYANI 2 , R. ALLEN 2 , C. EARLEY 2 , S.<br />
FERRE 1 ;
1 2<br />
Behavioral Neurosci Br., NIDA, IRP, NIH, DHHS, Baltimore, MD; Dept. of Neurol., Johns<br />
Hopkins Sch. of Med., Baltimore, MD<br />
Abstract: Iron deficiency in <strong>the</strong> brain leads to altered dopaminergic function, which could in<br />
turn lead to sensorimotor disorders such as Restless Legs Syndrome. However, <strong>the</strong> mech<strong>an</strong>ism<br />
that links both conditions is still unclear. Considering <strong>the</strong> strong modulation that adenosine<br />
exerts on dopamine signaling, one possibility could be ch<strong>an</strong>ges in adenosine receptor expression<br />
or function. We have found that iron deficiency in mice or in mammali<strong>an</strong> cultured cells produces<br />
<strong>an</strong> up-regulation of adenosine A2A receptors. In <strong>the</strong> striatum, A2A receptors are localized<br />
postsynaptically, in <strong>the</strong> dendritic field of GABAergic enkephalinergic neurons, <strong>an</strong>d also<br />
presynaptically, in <strong>the</strong> glutamatergic terminals that contact GABAergic dynorphinergic neurons.<br />
Striatal postsynaptic A2A receptor function c<strong>an</strong> be <strong>an</strong>alyzed by studying <strong>the</strong> ability of A2A<br />
receptor <strong>an</strong>tagonists to produce locomotor activity or to counteract striatal ERK phosphorylation<br />
induced by cortical electrical stimulation. Presynaptic A2A receptor function c<strong>an</strong> be <strong>an</strong>alyzed by<br />
<strong>the</strong> ability of A2A receptor <strong>an</strong>tagonists to block <strong>the</strong> motor output induced by cortical stimulation.<br />
In <strong>the</strong> present work we measured <strong>the</strong> ch<strong>an</strong>ges in striatal A2A receptor expression as well as<br />
biochemical, motor <strong>an</strong>d behavioral ch<strong>an</strong>ges in A2A receptor-modulated responses in Sprague-<br />
Dawley rats fed during three weeks with iron deficient diet (4 ppm) compared to rats fed with a<br />
control diet. The iron deficient diet increased <strong>the</strong> expression of tr<strong>an</strong>sferrin receptor throughout<br />
<strong>the</strong> brain <strong>an</strong>d produced <strong>an</strong> up-regulation of striatal A2A receptor. In iron-deficient <strong>an</strong>imals <strong>the</strong><br />
selective A2A receptor <strong>an</strong>tagonist MSX-3 induced a stronger locomotor activation <strong>an</strong>d it was also<br />
more potent at blocking striatal ERK phosphorylation <strong>an</strong>d <strong>the</strong> motor output (orofacial<br />
movements) induced by cortical electrical stimulation. These results indicate that brain iron<br />
deficiency induces a functional up-regulation of both pre- <strong>an</strong>d postsynaptic A2A receptor in <strong>the</strong><br />
rat striatum, which could be involved in <strong>the</strong> sensorimotor disorders associated with iron<br />
deficiency.<br />
Disclosures: C.R. Quiroz , None; V. Pearson, None; S. Guly<strong>an</strong>i, None; R. Allen, None; C.<br />
Earley, None; S. Ferre, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.5/EE20<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: CHDI Foundation inc.
NIDA IRP<br />
Title: Different pre- <strong>an</strong>d postsynaptic pharmacological profile of adenosine A2A receptor<br />
<strong>an</strong>tagonists<br />
Authors: M. ORRU 1 , C. QUIROZ 1 , V. BEAUMONT 2 , *S. FERRE 1 ;<br />
1 2<br />
Behavioral Neurosci. Br., NIDA, IRP, NIH, DHHS, Baltimore, MD; CHDI Mgmt., Los<br />
Angeles, CA<br />
Abstract: The striatum is <strong>the</strong> major input structure of <strong>the</strong> basal g<strong>an</strong>glia. Ninety percent of striatal<br />
neurons are GABAergic medium spiny neurons (MSNs) There are two subtypes of MSN,<br />
enkephalinergic <strong>an</strong>d dynorphinergic MSNs, which give rise to <strong>the</strong> indirect <strong>an</strong>d direct striatal<br />
efferent pathways, respectively. The striatum receives glutamatergic inputs <strong>from</strong> cortical, limbic<br />
<strong>an</strong>d thalamic areas. In <strong>the</strong> striatum, adenosine A2A receptors are localized postsynaptically, in <strong>the</strong><br />
dendritic field of enkephalinergic MSNs, where <strong>the</strong>y heteromerize with dopamine D2 receptors,<br />
<strong>an</strong>d presynaptically, in <strong>the</strong> glutamatergic terminals that contact dynorphinergic MSNs, where<br />
<strong>the</strong>y heteromerize with adenosine A1 receptors. Striatal postsynaptic A2A receptor function c<strong>an</strong><br />
be <strong>an</strong>alyzed by studying <strong>the</strong> ability of A2A receptor <strong>an</strong>tagonists to produce locomotor activity,<br />
while presynaptic A2A receptor function c<strong>an</strong> be <strong>an</strong>alyzed by <strong>the</strong> ability of A2A receptor<br />
<strong>an</strong>tagonists to block <strong>the</strong> motor output induced by electrical cortical stimulation. To study <strong>the</strong><br />
striatal presynaptic A2A receptor function, we recently established <strong>an</strong> experimental model by<br />
combining electrical stimulation in <strong>the</strong> cortical orofacial motor area <strong>an</strong>d electromyographic<br />
recording of <strong>the</strong> mastication muscles. This model allows determining a qu<strong>an</strong>titative variable<br />
(power correlation coefficient; PCC), which correlates with cortico-striatal glutamatergic<br />
neurotr<strong>an</strong>smission through <strong>the</strong> direct striatal efferent pathway. The aim of <strong>the</strong> present study was<br />
to <strong>an</strong>alyze, based on <strong>the</strong> methods outlined above, if several A2A receptor <strong>an</strong>tagonists would show<br />
different profiles as blockers of pre- <strong>an</strong>d postsynaptic striatal A2A receptors. In fact, <strong>the</strong><br />
comparison of <strong>the</strong> potencies of four A2A receptor <strong>an</strong>tagonists gave very different<br />
pharmacological profiles. The order of potencies of <strong>the</strong>se compounds at inducing motor<br />
activation was KW-6002>SCH-58261>MSX-3>SCH-442416, while <strong>the</strong> order of potencies at<br />
counteracting PCC was <strong>the</strong> opposite, SCH-442416>MSX-3>SCH-58261>KW-6002. The results<br />
indicate that KW-6002 <strong>an</strong>d SCH-442416 are quite selective post- <strong>an</strong>d presynaptic A2A receptor<br />
<strong>an</strong>tagonists, respectively. KW-6002 produced a signific<strong>an</strong>t locomotor activation at 0.3 mg/kg,<br />
while at 10 mg/kg, i.p., was still inactive at counteracting PCC. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, SCH-442416<br />
signific<strong>an</strong>tly counteracted PCC at 0.3 mg/kg, i.p., but <strong>the</strong> minimal locomotor-activating dose was<br />
3 mg/kg. The clinical import<strong>an</strong>ce of <strong>the</strong>se results is that drugs with <strong>an</strong> A2A receptor postsynaptic<br />
profile, like KW-6002, or a presynaptic A2A receptor profile, like SCH-442416, could be<br />
potentially useful in Parkinson’s disease <strong>an</strong>d Huntington’s disease, respectively.<br />
Disclosures: M. Orru, None; C. Quiroz, None; V. Beaumont, None; S. Ferre , None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.6/EE21<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: APIRE/Wyeth MD-PHD fellowship <strong>from</strong> Americ<strong>an</strong> Psychiatric Assn<br />
Title: Modulation of IPSCs in dorsal striatum by m-CPP<br />
Authors: *S. P. LIESKE 1 , A. BONCI 2 ;<br />
2 Neurol., 1 Univ. Cali<strong>for</strong>nia, S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Repetitive behaviors are a fundamental feature of multiple neurologic <strong>an</strong>d psychiatric<br />
disorders, including obsessive-compulsive disorder (OCD), Tourette syndrome, <strong>an</strong>d subst<strong>an</strong>ce<br />
abuse. Multiple lines of evidence have implicated <strong>the</strong> circuit through <strong>the</strong> basal g<strong>an</strong>glia as being<br />
of key import<strong>an</strong>ce in generating repetitive behaviors; of particular interest in this respect is <strong>the</strong><br />
subdivision of <strong>the</strong> dorsal striatum into “striosome” (“patch”) <strong>an</strong>d “matrix” compartments, with<br />
projections to <strong>the</strong> subst<strong>an</strong>tia nigra pars compacta (SNc), <strong>an</strong>d pars reticulata (SNr), respectively.<br />
An import<strong>an</strong>t role <strong>for</strong> serotonin in this system is suggested by several observations, among <strong>the</strong>m<br />
<strong>the</strong> well-known exacerbation of OCD symptoms by <strong>the</strong> serotonergic lig<strong>an</strong>d m-CPP. m-CPP has<br />
several different effects on <strong>the</strong> serotonergic signaling system, including acting as <strong>an</strong> agonist at<br />
multiple 5-HT receptor subtypes, <strong>an</strong>d increasing 5-HT levels via a direct effect on <strong>the</strong> 5-HT<br />
tr<strong>an</strong>sporter. Data will be presented examining <strong>the</strong> modulation of GABAergic IPSCs in medium<br />
spiny neurons (MSNs) of <strong>the</strong> dorsal striatum by m-CPP, with a focus on identifying <strong>the</strong><br />
particular serotonergic receptors involved. Miniature IPSC <strong>an</strong>alysis <strong>an</strong>d paired-pulse recordings<br />
to differentiate pre-synaptic <strong>an</strong>d post-synaptic effects will also be discussed. Contributions <strong>from</strong><br />
GABAergic interneurons will be differentiated electrophysiologically <strong>from</strong> IPSCs arising <strong>from</strong><br />
axon collaterals of MSNs. Finally, two distinct classifications of recorded MSNs will be<br />
considered: (1) as residing within <strong>the</strong> striosome vs. matrix, <strong>an</strong>d (2) as elements of <strong>the</strong> direct vs.<br />
indirect pathway.<br />
Disclosures: S.P. Lieske, Wyeth, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); A. Bonci, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 566.7/EE22<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: The Gatsby Charitable Foundation<br />
Medical Research Council<br />
Royal <strong>Society</strong><br />
Title: Differential responses of serotonin dorsal raphe neurons by noxious stimuli<br />
Authors: *J. V. SCHWEIMER, M. A. UNGLESS;<br />
Neurophysiol., MRC Clin. Sci. Ctr. - Imperial Col. London, London, United Kingdom<br />
Abstract: The serotonin system plays a fundamental role in a variety of behavioural <strong>an</strong>d<br />
cognitive functions, particularly in processing aversive stimuli. It has been suggested that<br />
serotonergic dorsal raphe neurons may encode a prediction-error rule <strong>for</strong> aversive stimuli <strong>an</strong>d act<br />
as <strong>an</strong> opponent system to <strong>the</strong> midbrain dopamine system, which encodes a prediction-error rule<br />
<strong>for</strong> appetitive stimuli.<br />
To test this hypo<strong>the</strong>sis, we used a combination of electrophysiological recordings <strong>an</strong>d <strong>the</strong><br />
juxtacellular labelling method. After finding a single spont<strong>an</strong>eously active neuron within <strong>the</strong><br />
dorsal raphe, we recorded baseline activity <strong>for</strong> 2-3 min to determine its physiological properties.<br />
<strong>When</strong> <strong>the</strong>se neurons exhibited typical patterns of serotonin neurons, that is broad action<br />
potentials <strong>an</strong>d slow clocklike firing rate or a stereotypic bursting pattern (doublets or triplets with<br />
approximately 10 ms interspike intervals), noxious footshocks (4 s duration, 5 mA, 20 Hz) were<br />
administered repeatedly. After recording, <strong>the</strong> individual neurons were labelled, <strong>an</strong>d<br />
neurochemically identified by immunohistochemistry <strong>for</strong> 5-hydroxytryptamine, tryptoph<strong>an</strong><br />
hydroxylase <strong>an</strong>d tyrosine hydroxylase. Two distinctive groups of serotonin neurons could be<br />
isolated. Identified clocklike serotonergic neurons showed phasic activation to <strong>the</strong> stimulus,<br />
which was strongest in <strong>the</strong> first 500 ms of <strong>the</strong> shock. These neurons were mainly located within<br />
rostral parts of <strong>the</strong> dorsal raphe nucleus, where mainly neurons projecting to <strong>the</strong> striatum are<br />
located. The o<strong>the</strong>r group consisted of serotonin neurons with a stereotypical bursting pattern as<br />
described previously by Hajos et al. (1996, 1997, 2007). Interestingly, <strong>the</strong>se serotonergic neurons<br />
were phasically inhibited by <strong>the</strong> footshocks; this response was also strongest in <strong>the</strong> first 500 ms.<br />
The bursting neurons have a wider rostral to caudal distribution within <strong>the</strong> dorsal raphe. These<br />
results support <strong>the</strong> hypo<strong>the</strong>sis that clocklike dorsal raphe serotonin neurons encode aversive<br />
in<strong>for</strong>mation, but also suggest that bursting serotonin neurons have distinct in<strong>for</strong>mation coding<br />
properties.<br />
Disclosures: J.V. Schweimer, None; M.A. Ungless, None.<br />
Poster
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.8/EE23<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NIH/NIA/IRP<br />
NIDA<br />
Title: Mild dietary iron deficiency alters locomotor responses to adenosine receptor <strong>an</strong>tagonists<br />
in mice: Implications <strong>for</strong> RLS<br />
Authors: *S. GULYANI 1 , R. WAN 1 , S. FERRE 2 , C. QUIROZ 2 , M. ORRU 2 , C. J. EARLEY 3 ,<br />
R. ALLEN 3 , M. P. MATTSON 1 ;<br />
1 Lab. of Neurosciences, NATIONAL INSTITUTE ON AGING/ NATIONAL INSTITUTES OF<br />
HEALTH, Baltimore, MD; 2 Natl. Inst. on Drug Abuse, Baltimore, MD; 3 Neurol., Johns Hopkins<br />
Univ. Sch. of Med., Baltimore, MD<br />
Abstract: Iron insufficiency is common, particularly in <strong>the</strong> elderly, pregn<strong>an</strong>t wom<strong>an</strong> <strong>an</strong>d inf<strong>an</strong>ts.<br />
Hemoglobin levels decline with age <strong>an</strong>d are associated with signific<strong>an</strong>t health problems in older<br />
individuals. Although hemoglobin (Hb) concentrations slightly below normal are common in <strong>the</strong><br />
elderly <strong>an</strong>d are often dismissed as having no clinical signific<strong>an</strong>ce, mild <strong>an</strong>emia has been<br />
increasingly shown to be associated with neurological <strong>an</strong>d cognitive disorders including Restless<br />
Legs Syndrome (RLS) <strong>an</strong>d attention deficit hyperactivity disorder (ADHD). Recent findings<br />
suggest that iron deficiency in <strong>the</strong> brain c<strong>an</strong> impair motor function by a mech<strong>an</strong>ism involving<br />
reduced dopaminergic signaling. We previously reported that iron deficiency results in <strong>an</strong> upregulation<br />
of adenosine A2A receptor signaling in cultured neurons <strong>an</strong>d in striatal neurons in<br />
vivo which may, in turn, result in down-regulation of dopamine D2 receptor signaling (Exp<br />
Neurol. <strong>2009</strong>; 215:236-42). In <strong>the</strong> present study we determined <strong>the</strong> effects of dietary iron<br />
deficiency on locomotor behavior in mice under basal conditions <strong>an</strong>d in <strong>the</strong> presence of<br />
<strong>an</strong>tagonists of A2R or A1 adenosine receptors. DBA/2J female mice were fed <strong>an</strong> iron deficient<br />
diet (7 ppm) <strong>an</strong>d control mice were fed a diet with 48 ppm iron <strong>for</strong> 2 months; <strong>the</strong> iron-deficient<br />
mice exhibited a 20% reduction in Hb levels. In iron-deficient mice <strong>the</strong> selective postsynaptic<br />
A2A receptor <strong>an</strong>tagonist KW60028 induced a stronger locomotor activation, whereas <strong>the</strong> A1<br />
receptor <strong>an</strong>tagonist CPT caused signific<strong>an</strong>tly less locomotor activation. These results suggest that<br />
mild dietary iron deficiency c<strong>an</strong> alter adenosine receptor-mediated locomotor behavior in a<br />
m<strong>an</strong>ner consistent with <strong>the</strong> increased involuntary locomotor activity that occurs in RLS.<br />
Disclosures: S. Guly<strong>an</strong>i, None; R. W<strong>an</strong>, None; S. Ferre, None; C. Quiroz, None; M. Orru,<br />
None; C.J. Earley, None; R. Allen, None; M.P. Mattson, None.
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.9/EE24<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: EU, FP6 MEST-CT-2005-019729<br />
EU, FP5 ‘Neurobotics’ 001917<br />
Swedish research council, VR-M -3026<br />
Swedish research council, VR-NT 621-2007-6049<br />
EU, FP7 'select <strong>an</strong>d act' 201716<br />
Karolinska Institutets Research funds<br />
ICT-STREP 216100 - LAMPETRA<br />
Title: The lamprey habenula; A sensory motor hub?<br />
Authors: M. R. JONES 1 , O. FLOROS 1 , X. ABALO 1 , B. ROBERTSON 1 , *S. GRILLNER 2 ;<br />
1 Nobel institute <strong>for</strong> Neurophysiology, Dept. of Neurosci., Karolinska Institutet, Stockholm,<br />
Sweden; 2 Karolinska Inst., S-171 77 Stockholm, Sweden<br />
Abstract: The Habenula is present in all vertebrates <strong>an</strong>d in mammals it receives input <strong>from</strong> <strong>the</strong><br />
basal g<strong>an</strong>glia, <strong>an</strong>d may play a role in cognitive <strong>an</strong>d motor functions. Our aim is determine which<br />
components of <strong>the</strong> habenula circuitry are present in <strong>the</strong> earliest group of vertebrates, <strong>the</strong> lamprey.<br />
Using <strong>an</strong>terograde <strong>an</strong>d retrograde tracing combined with immunohistochemistry we identified<br />
habenula afferents <strong>from</strong> hypothalamus, parapineal org<strong>an</strong>, <strong>an</strong>d olfactory bulbs <strong>an</strong>d a putative<br />
output region of <strong>the</strong> basal g<strong>an</strong>glia, referred to as <strong>the</strong> dorsal pallium. Afferent fibers<br />
immunoreactive <strong>for</strong> GABA innervated both <strong>the</strong> left <strong>an</strong>d right habenula, while enkephalin<br />
positive fibers were only seen innervating <strong>the</strong> right habenula. Habenula efferents were present in<br />
<strong>the</strong> hypothalamus, posterior tubercle <strong>an</strong>d <strong>the</strong> mammillary region, where <strong>the</strong>y directly contacted<br />
(close apposition) histamine, dopamine <strong>an</strong>d serotonin immunoreactive cells respectively. A<br />
second major efferent pathway passed through <strong>the</strong> fasciculus retroflexus to <strong>the</strong> pretectum,<br />
interpeduncular nucleus <strong>an</strong>d <strong>the</strong> reticular <strong>for</strong>mation. Histaminergic cells in <strong>the</strong> hypothalamus,
dopaminergic cells in <strong>the</strong> posterior tubercle project to <strong>the</strong> striatum, which also has a rich 5-HT<br />
innervation.<br />
The outputs of <strong>the</strong> lamprey habenula is similar to those of mammali<strong>an</strong> <strong>an</strong>d o<strong>the</strong>r vertebrates, with<br />
two major projection pathways; <strong>the</strong> medial habenula projecting to <strong>the</strong> interpeduncluar nucleus<br />
<strong>an</strong>d <strong>the</strong> lateral habenula projecting to <strong>the</strong> dopamine, histamine <strong>an</strong>d serotonin neurons. In contrast<br />
to <strong>the</strong> mammali<strong>an</strong> vertebrates <strong>the</strong> major inputs to <strong>the</strong> lamprey habenula are <strong>from</strong> <strong>the</strong> dorsal<br />
pallium, a putative output nucleus of <strong>the</strong> basal g<strong>an</strong>glia <strong>an</strong>d sensory input <strong>from</strong> <strong>the</strong> parapineal <strong>an</strong>d<br />
<strong>the</strong> olfactory bulbs. The Lamprey habenula, in contrast to <strong>the</strong> mammali<strong>an</strong> vertebrates, appears to<br />
link sensory systems to <strong>the</strong> basal g<strong>an</strong>glia.<br />
Disclosures: M.R. Jones, None; O. Floros, None; X. Abalo, None; B. Robertson, None; S.<br />
Grillner, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.10/EE25<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: The EU cortex training program, FP6 MEST-CT-2005-019729<br />
EU, FP5 ‘Neurobotics’ 001917<br />
ICT-STREP 216100 – LAMPETRA<br />
Swedish research council, VR-M -3026<br />
VR-NT 621-2007-6049<br />
Karolinska Institutets Research funds<br />
Select-<strong>an</strong>d-Act FP7 no 201716<br />
Title: The lamprey basal g<strong>an</strong>glia - A vertebrate blue-print<br />
Authors: *B. ROBERTSON, M. JONES, E. SAMUELSSON, R. HILL, J. HELLGREN, S.<br />
GRILLNER;<br />
Dept. of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Abstract: The basal g<strong>an</strong>glia constitute a key brain structure that plays a prominent role in motor<br />
functions in mammals as well as lamprey (Thompson et al., 2008; Ménard et al., 2007, 2008).<br />
The aim of <strong>the</strong> present study is to identify <strong>the</strong> different components of <strong>the</strong> lamprey basal g<strong>an</strong>glia,<br />
<strong>an</strong>d to what <strong>an</strong> extent <strong>the</strong>y correspond to those found in <strong>the</strong> mammali<strong>an</strong> basal g<strong>an</strong>glia.<br />
Anatomical tract tracing, immunohistochemistry <strong>an</strong>d cell attached electrophysiological<br />
recordings were employed to address this question. Two pallidal regions in <strong>the</strong> lamprey were<br />
identified; one region, potentially homologous to <strong>the</strong> mammali<strong>an</strong> globus pallidus, was located<br />
ventral to <strong>the</strong> eminentia thalami on <strong>the</strong> telencephalic/diencephalic border. It receives striatal<br />
input <strong>an</strong>d contains GABAergic projection neurons, of which those projecting to <strong>the</strong> tectum were<br />
shown to be tonically active. Neurons in this region were also immunoreactive <strong>for</strong> parvalbumin,<br />
<strong>an</strong>d receive striatal afferents <strong>an</strong>d enkephalin fibers. Separate subpopulations project to <strong>the</strong><br />
mesencephalic, <strong>the</strong> diencephalic <strong>an</strong>d <strong>the</strong> tectal regions. Ano<strong>the</strong>r region, in <strong>the</strong> midbrain, which<br />
c<strong>an</strong> be considered homologous to <strong>the</strong> subst<strong>an</strong>tia nigra pars reticulata receives input <strong>from</strong><br />
subst<strong>an</strong>ce P positive striatal neurons, sends GABAergic projections to <strong>the</strong> tectum <strong>an</strong>d projects to<br />
<strong>the</strong> diencephalic locomotor region, but not to <strong>the</strong> mesencephalic locomotor region. This midbrain<br />
region also contains parvalbumin immunopositive neurons <strong>an</strong>d is sparsely innervated by<br />
enkephalin fibers. Striatal neurons have <strong>the</strong> <strong>an</strong>atomical <strong>an</strong>d electrophysiological hallmarks of<br />
mammali<strong>an</strong> medium spiny neurons <strong>an</strong>d striatum receives similar dopaminergic, 5-HT <strong>an</strong>d<br />
histamine, as well as thalamic <strong>an</strong>d pallial (corresponding to cortex) inputs as in mammals<br />
(Pombal et al., 1997a,b; Ericsson et al., 2008). Our results <strong>an</strong>d previous results <strong>from</strong> our<br />
laboratory suggest that <strong>the</strong> basic features of <strong>the</strong> basal g<strong>an</strong>glia with regard to both structure <strong>an</strong>d<br />
function are conserved, including striatal/pallidal subdivisions. This design was thus present 530<br />
million years ago, when cyclostomes diverged <strong>from</strong> <strong>the</strong> main vertebrate line that is at <strong>the</strong> dawn<br />
of vertebrate evolution.<br />
References:<br />
1. Ericsson J, Wikström MA <strong>an</strong>d S. Grillner. (2008) FENS<br />
2. Ménard A, Auclair F, Bourcier-Lucas C, Grillner S, <strong>an</strong>d Dubuc R. (2007) J Comp Neurol<br />
501:260-273.<br />
3. Ménard, A <strong>an</strong>d Grillner S. (2008) J Neurophysiol 100: 1343-1353.<br />
4. Pombal MA, El M<strong>an</strong>ira A, Grillner S. 1997a. J Comp Neurol 386:71-91.<br />
5. Pombal MA, El M<strong>an</strong>ira A, Grillner S. 1997b. Brain Res 766:249-254.<br />
6. Thompson, RH, Ménard A, Pombal M, <strong>an</strong>d Grillner S. 2008. Eur J Neurosci 27:1452-1460.<br />
Disclosures: B. Robertson, None; M. Jones, None; E. Samuelsson, None; R. Hill, None; J.<br />
Hellgren, None; S. Grillner, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 566.11/EE26<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: CONACyT Gr<strong>an</strong>t 50427Q<br />
Title: Dopamine reduces <strong>the</strong> GABAergic input <strong>from</strong> globus pallidus to thalamic reticular<br />
nucleus via presynaptic D4 dopamine receptors<br />
Authors: D. GASCA-MARTINEZ 1 , A. HERNANDEZ 4 , A. SIERRA 2 , V. ANAYA-<br />
MARTINEZ 5 , R. VALDIOSERA 3 , B. FLORAN 3 , D. ERLIJ 3 , *J. ACEVES 6 ;<br />
1 Physiology, Biophysics <strong>an</strong>d Neurosciences, 2 Physiology, Biophysics <strong>an</strong>d Neurosci.,<br />
3 CINVESTAV-IPN, Mexico, DF, Mexico; 4 Inst. of Cell. <strong>an</strong>d Physiol., 5 FES Iztacala, UNAM,<br />
Mexico, DF, Mexico; 6 Ctr. Investigacion Del IPN, Mexico DF 07000, Mexico<br />
Abstract: The thalamic reticular nucleus (TRn) receives a signific<strong>an</strong>t dopaminergic projection<br />
<strong>from</strong> <strong>the</strong> pars compacta of subst<strong>an</strong>tia nigra (Anaya-Martinez et al., 2006) <strong>an</strong>d a GABAergic<br />
projection <strong>from</strong> <strong>the</strong> globus pallidus (As<strong>an</strong>uma, 1989). Although D4 dopamine receptors are<br />
present in TRn (Mrzljak et al., 1996), <strong>the</strong>ir physiological role is unknown. Here we have<br />
explored <strong>the</strong>ir role. Whole-cell recordings were made in brain slices <strong>from</strong> neonatal rats (14-21<br />
days old). Two types of recordings were made: locally evoked IPSCs <strong>an</strong>d spont<strong>an</strong>eous miniature<br />
IPSCs (mIPSCs). CNQX <strong>an</strong>d AP-5 were present in <strong>the</strong> medium to block EPSCs. TTX was also<br />
present when mIPSCs were recorded. In <strong>the</strong>se conditions, bicuculline blocked both evoked<br />
IPSCs as well as mIPSCs. Dopamine (30 µM) reduced <strong>the</strong> amplitude of <strong>the</strong> evoked IPSC by<br />
about 40%. From <strong>the</strong> dose-response curve <strong>an</strong> EC50 of 1.3 µM was calculated. The dopamine<br />
inhibition was associated with <strong>an</strong> increase in <strong>the</strong> paired-pulse ratio of <strong>the</strong> evoked IPSCs. In<br />
addition, dopamine reduced <strong>the</strong> frequency of <strong>the</strong> mIPSCs without affecting <strong>the</strong>ir amplitude. Both<br />
results indicate a presynaptic location of <strong>the</strong> receptors mediating <strong>the</strong> effect. The selective D4<br />
receptor <strong>an</strong>tagonist L-745,870, blocked <strong>the</strong> effect of dopamine on both evoked IPSCs <strong>an</strong>d<br />
mIPSCs, indicating that <strong>the</strong> effect was mediated by D4 receptors. The loss of pallidal neurons<br />
induced by kainic acid completely eliminated <strong>the</strong> effect of <strong>the</strong> activation of D4 receptors on both<br />
evoked IPSCs <strong>an</strong>d mIPSCs, indicating that <strong>the</strong> receptors were located on axon terminals <strong>from</strong> <strong>the</strong><br />
globus pallidus. Thus dopamine by regulating <strong>the</strong> inhibitory input <strong>from</strong> <strong>the</strong> globus pallidus to<br />
TRn may modulate <strong>the</strong> firing pattern of TRn neurons, <strong>the</strong>reby modulating <strong>the</strong> flow of sensory<br />
<strong>an</strong>d motor in<strong>for</strong>mation through <strong>the</strong> thalamus as well as wakening <strong>an</strong>d sleep states.<br />
Disclosures: D. Gasca-Martinez, None; A. Hern<strong>an</strong>dez, None; A. Sierra, None; V. Anaya-<br />
Martinez, None; R. Valdiosera, None; B. Flor<strong>an</strong>, None; D. Erlij, None; J. Aceves, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.12/EE27<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NIH Gr<strong>an</strong>t NS33538<br />
Title: Altered glutamate currents <strong>an</strong>d dopamine modulation in <strong>the</strong> striatum of a mouse model of<br />
schizophrenia<br />
Authors: *C. CEPEDA 1 , V. M. ANDRE 1 , D. M. CUMMINGS 1 , S. P. RAO 1 , S. M. GEE 1,2 , A.<br />
RAMSEY 2 , M. G. CARON 2 , M. S. LEVINE 1 ;<br />
1 2<br />
Mental Retardation Res. Ctr., Univ. Cali<strong>for</strong>nia, Los Angeles, CA; Dept Cell Biol., Duke Univ.,<br />
Durham, NC<br />
Abstract: N-methyl-D-aspartate (NMDA) receptors play <strong>an</strong> essential role in cognition <strong>an</strong>d<br />
blockade or deficits in NMDA receptor-function have been associated with <strong>the</strong> negative<br />
symptoms of schizophrenia. In a genetic mouse model of reduced NMDA receptor function<br />
generated by knock-down (KD) of <strong>the</strong> NR1 subunit, behavioral alterations similar to those<br />
observed in pharmacologically-induced <strong>an</strong>imal models of schizophrenia have been reported<br />
(Mohn et al., 1999). In <strong>the</strong> present study we compared glutamate receptor-mediated currents in<br />
acutely isolated striatal medium-sized spiny neurons (MSSNs), as well as spont<strong>an</strong>eous or evoked<br />
excitatory postsynaptic currents (EPSCs) in slices <strong>from</strong> NR1-KD mice <strong>an</strong>d <strong>the</strong>ir wild type (WT)<br />
control littermates at 3 months of age. Whole-cell patch clamp recordings in acutely isolated<br />
MSSNs <strong>from</strong> NR1-KD <strong>an</strong>d WT mice displayed similar passive membr<strong>an</strong>e properties<br />
(capacit<strong>an</strong>ce, input resist<strong>an</strong>ce, time const<strong>an</strong>t). Currents evoked by application of NMDA were<br />
severely reduced in NR1-KD MSSNs while currents evoked by α-amino-3-hydroxy-5-methyl-4isoxazolepropionic<br />
acid (AMPA) were signific<strong>an</strong>tly increased. Similarly, <strong>the</strong> frequency <strong>an</strong>d<br />
amplitude of spont<strong>an</strong>eous EPSCs at a holding potential of -70 mV (mediated primarily by<br />
AMPA receptors) were increased <strong>an</strong>d <strong>the</strong> kinetics of <strong>the</strong> currents were altered in MSSNs <strong>from</strong><br />
NR1-KDs. Spont<strong>an</strong>eous EPSCs displayed faster rise <strong>an</strong>d decay times <strong>an</strong>d reduced half-amplitude<br />
durations in cells <strong>from</strong> NR1-KD compared to WT mice. Evoked synaptic currents mediated by<br />
activation of NMDA receptors were smaller in cells <strong>from</strong> NR1-KD compared to WT mice.<br />
Dopamine D1 receptor modulation of synaptically-evoked NMDA currents was altered. In cells<br />
<strong>from</strong> WT mice <strong>the</strong> D1 agonist SKF 81297 increased peak current amplitude whereas in cells<br />
<strong>from</strong> NR1-KD mice <strong>the</strong> agonist decreased peak current amplitude. There was a trend toward<br />
decreased modulation of NMDA responses by <strong>the</strong> dopamine D1 receptor agonist in isolated<br />
MSSNs. While <strong>the</strong> reduction in NMDA receptor-mediated currents was expected, <strong>the</strong> increase in<br />
AMPA receptor-mediated responses was unexpected <strong>an</strong>d suggests compensatory mech<strong>an</strong>isms to<br />
counter deficits in NMDA receptor function. Ch<strong>an</strong>ges in AMPA responses <strong>an</strong>d dopamine<br />
receptor modulation of NMDA currents indicate that reduced function of NMDA receptors also<br />
is associated with alterations in AMPA <strong>an</strong>d dopamine receptor function.<br />
Disclosures: C. Cepeda, None; V.M. Andre, None; D.M. Cummings, None; S.P. Rao,<br />
None; S.M. Gee, None; A. Ramsey, None; M.G. Caron, None; M.S. Levine, None.
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.13/EE28<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NIH Gr<strong>an</strong>t NS33538<br />
Title: Dopamine modulation of excitatory currents in striatum is dictated by <strong>the</strong> expression of<br />
D1 or D2 receptors <strong>an</strong>d modified by endoc<strong>an</strong>nabinoids<br />
Authors: *V. M. ANDRE, C. CEPEDA, D. M. CUMMINGS, E. L. JOCOY, Y. E. FISHER, M.<br />
S. LEVINE;<br />
Mental Retardation Res. Ctr., UCLA, Los Angeles, CA<br />
Abstract: Striatal medium-sized spiny neurons (MSSNs) receive cortical <strong>an</strong>d thalamic<br />
glutamatergic inputs that are modulated by dopamine (DA). Two types of MSSNs c<strong>an</strong> be<br />
differentiated based on expression of DA receptors (DARs), peptide content, <strong>an</strong>d projections to<br />
output nuclei of <strong>the</strong> basal g<strong>an</strong>glia. D1R-expressing MSSNs give rise to <strong>the</strong> direct pathway<br />
whereas D2R-expressing MSSNs give rise to <strong>the</strong> indirect pathway. We used mice expressing<br />
enh<strong>an</strong>ced green fluorescent protein as a reporter gene to identify MSSNs containing D1R or D2R<br />
subtypes to examine DA modulation of spont<strong>an</strong>eous excitatory postsynaptic currents (sEPSCs)<br />
in slices <strong>an</strong>d N-methyl-D-aspartate (NMDA) <strong>an</strong>d α-amino-3-hydroxy-5-methyl-4isoxazolepropionic<br />
acid (AMPA) currents in acutely isolated MSSNs. Whole-cell patch clamp<br />
recordings in slices demonstrated a DAR specific modulation of sEPSCs. DA <strong>an</strong>d D1 agonists<br />
increased sEPSC frequency in D1R-expressing MSSNs whereas DA <strong>an</strong>d D2 agonists decreased<br />
sEPSC frequency in D2 cells. These effects were blocked by TTX indicating <strong>the</strong>y were<br />
presynaptically mediated. Fur<strong>the</strong>rmore, <strong>the</strong>y were fully (D1 cells) or partially (D2 cells)<br />
mediated through retrograde signaling via endoc<strong>an</strong>nabinoids (eCBs). A CB1 receptor agonist<br />
prevented <strong>the</strong> D1R-mediated increase in sEPSC frequency in D1 cells while a CB1 receptor<br />
<strong>an</strong>tagonist partially blocked <strong>the</strong> decrease in sEPSC frequency in D2 cells. Postsynaptically, low<br />
concentrations of a D1R agonist consistently increased NMDA <strong>an</strong>d AMPA currents in acutely<br />
isolated D1 cells while a D2R agonist decreased <strong>the</strong>se currents in D2 cells. These results show<br />
that both glutamate release <strong>an</strong>d postsynaptic excitatory currents are regulated in opposite<br />
directions by activation of D1 or D2 DARs. The direction of this regulation is also specific to D1<br />
<strong>an</strong>d D2R-expressing MSSNs. We suggest that activation of postsynaptic DARs control eCB<br />
mobilization, acting on presynaptic CB1Rs, thus modulating glutamate release differently in<br />
glutamate terminals making contact with D1 <strong>an</strong>d D2 cells. This differential modulation could be
elev<strong>an</strong>t to maintain <strong>the</strong> bal<strong>an</strong>ce of <strong>the</strong> direct <strong>an</strong>d indirect pathways to regulate basal g<strong>an</strong>glia<br />
output.<br />
Disclosures: V.M. Andre, None; C. Cepeda, None; D.M. Cummings, None; E.L. Jocoy,<br />
None; Y.E. Fisher, None; M.S. Levine, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.14/EE29<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Title: Adenosine A2A receptors modulate endoc<strong>an</strong>nabinoid release <strong>from</strong> indirect pathway<br />
striatal neurons<br />
Authors: *T. LERNER, A. C. KREITZER;<br />
Gladstone Institute/UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: The basal g<strong>an</strong>glia <strong>an</strong>d its primary input nucleus, <strong>the</strong> striatum, are critical <strong>for</strong><br />
motivation <strong>an</strong>d motor control. The striatum integrates in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> cortex, thalamus, <strong>an</strong>d<br />
midbrain, <strong>an</strong>d sends projections to downstream basal g<strong>an</strong>glia nuclei that regulate thalamocortical<br />
motor circuits. Striatal projection neurons, known as medium spiny neurons (MSNs), consist of<br />
two major classes: direct-pathway MSNs that express dopamine D1 receptors <strong>an</strong>d project<br />
directly to basal g<strong>an</strong>glia output nuclei, <strong>an</strong>d indirect-pathway MSNs that express dopamine D2<br />
receptors <strong>an</strong>d project to <strong>the</strong> globus pallidus. According to classical basal g<strong>an</strong>glia models,<br />
increased direct pathway activity facilitates movement, whereas increased indirect pathway<br />
activity inhibits movement. Import<strong>an</strong>tly, G-protein-coupled receptor (GPCR) signaling in direct-<br />
<strong>an</strong>d indirect-pathway MSNs plays a key role in regulating activity in <strong>the</strong>se circuits. Adenosine<br />
A2A receptors are GPCRs that are highly expressed in indirect-pathway MSNs, where <strong>the</strong>y<br />
functionally oppose <strong>the</strong> actions of D2 receptors. Although <strong>the</strong> role of dopamine receptors in<br />
regulating cellular <strong>an</strong>d synaptic plasticity in striatal MSNs has been studied extensively, <strong>the</strong><br />
mech<strong>an</strong>isms of A2A receptor modulation in <strong>the</strong> striatum are less well understood. We have<br />
examined <strong>the</strong> role of A2A receptors in plasticity of excitatory synapses onto indirect-pathway<br />
MSNs <strong>an</strong>d find that <strong>the</strong>y play a key role in regulating <strong>the</strong> induction of both short- <strong>an</strong>d long-term<br />
depression by modulating endoc<strong>an</strong>nabinoid release. A2A <strong>an</strong>tagonists facilitate endoc<strong>an</strong>nabinoid<br />
release, whereas A2A agonists block endoc<strong>an</strong>nabinoid release. We examine <strong>the</strong> downstream<br />
signaling mech<strong>an</strong>isms by which A2A receptors are able to exert <strong>the</strong>ir effects on indirect-pathway<br />
synaptic plasticity.
Disclosures: T. Lerner, None; A.C. Kreitzer, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.15/EE30<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: Parkinson’s Disease <strong>Society</strong> of <strong>the</strong> UK Gr<strong>an</strong>t G-0601<br />
Medical Research Council of <strong>the</strong> UK.<br />
Title: A qu<strong>an</strong>titative <strong>an</strong>alysis of <strong>the</strong> glutamatergic <strong>an</strong>d GABAergic somatodendritic innervation<br />
of individual dopaminergic neurons of <strong>the</strong> rat subst<strong>an</strong>tia nigra<br />
Authors: *P. HENNY, M. T. C. BROWN, P. J. MAGILL, J. P. BOLAM;<br />
Pharmacol., Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom<br />
Abstract: Nigrostriatal dopaminergic neurons play a fundamental role in motor function. High<br />
striatal dopamine facilitates movement, whereas a low dopaminergic tone is associated with<br />
hypoactivity <strong>an</strong>d bradykinesia. The level of released striatal dopamine depends on <strong>the</strong> activity of<br />
dopaminergic neurons, which is partly regulated by synaptic inputs. Indeed, in <strong>the</strong>se neurons<br />
activation of glutamate receptors <strong>an</strong>d decreases in GABAergic input underlie <strong>the</strong> switch <strong>from</strong> a<br />
tonic to a phasic firing pattern, which is associated with increased dopamine release. Similarly,<br />
GABA(A) receptor activation inhibits neuronal firing. To underst<strong>an</strong>d <strong>the</strong> mech<strong>an</strong>isms that<br />
underlie synaptic control of dopaminergic neuron activity, we studied <strong>the</strong> structural org<strong>an</strong>isation<br />
of <strong>the</strong> glutamatergic <strong>an</strong>d GABAergic innervation of <strong>the</strong> entire somatodendritic domain of single<br />
neurons. Following in vivo extracellular recording, single neurons in <strong>the</strong> subst<strong>an</strong>tia nigra were<br />
labelled with neurobiotin using <strong>the</strong> juxtacellular technique. Parasagittal tissue sections (50<br />
micrometers) were collected sequentially, <strong>an</strong>d labelled neurons were identified <strong>an</strong>d<br />
neurochemically characterised by fluorescent staining <strong>for</strong> neurobiotin <strong>an</strong>d immunolabelling <strong>for</strong><br />
tyrosine hydroxylase <strong>an</strong>d calbindin. Sections were <strong>the</strong>n processed to reveal neurobiotin <strong>an</strong>d <strong>the</strong><br />
vesicular tr<strong>an</strong>sporters <strong>for</strong> glutamate (VGluT2) or GABA (VGAT) using peroxidase reactions <strong>for</strong><br />
light <strong>an</strong>d electron microscopy. After light microscopic 3D reconstruction of <strong>the</strong> neurons,<br />
synapses onto labelled dendrites or somata were r<strong>an</strong>domly <strong>an</strong>d systematically sampled, <strong>an</strong>d<br />
examined in <strong>the</strong> electron microscope. Using stereology, synapses were <strong>the</strong>n counted <strong>an</strong>d <strong>the</strong>ir<br />
total number estimated. These data were combined with 3D structural data to examine synaptic<br />
density <strong>an</strong>d number as a function of location <strong>an</strong>d type of dendrite innervated. The results so far
indicate that: 1. single dopaminergic neurons receive between 8000 <strong>an</strong>d 9000 synaptic contacts.<br />
2. Overall, ~30% of synapses are glutamatergic <strong>an</strong>d ~65% are GABAergic. 3. Synaptic density<br />
increases with dist<strong>an</strong>ce <strong>from</strong> <strong>the</strong> cell body <strong>an</strong>d dendritic br<strong>an</strong>ch order, but is negatively<br />
correlated with dendritic caliber. 4. Import<strong>an</strong>t differences are observed between <strong>the</strong> distribution<br />
of glutamatergic <strong>an</strong>d GABAergic synapses along dendrites <strong>an</strong>d somata. It is suggested that <strong>the</strong><br />
spatial distribution of synapses plays <strong>an</strong> import<strong>an</strong>t role in how glutamate <strong>an</strong>d GABA influence<br />
dopaminergic neuron activity. Future work will test <strong>for</strong> differences between subpopulations of<br />
dopaminergic neuron (calbindin+ vs. calbindin-), as well as relationships between <strong>the</strong><br />
structural/synaptic org<strong>an</strong>ization <strong>an</strong>d activity of single neurons.<br />
Disclosures: P. Henny, None; M.T.C. Brown, None; P.J. Magill, None; J.P. Bolam, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.16/EE31<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: NIH Gr<strong>an</strong>ts F31 NS061528, R01 RR00165, CA21765, NS051537, NS042828, R01<br />
MH077298, <strong>an</strong>d PO1 NS44282<br />
Merit Award <strong>from</strong> <strong>the</strong> Department of Veter<strong>an</strong>s Affairs<br />
Americ<strong>an</strong> Leb<strong>an</strong>ese Syri<strong>an</strong> Associated Charities<br />
National Parkinson Foundation Center of Excellence at V<strong>an</strong>derbilt<br />
Title: Extra-cerebellar role <strong>for</strong> Cbln1: Modulation of dendritic spine density <strong>an</strong>d synapses in<br />
striatal medium spiny neurons<br />
Authors: *S. V. KUSNOOR 1 , J. PARRIS 2 , E. C. MULY 3,4,5 , J. I. MORGAN 2 , A. Y.<br />
DEUTCH 1 ;<br />
1 Prog Neuro, Dept Psych & Pharm, V<strong>an</strong>derbilt Univ. Med. Ctr., Nashville, TN; 2 Dept. of<br />
Developmental Neurobio., St. Jude Children’s Res. Hosp., Memphis, TN; 3 Dept. of Psychiatry<br />
<strong>an</strong>d Behavioral Sci., Emory Univ. Sch. of Med., Atl<strong>an</strong>ta, GA; 4 Atl<strong>an</strong>ta VAMC, Decatur, GA;<br />
5 Div. of Neurosci., Yerkes Primate Res. Ctr., Atl<strong>an</strong>ta, GA
Abstract: Cbln1 (precerebellin) is a secreted glycoprotein originally identified in <strong>the</strong> cerebellum<br />
that defines a subfamily (Cbln1-4) of <strong>the</strong> C1q/TNFα superfamily of proteins. Cbln1 has a<br />
heterogeneous distribution in brain, but <strong>the</strong> only site in which Cbln1 has been shown to have<br />
central effects is <strong>the</strong> cerebellar cortex, where it gets secreted by gr<strong>an</strong>ule cells <strong>an</strong>d regulates <strong>the</strong><br />
synaptic structure <strong>an</strong>d plasticity of gr<strong>an</strong>ule cell-Purkinje synapses. In cbln1 null mut<strong>an</strong>t mice,<br />
most Purkinje cell dendritic spines lack presynapatic partners <strong>an</strong>d have enlarged PSDs (Hirai et<br />
al. 2005). Neurons of <strong>the</strong> thalamic parafascicular nucleus (PF), which provides glutamatergic<br />
projections to <strong>the</strong> striatum, express high levels of Cbln1. We examined Cbln1 in thalamostriatal<br />
circuits <strong>an</strong>d determined if cbln1 knockout mice exhibit structural deficits in striatal neurons.<br />
Virtually all neurons of <strong>the</strong> PF expressed Cbln1-immunoreactivity (-ir), with only rare Cbln1-ir<br />
neurons seen in <strong>the</strong> central medial complex, <strong>the</strong> o<strong>the</strong>r thalamic region that projects to <strong>the</strong> dorsal<br />
striatum. In <strong>the</strong> striatum, Cbln1-ir processes were apposed to striatal medium spiny neuron<br />
(MSN) dendrites. Retrograde tract tracing revealed that all labeled PF cells were Cbln1-ir. We<br />
examined <strong>the</strong> structure of Golgi-impregnated MSNs in adult cbln1 knockout mice. MSN<br />
dendritic spine density was markedly increased in cbln1 -/- mice relative to wildtype littermates,<br />
but total dendritic length was unch<strong>an</strong>ged. Ultrastructural examination revealed that axospinous<br />
synaptic density of MSNs was increased in cbln1 -/- mice, with no ch<strong>an</strong>ge in PSD length. Thus,<br />
Cbln1 determines <strong>the</strong> synaptic org<strong>an</strong>ization of thalamostriatal neurons, with effects that are<br />
opposite to those seen in <strong>the</strong> cerebellum. These data suggest that Cbln1 may signal through<br />
different receptors or mech<strong>an</strong>isms in different brain regions.<br />
Disclosures: S.V. Kusnoor, None; J. Parris, None; E.C. Muly, None; J.I. Morg<strong>an</strong>,<br />
None; A.Y. Deutch, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.17/EE32<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: Brain Korea 21 Project <strong>for</strong> Medical Science, Yonsei University<br />
Title: Alteration of behavioral <strong>an</strong>d dopamine activities by high frequency stimulation of<br />
subthalamic nucleus in freely moveable rat parkinsoni<strong>an</strong> models with potable deep brain<br />
stimulator<br />
Authors: *D. LEE 1,2 , Y. HWANG 1,3 , J. OH 4 , S.-I. PARK 3,5 , Y. PARK 1 , S. KIM 5 , Z.-H. CHO 4 ,<br />
J. CHANG 1 ;
1 Dept. of Med. Sci. & Neurosurg., Yonsei Univ. Coll. of Med., Seoul, Republic of Korea; 2 Brain<br />
Korea 21 Project <strong>for</strong> Med. Sci., Yonsei Univ. Col. of Med., Seoul, Republic of Korea; 3 M.I.Tech<br />
Co., Ltd., Inst. of Interventional Med., Pyongtaek, Republic of Korea; 4 Neurosci. Res. Inst.,<br />
Gachon Univ., Coll. of Med. & Sci., Incheon, Republic of Korea; 5 Sch. of Electrical & Computer<br />
Eng., Seoul Nat'l Univ., Seoul, Republic of Korea<br />
Abstract: Although its functional mech<strong>an</strong>ism remains unknown, deep brain stimulation (DBS) is<br />
regarded as a most effective neurosurgical <strong>the</strong>rapy <strong>for</strong> patients with Parkinson’s disease. In order<br />
to evaluate <strong>the</strong> efficacy of deep brain stimulation in 6-hydroxydopamine (6-OHDA)-lesioned<br />
parkinsoni<strong>an</strong> rats, we studied rats with unilateral dopamine (DA) depletion by injection of 6-<br />
OHDA. We make portable stimulators <strong>for</strong> <strong>an</strong>imal Parkinson’s disease (PD) model to enable<br />
more natural behavior experiments. Stimulation parameters are selected <strong>from</strong> <strong>an</strong>imal stimulation<br />
test. Assumptions about <strong>the</strong> mediating effects of lesion of <strong>the</strong> nigrostriatal dopaminergic pathway<br />
by 6-OHDA <strong>an</strong>d <strong>the</strong> effects of STN-DBS are examined per<strong>for</strong>ming different motor specific<br />
behavior test. We also studied <strong>the</strong> tracer distributions of lig<strong>an</strong>ds using microPET <strong>for</strong> dopamine<br />
D2 receptors ([11C]raclopride) during STN-DBS to evaluate molecular ch<strong>an</strong>ges <strong>for</strong> DA<br />
tr<strong>an</strong>smission. Our results show that DBS of <strong>the</strong> STN in <strong>the</strong> dopamine-depleted rats decrease<br />
amphetamine-induced rotational behavior <strong>an</strong>d restore motor function in spont<strong>an</strong>eous behavior.<br />
There<strong>for</strong>e, our <strong>an</strong>imal DBS system could be a useful instrument <strong>for</strong> investigation of parkinsoni<strong>an</strong><br />
disease. Our microPET data <strong>from</strong> living parkinsoni<strong>an</strong> rats also provided results <strong>for</strong> <strong>an</strong> increased<br />
striatal DA concentration under STN-DBS. The results indicate that STN-DBS in a 6-OHDA<br />
induced hemipakinsoni<strong>an</strong> rat model may counteract some of dopamine release within <strong>the</strong><br />
striatum <strong>an</strong>d influence striatal dopaminergic metabolism.<br />
Disclosures: D. Lee, None; Y. Hw<strong>an</strong>g, None; J. Oh, None; S. Park, None; Y. Park, None; S.<br />
Kim, None; Z. Cho, None; J. Ch<strong>an</strong>g, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.18/EE33<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: CIHR Gr<strong>an</strong>t 15685<br />
Title: Facilitation of dentate gyrus population spike may involve septohippocampal GABAergic<br />
input
Authors: S. TAI, *L. LEUNG;<br />
Univ. Western Ontario, London, ON, C<strong>an</strong>ada<br />
Abstract: The medial septum (MS) is known to modulate <strong>the</strong> neural circuitry in <strong>the</strong><br />
hippocampus. It have been demonstrated that, in <strong>an</strong>aes<strong>the</strong>tized rats, MS stimulation prior to<br />
per<strong>for</strong><strong>an</strong>t path stimulation facilitated <strong>the</strong> population spike in <strong>the</strong> dentate gyrus <strong>an</strong>d such<br />
facilitation was unaffected by muscarinic <strong>an</strong>d nicotinic cholinergic <strong>an</strong>tagonists, suggesting that<br />
this facilitation may be mediated by septohippocampal GABAergic neurons. To study <strong>the</strong> effects<br />
of septohippocampal neurons in modulating neural circuitry in <strong>the</strong> dentate gyrus, selective lesion<br />
of septohippocampal GABAergic cells was made by infusion of orexin-saporin in <strong>the</strong> MS.<br />
Evoked field potentials were recorded in <strong>the</strong> dentate gyrus following stimulation of <strong>the</strong> medial<br />
per<strong>for</strong><strong>an</strong>t path in ureth<strong>an</strong>e-<strong>an</strong>es<strong>the</strong>tized rats using multich<strong>an</strong>nel silicon probes <strong>an</strong>d <strong>an</strong>alyzed as<br />
current source density. High-frequency stimulation of <strong>the</strong> pontis oralis (PnO) activated a<br />
hippocampal <strong>the</strong>ta rhythm. Theta peak power was attenuated in lesion rats as compared to<br />
control (sham lesion) rats. PnO stimulation resulted in a larger average increase in dentate<br />
population spike (pSpike) in control rats compared to lesion rats (26.4 ± 8.8 % vs 5.1 ± 8.7 %)<br />
but <strong>the</strong> difference was not signific<strong>an</strong>t (P = 0.1082; control n = 9, lesion n = 8). The number of<br />
choline acetyltr<strong>an</strong>sferase-immunopositive (cholinergic) cells in <strong>the</strong> MS was not signific<strong>an</strong>tly<br />
different (P = 0.88, unpaired t-test) between lesion <strong>an</strong>d control rats, while a signific<strong>an</strong>t decrease<br />
(about 50%) of <strong>the</strong> number of parvalbumin-immunopositive GABAergic cells in <strong>the</strong> MS was<br />
observed in lesion as compared to control rats (P
Title: Modulation of prelimbic cortex-nucleus accumbens pathway by presynaptic dopamine D1<br />
<strong>an</strong>d D2 receptors<br />
Authors: *Q. CUI, W.-H. YUNG;<br />
Physiol., The Chinese Univ. of Hong Kong, Hong Kong, China<br />
Abstract: Parkinson’s disease results <strong>from</strong> selective degeneration of dopamine neurons in <strong>the</strong><br />
midbrain, which is known to project to <strong>the</strong> cortex, dorsal striatum <strong>an</strong>d <strong>the</strong> ventral striatum<br />
structure known as <strong>the</strong> nucleus accumbens (NAc). Like <strong>the</strong> dorsal striatum, NAc contains two<br />
subpopulations of medium spiny neurons that express dopamine D1 or D2 receptors. Available<br />
evidence suggests that <strong>the</strong> projection <strong>from</strong> <strong>the</strong> prelimbic area of <strong>the</strong> prefrontal cortex to NAc is<br />
involved in working memory function <strong>an</strong>d dopamine depletion in ei<strong>the</strong>r or both areas may<br />
underlie working memory-related cognitive deficits observed in parkinsonism. However, <strong>the</strong><br />
exact distributions of dopamine receptors <strong>an</strong>d <strong>the</strong>ir functions in this projection are not clear.<br />
Making use of genetically engineered bacterial artificial chromosome (BAC) mice that<br />
selectively express GFP in D2-receptor expressing neurons, we addressed this issue by <strong>an</strong><br />
electrophysiological approach. Acute brain slices that preserve <strong>the</strong> prelimbic cortex-nucleus<br />
accumbens pathway were prepared. Whole-cell patch-clamp recordings were made <strong>from</strong><br />
visually-identified D2-receptor expressing <strong>an</strong>d non-expressing neurons in <strong>the</strong> NAc. Excitatory<br />
postsynaptic currents (EPSCs) were <strong>the</strong>n evoked by stimulating <strong>the</strong> prelimbic cortical area. In<br />
D2-receptor expressing neurons, application of dopamine D1 agonist SKF38393 (10µM)<br />
increased <strong>the</strong> amplitude of EPSCs (+4.67%, n=11, p=0.19) while dopamine D2 agonist<br />
quinpirole (10µM) inhibited <strong>the</strong> EPSCs (-8.09 %, n=16, p=0.04). Interestingly, opposite effects<br />
were obtained when <strong>the</strong> recordings were made <strong>from</strong> non-D2 receptor expressing (presumably<br />
D1) neurons. Thus, SKF38393 decreased <strong>the</strong> amplitude of EPSCs (-7.70 %, n=9, p=0.13) while<br />
quinpirole has a facilitatory action (+7.78 % of control, n=9, p=0.05). These effects were presynaptic<br />
in origin as revealed by a concomit<strong>an</strong>t ch<strong>an</strong>ge in <strong>the</strong> paired-pulse ratio. These data<br />
indicate that both D1 <strong>an</strong>d D2 receptor expressing neurons in <strong>the</strong> NAc receive excitatory inputs<br />
<strong>from</strong> <strong>the</strong> prelimbic area of <strong>the</strong> prefrontal cortex <strong>an</strong>d <strong>the</strong>se inputs were modulated by both D1 <strong>an</strong>d<br />
D2 receptors in a highly precise <strong>an</strong>d complementary pattern. These results are useful <strong>for</strong> fur<strong>the</strong>r<br />
investigation into <strong>the</strong> roles of dopamine in working memory function in both normal <strong>an</strong>d<br />
diseased states.<br />
Disclosures: Q. Cui, None; W. Yung, None.<br />
Poster<br />
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.20/EE35
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: Parkinson’s Disease <strong>Society</strong> Gr<strong>an</strong>t G-4067<br />
Parkinson’s Disease <strong>Society</strong> Gr<strong>an</strong>t G-0808<br />
BBSRC-Eli Lilly CASE Award<br />
IBRO Studentship<br />
Title: Bidirectional control of dopamine release probability by striatal muscarinic receptors<br />
Authors: *S. THRELFELL, M. A. CLEMENTS, I. PIENAAR, R. EXLEY, S. J. CRAGG;<br />
Dept. Physiology, Anat. <strong>an</strong>d Genet., Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom<br />
Abstract: Striatal dopamine (DA) <strong>an</strong>d acetylcholine (ACh) play import<strong>an</strong>t roles in regulating<br />
motivated behavior. Striatal ACh <strong>an</strong>d DA interact extensively through synchronous ch<strong>an</strong>ges in<br />
parent neuron activities <strong>an</strong>d reciprocal presynaptic regulation of <strong>the</strong>ir release. Reports that<br />
striatal muscarinic receptors (mAChRs) might govern DA signalling are however contradictory:<br />
while some report suppression of DA release by mAChRs, o<strong>the</strong>rs show facilitation. In this study<br />
we explored whe<strong>the</strong>r striatal mAChRs operate a variable activity-dependent control of DA<br />
release probability, similar to that operated by β2-subunit-containing(*) nicotinic receptors (β2*nAChRs).<br />
We used fast-sc<strong>an</strong> cyclic voltammetry at carbon-fiber microelectrodes in mouse<br />
striatal slices to investigate mAChR-regulation of synaptic DA tr<strong>an</strong>sients released by<br />
physiological firing frequencies. The mAChR agonist oxotremorine decreased DA release by<br />
single-pulse or low-frequency stimuli (4 pulses, 1-10 Hz) but increased release by high<br />
frequencies (>25 Hz), thus enh<strong>an</strong>cing <strong>the</strong> sensitivity of release to frequency in both caudateputamen<br />
(CPu) <strong>an</strong>d nucleus accumbens (NAc). These effects were precluded by prior application<br />
of <strong>the</strong> mAChR <strong>an</strong>tagonist atropine, <strong>an</strong>d could be mimicked <strong>an</strong>d prevented by inhibition of ACh<br />
input by <strong>the</strong> β2*-nAChR <strong>an</strong>tagonist DHβE. These effects were not prevented by prior application<br />
of GABA- <strong>an</strong>d glutamate-receptor <strong>an</strong>tagonists. These data reveal that striatal mAChRs exert<br />
strong, bidirectional control of DA release probability in <strong>an</strong> activity-dependent m<strong>an</strong>ner that<br />
promotes <strong>the</strong> sensitivity of DA release to presynaptic frequency. These mAChRs are located on<br />
cholinergic interneurons (i.e. autoreceptors) <strong>an</strong>d upon activation, eliminate ACh tone at β2*nAChRs<br />
on DA axons; mAChR activation may <strong>the</strong>re<strong>for</strong>e also reveal <strong>the</strong> ch<strong>an</strong>ges to DA signaling<br />
that result <strong>from</strong> pauses in cholinergic interneuron activity. MAChRs expressed by cholinergic<br />
neurons may provide a key target to influence ACh <strong>an</strong>d DA function in disorders including drug<br />
addiction <strong>an</strong>d Parkinson’s disease.<br />
Disclosures: S. Threlfell, None; M.A. Clements, None; I. Pienaar, None; R. Exley,<br />
None; S.J. Cragg, None.<br />
Poster
566. Basal G<strong>an</strong>glia: Tr<strong>an</strong>smitters <strong>an</strong>d Neuromodulation<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 566.21/EE36<br />
Topic: D.15.a. Tr<strong>an</strong>smitters <strong>an</strong>d neuromodulation<br />
Support: Wellcome Trust<br />
Title: Serotonergic control of striatal neurons<br />
Authors: *C. P. BLOMELEY, E. BRACCI;<br />
Univ. M<strong>an</strong>chester, M<strong>an</strong>chester, United Kingdom<br />
Abstract: The striatum is <strong>the</strong> largest nucleus of <strong>the</strong> basal g<strong>an</strong>glia, <strong>an</strong>d is crucially involved in<br />
motor control. In <strong>the</strong> past decade, it has emerged that interneurons, ra<strong>the</strong>r th<strong>an</strong> projection<br />
neurons, are <strong>the</strong> main target <strong>for</strong> extrinsic neuromodulators. We had found previously that 5hydroxytryptamine<br />
(5-HT, serotonin) exerted strong excitatory effects on cholinergic<br />
interneurons. Here we report that serotonin controls <strong>the</strong> state of excitability of GABAergic<br />
interneurons in a type-selective m<strong>an</strong>ner. Bath application of serotonin (30 micromolar) elicited<br />
slow, reversible depolarizations in fast spiking interneurons. Similar effects were observed using<br />
conventional whole-cell <strong>an</strong>d gramicidin per<strong>for</strong>ated-patch techniques. The serotonin effects were<br />
mediated by 5-HT2C receptors as <strong>the</strong>y were reversed by <strong>the</strong> 5-HT2 receptor <strong>an</strong>tagonist<br />
ket<strong>an</strong>serin <strong>an</strong>d by <strong>the</strong> selective 5-HT2C receptor <strong>an</strong>tagonist RS 102221. Serotonin also affected<br />
current-evoked spike frequency <strong>an</strong>d produced spont<strong>an</strong>eous firing in a minority of responsive<br />
neurons. In voltage-clamped neurons, serotonin suppressed a current that reversed around -100<br />
mV <strong>an</strong>d displayed a marked inward rectification. Consistently, <strong>the</strong> effects of serotonin were<br />
completely occluded by low concentrations of extracellular barium, which selectively blocks<br />
Kir2 ch<strong>an</strong>nels. The effects of serotonin on low-threshold spike interneurons were different, <strong>an</strong>d<br />
involved a complex modulation of membr<strong>an</strong>e properties that resulted in increased or decreased<br />
excitability depending on <strong>the</strong> initial state of <strong>the</strong> interneuron, <strong>an</strong>d on <strong>the</strong> type of current steps used<br />
top elicit spikes. Collectively, <strong>the</strong>se results indicate that serotonin, released in <strong>the</strong> striatum by<br />
fibers originating in <strong>the</strong> raphe nuclei, exerts a powerful <strong>an</strong>d complex control over <strong>the</strong> operation<br />
of this nucleus, by differentially affecting all <strong>the</strong> interneuronal types.<br />
Disclosures: C.P. Blomeley, None; E. Bracci, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.1/EE37<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NINDS 5K01NS49040<br />
Title: Hum<strong>an</strong> subthalamic neuron activity related to submovements during visuomotor learning<br />
Authors: *C. OJAKANGAS, M. J. CIESLAK, R. PENN;<br />
Dept Surgery/Neurosurgery, Univ. Chicago, Chicago, IL<br />
Abstract: Although known to be related to movement, <strong>the</strong> specifics of how <strong>the</strong> subthalamic<br />
nucleus (STN) functions is unclear. We are studying <strong>the</strong> STN <strong>an</strong>d its role in motor learning<br />
during neurosurgery <strong>for</strong> Deep Brain Stimulator impl<strong>an</strong>tation. Single <strong>an</strong>d multi-unit neuronal<br />
activity <strong>an</strong>d kinematics were recorded <strong>an</strong>d <strong>an</strong>alyzed <strong>from</strong> 7 Parkinson patients during<br />
intraoperative experiments approved by <strong>the</strong> University of Chicago's Institutional Review Board.<br />
The task involved simple visuomotor targeted arm movements using a digitizing tablet <strong>an</strong>d pen<br />
to bring a cursor to a target during a "Learning" condition (L) <strong>an</strong>d a "Non-Learning" condition<br />
(NL). Off-line discrimination <strong>an</strong>d <strong>an</strong>alysis of kinematics, muscle activity <strong>an</strong>d neural activity<br />
<strong>from</strong> single- <strong>an</strong>d multi-neuronal units were per<strong>for</strong>med. Thirty to sixty percent of units showed<br />
signific<strong>an</strong>tly different firing rates in L compared to NL (p< 0.05, 2-way ANOVA). Also, <strong>for</strong> <strong>the</strong><br />
different conditions presented in both <strong>the</strong> L <strong>an</strong>d NL phase, neurons showed signific<strong>an</strong>tly<br />
different firing rate only during <strong>the</strong> L phase (p
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.2/EE38<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: New Zeal<strong>an</strong>d Health Research Council<br />
Title: Altered neuronal activity in <strong>the</strong> striatum of <strong>the</strong> genetically hypertensive rat, a putative<br />
<strong>an</strong>imal model of ADHD<br />
Authors: *C. PERK 1 , B. HYLAND 2 , J. WICKENS 3 ;<br />
1 Dept. of Anat. <strong>an</strong>d Structural Biol., Univ. of Otago, Dunedin, New Zeal<strong>an</strong>d; 2 Univ. Otago Med.<br />
Sch., Dunedin, New Zeal<strong>an</strong>d; 3 Okinawa Instituteof Sci. <strong>an</strong>d Technol., Uruma, Jap<strong>an</strong><br />
Abstract: Abnormal processing of signals related to reward may underpin <strong>the</strong> altered<br />
behavioural sensitivity to reward that is seen in attention deficit hyperactivity disorder (ADHD).<br />
The indirect dopamine agonist amphetamine (AMPH) <strong>an</strong>d related drugs are effective in<br />
treatment of ADHD but <strong>the</strong>ir effect on neural processing in this condition remains unclear. We<br />
investigated in<strong>for</strong>mation processing at <strong>the</strong> neuronal level in <strong>the</strong> striatum of genetically<br />
hypertensive (GH) rats, a strain recently shown to exhibit behavioural characteristics seen in<br />
hum<strong>an</strong> ADHD, <strong>an</strong>d compared this with data <strong>from</strong> Wistar controls. Rats were trained in 2<br />
contextual tasks. In one, <strong>an</strong> auditory cue (4.5 kHz, 0.5 s duration) delivered in <strong>the</strong> presence of <strong>an</strong><br />
illuminated room light (light-on condition) was never followed by reward. In <strong>the</strong> o<strong>the</strong>r, <strong>the</strong> same<br />
cue delivered in <strong>the</strong> absence of <strong>the</strong> room light (dark condition) was always followed by a reward<br />
(~0.025ml of saccharin-sweetened water). Between behavioural experiments, cells were recorded<br />
while <strong>an</strong>imals rested <strong>for</strong> two 5 min periods, first in <strong>the</strong> light, <strong>the</strong>n in <strong>the</strong> dark. The paradigm was<br />
<strong>the</strong>n repeated 20 mins after <strong>the</strong> administration of saline or d-amphetamine (i.p., 0.5 mg/kg). Data<br />
is expressed as medi<strong>an</strong> ± SD medi<strong>an</strong> except where noted. During pre-treatment recordings taken<br />
at rest, a total of 166 cells were recorded (WI: n = 95, GH: n = 71). GH rat neurons had a<br />
signific<strong>an</strong>tly lower firing rate (GH 0.52 ± 5.21 Hz, WI 1.28 ± 3.93 Hz; p = 0.016). Additionally,<br />
<strong>an</strong>alysis of neuronal burst firing characteristics showed that cells <strong>from</strong> GH rats also had a lower<br />
intra-burst frequency (GH: 15.56 ± 78.96 Hz, WI: 22.84 ± 99.96 Hz; p = 0.037), a longer me<strong>an</strong><br />
burst duration (GH: 293.72 ± 688.00ms WI: 190.72 ± 1155.84ms; p = 0.023), <strong>an</strong>d a longer me<strong>an</strong><br />
inter-burst interval (GH: 10.53 ± 10.73s WI: 4.67 ± 13.56s; p = 0.048). During pre-treatment<br />
behavioural experiments a total of 173 cells were well-isolated (WI: n = 98, GH: n = 75). There<br />
was a signific<strong>an</strong>t effect of strain (p = 0.001) on <strong>the</strong> amplitude of tone-related excitations relative<br />
to baseline (WI: 13.04 ± 22.49 Hz, GH: 7.68 ± 7.86; me<strong>an</strong> ± SD). Treatment with AMPH did not<br />
signific<strong>an</strong>tly alter <strong>an</strong>y between-strain differences, i.e. did not normalise GH rat data to that of WI<br />
<strong>an</strong>imals. These results suggest that <strong>the</strong>re is altered in<strong>for</strong>mation processing in <strong>the</strong> GH rat striatum,<br />
but do not provide support <strong>for</strong> <strong>the</strong> hypo<strong>the</strong>sis that amphetamine <strong>an</strong>d related treatments may<br />
normalize <strong>the</strong>se aspects of neural activity.<br />
Disclosures: C. Perk, None; B. Hyl<strong>an</strong>d, None; J. Wickens, None.
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.3/EE39<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NIH Gr<strong>an</strong>t T32 NS07222<br />
Title: A rat stop-signal task <strong>for</strong> electrophysiological investigation of movement inhibition<br />
Authors: *D. K. LEVENTHAL 1 , A. NEARY 2 , J. PETTIBONE 2 , J. BERKE 2 ;<br />
1 Neurol., 2 Psychology, Univ. of Michig<strong>an</strong>, Ann Arbor|910006186|0, Ann Arbor, MI<br />
Abstract: The ability to inhibit pl<strong>an</strong>ned actions is <strong>an</strong> essential component of motor control. Such<br />
inhibition has been widely studied using stop-signal tasks, in which cued actions-in-preparation<br />
must be counterm<strong>an</strong>ded on a subset of trials. Per<strong>for</strong>m<strong>an</strong>ce in stop-signal tasks is well fit by<br />
cognitive models in which distinct GO <strong>an</strong>d STOP processes "race" <strong>for</strong> completion, with <strong>the</strong><br />
outcome of <strong>the</strong> race determining whe<strong>the</strong>r actions are successfully counterm<strong>an</strong>ded. Lesion <strong>an</strong>d<br />
imaging studies have suggested that distinct pathways through cortical-basal g<strong>an</strong>glia circuits<br />
subserve <strong>the</strong>se distinct GO <strong>an</strong>d STOP processes. However, this <strong>the</strong>ory has not yet been tested<br />
using electrophysiological methods, which have <strong>the</strong> required temporal resolution to observe <strong>the</strong><br />
hypo<strong>the</strong>sized "race". To this end, we have developed a stop-signal task <strong>for</strong> rats that c<strong>an</strong> be used<br />
toge<strong>the</strong>r with high-density recordings <strong>from</strong> multiple basal g<strong>an</strong>glia stations simult<strong>an</strong>eously.<br />
Building on a choice reaction time task used in our laboratory, we have developed a task where<br />
<strong>the</strong> rat must first poke <strong>an</strong>d hold its nose in a port, <strong>the</strong>n make simple left or right movements<br />
depending on <strong>an</strong> instruction cue (<strong>the</strong> GO tone). On some trials, a burst of white noise (<strong>the</strong> stopsignal)<br />
after <strong>the</strong> GO tone signals <strong>the</strong> rat to maintain its position in <strong>the</strong> original nose-poke port.<br />
The combination of <strong>an</strong> initial choice (left vs right) component makes this version more<br />
comparable to hum<strong>an</strong> <strong>an</strong>d monkey studies th<strong>an</strong> prior rat stop-signal tasks, <strong>an</strong>d <strong>the</strong> requirement of<br />
no head movement on STOP trials aids <strong>the</strong> interpretability of electrophysiological signals. Rats<br />
typically achieved 70-80% success rates on GO trials, comparable to our previous results with<br />
choice reaction time tasks. Consistent with race models, <strong>the</strong> probability of successful stopping<br />
decreased as <strong>the</strong> stop-signal delay (SSD) increased, though <strong>the</strong> level of success varied between<br />
rats. In line with results <strong>from</strong> o<strong>the</strong>r groups, <strong>the</strong> inferred stop-signal reaction time (SSRT) also<br />
decreased with SSD <strong>for</strong> all rats. We have now impl<strong>an</strong>ted several rats with 21-tetrode assemblies,<br />
targeting frontal cortex, striatum, subthalamic nucleus, <strong>an</strong>d subst<strong>an</strong>tia nigra pars reticulata. Task<br />
per<strong>for</strong>m<strong>an</strong>ce was essentially unch<strong>an</strong>ged between pre- <strong>an</strong>d post-impl<strong>an</strong>t test sessions, with <strong>the</strong><br />
exception of slightly increased movement times. These preliminary results demonstrate <strong>the</strong>
viability of this task <strong>an</strong>d recording arr<strong>an</strong>gement <strong>for</strong> <strong>the</strong> electrophysiological investigation of<br />
basal g<strong>an</strong>glia circuits in action initiation <strong>an</strong>d c<strong>an</strong>cellation.<br />
Disclosures: D.K. Leventhal, None; A. Neary, None; J. Pettibone, None; J. Berke, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.4/EE40<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Intramural Research Program at NEI, NIH<br />
Title: The rostral part of <strong>the</strong> external pallidum <strong>an</strong>d <strong>the</strong> ventral pallidum convey rewardassociated<br />
visuomotor in<strong>for</strong>mation<br />
Authors: *Y. TACHIBANA, O. HIKOSAKA;<br />
Lab. of Sensorimot. Res., NEI, Be<strong>the</strong>sda, MD<br />
Abstract: The external pallidum is considered to be <strong>an</strong> interface connecting <strong>the</strong> input nuclei to<br />
<strong>the</strong> output nuclei of <strong>the</strong> basal g<strong>an</strong>glia. It receives excitatory inputs mainly <strong>from</strong> <strong>the</strong> subthalamic<br />
nucleus <strong>an</strong>d inhibitory inputs <strong>from</strong> <strong>the</strong> striatum, <strong>an</strong>d sends GABAergic inhibitory projections<br />
mainly to <strong>the</strong> internal pallidum <strong>an</strong>d <strong>the</strong> subst<strong>an</strong>tia nigra pars reticulata. The caudal part of <strong>the</strong><br />
external pallidum is known to be a ‘skeletomotor’ region, <strong>an</strong>d has been well-characterized with<br />
reference to Parkinson’s disease. In contrast, physiological evidence is still lacking regarding <strong>the</strong><br />
rostral part of <strong>the</strong> external pallidum, which receives inputs <strong>from</strong> <strong>the</strong> ‘visuomotor’ region of <strong>the</strong><br />
caudate (Hikosaka et al., 1989; Hedreen <strong>an</strong>d DeLong, 1991). To investigate <strong>the</strong> functions of <strong>the</strong><br />
rostral external pallidum, we recorded neuronal activity in this region using a reward-biased<br />
memory-guided saccade task. The recording sites included <strong>the</strong> region ventral to <strong>the</strong> <strong>an</strong>terior<br />
commissure which is often called <strong>the</strong> ventral pallidum. We found that neurons in <strong>the</strong> rostral<br />
pallidum responded to <strong>the</strong> saccade task in a complex m<strong>an</strong>ner with different combinations of<br />
visual <strong>an</strong>d saccadic responses <strong>an</strong>d different combinations of excitatory <strong>an</strong>d inhibitory responses.<br />
The direction selectivities of <strong>the</strong>se responses were, on <strong>the</strong> average, weaker th<strong>an</strong> those reported<br />
<strong>for</strong> neurons in <strong>the</strong> o<strong>the</strong>r basal g<strong>an</strong>glia nuclei (e.g., caudate nucleus, subst<strong>an</strong>tia nigra pars<br />
reticulata). The visual-saccadic responses were sometimes modulated by <strong>the</strong> expected reward<br />
size, ei<strong>the</strong>r positively or negatively. Similar features were found in neurons in <strong>the</strong> ventral<br />
pallidum, but <strong>the</strong>ir activity tended to be determined, ra<strong>the</strong>r th<strong>an</strong> modulated, by <strong>the</strong> expected<br />
reward size. We also found differences in electrophysiological properties. Neurons in <strong>the</strong> caudal
external pallidum are known to exhibit high frequency firing with frequent long-lasting pauses.<br />
In contrast, neurons in <strong>the</strong> rostral external pallidum <strong>an</strong>d <strong>the</strong> ventral pallidum tended to show<br />
lower frequency firing with fewer pauses. Their spike durations tended to be longer th<strong>an</strong> those in<br />
<strong>the</strong> caudal pallidum. These electrophysiological differences occurred in a graded m<strong>an</strong>ner in <strong>the</strong><br />
rostrocaudal axis. Based on <strong>the</strong>se results we hypo<strong>the</strong>size that (1) <strong>the</strong>re are physiological <strong>an</strong>d<br />
functional gradients within <strong>the</strong> external pallidum, <strong>an</strong>d (2) <strong>the</strong> ventral pallidum is a continuum of<br />
<strong>the</strong> external pallidum.<br />
Disclosures: Y. Tachib<strong>an</strong>a, None; O. Hikosaka, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.5/EE41<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Supported by DICBR of NIAAA<br />
Title: Neural correlates of ultrafast action sequence generation in basal g<strong>an</strong>glia circuits<br />
Authors: X. JIN 1 , *R. M. COSTA 1,2 ;<br />
1 2<br />
NIAAA/NIH, Be<strong>the</strong>sda, MD; Champalimaud Neurosci. Programme at Inst. Gulbenki<strong>an</strong> de<br />
Ciência, Oeiras, Portugal<br />
Abstract: Org<strong>an</strong>ism’s behavior c<strong>an</strong> be org<strong>an</strong>ized as action sequences, where a series of<br />
movements are arr<strong>an</strong>ged with a specific order, timing, <strong>an</strong>d duration. Hum<strong>an</strong> l<strong>an</strong>guage <strong>an</strong>d <strong>an</strong>imal<br />
vocalization are special examples of such type of sequences because of <strong>the</strong> extremely fast <strong>an</strong>d<br />
precise control that <strong>the</strong>y require during speech production. Basal g<strong>an</strong>glia circuits have been<br />
implicated in learning, generating <strong>an</strong>d monitoring action sequences. Here we trained mice to<br />
per<strong>for</strong>m <strong>an</strong> oper<strong>an</strong>t conditioning task where <strong>the</strong> <strong>an</strong>imals need to press a lever in <strong>an</strong> ultrafast<br />
m<strong>an</strong>ner while monitoring <strong>the</strong> activity in basal g<strong>an</strong>glia circuits. Under a differential rein<strong>for</strong>cement<br />
schedule, mice were rein<strong>for</strong>ced only when a fixed number of presses were accomplished within a<br />
defined time window, i.e. a limited-time fixed-ratio. With training, mice gradually learned to<br />
execute a sequence of 4 or 8 presses faster <strong>an</strong>d faster <strong>from</strong> free-oper<strong>an</strong>t to 1, 2, 4, <strong>an</strong>d finally up<br />
to surprisingly 8 Hz. This paradigm allowed us to establish a model to investigate <strong>the</strong> neural<br />
correlates of ultrafast action sequence learning <strong>an</strong>d generation in genetic tractable mammals. We<br />
impl<strong>an</strong>ted microelectrode arrays to simult<strong>an</strong>eously record <strong>the</strong> neural activity in at least two brain<br />
areas including <strong>the</strong> dorsal striatum (both dorsomedial <strong>an</strong>d dorsolateral), <strong>the</strong> subst<strong>an</strong>tia nigra pars
compacta (SNc), <strong>the</strong> globus pallidus (GP) <strong>an</strong>d <strong>the</strong> primary motor cortex (MI) while <strong>the</strong> mice<br />
learned <strong>an</strong>d per<strong>for</strong>med <strong>the</strong> task. The identification of putative major cell types in each brain areas<br />
was done by wave<strong>for</strong>m (spike width vs. spike amplitude), baseline firing rate, <strong>an</strong>d response to<br />
specific drugs (e.g. quinpirole). There were three major types of neuronal activities commonly<br />
observed in basal g<strong>an</strong>glia circuits. The first type showed a phasic burst of discharge only be<strong>for</strong>e<br />
<strong>the</strong> first press of each action sequence but not during <strong>the</strong> rest of <strong>the</strong> presses. The second type<br />
showed a phasic decrease in firing rate throughout <strong>the</strong> entire action sequence. The third type<br />
showed a sustained increase in firing rate during <strong>the</strong> entire sequence of presses. These types of<br />
neural activity could subserve different roles in <strong>the</strong> initiation, permission, <strong>an</strong>d monitoring of<br />
<strong>the</strong>se rapid actions sequences. Interestingly, we found <strong>the</strong>se three different response types were<br />
preferentially m<strong>an</strong>ifested in putatively different cell types, indicating different functional roles<br />
<strong>for</strong> each cell type in <strong>the</strong>se brain areas. We also observed neural correlates of sequence variability<br />
in <strong>the</strong>se circuits. These data emphasizes <strong>the</strong> critical role of basal g<strong>an</strong>glia circuits in sequence<br />
learning <strong>an</strong>d generation, <strong>an</strong>d shed light on possible neural mech<strong>an</strong>isms underlying <strong>the</strong> learning<br />
<strong>an</strong>d execution of very fast action sequences.<br />
Disclosures: X. Jin, None; R.M. Costa, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.6/EE42<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Indi<strong>an</strong>a University - Purdue University Indi<strong>an</strong>apolis RSFG<br />
Title: Dynamics <strong>an</strong>d network mech<strong>an</strong>isms of intermittent synchronous oscillations in<br />
subthalamic nucleus in Parkinson’s disease<br />
Authors: C. PARK 1 , R. M. WORTH 2 , *L. L. RUBCHINSKY 3 ;<br />
1 Ma<strong>the</strong>matical Sci., Indi<strong>an</strong>a Univ. - Purdue Univ. Indi<strong>an</strong>apolis, Indi<strong>an</strong>apolis, IN; 2 Neurolog.<br />
Surgery, Indi<strong>an</strong>a Univ. Sch. of Med., Indi<strong>an</strong>apolis, IN; 3 IUPUI & Indi<strong>an</strong>a Univ. Sch. Med.,<br />
Indi<strong>an</strong>apolis, IN<br />
Abstract: The low-dopamine state as is seen in Parkinson’s disease (PD) is marked by <strong>an</strong><br />
increase in oscillatory <strong>an</strong>d synchronous activity in <strong>the</strong> beta b<strong>an</strong>d. While causal relationship<br />
between this activity <strong>an</strong>d PD motor symptoms is not completely certain, this activity is probably<br />
closely related to <strong>the</strong> pathologies of motor behavior. The present study explores this dynamical
nature of synchronous beta oscillations in PD as well as its potential mech<strong>an</strong>isms. We<br />
simult<strong>an</strong>eously record spikes <strong>an</strong>d LFP <strong>from</strong> STN of PD patients, <strong>an</strong>alyze <strong>the</strong> phase<br />
synchronization between <strong>the</strong>se signals as it develops in time <strong>an</strong>d use computational models to<br />
explore network mech<strong>an</strong>isms of <strong>the</strong> observed dynamics. To explore synchrony patterns on a fine<br />
time scale we <strong>an</strong>alyzed <strong>the</strong> first-return plots of <strong>the</strong> phase differences between recorded signals.<br />
The results of <strong>the</strong> <strong>an</strong>alysis reveal a complicated temporal structure of synchronization of betab<strong>an</strong>d<br />
oscillations in STN in PD. Synchronized dynamics is interrupted by desynchronization<br />
events, which are irregular, although not completely r<strong>an</strong>dom, with a predomin<strong>an</strong>ce of short<br />
desynchronization events. The signals go out of phase <strong>for</strong> just one cycle of oscillations more<br />
often th<strong>an</strong> <strong>for</strong> two or a larger number of cycles. The ch<strong>an</strong>ces of longer desynchronization events<br />
decrease with <strong>the</strong> duration of <strong>the</strong>se events. An alternative scenario (longer but less frequent<br />
desynchronization events) would produce <strong>the</strong> same average degree of synchronization, but is not<br />
supported by <strong>the</strong> data <strong>an</strong>alysis. The domin<strong>an</strong>ce of <strong>the</strong> short desynchronization events indicates<br />
that even though <strong>the</strong> synchronization in parkinsoni<strong>an</strong> basal g<strong>an</strong>glia is fragile enough to be<br />
frequently destabilized, it has <strong>the</strong> ability to reestablish itself very quickly, which may be<br />
import<strong>an</strong>t <strong>for</strong> <strong>the</strong> development of <strong>the</strong> <strong>the</strong>rapeutically relev<strong>an</strong>t methods to suppress this<br />
synchrony.<br />
The computational investigation of conduct<strong>an</strong>ce-based models of subthalamo-pallidal circuits<br />
allowed us to identify <strong>the</strong> parameter domains, where <strong>the</strong> model network without <strong>an</strong>y external<br />
input or plasticity effects reproduces imperfect synchronization with <strong>the</strong> same characteristic fine<br />
temporal structure. The parameter values correspond to <strong>the</strong> moderately strong synaptic strengths<br />
in <strong>the</strong> model, which is a realistic case <strong>for</strong> PD (in a healthy state <strong>the</strong>se synaptic connections would<br />
be inhibited by dopaminergic action). While a variety of mech<strong>an</strong>isms may contribute to <strong>the</strong><br />
observed specific variability in synchrony on short time scales, <strong>the</strong> computational results indicate<br />
that this variability c<strong>an</strong> be generated intrinsically in pallido-subthalamic circuits without <strong>an</strong>y<br />
external inputs due to moderately large strength of synaptic coupling.<br />
Disclosures: C. Park, None; R.M. Worth, None; L.L. Rubchinsky, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.7/EE43<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: EU Gr<strong>an</strong>t IST-2000-2817<br />
EU Gr<strong>an</strong>t IST-2005-27268
BMBF-MOS<br />
Volkswagen Foundation<br />
Title: Effective connectivity within a vibrissae related thalamo-cortico-striatal cell assembly<br />
Authors: *E. C. SYED, A. SHAROTT, A. K. ENGEL, A. KRAL;<br />
Dept. of Neurophysiol. <strong>an</strong>d Pathophysiology, Univ. Med. Ctr. Hamburg-, Hamburg, Germ<strong>an</strong>y<br />
Abstract: The vibrissae sensory system follows a well described pathway <strong>from</strong> <strong>the</strong> mystacial<br />
pad via thalamus to cortex, which <strong>the</strong>n projects to distributed areas of <strong>the</strong> striatum. Thus,<br />
<strong>an</strong>atomical connections predict that vibrissae specific sensory activity in striatum is likely to be<br />
relayed via <strong>the</strong> barrel cortex <strong>from</strong> <strong>the</strong> thalamus. This is of particular import<strong>an</strong>ce <strong>for</strong> <strong>the</strong> role of<br />
barrel cortex in active whisker guided behaviour.<br />
The aims of this study were three-fold: Firstly to assess how a strong oscillatory sensory input<br />
affects <strong>the</strong> activity of neural assemblies throughout <strong>the</strong> sensory pathway to <strong>the</strong> basal g<strong>an</strong>glia;<br />
secondly, to observe how sensory in<strong>for</strong>mation is tr<strong>an</strong>smitted <strong>from</strong> thalamus to striatum via cortex<br />
at a population level; thirdly, to establish <strong>the</strong> role of cortical activity in mediating thalamostriatal<br />
vibrissae specific functional connectivity.<br />
We recorded local field potentials (LFPs) <strong>an</strong>d multi-units <strong>from</strong> multiple sites in thalamus, barrel<br />
cortex <strong>an</strong>d dorso-lateral striatum using silicon-based electrodes in 19 male Brown Norway rats.<br />
Firstly, we characterized <strong>the</strong> responses to <strong>the</strong> sensory stimulation in each of <strong>the</strong>se three<br />
structures. Secondly, we qu<strong>an</strong>tified <strong>the</strong> sensory-evoked covari<strong>an</strong>ce between thalamus <strong>an</strong>d cortex,<br />
cortex <strong>an</strong>d striatum <strong>an</strong>d thalamus <strong>an</strong>d striatum. Thirdly, we reversibly abolished <strong>the</strong> activity in<br />
barrel cortex in order to observe <strong>the</strong> effect on <strong>the</strong> thalamic <strong>an</strong>d striatal sensory responses.<br />
Oscillatory stimulation of <strong>the</strong> rat vibrissae caused a signific<strong>an</strong>t qualitative increase in phase<br />
locking between thalamic <strong>an</strong>d striatal LFPs at <strong>the</strong> stimulation frequency. Phase locking between<br />
thalamic <strong>an</strong>d cortical LFPs <strong>an</strong>d between cortical <strong>an</strong>d striatal LFPs was also signific<strong>an</strong>tly<br />
streng<strong>the</strong>ned both qualitatively <strong>an</strong>d qu<strong>an</strong>titatively. Phase locking of one cortical LFP with both<br />
one thalamic <strong>an</strong>d one striatal LFP did not, however, predict signific<strong>an</strong>t phase locking between<br />
<strong>the</strong> thalamic <strong>an</strong>d striatal LFP. Following abolishment of cortical activity, sensory modulation of<br />
phase locking between thalamic <strong>an</strong>d cortical LFPs remained unaffected while <strong>the</strong> sensoryinduced<br />
increase in phase locking between striatal LFPs <strong>an</strong>d both cortical <strong>an</strong>d thalamic LFPs was<br />
no longer signific<strong>an</strong>t.<br />
Thus we reveal functional circuits, which are modulated by sensory stimulation, assess <strong>the</strong>ir<br />
distribution with regard to known <strong>an</strong>atomical org<strong>an</strong>ization <strong>an</strong>d reveal directionality of sensory<br />
specific in<strong>for</strong>mation tr<strong>an</strong>sfer over at least one synapse within a cell assembly.<br />
Disclosures: E.C. Syed, None; A. Sharott, None; A.K. Engel, None; A. Kral, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.8/EE44<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Title: Different representation of action comm<strong>an</strong>d in <strong>the</strong> dorsal <strong>an</strong>d ventral striatum<br />
Authors: *M. ITO 1 , K. DOYA 1,2 ;<br />
1 OIST, Okinawa, Jap<strong>an</strong>; 2 ATR, Kyoto, Jap<strong>an</strong><br />
Abstract: The striatum is <strong>an</strong> input structure of <strong>the</strong> basal g<strong>an</strong>glia that contributes to <strong>the</strong> process of<br />
<strong>the</strong> decision making. Previous lesion <strong>an</strong>d imaging studies suggested that <strong>the</strong> dorsal striatum (DS)<br />
contributes to action selection while <strong>the</strong> ventral striatum (VS) to reward prediction <strong>an</strong>d<br />
motivation. However, it is still unclear whe<strong>the</strong>r <strong>an</strong>d how <strong>the</strong> neuronal representations are<br />
different in VS <strong>an</strong>d DS.<br />
In this study, we recorded neuronal activity <strong>from</strong> <strong>the</strong> dorsolateral striatum (DLS), <strong>the</strong><br />
dorsomedial striatum (DMS), <strong>an</strong>d VS of rats per<strong>for</strong>ming a choice task consisting of two trial<br />
types; fixed-reward trials <strong>an</strong>d varied-reward trials. In each trial, a rat should keep nose poking till<br />
<strong>the</strong> end of a tone cue <strong>an</strong>d <strong>the</strong>n select a left- or right-hole poking. A sucrose pellet was delivered<br />
stochastically depending on <strong>the</strong> presented tone <strong>an</strong>d <strong>the</strong> selected action. In fixed-reward trials, one<br />
of two tones was presented <strong>an</strong>d <strong>the</strong> reward probability <strong>for</strong> each action after two tones was fixed<br />
through <strong>the</strong> experiments; (left, right)=(50%, 0%) <strong>for</strong> tone L <strong>an</strong>d (0%, 50%) <strong>for</strong> tone R. In variedreward<br />
trials, only one tone (tone V) was used as a cue <strong>an</strong>d <strong>the</strong> reward probability <strong>for</strong> each action<br />
was varied among 10%, 50%, <strong>an</strong>d 90% in a block-wise m<strong>an</strong>ner.<br />
Behavioral <strong>an</strong>alysis showed <strong>the</strong> latency <strong>from</strong> <strong>the</strong> offset of <strong>the</strong> tone cue to <strong>the</strong> initiation of <strong>the</strong><br />
action was signific<strong>an</strong>tly longer in varied-reward th<strong>an</strong> in fixed-reward trials. An extinction test, in<br />
which no pellets were delivered during a sequence of five trials with <strong>the</strong> same tone, revealed that<br />
<strong>the</strong> extinction of pre-learned choice was signific<strong>an</strong>tly slower <strong>for</strong> tone L <strong>an</strong>d tone R th<strong>an</strong> <strong>for</strong> tone<br />
V. These results suggest that <strong>the</strong> rats’ choice behavior were more habituated in fixed-reward th<strong>an</strong><br />
in varied reward trials.<br />
We <strong>an</strong>alyzed <strong>the</strong> neural firing during one second be<strong>for</strong>e <strong>the</strong> initiation of <strong>the</strong> action. 51% (49/96),<br />
47% (33/70) <strong>an</strong>d 40% (27/68) of <strong>the</strong> recorded neurons <strong>from</strong> DLS, DMS <strong>an</strong>d VS, respectively,<br />
represented action comm<strong>an</strong>d, with signific<strong>an</strong>tly different firing <strong>for</strong> different action to be taken.<br />
The average in<strong>for</strong>mation of action comm<strong>an</strong>d was signific<strong>an</strong>tly higher in DS th<strong>an</strong> in VS (0.040,<br />
0.037, <strong>an</strong>d 0.012 bit/s <strong>for</strong> DLS, DMS, <strong>an</strong>d VS neurons). We compared <strong>the</strong> in<strong>for</strong>mation of action<br />
comm<strong>an</strong>d in fixed-reward <strong>an</strong>d varied-reward trials <strong>an</strong>d found that 20% (10/49), 33% (11/33), <strong>an</strong>d<br />
7% (2/27) in DLS, DMS, <strong>an</strong>d VS neurons, respectively, had more action-comm<strong>an</strong>d in<strong>for</strong>mation<br />
in fixed-reward trials. We found no neurons of <strong>the</strong> opposite type, showing higher actioncomm<strong>an</strong>d<br />
in<strong>for</strong>mation in varied-reward trials, in <strong>an</strong>y regions. Our findings suggested that action<br />
selection involves <strong>the</strong> dorsal striatum more th<strong>an</strong> <strong>the</strong> ventral striatum, <strong>an</strong>d that habitual actions are<br />
represented more strongly in <strong>the</strong> dorsal striatum.<br />
Disclosures: M. Ito, None; K. Doya, None.
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.9/EE45<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NIMH Gr<strong>an</strong>t MH60379<br />
ONR N00014-04-1-0208<br />
Friends of <strong>the</strong> MIBR Graduate Student Fellowship<br />
Title: Projection neurons are entrained to different local field frequencies in dorsomedial <strong>an</strong>d<br />
dorsolateral striatum<br />
Authors: *C. A. THORN 1,2 , A. M. GRAYBIEL 1,3 ;<br />
1 2 3<br />
McGovern Inst. Brain Rese, Dept. of Electrical Eng. <strong>an</strong>d Comp. Sci., Dept. of Brain <strong>an</strong>d<br />
Cognitive Sci., MIT, Cambridge, MA<br />
Abstract: We have previously shown that striatal <strong>the</strong>ta rhythms in local field potentials (LFPs)<br />
are tightly coordinated across <strong>the</strong> medial <strong>an</strong>d lateral striatum, despite <strong>the</strong> fact that <strong>the</strong>se regions<br />
are parts of distinct cortico-basal g<strong>an</strong>glia loops (DeCoteau et al, J Neurophys 2007). Moreover,<br />
striatal rhythms are coordinated with hippocampal <strong>the</strong>ta during learning <strong>an</strong>d task per<strong>for</strong>m<strong>an</strong>ce,<br />
suggesting a ch<strong>an</strong>ge in global brain state <strong>an</strong>d communication between dist<strong>an</strong>t structures during<br />
task per<strong>for</strong>m<strong>an</strong>ce (DeCoteau et al, PNAS 2007; Tort et al, PNAS 2008). Spike-LFP coherence is<br />
prominent in <strong>an</strong>aes<strong>the</strong>tized preparations (Sharrott et al, J Neurosci <strong>2009</strong>), <strong>an</strong>d ventral striatal<br />
projection neurons are entrained to hippocampal <strong>the</strong>ta in behaving <strong>an</strong>imals (Berke et. al, Neuron<br />
2004), but such entrainment is less common (ca. 15-20%) in dorsal <strong>an</strong>d lateral striatal regions<br />
(DeCoteau et al, 2007; Berke et al, 2004). To test whe<strong>the</strong>r spike-LFP synchronization in <strong>the</strong><br />
dorsal striatum is region-specific <strong>an</strong>d task-relev<strong>an</strong>t, we simult<strong>an</strong>eously recorded single unit<br />
activity <strong>an</strong>d LFPs <strong>from</strong> <strong>the</strong> dorsomedial <strong>an</strong>d dorsolateral striatum as rats were trained on a Tmaze<br />
task. Here, we report task-modulated entrainment of striatal projection neurons to delta- (2-<br />
7 Hz) <strong>an</strong>d <strong>the</strong>ta- (5-12 Hz) b<strong>an</strong>d frequency components of <strong>the</strong> LFP. Consistent with previous<br />
studies, we found a task-dependent increase in delta- <strong>an</strong>d <strong>the</strong>ta-b<strong>an</strong>d power both medially <strong>an</strong>d<br />
laterally, <strong>an</strong>d <strong>an</strong> increase in LFP coherence between <strong>the</strong>se two regions in both frequency b<strong>an</strong>ds.<br />
Single units in <strong>the</strong> dorsomedial striatum were preferentially entrained to <strong>the</strong> <strong>the</strong>ta-b<strong>an</strong>d rhythms<br />
mid-task (>25% measured <strong>from</strong> locomotion onset through turn start), while dorsolateral striatal<br />
neurons were preferentially entrained to delta-b<strong>an</strong>d rhythms (ca. 35% during turning), with a
smaller subset entrained to <strong>the</strong>ta-b<strong>an</strong>d oscillations (ca. 15% around cue onset <strong>an</strong>d turn start). The<br />
percentage of units entrained to <strong>the</strong>se rhythms in each region was modulated across trial-time,<br />
with approximately twice as m<strong>an</strong>y units entrained in-task as were entrained during <strong>the</strong> pre-task<br />
baseline period. These results raise <strong>the</strong> possibility that <strong>the</strong> functions of <strong>the</strong> dorsomedial <strong>an</strong>d<br />
dorsolateral striatum are distinguished not only by <strong>the</strong>ir respective spike activity patterns but also<br />
by <strong>the</strong>ir patterns of spike-LFP coherence. These differences in spike-LFP coherence by medial<br />
<strong>an</strong>d lateral striatal projection neurons may relate to <strong>the</strong> different functional contributions of <strong>the</strong>ir<br />
respective cortico-basal g<strong>an</strong>glia loops during successful task acquisition <strong>an</strong>d per<strong>for</strong>m<strong>an</strong>ce.<br />
Disclosures: C.A. Thorn, None; A.M. Graybiel, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.10/EE46<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NIH gr<strong>an</strong>t R01-MH60379 (to AMG)<br />
NIH gr<strong>an</strong>t F32-MH085454 (to KSS)<br />
Title: Reward devaluation in a T-maze task shows dynamics of stimulus-response habit<br />
<strong>for</strong>mation<br />
Authors: *K. S. SMITH, A. M. GRAYBIEL;<br />
McGovern Inst. Brain Res., M.I.T., Cambridge, MA<br />
Abstract: With practice <strong>an</strong>d rehearsal, learned procedures c<strong>an</strong> eventually develop into habits <strong>an</strong>d<br />
be ‘chunked’ as cohesive, rapidly executable units. Our laboratory has identified task-bracketing<br />
neuronal firing patterns that develop in <strong>the</strong> sensorimotor striatum of rats learning a conditional Tmaze<br />
task, <strong>an</strong>d has suggested that <strong>the</strong>se may be related to <strong>the</strong> process of habit <strong>for</strong>mation. Here<br />
we used a reward devaluation protocol to test <strong>for</strong>mally whe<strong>the</strong>r this maze learning coincides with<br />
a shift <strong>from</strong> goal-directed (action-outcome mediated) responding to goal-independent (habitual)<br />
responding. Six rats learned to traverse a maze runway after a start signal (white noise) <strong>an</strong>d to<br />
turn down left or right end-arms according to instruction cues (1 or 8 kHz tones) <strong>for</strong> chocolate or<br />
sucrose reward paired with that arm <strong>an</strong>d cue. One of <strong>the</strong> rewards was <strong>the</strong>n devalued in <strong>the</strong><br />
homecage by pairings with lithium chloride injection, ei<strong>the</strong>r early after learning (after 2 days of<br />
at least 72.5% correct per<strong>for</strong>m<strong>an</strong>ce) or after overtraining (>10 days at >72.5% correct).
Afterwards, rats received <strong>an</strong> extinction session in which rewards were omitted, followed by<br />
several regular sessions in which rewards were again provided. In extinction, rats devalued early<br />
after task acquisition showed a per<strong>for</strong>m<strong>an</strong>ce decrement on <strong>the</strong> devalued arm only, but rats<br />
devalued after overtraining failed to show <strong>an</strong>y decrement. <strong>When</strong> reward-based training resumed,<br />
early-devalued rats rapidly adjusted behaviors to avoid <strong>the</strong> devalued arm despite <strong>the</strong> cue<br />
instruction. Overtrained rats continued to enter <strong>the</strong> devalued arm when so cued a larger number<br />
of trials, despite actually consuming <strong>the</strong> reward only a quarter of <strong>the</strong> time, as though <strong>the</strong>y were<br />
slow to integrate <strong>the</strong> ch<strong>an</strong>ge in outcome value into per<strong>for</strong>m<strong>an</strong>ce strategy. Later, in two of <strong>the</strong><br />
overtrained rats, <strong>the</strong> reward not initially devalued was also devalued. Within <strong>the</strong> next training<br />
session, <strong>the</strong>se rats proceeded to sample both rewards, <strong>the</strong>n sampled both arms but did not<br />
consume <strong>the</strong> rewards, <strong>an</strong>d ultimately stopped entering ei<strong>the</strong>r arm. However, <strong>the</strong>y continued to<br />
traverse <strong>the</strong> initial runway after <strong>the</strong> start signal on all trials, <strong>an</strong>d in over a quarter of <strong>the</strong> trials r<strong>an</strong><br />
down <strong>the</strong> runway, stopped at <strong>the</strong> choice point, <strong>an</strong>d <strong>the</strong>n returned back to <strong>the</strong> start. We conclude<br />
(1) that overtraining on our conditional T-maze task coincides with in <strong>the</strong> development of a<br />
stimulus-response habit, <strong>an</strong>d (2) that <strong>the</strong> initial phase of habitual behavior in <strong>the</strong> task may be<br />
more outcome-insensitive <strong>an</strong>d habitual th<strong>an</strong> <strong>the</strong> approach <strong>an</strong>d consumption phases. The<br />
<strong>for</strong>mation of task-bracketing patterns of neural firing in <strong>the</strong> sensorimotor striatum thus parallels a<br />
process of habit <strong>for</strong>mation <strong>an</strong>d may signal chunking of valued action sequences.<br />
Disclosures: K.S. Smith, None; A.M. Graybiel, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.11/EE47<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NIH/NIMH R01-MH60379<br />
ONR N00014-04-0208<br />
Title: Comparison of training-induced activity in <strong>the</strong> dorsolateral striatum during training on<br />
three versions of a T-maze task<br />
Authors: *T. D. BARNES 1 , D. HU 1 , J. .-B. MAO 1 , Y. KUBOTA 1 , M. HOWE 1 , D. Z. JIN 2 , A.<br />
GRAYBIEL 1 ;<br />
1 MIT, Cambridge, MA; 2 Pennsylv<strong>an</strong>ia State Univ., Univeristy Park, PA
Abstract: We have previously trained rats to turn left or right in a T-maze based on a tone that<br />
sounded at mid-run <strong>an</strong>d remained on until rats turned down <strong>the</strong> instructed arm <strong>an</strong>d reached a goal<br />
that was baited with chocolate <strong>for</strong> correct turns. As rats learned this task, <strong>the</strong> ensemble firing<br />
rates of putative output neurons of <strong>the</strong> dorsolateral striatum shifted <strong>from</strong> a pattern in which firing<br />
was distributed throughout <strong>the</strong> entire time of <strong>the</strong> T-maze runs, to a more stereotypical pattern<br />
with strong responses at <strong>the</strong> beginning <strong>an</strong>d end of <strong>the</strong> runs <strong>an</strong>d weaker firing during mid-run.<br />
Here, we trained rats on two vari<strong>an</strong>ts of <strong>the</strong> T-maze task <strong>an</strong>d recorded with tetrodes <strong>from</strong> <strong>the</strong><br />
dorsolateral striatum during <strong>the</strong> acquisition <strong>an</strong>d overtraining on <strong>the</strong>se tasks to determine whe<strong>the</strong>r<br />
<strong>the</strong> learning-related activity pattern is susceptible to m<strong>an</strong>ipulations of <strong>the</strong> timing of cue<br />
presentation or learning <strong>the</strong> stimulus-response association. In <strong>the</strong> first vari<strong>an</strong>t, we moved <strong>the</strong><br />
presentation of <strong>the</strong> instruction tones <strong>from</strong> mid-run to <strong>the</strong> beginning of <strong>the</strong> trial (be<strong>for</strong>e <strong>the</strong><br />
opening of a start gate), <strong>an</strong>d tested whe<strong>the</strong>r pre-trial instruction would allow <strong>the</strong> rat to chunk <strong>the</strong><br />
entire trial <strong>an</strong>d result in <strong>the</strong> task-boundary activity pattern being more robust. In <strong>the</strong> rats trained<br />
on this task version, we found a greatly heightened pattern of activity accentuating <strong>the</strong> beginning<br />
<strong>an</strong>d end of <strong>the</strong> maze run. We tested whe<strong>the</strong>r <strong>the</strong> patterns of firing were consistently related to<br />
speed or acceleration of running across <strong>the</strong> maze runs, <strong>an</strong>d found that <strong>the</strong> correlations were<br />
positive early in <strong>the</strong> runs but negative mid-run <strong>an</strong>d late in <strong>the</strong> runs, <strong>an</strong>d thus possibly state<br />
dependent. In <strong>the</strong> second task vari<strong>an</strong>t, all aspects of <strong>the</strong> task were identical to those in <strong>the</strong><br />
original task, except that rats received reward according to a pre-determined schedule based on<br />
<strong>the</strong> average per<strong>for</strong>m<strong>an</strong>ce of o<strong>the</strong>r rats trained on <strong>the</strong> original task. In this non-associative version<br />
of <strong>the</strong> task, <strong>the</strong> rats’ running speeds <strong>an</strong>d <strong>the</strong> probability of rewards were similar to those of rats<br />
run on <strong>the</strong> st<strong>an</strong>dard associative task, allowing assessment of whe<strong>the</strong>r associative learning was<br />
responsible <strong>for</strong> <strong>the</strong> training-induced ch<strong>an</strong>ges in striatal firing. We found that <strong>the</strong> beginning <strong>an</strong>d<br />
end firing pattern in <strong>the</strong> dorsolateral striatum was markedly weaker th<strong>an</strong> that recorded in rats<br />
per<strong>for</strong>ming <strong>the</strong> st<strong>an</strong>dard task. These findings suggest that <strong>the</strong> development of <strong>the</strong> task-bracketing<br />
pattern of activity in <strong>the</strong> dorsolateral striatum is modulated by associative learning <strong>an</strong>d is<br />
augmented under conditions favoring chunking of sequential action sequences.<br />
Disclosures: T.D. Barnes, None; D. Hu, None; J.-. Mao, None; Y. Kubota, None; M. Howe,<br />
None; D.Z. Jin, None; A. Graybiel, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.12/EE48<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NIMH RO1 MH60379
ONR N00014-04-1-0208<br />
Title: Dynamic modulation of ensemble activity in <strong>the</strong> nucleus accumbens during T-maze<br />
learning<br />
Authors: *H. E. ATALLAH, M. H. HOWE, A. M. GRAYBIEL;<br />
Dept. Brain & Cog. Sci. & McGovern Inst. <strong>for</strong> Brain Res., MIT, Cambridge, MA<br />
Abstract: The nucleus accumbens (NAc) is critical <strong>for</strong> rein<strong>for</strong>cement learning, yet <strong>the</strong> unique<br />
contribution of this structure is still unclear. We used chronic multi-tetrode recording methods to<br />
monitor <strong>the</strong> spiking activity of medium spiny neurons (MSNs) in <strong>the</strong> core of <strong>the</strong> nucleus<br />
accumbens as rats acquired a conditional T-maze task. The task requires that <strong>the</strong> rat, after<br />
presentation of <strong>an</strong> initial warning click, run down <strong>the</strong> straight arm <strong>an</strong>d enter ei<strong>the</strong>r <strong>the</strong> right or<br />
left end-arm of <strong>the</strong> maze, depending on which one of two auditory tones is presented, to receive<br />
a chocolate reward on <strong>the</strong> instructed side. The recording data suggest that <strong>the</strong> population activity<br />
of <strong>the</strong> MSNs is modulated dynamically during learning <strong>an</strong>d that <strong>the</strong> NAc ensemble activity<br />
differs <strong>from</strong> that recorded in this lab in <strong>the</strong> dorsolateral <strong>an</strong>d dorsomedial striatum in similar Tmaze<br />
tasks. The strongest NAc population response occurred at goal-reaching/reward attainment<br />
early in training. In contrast sharp, phasic activity in response to <strong>the</strong> warning click developed<br />
with training. These population responses reflected <strong>the</strong> activity of at least three classes of eventrelated<br />
units which comprised less th<strong>an</strong> half of total number of MSNs: One group responded<br />
exclusively, or nearly so, when <strong>the</strong> rat reached <strong>the</strong> goal on rewarded trials. These neurons<br />
reduced <strong>the</strong>ir firing as learning (measured by % correct per<strong>for</strong>m<strong>an</strong>ce) progressed. A second<br />
group responded phasically to <strong>the</strong> initial warning cue. These responses mainly emerged when <strong>the</strong><br />
rats reached <strong>the</strong> learning criterion <strong>an</strong>d became more robust as training progressed. A third group<br />
of units ramped up <strong>the</strong>ir activity <strong>from</strong> around <strong>the</strong> middle of <strong>the</strong> maze-runs to goal-reaching in<br />
both rewarded <strong>an</strong>d unrewarded trials <strong>an</strong>d <strong>the</strong>n abruptly stopped firing. These patterns of NAc<br />
activity have commonalities with <strong>the</strong> reward prediction error coding typically assigned to<br />
dopamine-containing neurons <strong>an</strong>d st<strong>an</strong>d in contrast to <strong>the</strong> learning-related patterns that we have<br />
reported in more dorsal striatal regions.<br />
Disclosures: H.E. Atallah, None; M.H. Howe, None; A.M. Graybiel, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.13/EE49<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior
Support: DA 06886<br />
DA 04551<br />
T32 Training<br />
Title: Absence of cue-evoked firing in dorsolateral striatum neurons during <strong>the</strong> <strong>for</strong>mation <strong>an</strong>d<br />
expression of habitual behavior<br />
Authors: *S. MA 1,2 , D. H. ROOT 1 , C. TANG 2 , A. P. PAWLAK 1 , D. J. BARKER 1 , M. O.<br />
WEST 1 ;<br />
1 Psychology, Rutgers Univ., Piscataway, NJ; 2 Ctr. <strong>for</strong> Neurosciences, The Feinstein Inst. <strong>for</strong><br />
Med. Research,North Shore-Long Isl<strong>an</strong>d Jewish Hlth. Syst., M<strong>an</strong>hasset, NY<br />
Abstract: The sensorimotor (dorsolateral) striatum has been hypo<strong>the</strong>sized to be import<strong>an</strong>t <strong>for</strong><br />
stimulus-response habits, since lesions to this region disrupt habit <strong>for</strong>mation. Thus, if <strong>the</strong><br />
dorsolateral striatum is crucial <strong>for</strong> habit learning, single neurons of <strong>the</strong> region should be sensitive<br />
to both <strong>the</strong> reward related stimulus as well as <strong>the</strong> habitual response in a paradigm that allows<br />
habitual behavior to be acquired. In o<strong>the</strong>r words, <strong>the</strong>se neurons should receive sensory inputs <strong>an</strong>d<br />
exert impact on downstream motor neurons involved in <strong>the</strong> behavioral response. A previous<br />
study in our laboratory examined <strong>the</strong> neural activity of dorsolateral striatum neurons during a<br />
learned vertical head movement task <strong>for</strong> water reward, predicted by a tone cue. Neurons were<br />
categorized by <strong>the</strong>ir unconditioned directional correlates with head movements (e.g., upwards,<br />
downwards). Neurons with apparently no sensory correlate were also recorded over training<br />
days. Habit <strong>for</strong>mation was tested by reward satiety early <strong>an</strong>d late in training. We have previously<br />
shown that in this population of neurons, as <strong>an</strong>imals became habitual (i.e., resist<strong>an</strong>t to reward<br />
devaluation), <strong>the</strong> vast majority of neurons decrease firing rates to <strong>the</strong> learned vertical head<br />
movement. A minority of neurons remained or increased correlations with <strong>the</strong> head movement.<br />
The present study reexamined <strong>the</strong> dorsolateral striatal neurons across training days <strong>for</strong> possible<br />
cue evoked activity while <strong>an</strong>imals became habitual.<br />
Disclosures: S. Ma, None; D.H. Root, None; C. T<strong>an</strong>g, None; A.P. Pawlak, None; D.J.<br />
Barker, None; M.O. West, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.14/EE50
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: NS045962<br />
Title: Altered activity of striatal medium spiny neurons in <strong>the</strong> chronic parkinsoni<strong>an</strong> state<br />
Authors: *S. UTHAYATHAS 1 , J. S. WHITHEAR 1 , S. M. PAPA 2 ;<br />
1 Yerkes Natl. Primate Res. Ctr., 2 Neurol., Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Medium spiny neurons (MSNs) play a major role in motor function because <strong>the</strong>y<br />
integrate inputs arriving <strong>from</strong> widely distributed cortical areas <strong>an</strong>d provide striatal outputs to<br />
o<strong>the</strong>r basal g<strong>an</strong>glia regions critically involved in movement control. Previously, we have studied<br />
<strong>the</strong> MSN activity patterns (bursts, pauses, <strong>an</strong>d dispersion) with single cell recordings in MPTPtreated<br />
monkeys during <strong>the</strong> tr<strong>an</strong>sition to different motor states (‘off’, ‘on’, <strong>an</strong>d ‘on-withdyskinesias’)<br />
following s.c. levodopa administration. In <strong>the</strong> present report, we extend our<br />
previous studies with additional <strong>an</strong>alyses of MSN activity patterns in parkinsoni<strong>an</strong> monkeys. We<br />
<strong>an</strong>alyzed <strong>the</strong> oscillatory activity of 140 MSNs separated in two groups according to clearly<br />
distinguished bursty (B-MSNs, 48) <strong>an</strong>d non-bursty (NB-MSNs, 92) activity patterns.<br />
Autocorrelograms were computed after spike sorting <strong>an</strong>alysis <strong>an</strong>d confirmation that <strong>the</strong> same<br />
units were isolated in <strong>the</strong> tr<strong>an</strong>sition to motor states. In <strong>the</strong> ‘off’ state, most NB-MSNs did not<br />
exhibit oscillatory activity. However, 15% of B-MSNs exhibited oscillations in slow frequency<br />
b<strong>an</strong>ds (~3-5 Hz). Thus, B-MSN oscillations associated with parkinsoni<strong>an</strong> motor disability are<br />
discrete. MSNs with oscillatory activity in <strong>the</strong> ‘off’ state frequently maintained <strong>the</strong> oscillation or<br />
tr<strong>an</strong>sitioned to non-oscillatory activity after dopamine inputs in <strong>the</strong> ‘on’ state. Results of<br />
autocorrelation <strong>an</strong>alysis toge<strong>the</strong>r with burst, pause <strong>an</strong>d dispersion <strong>an</strong>alyses indicate that <strong>the</strong><br />
chronic parkinsoni<strong>an</strong> state is characterized by profound alteration of <strong>the</strong> MSN activity, which is<br />
much faster <strong>an</strong>d predomin<strong>an</strong>tly irregular with signific<strong>an</strong>t bursting <strong>an</strong>d pausing. Only few bursty<br />
MSNs exhibit clear oscillation in <strong>the</strong> frequency of parkinsoni<strong>an</strong> tremor, <strong>an</strong>d <strong>the</strong>se “tremor<br />
MSNs” could be <strong>an</strong>alogous to tremor-related units in o<strong>the</strong>r basal g<strong>an</strong>glia areas. As reported<br />
earlier, <strong>the</strong> “on” state is associated with signific<strong>an</strong>t decreases in burst activity; however,<br />
numerous units, particularly those with characteristic frequency ch<strong>an</strong>ges, increase pause rates.<br />
MSNs with levodopa-induced firing decreases corresponding to D2 receptor-mediated responses<br />
increase pause rates <strong>an</strong>d dispersion. Fur<strong>the</strong>rmore, MSNs with bidirectional ch<strong>an</strong>ges of frequency,<br />
which are known to be associated with <strong>the</strong> expression of dyskinesias, subst<strong>an</strong>tially increase<br />
pause rates <strong>an</strong>d dispersion. In conclusion, increased excitability <strong>an</strong>d irregularity with pausing <strong>an</strong>d<br />
bursting, but not oscillation of firing, seem to subst<strong>an</strong>tially contribute to <strong>the</strong> altered activity<br />
patterns of MSNs that characterize <strong>the</strong> chronic parkinsoni<strong>an</strong> state.<br />
Disclosures: S. Uthayathas, None; J.S. Whi<strong>the</strong>ar, None; S.M. Papa, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.15/EE51<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: EU Marie Curie Training Network Programme Gr<strong>an</strong>t MRTN-CT-2005-019247.<br />
Title: Entrainment of STN neurons by cortical beta oscillations in Parkinson's disease<br />
Authors: *A. SHAROTT 1 , A. GULBERTI 1 , W. HAMEL 2 , J. A. KOEPPEN 2 , S. ZITTEL 3 , U.<br />
HIDDING 3 , C. GERLOFF 3 , M. WESTPHAL 2 , A. K. ENGEL 1 , C. K. E. MOLL 1 ;<br />
1 Dept. of Neurophys. <strong>an</strong>d Pathophys., Univ. Med. Ctr. Hamburg-Eppendorf, Hamburg,<br />
Germ<strong>an</strong>y; 2 Dept. of Neurosurg., 3 Dept of Neurol., Univ. Med. Ctr. Hamburg-Eppendorf,<br />
Hamburg, Germ<strong>an</strong>y<br />
Abstract: Synchronized oscillations in <strong>the</strong> β r<strong>an</strong>ge (10-30Hz) within cortex-basal g<strong>an</strong>glia<br />
circuits have been proposed as a pathophysiological mech<strong>an</strong>ism in Parkinson’s disease (PD).<br />
Much evidence supporting this idea has come <strong>from</strong> recordings of PD patients after <strong>the</strong><br />
impl<strong>an</strong>tation of electrodes <strong>for</strong> deep brain stimulation (DBS) of <strong>the</strong> subthalamic nucleus (STN).<br />
Local field potentials (LFPs) recorded postoperatively through <strong>the</strong> <strong>the</strong>rapeutic DBS electrodes<br />
display β oscillations that are coherent with <strong>the</strong> frontocentral EEG <strong>an</strong>d are ameliorated by<br />
dopaminergic medication. Such studies indicate that STN oscillations are driven by cortex, but<br />
this has not been verified at <strong>the</strong> level of single neurons. Here we compared <strong>the</strong> relationships<br />
between cortical <strong>an</strong>d STN (units+LFP) signals using intraoperative recording.<br />
Simult<strong>an</strong>eous recordings of cortex <strong>an</strong>d STN were made in 13 patients undergoing stereotactic<br />
impl<strong>an</strong>tation of DBS electrodes <strong>for</strong> <strong>the</strong> treatment of adv<strong>an</strong>ced PD. EEG recordings were<br />
referenced to give one frontocentral <strong>an</strong>d two lateral dipoles. Electrocorticographic signals<br />
(ECoG) were recorded using a monopolar epidural electrode beneath <strong>the</strong> cr<strong>an</strong>iotomy. STN units<br />
(n = 256) were recorded <strong>from</strong> 5 simult<strong>an</strong>eously adv<strong>an</strong>ced microelectrodes (interelectrode<br />
dist<strong>an</strong>ce: 2mm). STN-LFPs were taken <strong>from</strong> macroelectrodes 3mm above <strong>the</strong> microelectrode tip<br />
on three trajectories. Patients were included only if (i) 10 STN units had been recorded <strong>for</strong> over<br />
60s each, (ii) <strong>the</strong>re were units that displayed oscillations <strong>an</strong>d (iii) <strong>the</strong>re was signific<strong>an</strong>t coherence<br />
between <strong>the</strong> frontocentral EEG <strong>an</strong>d STN-LFP in <strong>the</strong> β r<strong>an</strong>ge.<br />
Analysis of spectral relationships of physiologically identified STN-LFPs verified that patients<br />
displayed core features of β oscillations in relation to <strong>the</strong> EEG described previously. Coherence<br />
was signific<strong>an</strong>tly higher with frontal th<strong>an</strong> lateral ch<strong>an</strong>nels <strong>an</strong>d phase <strong>an</strong>d directed tr<strong>an</strong>sfer<br />
function <strong>an</strong>alyses showed <strong>the</strong> EEG/ECoG leading <strong>the</strong> STN-LFP by around 20ms. Around 30% of<br />
STN single <strong>an</strong>d multi-units were signific<strong>an</strong>tly coherent with <strong>the</strong> frontocentral EEG <strong>an</strong>d showed<br />
<strong>the</strong> same <strong>an</strong>atomical <strong>an</strong>d directional relationships to <strong>the</strong> EEG in <strong>the</strong> β r<strong>an</strong>ge. The delay derived<br />
<strong>from</strong> <strong>the</strong> phase spectra, however, was longer <strong>for</strong> units, 30-90ms, th<strong>an</strong> <strong>for</strong> LFPs. Phase histograms<br />
<strong>an</strong>d spike triggered averaging confirmed that oscillations in <strong>the</strong> STN neurons lagged cortical β<br />
oscillations, whereas those <strong>for</strong> <strong>the</strong> STN-LFP were centered on zero.<br />
These results show that cortical β oscillations precede those STN neurons in PD patients off
medication <strong>an</strong>d suggest that STN-LFPs provide accurate directional in<strong>for</strong>mation, but not precise<br />
temporal relations of STN neurons to cortex at β frequencies.<br />
Disclosures: A. Sharott, None; A. Gulberti, None; W. Hamel, None; J.A. Koeppen, None; S.<br />
Zittel, None; U. Hidding, None; C. Gerloff, None; M. Westphal, None; A.K. Engel,<br />
None; C.K.E. Moll, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.16/EE52<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Dystonia Medical Research Foundation Research Gr<strong>an</strong>t<br />
Doris Duke Charitable Foundation Award #2007084<br />
Title: Cortical local field potentials distinguish Parkinson’s disease, primary dystonia, <strong>an</strong>d<br />
essential tremor<br />
Authors: *A. L. CROWELL 1 , J. L. OSTREM 1,2 , S. A. SHIMAMOTO 1 , D. A. LIM 1,2 , P. A.<br />
STARR 1,2 ;<br />
1 Univ. of Cali<strong>for</strong>nia, S<strong>an</strong> Fr<strong>an</strong>cisco, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 2 Parkinson's Dis. Research, Educ. <strong>an</strong>d<br />
Clin. Center, SFVAMC, S<strong>an</strong> Fr<strong>an</strong>cisco, CA<br />
Abstract: Synchronized oscillatory activity plays a role in <strong>the</strong> large-scale org<strong>an</strong>ization of neural<br />
networks <strong>an</strong>d may play a role in disease pathogenesis. One hypo<strong>the</strong>sis of Parkinson’s disease<br />
(PD) pathophysiology highlights <strong>the</strong> role of excessive beta b<strong>an</strong>d (13-30 Hz) synchrony in <strong>the</strong><br />
basal g<strong>an</strong>glia-thalamocortical motor circuit. Evidence <strong>for</strong> this hypo<strong>the</strong>sis is based largely on<br />
local field potential (LFP) recordings in <strong>the</strong> subthalamic nucleus (STN), while LFP recordings of<br />
<strong>the</strong> motor cortex have not been per<strong>for</strong>med. In addition, it is unclear to what extent excessive beta<br />
oscillations are unique to PD versus shared by o<strong>the</strong>r movement disorders of basal g<strong>an</strong>glia origin.<br />
In this study, we used invasive cortical recording to test <strong>the</strong> hypo<strong>the</strong>sis that oscillation<br />
frequencies in <strong>the</strong> primary motor (M1) <strong>an</strong>d primary sensory (S1) cortical LFPs distinguish<br />
between <strong>the</strong> three most common movement disorders.<br />
Local field potentials were recorded by subdural electrocorticography <strong>from</strong> M1 <strong>an</strong>d S1 in<br />
patients undergoing deep brain stimulation surgery <strong>for</strong> rigid-akinetic PD, primary cr<strong>an</strong>iocervical<br />
dystonia, or essential tremor (ET). These signals were recorded during alternating periods of rest
<strong>an</strong>d movement, using a paradigm designed to maximize cortical beta b<strong>an</strong>d oscillations. Electrode<br />
localization was confirmed by intraoperative imaging <strong>an</strong>d by somatosensory evoked potentials.<br />
Power spectral density in physiologically relev<strong>an</strong>t frequency b<strong>an</strong>ds was evaluated by repeated<br />
measures <strong>an</strong>alysis of vari<strong>an</strong>ce with disease state as a fixed factor.<br />
We found that both PD <strong>an</strong>d dystonia patients have high power in <strong>the</strong> beta b<strong>an</strong>d frequency r<strong>an</strong>ge<br />
(13-30Hz), whereas <strong>the</strong> domin<strong>an</strong>t oscillatory frequency r<strong>an</strong>ge <strong>for</strong> ET patients is 4-12Hz. Within<br />
<strong>the</strong> beta b<strong>an</strong>d, PD patients have slightly lower oscillation frequencies th<strong>an</strong> dystonia patients. M1<br />
<strong>an</strong>d S1 activities were similar. Frequency distribution patterns were similar under rest <strong>an</strong>d<br />
movement conditions. Dystonia patients, however, showed signific<strong>an</strong>tly less movement-related<br />
desynchronization in most frequency b<strong>an</strong>ds compared to <strong>the</strong> o<strong>the</strong>r patient groups.<br />
These data show that primary dystonia <strong>an</strong>d PD both are characterized by excess beta b<strong>an</strong>d<br />
oscillations in somatosensory cortices. In contrast, oscillatory activity in ET, a disorder thought<br />
not to involve basal g<strong>an</strong>glia, is restricted to lower frequencies. This suggests similarities in <strong>the</strong><br />
circuit abnormalities <strong>for</strong> dystonia <strong>an</strong>d PD at <strong>the</strong> cortical level, although <strong>the</strong>se two disorders c<strong>an</strong><br />
be distinguished by differential activity within <strong>the</strong> beta b<strong>an</strong>d <strong>an</strong>d by degree of movement-related<br />
desynchronization. These findings may contribute to <strong>the</strong> development of cortically based<br />
<strong>the</strong>rapies <strong>for</strong> movement disorders.<br />
Disclosures: A.L. Crowell, None; J.L. Ostrem, None; S.A. Shimamoto, None; D.A. Lim,<br />
None; P.A. Starr, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.17/EE53<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Title: Physiology of hum<strong>an</strong> striatal neurons in Parkinson´s disease <strong>an</strong>d dystonia<br />
Authors: C. K. E. MOLL 1 , A. SHAROTT 1 , W. HAMEL 2 , J. KOEPPEN 2 , J. SARNTHEIN 4 , A.<br />
GULBERTI 1 , *G. A. ENGLER 5 , C. BUHMANN 3 , A. MÜNCHAU 3 , C. GERLOFF 3 , M.<br />
WESTPHAL 2 , A. K. ENGEL 1 ;<br />
1 Dept. of Neurophysiol. <strong>an</strong>d Pathophysiology, 2 Dept. of Neurosurg., 3 Dept. of Neurol., Univ.<br />
Med. Ctr. Hamburg-Eppendorf, Hamburg, Germ<strong>an</strong>y; 4 Univ. Hosp. Zurich, Neurosurgery Clinic,<br />
Switzerl<strong>an</strong>d; 5 Univ. Med. Ctr. Hamburg, Hamburg, Germ<strong>an</strong>y<br />
Abstract: The striatum is centrally implicated in movement control <strong>an</strong>d considered a key<br />
structure in <strong>the</strong> pathophysiology of different movement disorders, such as Parkinsons disease
(PD) <strong>an</strong>d dystonia (DYS). However, how pathology affects neuronal signalling within <strong>the</strong> hum<strong>an</strong><br />
striatal microcircuitry is unknown. We identified three presumably different neuronal<br />
populations in microelectrode-recordings <strong>from</strong> <strong>the</strong> striatum of PD <strong>an</strong>d DYS patients undergoing<br />
deep brain stimulation surgery. Here we show that a subset of presumed medium-spiny<br />
projection neurons in PD but not in DYS display excessive activity levels. In contrast to <strong>the</strong><br />
predictions of <strong>the</strong> acetylcholine-dopamine hypo<strong>the</strong>sis, we found that <strong>the</strong> tonically active<br />
cholinergic interneurons (TANs) are not hyperactive in PD but even have reduced firing rates<br />
compared to TANs recorded in vivo <strong>from</strong> MPTP treated monkeys. Putative GABAergic<br />
interneurons were identified based on <strong>the</strong>ir unique bursting behaviour, strongly resembling low<br />
threshold spiking (LTS) described in thalamic projection neurons. These neurons were similarly<br />
active (in fact also tonically active) in both diseases. The present results suggest major<br />
differences of striatal outflow in PD vs. DYS, despite comparable activity in interneuronal<br />
circuits. Moreover, our finding of differential attention-dependent responses suggests<br />
functionally distinct contributions of specific striatal cell types to saliency processing.<br />
Disclosures: C.K.E. Moll, None; A. Sharott, None; W. Hamel, None; J. Koeppen, None; J.<br />
Sarn<strong>the</strong>in, None; A. Gulberti, None; G.A. Engler, None; C. Buhm<strong>an</strong>n, None; A. Münchau,<br />
None; C. Gerloff, None; M. Westphal, None; A.K. Engel, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.18/EE54<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: BMBF 01 GQ0733<br />
Title: Temporal relations of acoustically evoked potentials in auditory cortex <strong>an</strong>d striatum of <strong>the</strong><br />
Mongoli<strong>an</strong> gerbil<br />
Authors: M. L. WOLDEIT, *A. L. SCHULZ, F. W. OHL;<br />
Inst. Neurobiol, Magdeburg, Germ<strong>an</strong>y<br />
Abstract: Sensory gating is a process by which a (weak) sensory event modulates evoked<br />
neuronal signals to subsequent sensory stimuli of <strong>the</strong> same or different sensory modality. This<br />
process is known to be disturbed in a number of neuropsychiatric disorders, e.g. schizophrenia or<br />
obsessive compulsive disorder. Demonstration of disturb<strong>an</strong>ces in gating, e.g. using paired-pulse<br />
protocols, is also indicative of emotional <strong>an</strong>d cognitive impairments. Gating could be a filter
mech<strong>an</strong>ism thought to prevent <strong>the</strong> brain <strong>from</strong> <strong>an</strong> overflow of distracting in<strong>for</strong>mation.<br />
In <strong>the</strong> context of <strong>an</strong> auditory discrimination experiment with trains of frequency-modulated<br />
tones, we measured local field potentials simult<strong>an</strong>eously in <strong>the</strong> striatum <strong>an</strong>d <strong>the</strong> primary auditory<br />
cortex in awake <strong>an</strong>d behaving gerbils (Meriones unguiculatus). We observed different stimulus<br />
coupling dynamics in cortex <strong>an</strong>d striatum: While in <strong>the</strong> auditory cortex evoked potentials were<br />
robustly coupled to each individual stimulus within <strong>the</strong> train, field potentials in <strong>the</strong> striatum were<br />
elicited only by <strong>the</strong> first stimulus of a train. This let us to investigate qu<strong>an</strong>titatively <strong>the</strong> temporal<br />
dynamics of evoked potentials in both areas <strong>an</strong>d <strong>the</strong>ir coupling to auditory stimuli. Specifically,<br />
we varied <strong>the</strong> inter-tone intervals within <strong>the</strong> trains, to determine <strong>the</strong> upper boundary of sensory<br />
gating in <strong>the</strong> striatum. Fur<strong>the</strong>rmore, we found that <strong>the</strong> latency of <strong>the</strong> first peak of <strong>the</strong> evoked<br />
potential in <strong>the</strong> striatum was signific<strong>an</strong>tly earlier (~40 ms) th<strong>an</strong> in <strong>the</strong> auditory cortex (~50 ms).<br />
This result indicates that at least <strong>the</strong> early part of <strong>the</strong> auditory response in <strong>the</strong> striatum is not<br />
driven via <strong>the</strong> auditory cortex.<br />
Disclosures: M.L. Woldeit, None; A.L. Schulz, None; F.W. Ohl, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.19/EE55<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Title: Population coding of stimulus value by neurons in <strong>the</strong> medial <strong>an</strong>d ventral striatum<br />
Authors: *E. Y. KIMCHI 1 , M. LAUBACH 2 ;<br />
1 Interdepartmental Neurosci Program, 2 John B. Pierce Lab., Yale Univ. Sch. Med., New Haven,<br />
CT<br />
Abstract: We have previously described a dynamic coding of action selection by <strong>the</strong> striatum<br />
(Kimchi <strong>an</strong>d Laubach, J Neurosci, <strong>2009</strong>). Subjects switched <strong>from</strong> making No-Go to Go<br />
responses following a positive ch<strong>an</strong>ge in <strong>the</strong> reward value of a stimulus. This was preceded by<br />
dynamic modulations of spike activity in <strong>the</strong> medial, but not <strong>the</strong> ventral, striatum. These results<br />
suggest that <strong>the</strong> medial striatum biases <strong>an</strong>imals to collect rewards to potentially valuable stimuli.<br />
Here, we report new data, collected during <strong>the</strong> same task, <strong>from</strong> sessions in which <strong>the</strong> stimulus<br />
underwent a negative ch<strong>an</strong>ge in reward value (<strong>from</strong> rewarded to unrewarded). The same methods<br />
<strong>for</strong> qu<strong>an</strong>tifying dynamic ch<strong>an</strong>ges in neuronal activity were used (Bayesi<strong>an</strong> decoding of Go<br />
responding based on firing rates during <strong>the</strong> RT period, ch<strong>an</strong>ge-point <strong>an</strong>alysis of behavioral<br />
responding <strong>an</strong>d neuronal predictions of responding). As in our previous study, we found that
m<strong>an</strong>y striatal neurons (>20%) showed rapid <strong>an</strong>d major ch<strong>an</strong>ges in firing rates following <strong>the</strong><br />
negative ch<strong>an</strong>ge in reward value <strong>an</strong>d rats rapidly switched <strong>from</strong> making Go responses to making<br />
No-Go responses after <strong>the</strong> switch in stimulus set. As in our previous paper, neurons in <strong>the</strong> medial<br />
striatum showed altered spike activity earlier in <strong>the</strong> trial, compared to neurons in <strong>the</strong> ventral<br />
striatum (or nucleus accumbens). Plots of posterior probabilities, <strong>from</strong> decoding <strong>an</strong>alysis, <strong>for</strong><br />
groups of simult<strong>an</strong>eously recorded neurons showed that <strong>the</strong>re was considerable trial-to-trial<br />
variability across neurons. Partial correlation <strong>an</strong>alysis was used to measure correlations between<br />
posterior probabilities <strong>for</strong> simult<strong>an</strong>eously recorded pairs of neurons <strong>an</strong>d to control <strong>for</strong> Go <strong>an</strong>d<br />
No-Go responding. Correlations beween pairs of neurons were ra<strong>the</strong>r low (
A brain area that has been closely linked to <strong>the</strong> latter states are <strong>the</strong> basal g<strong>an</strong>glia. Neuronal<br />
responses of <strong>the</strong> striatum <strong>an</strong>d nucleus accumbens were correlated to reward, prediction of<br />
reward, prediction error evaluation <strong>an</strong>d to <strong>the</strong> control of motor output.<br />
Our first goal was to reproduce results of electrophysiological neuronal signals in <strong>the</strong> striatum of<br />
<strong>the</strong> Mongoli<strong>an</strong> gerbil in <strong>an</strong> auditory discrimination paradigm. There<strong>for</strong>e learning behavior <strong>an</strong>d<br />
per<strong>for</strong>m<strong>an</strong>ce were studied in a GO/(NO-GO) task aiming at <strong>the</strong> avoid<strong>an</strong>ce of a mild foot shock<br />
by crossing a hurdle in a two-way shuttle box. Hurdle crossings were registered as conditioned<br />
responses to <strong>the</strong> rein<strong>for</strong>ced stimulus (CS+) <strong>an</strong>d as false alarms in response to <strong>the</strong> unrein<strong>for</strong>ced<br />
stimulus (CS-). Rising <strong>an</strong>d falling linearly frequency-modulated (FM) tones served as <strong>the</strong><br />
respective conditioned stimuli CS+ <strong>an</strong>d CS-. After <strong>the</strong> <strong>an</strong>imals reached a predefined per<strong>for</strong>m<strong>an</strong>ce<br />
level <strong>the</strong> contingency of <strong>the</strong> FM tones was reversed. During <strong>the</strong> behavioral task local field<br />
potentials were measured simult<strong>an</strong>eously in <strong>the</strong> striatum <strong>an</strong>d <strong>the</strong> primary auditory cortex of <strong>the</strong><br />
gerbil. In a second step we investigated <strong>the</strong> interaction between <strong>the</strong> local field potentials in <strong>the</strong><br />
AC <strong>an</strong>d <strong>the</strong> striatum in order to make prediction on a single trial basis <strong>from</strong> <strong>the</strong> behavioral<br />
per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d <strong>the</strong> neuronal signals in <strong>the</strong> striatum to <strong>the</strong> plastic properties of <strong>the</strong> AC.<br />
Disclosures: A.L. Schulz, None; M.L. Woldeit, None; V. Korz, None; F.W. Ohl, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.21/EE57<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: CNRS<br />
Title: Two-stage action selection in a computational model of <strong>the</strong> basal g<strong>an</strong>glia<br />
Authors: *M. GUTHRIE, B. BIOULAC, C. GROSS, T. BORAUD;<br />
Univ. Bordeaux, Bordeaux, Fr<strong>an</strong>ce<br />
Abstract: There is continued debate about <strong>the</strong> role of cortex-basal g<strong>an</strong>glia loops in decision<br />
making <strong>an</strong>d action selection processes. In particular, it has been shown that <strong>the</strong>re are several<br />
parallel functional loops through <strong>the</strong> basal g<strong>an</strong>glia connecting back to distinct areas of cortex.<br />
The picture of segregated loops is complicated by studies showing axonal divergence that imply<br />
that <strong>the</strong> loops are not completely segregated. Here we propose a unified computational model of<br />
cortical - basal g<strong>an</strong>glia loops that has two functional loops, one cognitive <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r motor.<br />
There are two pathways through each loop, <strong>the</strong> direct pathway through <strong>the</strong> striatum to <strong>the</strong> GPi
<strong>an</strong>d <strong>the</strong> hyperdirect pathway via <strong>the</strong> STN to <strong>the</strong> GPi. The separate loops interact only via<br />
divergent projections to GPi <strong>from</strong> striatum <strong>an</strong>d STN.<br />
The model is used to simulate a two-choice decision task that has been shown, in monkeys, to<br />
have two distinct phases; a first stage in which a target is selected <strong>an</strong>d a second in which a<br />
direction of movement is selected. The first loop in <strong>the</strong> model is considered to be more cognitive,<br />
receives cortical input regarding <strong>the</strong> value of <strong>the</strong> two targets shown <strong>an</strong>d acts in deciding which of<br />
<strong>the</strong> two targets to select. The second loop is considered to be more motor related <strong>an</strong>d receives<br />
cortical input regarding <strong>the</strong> two possible directions of movement that could be taken to reach a<br />
target (although <strong>the</strong>re is no in<strong>for</strong>mation on <strong>the</strong> value of <strong>the</strong> targets directly available to structures<br />
in this loop) <strong>an</strong>d acts to choose <strong>the</strong> direction of movement. The two model loops per<strong>for</strong>m <strong>the</strong> two<br />
stages of <strong>the</strong> task in parallel, with <strong>the</strong> decision in <strong>the</strong> target selection phase feeding <strong>for</strong>ward via<br />
<strong>the</strong> divergent projections to influence <strong>the</strong> decision in <strong>the</strong> direction selection phase. Structures in<br />
<strong>the</strong> cortex <strong>an</strong>d basal g<strong>an</strong>glia are modeled using spiking Izhikevich neurons of appropriate types<br />
<strong>an</strong>d in numerically appropriate ratios.<br />
Using this <strong>an</strong>atomically grounded architecture, <strong>the</strong> model is able to per<strong>for</strong>m a choice between<br />
two alternatives <strong>an</strong>d to reproduce population responses of neurons in <strong>the</strong> basal g<strong>an</strong>glia that were<br />
obtained in monkeys per<strong>for</strong>ming this free double choice task.<br />
Disclosures: M. Guthrie, None; B. Bioulac, None; C. Gross, None; T. Boraud, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.22/EE58<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Ministry of Science, Culture & Sport, Israel<br />
<strong>the</strong> Ministry of Research, Fr<strong>an</strong>ce<br />
Fr<strong>an</strong>ce-Israel Laboratory of Neuroscience (FILN-LEA)<br />
Title: Modelling learning of <strong>the</strong> two-armed b<strong>an</strong>dit reward schedule by primates<br />
Authors: S. LAQUITAINE 1 , Y. LOEWENSTEIN 2 , *T. BORAUD 1 ;<br />
1 Lab. M.A.C., CNRS - Univ. Bx2, Bordeaux, Fr<strong>an</strong>ce; 2 Hebrew Univ. of Jerusalem, Jerusalem,<br />
Israel
Abstract: It is now widely believed that decisions are guided by a small number of internal<br />
subjective variables that determine choice preference. The process of learning m<strong>an</strong>ifests as a<br />
ch<strong>an</strong>ge in <strong>the</strong> state of <strong>the</strong>se variables. It is not clear how to find <strong>the</strong> neural correlates of <strong>the</strong>se<br />
variables, in particular because <strong>the</strong>ir state c<strong>an</strong>not be directly measured or controlled by <strong>the</strong><br />
experimenter. Ra<strong>the</strong>r, <strong>the</strong>se variables reflect <strong>the</strong> history of <strong>the</strong> subject’s actions <strong>an</strong>d reward<br />
experience. We seek to construct a behavioral model that captures <strong>the</strong> dynamics of learning <strong>an</strong>d<br />
decision making, such that <strong>the</strong> internal variables of this model will serve as a proxy <strong>for</strong> <strong>the</strong><br />
subjective variables. We use <strong>the</strong> <strong>the</strong>ory of rein<strong>for</strong>cement learning in order to find a behavioral<br />
model that best captures <strong>the</strong> learning dynamics of monkeys in a two-armed b<strong>an</strong>dit reward<br />
schedule. We consider two families of learning algorithms: value function estimation <strong>an</strong>d direct<br />
policy optimization. In <strong>the</strong> <strong>for</strong>mer, <strong>the</strong> values of <strong>the</strong> alternative actions are learned <strong>an</strong>d <strong>the</strong> policy<br />
is determined by <strong>the</strong>se values; in <strong>the</strong> latter, <strong>the</strong> policy is directly updated based on actions <strong>an</strong>d <strong>the</strong><br />
resulting rewards.<br />
Our preliminary results suggest that <strong>the</strong> learning behavior in monkeys is well described by <strong>the</strong><br />
simplest gradient-based direct-policy algorithm, <strong>the</strong> linear reward-inaction algorithm.<br />
Disclosures: S. Laquitaine, None; Y. Loewenstein, None; T. Boraud, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.23/EE59<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Title: The neuronal activity stream associated with a reward-based choice task is synchronized<br />
during <strong>the</strong> tr<strong>an</strong>sition between <strong>the</strong> striatum <strong>an</strong>d <strong>the</strong> globus pallidus pars interna<br />
Authors: *A. GARENNE 1,2 , B. PASQUEREAU 4 , A. NADJAR 1,3,5 , B. BIOULAC 6,1,2 , C.<br />
GROSS 6,1,2 , T. BORAUD 1,2 ;<br />
1 Univ. Bordeaux 2, Bordeaux, Fr<strong>an</strong>ce; 2 Umr 5227, 3 Umr 5226, CNRS, Bordeaux, Fr<strong>an</strong>ce;<br />
4 Neurobio. <strong>an</strong>d Ctr. <strong>for</strong> <strong>the</strong> Neural Basis of Cognition, Univ. of Pittsburgh, Pittsburgh, PA; 5 Umr<br />
1286, INRA, Bordeaux, Fr<strong>an</strong>ce; 6 CHU Pellegrin, Bordeaux, Fr<strong>an</strong>ce<br />
Abstract: As pointed out by electrophysiological data recorded in vivo <strong>from</strong> monkeys, basal<br />
g<strong>an</strong>glia nuclei like striatum or GPi (globus pallidus pars interna) are involved in decision making<br />
under probabilistic conditions (i.e.: when dealing with multiple choice tests weighted by different<br />
reward probabilities). To <strong>an</strong>alyze <strong>the</strong>se data <strong>an</strong>d to have <strong>an</strong> overall view of <strong>the</strong> neuron dynamics<br />
associated with a choice task in 2 trained monkeys, we have investigated <strong>the</strong> temporal outline of
peri-event time histograms (PETH). The considered events are all <strong>the</strong> different milestones of <strong>the</strong><br />
task (e.g. cue presentation to <strong>the</strong> monkey). There<strong>for</strong>e, to underst<strong>an</strong>d <strong>the</strong> respective involvement<br />
of striatum <strong>an</strong>d GPi in <strong>the</strong>se dynamics <strong>an</strong>d <strong>the</strong>ir relationship to <strong>the</strong> experimental conditions, we<br />
have implemented <strong>an</strong> algorithm which c<strong>an</strong> automatically compute <strong>the</strong> PETH, using <strong>an</strong> eventsequence<br />
detection algorithm. Since <strong>the</strong> trigger <strong>for</strong> some steps of <strong>the</strong> choice processes is<br />
controlled by <strong>the</strong> <strong>an</strong>imal itself <strong>an</strong>d this generates high variability in <strong>the</strong>ir time interval duration,<br />
<strong>an</strong> additional procedure of time <strong>an</strong>d amplitude normalization of <strong>the</strong> PETHs has been applied.<br />
Using this tool, we have per<strong>for</strong>med new <strong>an</strong>alyses on <strong>the</strong> data of a previous publication by our<br />
team. We have <strong>the</strong>n applied two strategies to investigate neuronal subpopulations properties<br />
along with <strong>the</strong>ir dynamics. The first one makes use of <strong>the</strong> PETH correlation matrixes. We have<br />
computed <strong>the</strong> average level of correlation among GPi PETH <strong>an</strong>d striatum PETH. We have thus<br />
found that GPi PETHs were signific<strong>an</strong>tly highly correlated compared to striatum PETHs which<br />
conversely exhibits a large variability of profiles in both monkeys. This first result thus reveals a<br />
robust dissociation between striatum <strong>an</strong>d GPi neuronal activities. The second strategy relies on<br />
<strong>an</strong> interval-based <strong>an</strong>alysis of <strong>the</strong> PETH <strong>an</strong>d computes statistically signific<strong>an</strong>t increase, stability<br />
or decrease in <strong>the</strong> average level of neuronal activity between <strong>the</strong> task events. This approach both<br />
reduces dimensionality of <strong>the</strong> data <strong>an</strong>d makes clearer dynamic trends among PETHs. Our first<br />
investigations reveal that (i) GPi neurons profile are much less versatile th<strong>an</strong> striatum neurons<br />
profiles <strong>an</strong>d that (ii) <strong>the</strong> GPi neurons involved in <strong>the</strong> decision phase are involved in <strong>the</strong> static<br />
motor phase of <strong>the</strong> task as well. Our data rein<strong>for</strong>ce <strong>the</strong> hypo<strong>the</strong>sis that basal g<strong>an</strong>glia are involved<br />
in decision making <strong>an</strong>d suggest that <strong>the</strong> underlying mech<strong>an</strong>ism are supported by competition<br />
processes in <strong>the</strong> network between <strong>the</strong> striatum <strong>an</strong>d <strong>the</strong> GPi.<br />
Disclosures: A. Garenne, None; B. Pasquereau, None; A. Nadjar, None; B. Bioulac,<br />
None; C. Gross, None; T. Boraud, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.24/EE60<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Intramural Research Program, NINDS, NIH<br />
Title: Functional correlates of exaggerated oscillatory activity in basal g<strong>an</strong>glia <strong>an</strong>d impairment<br />
of motor activity in hemiparkinsoni<strong>an</strong> rats
Authors: E. BRAZHNIK, I. AVILA, N. NOVIKOV, D. A. BERGSTROM, *J. R. WALTERS;<br />
Neurophys/Pharmacol Sec, NIH NINDS, Be<strong>the</strong>sda, MD<br />
Abstract: Increased oscillatory activity in <strong>the</strong> basal g<strong>an</strong>glia is associated with movement<br />
disability in Parkinson’s disease. Recent studies <strong>from</strong> this lab in awake hemiparkinsoni<strong>an</strong> rats<br />
have detected a selective increase in high beta oscillatory activity (25-40 Hz) in <strong>the</strong> dopamine<br />
(DA) cell lesioned basal g<strong>an</strong>glia output during walking. The present study fur<strong>the</strong>r explores<br />
correlates between high beta <strong>an</strong>d gamma (45-60 Hz) r<strong>an</strong>ge activity in <strong>the</strong> basal g<strong>an</strong>glia <strong>an</strong>d<br />
behavioral state. Single neurons <strong>an</strong>d local field potentials (LFPs) in subst<strong>an</strong>tia nigra pars<br />
reticulata (SNpr, N=7) <strong>an</strong>d globus pallidus (GPe, N=4) <strong>an</strong>d EMG activity were simult<strong>an</strong>eously<br />
recorded <strong>from</strong> lesioned <strong>an</strong>d non-lesioned hemispheres in hemiparkinsoni<strong>an</strong> rats during rest <strong>an</strong>d<br />
walking ipsilateral to lesion in a circular treadmill; contralateral walking was impaired. LFP<br />
power in <strong>the</strong>se two regions was examined during different states (rest, alert, walking, under<br />
ureth<strong>an</strong>e <strong>an</strong>es<strong>the</strong>sia), single <strong>an</strong>d multiple walking episodes, <strong>an</strong>d over multiple days. SNpr<br />
exaggerated high beta oscillations were fully emerged 7 days post lesion in <strong>the</strong> lesioned<br />
hemisphere during walking with greater power in lesioned vs non-lesioned SNpr (ratio:<br />
17.5±6.6). High beta power remained const<strong>an</strong>t 4-8 weeks post lesion. Power was greater at<br />
walking onset <strong>an</strong>d diminished continually during walking episodes (to 50% at 5 th min). High beta<br />
power during alert inactive state <strong>an</strong>d under ureth<strong>an</strong>e <strong>an</strong>es<strong>the</strong>sia (evoked by pinching tail) was<br />
also signific<strong>an</strong>tly greater in <strong>the</strong> lesioned vs non-lesioned SNpr (ratios: 13.9±7.6 <strong>an</strong>d 4.3±1.3).<br />
Results showed a subtle but signific<strong>an</strong>t decrease in peak frequency during alert inactive state<br />
(29.3±0.4 Hz) relative to walk (36.5±0.2 Hz) that fur<strong>the</strong>r declined under ureth<strong>an</strong>e (21.8±0.5 Hz).<br />
Similar recordings <strong>from</strong> <strong>the</strong> GPe showed only modest oscillatory activity in <strong>the</strong> high beta r<strong>an</strong>ge<br />
in <strong>the</strong> lesioned hemisphere <strong>an</strong>d prominent oscillatory activity in <strong>the</strong> gamma r<strong>an</strong>ge in both<br />
hemispheres during walking. Gamma oscillatory activity was not notable in <strong>the</strong> SNpr. L-dopa<br />
treatment signific<strong>an</strong>tly reduced high beta power in <strong>the</strong> SNpr <strong>an</strong>d GPe LFPs (by ~80%) on <strong>the</strong><br />
lesioned side <strong>an</strong>d improved contralateral walking. A serotonin (5HT)-1A agonist reversed Ldopa’s<br />
effects on SNpr high beta power <strong>an</strong>d walking, whereas a 5HT-1A <strong>an</strong>tagonist restored<br />
<strong>the</strong>se effects, consistent with evidence that 5HT terminals in <strong>the</strong> CNS are involved in DA<br />
conversion <strong>from</strong> L-dopa <strong>an</strong>d release after DA cell death. Results support <strong>the</strong> view that increased<br />
beta r<strong>an</strong>ge synchronization in basal g<strong>an</strong>glia output due to loss of DA leads to motor dysfunction<br />
<strong>an</strong>d may provide insight into mech<strong>an</strong>isms underlying <strong>the</strong> efficacy of <strong>an</strong>tiparkinsoni<strong>an</strong> <strong>the</strong>rapies.<br />
Disclosures: E. Brazhnik, None; I. Avila, None; N. Novikov, None; D.A. Bergstrom,<br />
None; J.R. Walters, None.<br />
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 567.25/EE61<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: Intramural Research Program, NINDS, NIH<br />
Title: Correlations between basal g<strong>an</strong>glia subst<strong>an</strong>tia nigra pars reticulata (SNpr) neuronal output<br />
<strong>an</strong>d motor activity in a rodent model of Parkinson’s disease<br />
Authors: *I. AVILA 1 , D. A. BERGSTROM 1 , E. BRAZHNIK 1 , L. C. PARR-BROWNLIE 1 , E.<br />
CASTAÑEDA 2 , J. R. WALTERS 1 ;<br />
1 NPS, NINDS-NIH, Be<strong>the</strong>sda, MD; 2 Dept. of Psych., UTEP, El Paso, TX<br />
Abstract: To identify <strong>the</strong> sequence of neural events linking alterations in basal g<strong>an</strong>glia activity<br />
to motor dysfunction in Parkinson’s disease, it would be helpful to elucidate conditions under<br />
which ch<strong>an</strong>ges in <strong>the</strong>se two domains are correlated. The present study utilizes <strong>the</strong> disequilibrium<br />
in dopamine receptor innervation <strong>an</strong>d sensitivity between dopamine cell lesioned <strong>an</strong>d nonlesioned<br />
hemispheres to qu<strong>an</strong>tify relationships between ch<strong>an</strong>ges in SNpr neuronal output <strong>an</strong>d<br />
motor behavior. To determine how rotation rates correlate with firing rate in SNpr neuronal<br />
populations in rats with unilateral dopamine cell lesions, firing rates were recorded with<br />
chronically impl<strong>an</strong>ted electrodes in <strong>the</strong> SNpr in lesioned <strong>an</strong>d non-lesioned hemispheres while<br />
rotational behaviors were induced with acute <strong>an</strong>d chronic administration of L-DOPA (25 mg/kg)<br />
or d-amphetamine (5 mg/kg). Rotation rates were scored <strong>from</strong> videotaped sessions following<br />
electrophysiological recordings. Repeated L-DOPA treatments signific<strong>an</strong>tly increased <strong>the</strong><br />
intensity of rotations: peak rotations on day 1 of L-DOPA (acute treatment, 7 days post-lesion)<br />
were 12 ± 3 rpm <strong>an</strong>d on day 14 of chronic L-DOPA treatments were 30 ± 4 rpm. However, in <strong>the</strong><br />
lesioned hemisphere, ratios of me<strong>an</strong> SNpr rate decrease to me<strong>an</strong> rotation rate increase were not<br />
signific<strong>an</strong>tly different between acute <strong>an</strong>d chronic treatments: a 10% decrease in me<strong>an</strong> firing rate<br />
corresponded to 3 <strong>an</strong>d 4 rpm increases in rotation rates, respectively. In contrast, in <strong>the</strong> nonlesioned<br />
hemisphere, SNpr firing rates showed increases during peak rotation that were not<br />
signific<strong>an</strong>tly different after acute (78%) <strong>an</strong>d chronic (53%) L-DOPA treatment. Fur<strong>the</strong>r <strong>an</strong>alyses<br />
examined whe<strong>the</strong>r <strong>the</strong> % SNpr neurons responding to L-DOPA with rate increases vs. rate<br />
decreases remained consistent across treatment conditions, <strong>an</strong>d whe<strong>the</strong>r rotational behavior was<br />
better correlated with rate ch<strong>an</strong>ge in a specific SNpr neuronal subpopulation. In <strong>the</strong> lesioned<br />
hemisphere, more SNpr neurons showed a signific<strong>an</strong>t decrease in rate (>15% baseline) after<br />
chronic L-DOPA treatment (80%) th<strong>an</strong> after acute treatment (50%), arguing <strong>for</strong> a continuum of<br />
response sensitivity ra<strong>the</strong>r th<strong>an</strong> a set of subpopulations with distinct responses. While L-DOPAinduced<br />
contralateral rotation correlated with me<strong>an</strong> SNpr rate ch<strong>an</strong>ge in <strong>the</strong> lesioned hemisphere,<br />
preliminary results indicate that d-amphetamine-induced ipsilateral rotation correlates with me<strong>an</strong><br />
rate in only <strong>the</strong> SNpr subpopulation showing rate decreases in <strong>the</strong> non-lesioned hemisphere.<br />
These results establish a base <strong>for</strong> examination of downstream sites <strong>for</strong> comparable correlations.<br />
Disclosures: I. Avila, None; D.A. Bergstrom, None; E. Brazhnik, None; L.C. Parr-Brownlie,<br />
None; E. Castañeda, None; J.R. Walters, None.
Poster<br />
567. Basal G<strong>an</strong>glia: Electrophysiology <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 567.26/EE62<br />
Topic: D.15.d. Systems physiology <strong>an</strong>d behavior<br />
Support: US NIH DE40872<br />
C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research MOP 42505<br />
Title: Event-related desynchronization of cortical oscillatory activity in patients with multiple<br />
system atrophy<br />
Authors: *R. LEVY 1,2 , A. M. LOZANO 3 , A. E. LANG 4 , J. O. DOSTROVSKY 5 ;<br />
1 2 3<br />
Calgary, AB, C<strong>an</strong>ada; Clin. Neurosci., Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada; Neurosurg.,<br />
4 5<br />
Neurol., The Toronto Western Res. Inst., Toronto, ON, C<strong>an</strong>ada; Physiol., Univ. of Toronto,<br />
Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Multiple system atrophy (MSA) is a progressive neurodegenerative disease<br />
characterized by parkinsonism (MSA-P), cerebellar (MSA-C) <strong>an</strong>d autonomic deficits. In<br />
Parkinson’s disease (PD), impaired modulation of motor cortical <strong>an</strong>d subcortical oscillations has<br />
been implicated in <strong>the</strong> pathophysiology of akinesia <strong>an</strong>d bradykinesia. Event-related<br />
desynchronization (ERD) <strong>an</strong>d event-related synchronization (ERS) of cortical oscillatory<br />
rhythms preceding, during, <strong>an</strong>d following a self paced movement is a marker of cortical<br />
activation. In normal subjects ERD of <strong>the</strong> mu rhythm (8-12 Hz) occurs 1.5 to 2 s be<strong>for</strong>e a<br />
voluntary movement. In contrast, mu <strong>an</strong>d beta r<strong>an</strong>ge (13-35 Hz) oscillations demonstrate a<br />
decreased ERD latency of 0.5 to 0.6 s in untreated patients with PD versus 1.0 to 1.4 s in those<br />
treated with levodopa. The pattern of ERD of cortical oscillations in MSA has not been studied.<br />
Motor cortical rhythms were recorded <strong>from</strong> subdural strip electrodes in three patients with MSA-<br />
P while taking <strong>the</strong>ir regular dopaminergic medications. Patients per<strong>for</strong>med <strong>an</strong> externally cued<br />
wrist extension movement to a ready cue which was followed by a go cue. Recordings were also<br />
obtained during imagined wrist extension movements to <strong>the</strong> same cues. Lastly, patients<br />
per<strong>for</strong>med self paced wrist extension. ERD <strong>an</strong>d ERS were examined in subject specific<br />
frequency b<strong>an</strong>ds. All patients showed movement-related ERD in subject specific frequency<br />
b<strong>an</strong>ds below ~40 Hz in both self paced <strong>an</strong>d externally cued conditions. The ERD latency<br />
preceding self cued movement was reduced (
occurring in untreated PD patients. The findings suggest that cortical activation in patients with<br />
MSA is diminished, may be related to pathophysiological ch<strong>an</strong>ges occurring in <strong>the</strong> basal g<strong>an</strong>glia<br />
<strong>an</strong>d correlates with <strong>the</strong> poor clinical response that <strong>the</strong>se patients typically obtain with<br />
dopaminergic <strong>the</strong>rapy.<br />
Disclosures: R. Levy, None; A.M. Loz<strong>an</strong>o, None; A.E. L<strong>an</strong>g, None; J.O. Dostrovsky, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.1/EE63<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: DFG 0923-102<br />
NWF-08/07<br />
Title: Reorg<strong>an</strong>ization of movement-related intracortical inhibition (SICI) in acute to subacute<br />
stroke<br />
Authors: *G. LIUZZI, V. HÖRNIß, K. HEISE, J. HOPPE, M. ZIMMERMAN, C. GERLOFF,<br />
F. C. HUMMEL;<br />
Univ. Hosp Hamburg, Dept of Neurol., Hamburg, Germ<strong>an</strong>y<br />
Abstract: Background: The preparation of a voluntary movement is accomp<strong>an</strong>ied by a reduction<br />
of intracortical inhibition in <strong>the</strong> primary motor cortex (M1). It has been proposed that deficient<br />
control of intracortical inhibitory circuits contributes to impaired movement control of <strong>the</strong> paretic<br />
h<strong>an</strong>d in chronic stroke. However, it is not known how <strong>the</strong>se reorg<strong>an</strong>izational ch<strong>an</strong>ges in<br />
intracortical inhibition develop in <strong>the</strong> early period after stroke.<br />
Methods: We investigated GABAergic short intracortical inhibition (SICI) in <strong>the</strong> ipsilesional M1<br />
of patients with acute subcortical stroke (n=11; 63.2 +/- 3.0 years). SICI was obtained in a simple<br />
reaction time (RT) paradigm at four different time points during movement preparation (20, 50,<br />
80, 95% of RT), while patients moved <strong>the</strong> affected h<strong>an</strong>d. Measurements were undertaken at 5<br />
days, 6 weeks <strong>an</strong>d 3 months after stroke.<br />
Results: The main novel finding was that SICI during movement preparation was comparable in<br />
<strong>the</strong> acute stage (day 5 <strong>an</strong>d 6 weeks) compared to age-matched healthy controls. However, at 3<br />
months after stroke, SICI was disinhibited <strong>an</strong>d less modulated compared to controls (p=0.005).<br />
Conclusions: Our study complements previous findings of deficient movement-related SICI in
chronic stroke. The present data support <strong>the</strong> view that SICI does not reorg<strong>an</strong>ize until <strong>the</strong> subacute<br />
period after stroke (several weeks), indicating a potential target <strong>for</strong> <strong>the</strong>rapeutic interventions in<br />
early stroke.<br />
Disclosures: G. Liuzzi, None; V. Hörniß, None; K. Heise, None; J. Hoppe, None; M.<br />
Zimmerm<strong>an</strong>, None; C. Gerloff, None; F.C. Hummel, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.2/EE64<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIDRR H133G070089<br />
Title: Finger/Wrist flexor <strong>an</strong>d biceps activity increases as a function of limb loading <strong>an</strong>d reach in<br />
chronic hemiparetic stroke<br />
Authors: *L. C. MILLER, A. H. A. STIENEN, J. P. A. DEWALD;<br />
Northwestern Univ., Chicago, IL<br />
Abstract: The expression of <strong>the</strong> flexion synergy in <strong>the</strong> paretic upper limb of moderately to<br />
severely affected hemiparetic stroke survivors may cause disabling coupling between shoulder<br />
abductors <strong>an</strong>d elbow <strong>an</strong>d wrist/finger flexors. This would explain <strong>the</strong> hypertonia in <strong>the</strong><br />
finger/wrist flexors frequently observed during upper limb movements following stroke. We<br />
have previously shown that isometric finger/wrist <strong>for</strong>ces increase with increasing shoulder<br />
abduction activity, but <strong>the</strong> muscle activity underlying <strong>the</strong>se <strong>for</strong>ces has not been measured. This<br />
study measured electromyography (EMG) <strong>from</strong> 9 muscles in <strong>the</strong> paretic <strong>an</strong>d non-paretic upper<br />
limbs during lifting <strong>an</strong>d reaching tasks per<strong>for</strong>med with various levels of concurrent shoulder<br />
abduction activity.<br />
8 particip<strong>an</strong>ts with chronic hemiparetic stroke placed <strong>the</strong>ir arm in <strong>the</strong> Arm Coordination Training<br />
3-D (ACT-3D) device, which c<strong>an</strong> alter shoulder abduction torques required to lift <strong>the</strong> limb. They<br />
were instructed to move <strong>the</strong> limb into a home position of 85° shoulder abduction, 90° elbow<br />
flexion, <strong>an</strong>d 40° shoulder flexion <strong>an</strong>d to hold <strong>for</strong> 1s. After holding <strong>the</strong> home position <strong>for</strong> 1s, a<br />
virtual target appeared on <strong>the</strong> feedback screen, located in <strong>the</strong> sagittal pl<strong>an</strong>e through <strong>the</strong> shoulder<br />
<strong>an</strong>d at a dist<strong>an</strong>ce equal to <strong>the</strong> length of <strong>the</strong> arm. <strong>When</strong> <strong>the</strong> target appeared, particip<strong>an</strong>ts reached in<br />
<strong>the</strong> horizontal pl<strong>an</strong>e as fast <strong>an</strong>d as far as possible toward <strong>the</strong> target <strong>an</strong>d held <strong>the</strong> maximal reach<br />
position <strong>for</strong> 2s. 11 trials were completed at each of 7 active shoulder support levels: while
supported on <strong>the</strong> ACT3D haptic table <strong>an</strong>d while lifting up at 0, 5, 15, 25, 35, <strong>an</strong>d 50% of<br />
maximum shoulder abduction <strong>for</strong>ce. Surface EMG data were recorded over <strong>the</strong> following<br />
muscles: flexor digitorum superficialis (FDS), extensor digitorum communis (EDC), biceps<br />
brachii (BB), triceps brachii (long <strong>an</strong>d lateral), deltoid (<strong>an</strong>terior, middle, posterior (PDL)), <strong>an</strong>d<br />
pectoralis major.<br />
Two-way repeated measures ANOVA revealed signific<strong>an</strong>t main effects of support level <strong>an</strong>d limb<br />
on FDS, BB, <strong>an</strong>d PDL activity during lift <strong>an</strong>d at <strong>the</strong> end of reach, as well as signific<strong>an</strong>t<br />
interactions between support level <strong>an</strong>d limb. Additionally, ratios of EDC/FDS activity calculated<br />
during maximum volitional wrist flexion (.78 ± .36) were signific<strong>an</strong>tly different that those during<br />
<strong>the</strong> lift (.17 ± .04) <strong>an</strong>d reach (.23 ± .13) phases (p = 0.01). These results demonstrate that<br />
shoulder abduction loading of <strong>the</strong> paretic upper limb during dynamic movements incrementally<br />
increases BIC, FDS, <strong>an</strong>d PDL activity that occurs in chronic hemiparetic stroke, in accord<strong>an</strong>ce<br />
with <strong>the</strong> flexion synergy, <strong>an</strong>d that EDC/FDS ratios at <strong>the</strong> wrist are higher during volitional<br />
flexion th<strong>an</strong> during shoulder abduction coupled flexion.<br />
Disclosures: L.C. Miller, None; A.H.A. Stienen, None; J.P.A. Dewald, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.3/EE65<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: AHA 0815648G<br />
Title: Negative work symmetry during pedaling with increased body weight support <strong>for</strong> people<br />
post-stroke<br />
Authors: *J. K. BURGESS, D. A. BROWN;<br />
Dept of Physical Therapy <strong>an</strong>d Hum<strong>an</strong> Movement Sci., Northwestern Univ., Chicago, IL<br />
Abstract: Motor disability associated with stroke is characterized by a degradation of motor<br />
function on <strong>the</strong> side of <strong>the</strong> body contralateral to <strong>the</strong> injury. This asymmetry of function causes<br />
movement challenges such as maintaining proper bal<strong>an</strong>ce during locomotion, attaining a normal<br />
walking speed, <strong>an</strong>d muscle weakness. To address <strong>the</strong>se problems, patients post-stroke often<br />
participate in rehabilitation paradigms that include body weight support during walking. These<br />
lowered weight conditions might allow <strong>for</strong> better controlled weight tr<strong>an</strong>sfer, improved timing of<br />
gait phases <strong>an</strong>d faster gait speed. However, <strong>the</strong>re is limited underst<strong>an</strong>ding about how <strong>the</strong> body
controls its own load in maintaining <strong>an</strong> upright posture during a locomotion. Exploring <strong>the</strong><br />
biomech<strong>an</strong>ical effects of weight support during post-stroke gait would benefit improved<br />
rehabilitation outcomes.<br />
Previous studies with <strong>the</strong> upper limb have suggested that relieving <strong>the</strong> load dem<strong>an</strong>ds of a task <strong>for</strong><br />
someone with stroke leads to improved independent joint control (Beer 2000). Inappropriate<br />
integration of peripheral input, such as <strong>from</strong> load-related signals, leads to poor modulation of<br />
descending control of gait (Deitz 2002). A pedaling paradigm incorporated with body weight<br />
support, as used in this study, was utilized to provide a complex but highly controlled kinematic<br />
locomotor pattern (Brown 1998). Pedaling is often easier <strong>for</strong> post-stroke subjects to complete<br />
compared with walking. This is often a consequence of <strong>the</strong> nonparetic leg compensating <strong>for</strong> <strong>the</strong><br />
impairment of <strong>the</strong> paretic leg during <strong>the</strong> pedaling cycle. In this study, we examined <strong>the</strong> effect of<br />
body weight support on <strong>the</strong> symmetry of <strong>the</strong> net mech<strong>an</strong>ical work produced across <strong>the</strong> pedaling<br />
cycle between legs to examine this possible compensation. Negative work is exaggerated on <strong>the</strong><br />
upstroke of <strong>the</strong> paretic leg <strong>for</strong> people post-stroke as a result of inappropriate muscle control.<br />
In this experiment, subjects post-stroke <strong>an</strong>d healthy age-matched subjects pedaled at 40rpm <strong>for</strong><br />
30s in a custom ergometer that relieved 20-60% of <strong>the</strong>ir body weight in 5% increments. The<br />
ergometer allowed <strong>for</strong> free movement along <strong>the</strong> cr<strong>an</strong>k axis <strong>an</strong>d subjects were instructed to<br />
maintain <strong>the</strong> position of <strong>the</strong> backboard as steady as possible. EMG activity of 5 muscles of <strong>the</strong><br />
leg were also measured during all trials. Results currently suggest that increasing body weight<br />
support while pedaling increases <strong>the</strong> symmetry of negative work production between paretic <strong>an</strong>d<br />
nonparetic legs during <strong>the</strong> upswing phase of pedaling. While this data might indicate a decrease<br />
in <strong>the</strong> nonparetic leg compensating <strong>for</strong> <strong>the</strong> paretic leg, <strong>the</strong>re is evidence to suggest that <strong>the</strong><br />
paretic leg increases its work output relative to <strong>the</strong> task dem<strong>an</strong>ds.<br />
Disclosures: J.K. Burgess, None; D.A. Brown, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.4/EE66<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Title: Muscle synergies in stroke patients during walking<br />
Authors: *L. GIZZI 1,2 , J. FELDBÆK NIELSEN 3 , F. FELICI 1 , G. FILLIGOI 4 , D. FARINA 2 ;<br />
1 DiSMUS, Univ. Degli Studi Di Roma Foro Italico, Rome, Italy; 2 Smi, Aalborg Universitet,<br />
Aalborg, Denmark; 3 Regionhospital Hammel Neurocenter, Aarhus Universitet, Hammel,<br />
Denmark; 4 Univ. degli studi di Roma Sapienza, Rome, Italy
Abstract: It has been hypo<strong>the</strong>sized that <strong>the</strong> central nervous system (CNS) c<strong>an</strong> accomplish <strong>the</strong><br />
control of a large number of degrees of freedom <strong>for</strong> joint movements by me<strong>an</strong>s of a small<br />
alphabet of control signals [1], which reduces <strong>the</strong> computational load. Evidence of this lowdimensional<br />
control strategy has been early found in invertebrates <strong>an</strong>d lower vertebrates <strong>an</strong>d<br />
recently in primates, <strong>an</strong>d hum<strong>an</strong>s [1]. Hum<strong>an</strong> upper <strong>an</strong>d lower limb synergies have been assessed<br />
during several motor tasks, but no evidence of a modular org<strong>an</strong>ization of motor control has been<br />
provided in terms of walking muscle activation patterns in stroke patients. There<strong>for</strong>e, <strong>the</strong> aim of<br />
this study was <strong>the</strong> assessment of muscle synergies in early post-stroke patients as compared with<br />
healthy controls. Being stroke a central lesion, it was hypo<strong>the</strong>sized that spinal synergies are<br />
similar in stroke patients <strong>an</strong>d healthy individuals but that <strong>the</strong> motor primitives (control signals)<br />
differ. Twelve early stroke patients (age r<strong>an</strong>ge 28-64 years) <strong>an</strong>d twelve age-matched healthy<br />
controls (25-60 years) participated in <strong>the</strong> study. EMG signals were recorded with Ag-AgCl<br />
electrodes mounted in single differential mode <strong>from</strong> a total of 32 muscles of both sides of <strong>the</strong><br />
body. The acquisition rate was 2048 samples/s <strong>an</strong>d signals were filtered (b<strong>an</strong>dpass 20-400Hz)<br />
<strong>an</strong>d <strong>the</strong>n full-wave rectified <strong>an</strong>d low pass filtered (10Hz) in order to apply a matrix factorization<br />
technique <strong>for</strong> synergy extraction [2]. Stride identification was per<strong>for</strong>med by me<strong>an</strong>s of a VICON<br />
stereophotogrammetry system (acquisition rate 100 Hz) using 8 markers. The synergies extracted<br />
<strong>from</strong> healthy subjects were compared to those extracted <strong>from</strong> <strong>the</strong> patients. Reconstruction quality<br />
was evaluated by computing <strong>the</strong> coefficient of determination between <strong>the</strong> observed signals <strong>an</strong>d<br />
those obtained with <strong>the</strong> extracted synergies. The results showed that 4 synergies were sufficient<br />
to explain of <strong>the</strong> muscular activation pattern in both patients <strong>an</strong>d control subjects. This result<br />
shows that <strong>the</strong> muscular activation pattern during walking c<strong>an</strong> be reduced to a dimensionality of<br />
four in stroke patients, indicating that walking c<strong>an</strong> be described by a modular org<strong>an</strong>ization with a<br />
small alphabet of control signals in both stroke patients <strong>an</strong>d healthy subjects.<br />
REFERENCES:<br />
[1] Saltiel P, Wyler-Duda K, d’Avella A, Tresch MC, Bizzi E (2001) Muscle synergies encoded<br />
within <strong>the</strong> spinal cord: evidence <strong>from</strong> focal intraspinal NMDA iontophoresis in <strong>the</strong> frog. J<br />
Neurophysiol 85:605- 619.<br />
[2] Tresch M.C., Cheung V.C., d'Avella A. (2006) Matrix factorization algorithms <strong>for</strong> <strong>the</strong><br />
identification of muscle synergies: evaluation on simulated <strong>an</strong>d experimental data sets, J<br />
Neurophysiol. 95(4):2199-212<br />
Disclosures: L. Gizzi, None; J. Feldbæk Nielsen, None; F. Felici, None; G. Filligoi, None; D.<br />
Farina, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.5/EE67
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH: R01 HD045639<br />
NSF: BCS-0450218<br />
ONR: N00014-05-1-0844<br />
Title: Ch<strong>an</strong>ges in variability in motor learning in patients with Parkinson’s disease<br />
Authors: L. K. PENDT 1 , H. MULLER 3 , *D. STERNAD 2 ;<br />
1 2 3<br />
Biol., Biology, Elec & Computer Eng, Nor<strong>the</strong>astern Univ., Boston, MA; Movement Sci.,<br />
Justus-Liebig Univ., Giessen, Germ<strong>an</strong>y<br />
Abstract: Parkinson’s disease (PD) is m<strong>an</strong>ifested not only in several impairments of motor<br />
control but also in problems in motor learning. <strong>When</strong> practicing a task, PD patients c<strong>an</strong> improve<br />
even though <strong>the</strong>ir improvement rate is slower, particularly in early learning stages, <strong>an</strong>d<br />
per<strong>for</strong>m<strong>an</strong>ce remains below those of healthy subjects. Given that PD patients c<strong>an</strong>not reduce<br />
variability to <strong>the</strong> same amount as healthy subjects, we use variability as a window <strong>for</strong> uncovering<br />
underlying mech<strong>an</strong>isms. In previous work we developed a method that distinguishes three routes<br />
to per<strong>for</strong>m<strong>an</strong>ce improvement in redund<strong>an</strong>t tasks where execution variables are redund<strong>an</strong>t with<br />
respect to <strong>the</strong> result variables. The functional relation between result <strong>an</strong>d execution defines <strong>the</strong><br />
solution m<strong>an</strong>ifold. Distributional properties of variability in execution variables with respect to<br />
<strong>the</strong> solution m<strong>an</strong>ifold c<strong>an</strong> be parsed into three different contributions: variability due to<br />
exploiting error-toler<strong>an</strong>t solutions, due to reducing stochastic noise, <strong>an</strong>d due to covariation<br />
between execution variables.<br />
To investigate whe<strong>the</strong>r <strong>the</strong> learning deficits in PD patients is specifically due to one of <strong>the</strong>se<br />
components, we studied 19 mild to moderate, non-demented PD patients <strong>an</strong>d 19 age <strong>an</strong>d gender<br />
matched control subjects. Both groups practiced a virtual throwing task over 5 days with 200<br />
trials each day. Subjects have to accurately throw a ball suspended to a pole to hit a target<br />
located at <strong>the</strong> o<strong>the</strong>r side of <strong>the</strong> pole (skittles). This experimental task is redund<strong>an</strong>t as <strong>the</strong> two<br />
execution variables, position <strong>an</strong>d velocity at ball release, fully determine <strong>the</strong> elliptic ball<br />
trajectory <strong>an</strong>d <strong>the</strong> one result variable, error to target. The individual contributions of <strong>the</strong><br />
components Toler<strong>an</strong>ce, Noise <strong>an</strong>d Covariation to <strong>the</strong> throwing per<strong>for</strong>m<strong>an</strong>ce was determined <strong>an</strong>d<br />
compared between groups.<br />
As expected, patients were able to learn <strong>the</strong> task, although <strong>the</strong>y per<strong>for</strong>med worse th<strong>an</strong> <strong>the</strong> control<br />
group; specifically, <strong>the</strong>y showed less per<strong>for</strong>m<strong>an</strong>ce retention across practice days. Differences in<br />
per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d retention were due to <strong>the</strong> component Toler<strong>an</strong>ce, i.e. PD patients could not find<br />
equally successful per<strong>for</strong>m<strong>an</strong>ce strategies <strong>an</strong>d <strong>the</strong>y needed on average 70 trials more th<strong>an</strong> <strong>the</strong><br />
control group to achieve <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce level of <strong>the</strong> previous day. However, patients reduced<br />
Noise by <strong>the</strong> same amount as <strong>the</strong> control group. Nei<strong>the</strong>r of <strong>the</strong> two groups used Covariation to<br />
improve <strong>the</strong>ir throwing per<strong>for</strong>m<strong>an</strong>ce.<br />
These results show that learning deficits in Parkinson disease c<strong>an</strong> be ascribed to <strong>the</strong> Toler<strong>an</strong>ce<br />
component. Given that Toler<strong>an</strong>ce contributes to improvements early in practice, this result<br />
fur<strong>the</strong>r elucidates why PD patients have problems in early learning stages.
Disclosures: L.K. Pendt, None; H. Muller, None; D. Sternad, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.6/EE68<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: Falk Neurorehabilitation Medical Trust<br />
Title: Stroke impairs strength <strong>an</strong>d control of motor output during sub-maximal fatiguing<br />
contractions<br />
Authors: *A. S. HYNGSTROM 1 , T. L. ONUSHKO 2 , R. HEITZ 2 , A. A. RUTKOWSKI 1 , S. K.<br />
HUNTER 1 , B. D. SCHMIT 2 ;<br />
1 Physical Therapy, 2 Biomed. Engin., Marquette Univ., Milwaukee, WI<br />
Abstract: Chronic stroke c<strong>an</strong> lead to marked decrements in muscle function of <strong>the</strong> paretic leg<br />
(PL) compared with <strong>the</strong> non-paretic leg (NPL). In particular, alterations in muscle fatigability in<br />
people post-stroke could ultimately impact functional tasks such as walking. The purpose of this<br />
study was to compare <strong>the</strong> time to task failure <strong>an</strong>d fluctuations in motor output of <strong>the</strong> PL <strong>an</strong>d NPL<br />
<strong>for</strong> a sub-maximal isometric fatiguing contraction during hip flexion. Hip flexion torques of <strong>the</strong><br />
PL <strong>an</strong>d NPL were measured in 6 chronic stroke subjects (me<strong>an</strong> = 54 ± 7 years old) using a<br />
servomotor drive system equipped with leg braces instrumented with <strong>for</strong>ce tr<strong>an</strong>sducers. Subjects<br />
were supine with <strong>the</strong> hip in 20 degrees of flexion <strong>an</strong>d <strong>the</strong> contralateral hip held at 0 degrees.<br />
Knees <strong>an</strong>d <strong>an</strong>kles were restrained in a neutral position using braces <strong>an</strong>d a strap was placed across<br />
<strong>the</strong> pelvis to minimize pelvic rotation. Maximal voluntary contractions (MVCs) of <strong>the</strong> hip flexors<br />
were per<strong>for</strong>med <strong>for</strong> each leg separately be<strong>for</strong>e <strong>an</strong>d after <strong>an</strong> isometric fatiguing contraction of <strong>the</strong><br />
PL sustained at 20% MVC of hip flexion until failure to sustain <strong>the</strong> required torque. This<br />
protocol was repeated in <strong>the</strong> NPL. Fluctuations in motor output were qu<strong>an</strong>tified over intervals of<br />
10% task duration as <strong>the</strong> normalized st<strong>an</strong>dard deviation of <strong>the</strong> me<strong>an</strong> torque [coefficient of<br />
variation (CV)] during <strong>the</strong> sub-maximal fatiguing contraction. Hip flexion MVC of <strong>the</strong> PL (82.8<br />
± 48 Nm) was weaker th<strong>an</strong> <strong>the</strong> non-paretic limb (119.1 ± 72 Nm). Despite being weaker, <strong>the</strong> PL<br />
had on average a 54% longer time to task failure (285.8 ± 216.7 s vs. 165.2 s ± 94.5 s). The<br />
average CV during <strong>the</strong> fatiguing contraction was greater <strong>for</strong> <strong>the</strong> PL (0.36 ± .45) as compared to<br />
<strong>the</strong> NPL (0.08 ± 0.03). Similar to healthy controls, <strong>the</strong> CV of <strong>the</strong> NPL increased as a function of<br />
time <strong>from</strong> <strong>the</strong> start to <strong>the</strong> end of <strong>the</strong> fatiguing contraction (0.07 to 0.11). In contrast, <strong>the</strong> CV <strong>for</strong><br />
<strong>the</strong> PL was larger at <strong>the</strong> start of <strong>the</strong> fatiguing contraction (0.66) compared with <strong>the</strong> end of <strong>the</strong>
task (0.11). There was a positive correlation between <strong>the</strong> symmetry of MVC magnitude (paretic<br />
MVC magnitude paretic/non-paretic MVC magnitude) <strong>an</strong>d <strong>the</strong> CV of <strong>the</strong> paretic (r 2 =0.32) <strong>an</strong>d<br />
non-paretic limb (r 2 =0.80). These preliminary findings indicate that strength <strong>an</strong>d control of motor<br />
output of <strong>the</strong> PL is impaired during a low-<strong>for</strong>ce fatiguing contraction, which <strong>for</strong>ms <strong>the</strong><br />
foundation of functional tasks such as walking. Fur<strong>the</strong>rmore, greater strength decrements of <strong>the</strong><br />
PL with stroke may serve as a potentially import<strong>an</strong>t indicator of loss of motor control during submaximal<br />
tasks with <strong>the</strong> hip flexors.<br />
Disclosures: A.S. Hyngstrom, None; T.L. Onushko, None; R. Heitz, None; A.A. Rutkowski,<br />
None; S.K. Hunter, None; B.D. Schmit, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.7/EE69<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH Gr<strong>an</strong>t R01-NS052509<br />
Title: Effects of tendon vibration on m<strong>an</strong>ual tracking tasks after stroke<br />
Authors: *M. O. CONRAD, R. A. SCHEIDT, B. D. SCHMIT;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: Stroke survivors commonly experience hemiparesis in <strong>the</strong> arm resulting in movements<br />
that lack coordination, have a reduced r<strong>an</strong>ge of motion, <strong>an</strong>d consist of poor endpoint control. In<br />
prior studies we established that a non-specific vibratory stimulus applied to <strong>the</strong> wrist<br />
musculature improves arm stability at <strong>the</strong> end of rapid point-to-point arm movements. In <strong>the</strong><br />
current study, we hypo<strong>the</strong>sized that tendon vibration applied at <strong>the</strong> wrist musculature would<br />
improve per<strong>for</strong>m<strong>an</strong>ce of a multijoint tracking task requiring integration of error feedback with<br />
<strong>the</strong> cortical pl<strong>an</strong>ning <strong>an</strong>d control of movement.<br />
Ten chronic stroke <strong>an</strong>d five neurologically intact subjects were seated as <strong>the</strong>y tracked a target<br />
through a horizontal figure-8 pattern while grasping <strong>the</strong> m<strong>an</strong>ipul<strong>an</strong>dum of a pl<strong>an</strong>ar robot. Stroke<br />
subjects completed a total of 36 trials (12 trials x 3 blocks) with <strong>the</strong>ir hemiparetic arm, while <strong>the</strong><br />
control group completed <strong>the</strong> experiment using <strong>the</strong>ir domin<strong>an</strong>t side. During <strong>the</strong> middle block of<br />
trials, custom made tendon vibrators (Faulhaber Group, Clearwater, FL) applied 70 Hz vibration<br />
to <strong>the</strong> <strong>for</strong>earm flexor musculature. Position, velocity <strong>an</strong>d electromyography data were evaluated<br />
to compare <strong>the</strong> quality of arm movements during pre-vibration, vibration <strong>an</strong>d post-vibration
trials.<br />
Despite tracking a target that moved at a const<strong>an</strong>t velocity, h<strong>an</strong>d trajectories made by both stroke<br />
<strong>an</strong>d control subjects appeared to be segmented into submovements. Such submovements were<br />
identifiable in t<strong>an</strong>gential velocity data as m<strong>an</strong>y single peaked, bell shaped speed pulses. <strong>When</strong><br />
tendon vibration was applied at <strong>the</strong> wrist musculature, stroke subjects experienced signific<strong>an</strong>tly<br />
improved tracking per<strong>for</strong>m<strong>an</strong>ce. Data supporting <strong>the</strong> improved quality of h<strong>an</strong>d movement<br />
included decreased h<strong>an</strong>d path lengths, a decrease in <strong>the</strong> st<strong>an</strong>dard deviation of h<strong>an</strong>d velocity <strong>an</strong>d<br />
increased endpoint stability. Improved tracking per<strong>for</strong>m<strong>an</strong>ce was accomp<strong>an</strong>ied by decreases in<br />
<strong>the</strong> muscle activity required to complete <strong>the</strong> task.<br />
Possible mech<strong>an</strong>isms behind improved movement control in response to tendon vibration may<br />
include improved sensorimotor integration in <strong>the</strong> brain, improved cortical modulation of spinal<br />
reflex activity or enh<strong>an</strong>ced cortical-cerebellar communication. This study is clinically relev<strong>an</strong>t in<br />
that it provides a real-time <strong>the</strong>rapeutic application that c<strong>an</strong> be used to increase stability in <strong>the</strong><br />
hemiparetic arm. Supported by NIH R01-NS052509.<br />
Disclosures: M.O. Conrad, None; R.A. Scheidt, None; B.D. Schmit, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.8/EE70<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: CREST of JST<br />
JSPS Research Fellowships <strong>for</strong> Young Scientists<br />
Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research on Priority Areas—Integrative Brain Research,<br />
<strong>from</strong> <strong>the</strong> MEXT<br />
Title: Ch<strong>an</strong>ges in regional brain activities involved in recovery of dexterous h<strong>an</strong>d movements<br />
after lesion of <strong>the</strong> primary motor cortex: PET study with macaque monkeys<br />
Authors: *Y. MURATA 1,2 , N. HIGO 1,3 , Y. NISHIMURA 3,4,5 , T. OISHI 3,6 , H. TSUKADA 3,7 , T.<br />
ISA 3,4,8 , H. ONOE 3,9 ;<br />
1 AIST, Tsukuba, Jap<strong>an</strong>; 2 JSPS, Tokyo, Jap<strong>an</strong>; 3 CREST, JST, Kawaguchi, Jap<strong>an</strong>; 4 Dept. Dev.<br />
Physiol., Natl. Inst. Physiol. Sci. (NIPS), Okazaki, Jap<strong>an</strong>; 5 Dept. Physiol. Biophys. &<br />
Washington Natl. Pri. Res. Ctr., Univ. Washington, Seattle, WA; 6 Dept. Cell. Mol. Biol., Primate
Res. Inst., Kyoto Univ., Inuyama, Jap<strong>an</strong>; 7 Central Res. Lab., Hamamatsu Photonics K. K.,<br />
Hamamatsu, Jap<strong>an</strong>; 8 Dept. Physiol. Sci. Grad., Univ. Adv. Stud., Hayama, Jap<strong>an</strong>; 9 Func. Probe<br />
Res. Lab., CMIS, RIKEN, Kobe, Jap<strong>an</strong><br />
Abstract: We previously reported that intensive motor training after <strong>the</strong> primary motor cortex<br />
(M1) lesion c<strong>an</strong> promote recovery of m<strong>an</strong>ual dexterity including precision grip in macaque<br />
monkeys (Murata et al., 2008, J Neurophysiol.). We also reported that expression of a plasticityrelated<br />
molecule, GAP-43, increased in <strong>the</strong> ventral premotor areas (PMv) after M1 lesion,<br />
suggesting that reorg<strong>an</strong>ization of neural circuits may occur in PMv (Higo et al., 2006, Neurosci<br />
Res Suppl). To clarify brain regions involved in functional compensation of dexterous h<strong>an</strong>d<br />
movements after M1 lesion, we measured regional cerebral blood flow (rCBF), as <strong>an</strong> index of<br />
brain activity, during per<strong>for</strong>ming a precision grip task using positron emission tomography<br />
(PET) with H2 15 O in two rhesus monkeys (Macaca mulatta). In <strong>the</strong> precision grip task, monkeys<br />
gripped a small cubic knob of a plastic disk, <strong>an</strong>d retrieved it <strong>from</strong> a tube through a slit aperture.<br />
A detailed map of h<strong>an</strong>d representation in M1 was determined using intracortical<br />
microstimulation (ICMS), followed by microinjection of ibotenic acid into <strong>the</strong> digit region.<br />
Immediately after injection, a complete impairment of digit movements was observed in <strong>the</strong><br />
contralateral h<strong>an</strong>d. After lesion, monkeys underwent a post-lesion training using <strong>the</strong> precision<br />
grip task (30 min/day, five days a week). During <strong>the</strong> first month after lesion, <strong>the</strong> success rate <strong>an</strong>d<br />
<strong>the</strong> use of precision grip during <strong>the</strong> task were recovered to almost <strong>the</strong> same level as be<strong>for</strong>e lesion.<br />
A series of PET sc<strong>an</strong>s was conducted be<strong>for</strong>e lesion, during <strong>the</strong> early recovery period (1 - 2<br />
months after lesion), <strong>an</strong>d during <strong>the</strong> late recovery period (3 - 4 months after lesion). Be<strong>for</strong>e<br />
lesion, per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> precision grip task was associated with <strong>an</strong> increase in rCBF in <strong>the</strong><br />
contralateral M1, primary somatosensory area (S1), <strong>an</strong>d <strong>an</strong>terior intraparietal area (AIP). We<br />
<strong>the</strong>n compared rCBF be<strong>for</strong>e <strong>an</strong>d after lesion to identify brain regions which were involved in<br />
functional compensation during <strong>the</strong> recovery period. After M1 lesion, signific<strong>an</strong>t increases of<br />
rCBF during <strong>the</strong> early recovery period were observed in <strong>the</strong> bilateral PMv. An increase of rCBF<br />
in <strong>the</strong> ipsi-lesional PMv was also observed during <strong>the</strong> late recovery period while increased area<br />
was smaller th<strong>an</strong> that during <strong>the</strong> early recovery period. The increase of activity in PMv is<br />
consistent with our previous finding of GAP-43 gene expression. These results strongly suggest<br />
that bilateral PMv plays a critical role in compensation of dexterous h<strong>an</strong>d movements after M1<br />
lesion in a time-dependent m<strong>an</strong>ner.<br />
Disclosures: Y. Murata, None; N. Higo, None; Y. Nishimura, None; T. Oishi, None; H.<br />
Tsukada, None; T. Isa, None; H. Onoe, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 568.9/EE71<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: Americ<strong>an</strong> Heart Association-Midwest Affiliate<br />
Title: Corticomotor excitability during pedaling post-stroke<br />
Authors: *S. M. SCHINDLER-IVENS, K. ONDISHKO, S. KNOBLAUCH, J. WIESER;<br />
Dept Physical Therapy, Marquette Univ., Milwaukee, WI<br />
Abstract: Abnormal muscle timing contributes to impaired locomotion post-stroke, but <strong>the</strong><br />
mech<strong>an</strong>isms are unclear. During pedaling, neurologically intact (NI) extensor muscles such as<br />
<strong>the</strong> soleus (SO) are active during <strong>the</strong> downstroke (1st 180 deg) <strong>an</strong>d are relatively inactive during<br />
<strong>the</strong> upstroke (2nd 180 deg). In contrast, paretic (PAR) extensor muscles display inadequate<br />
activity during downstroke <strong>an</strong>d extr<strong>an</strong>eous activity during upstroke. Our aim was to determine<br />
whe<strong>the</strong>r inappropriate activation of PAR extensor muscles is associated with altered<br />
corticomotor excitability. Tr<strong>an</strong>scr<strong>an</strong>ial magnetic stimulation (TMS) was used to probe<br />
corticomotor excitability to <strong>the</strong> SO during pedaling. We hypo<strong>the</strong>sized that, if ch<strong>an</strong>ges in<br />
corticomotor excitability contribute to inappropriate extensor muscle activity, <strong>the</strong>n responses to<br />
TMS would be abnormally modulated during pedaling. Thirteen individuals with chronic poststroke<br />
hemiparesis <strong>an</strong>d 12 NI individuals pedaled a bicycle ergometer at 40 RPM while TMS<br />
was used to elicit SO motor evoked potentials (MEPs) at 11 different positions in <strong>the</strong> pedaling<br />
cycle. SO MEPs <strong>an</strong>d background muscle activity were recorded <strong>an</strong>d normalized to M-max.<br />
Comparison was made between <strong>the</strong> amplitude of MEPs during pedaling <strong>an</strong>d tonic pl<strong>an</strong>tar flexion<br />
at comparable levels of SO activity <strong>an</strong>d equivalent cr<strong>an</strong>k positions. Two-way ANOVA <strong>an</strong>d<br />
simple effects <strong>an</strong>alyses were used to detect differences between conditions at p
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.10/EE72<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH Gr<strong>an</strong>t R01-NS052509<br />
Title: Asymmetry in acceleration-deceleration phases of 3D targeted reaching in hemiparetic<br />
stroke<br />
Authors: *K. SRUNGBOONMEE 1 , B. SCHMIT 2 ;<br />
2 Biomed. Engin., 1 Marquette Univ., Milwaukee, WI<br />
Abstract: In individuals with hemiparetic stroke, hyperexcitable reflexes <strong>an</strong>d spasticity affect<br />
<strong>the</strong> quality of arm movement. In particular, co-contraction of muscle throughout <strong>the</strong> arm c<strong>an</strong><br />
contribute to decreased smoothness of arm movements. Previous studies examining 2D<br />
multijoint reaching have shown <strong>the</strong> h<strong>an</strong>d speed profile is asymmetrical. Specifically, <strong>the</strong><br />
deceleration phase contains more “ripples” th<strong>an</strong> in <strong>the</strong> acceleration phase of <strong>the</strong> movement. In<br />
<strong>the</strong> current study we examined this phenomenon <strong>for</strong> 3D targeted reaching movements in healthy<br />
<strong>an</strong>d post-stroke individuals. The objective of this study was to characterize <strong>the</strong> asymmetry of <strong>the</strong><br />
h<strong>an</strong>d speed in stroke survivors <strong>an</strong>d <strong>the</strong> smoothness of movement in <strong>the</strong> hemiparetic reach.<br />
Eleven chronic stroke subjects with upper extremity hemiparesis <strong>an</strong>d five neurologically intact<br />
control subjects participated in this study. Six targets were presented horizontally on a board in 2<br />
rows (at <strong>the</strong> shoulder <strong>an</strong>d waist-height positions) <strong>an</strong>d vertically in 3 columns (50 cm apart). The<br />
subject’s paretic side was aligned with respect to <strong>the</strong> center of <strong>the</strong> board. Visual <strong>an</strong>d auditory<br />
cues were used to indicate <strong>the</strong> start of <strong>the</strong> task, <strong>an</strong>d <strong>the</strong> targets were presented in r<strong>an</strong>dom order.<br />
Subjects were instructed to reach as fast as possible towards each target. Kinematic data were<br />
collected using a camera-based tracking system. The wrist (i.e. endpoint) trajectory smoothness<br />
was qu<strong>an</strong>tified using a normalized jerk score (NJS).<br />
In both stroke <strong>an</strong>d control subjects, asymmetrical speed profiles were observed during 3D<br />
targeted reaching. The NJS of <strong>the</strong> deceleration phase was higher th<strong>an</strong> in <strong>the</strong> acceleration phase<br />
<strong>for</strong> both groups, but more pronounced in stroke. Stroke subjects’ endpoint trajectories had a<br />
higher NJS th<strong>an</strong> controls <strong>an</strong>d <strong>the</strong>ir NJS was correlated with <strong>the</strong>ir Fugl-Meyer score (p
The speed profile asymmetry was observed in both stroke <strong>an</strong>d healthy individuals, where <strong>the</strong><br />
deceleration phase had higher jerk th<strong>an</strong> <strong>the</strong> acceleration phase. This was more pronounced in <strong>the</strong><br />
speed profile of <strong>the</strong> hemiparetic arm compared to controls. Co-contraction in stroke subjects<br />
could be a contributing factor to <strong>the</strong> non-smooth movement.<br />
Disclosures: K. Srungboonmee, None; B. Schmit, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.11/EE73<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: Natural Sciences <strong>an</strong>d Engineering Research Council of C<strong>an</strong>ada<br />
Americ<strong>an</strong> Heart Association Gr<strong>an</strong>t in Aid<br />
Americ<strong>an</strong> Heart Association Beginning Gr<strong>an</strong>t in Aid<br />
National Institutes of Health<br />
Title: Neural reorg<strong>an</strong>ization in chronic stroke<br />
Authors: *C. M. CIRSTEA, W. M. BROOKS, S. C. CRACIUNAS, A. E. POPESCU, L. J.<br />
HUTFLES, C. R. SAVAGE, R. J. NUDO;<br />
Univ. K<strong>an</strong>sas Med. Ctr., K<strong>an</strong>sas City, KS<br />
Abstract: The development of effective novel stroke <strong>the</strong>rapies depends on successful tr<strong>an</strong>slation<br />
of c<strong>an</strong>didate interventions <strong>from</strong> <strong>an</strong>imal models to hum<strong>an</strong>s. Despite success in <strong>an</strong>imal models,<br />
m<strong>an</strong>y <strong>the</strong>rapeutic agents have not produced comparable results in clinical trials. Animal models<br />
do, however, provide insights into <strong>the</strong> cellular/molecular events underlying neural reorg<strong>an</strong>ization<br />
post-stroke, measurements difficult to obtain in <strong>the</strong> living hum<strong>an</strong>s. Insights into <strong>the</strong>se events in<br />
hum<strong>an</strong>s could be provided by magnetic reson<strong>an</strong>ce spectroscopy (1H-MRS).<br />
The goals of this study were: (i) to qu<strong>an</strong>tify concentrations of certain molecules<br />
(neurochemicals) in hum<strong>an</strong> cerebral tissue in remote motor areas (primary motor cortex, M1;<br />
premotor cortex, PM; supplementary motor area, SMA, 1H-MRS): <strong>an</strong>d (ii) to determine whe<strong>the</strong>r<br />
<strong>the</strong>se neurochemicals correlate with <strong>the</strong> clinical motor severity in subcortical stroke survivors.<br />
We studied neural reorg<strong>an</strong>ization <strong>from</strong> several perspectives: neuronal (N-acetylaspartate, NAA),
glial (myoinositol, mIn), <strong>an</strong>d glutamatergic (glutamate, Glu) system. The 1H-MRS<br />
measurements were guided by functional MRI to precisely identify h<strong>an</strong>d representation in remote<br />
motor areas. Gray matter (GM) volume measurements were used to control <strong>for</strong> atrophy effects<br />
upon calculated neurometabolite concentrations.<br />
The preliminary findings indicate that <strong>the</strong> neurochemical status of radiologically normalappearing<br />
of M1, PM <strong>an</strong>d SMA is altered in chronic phases of subcortical stroke. Specifically, in<br />
<strong>the</strong> injured hemisphere, we found neuronal (low NAA) <strong>an</strong>d glutamatergic (low Glu) alterations<br />
in M1, neuronal <strong>an</strong>d glial (high mIn) alterations in PM, <strong>an</strong>d neuronal <strong>an</strong>d glutamatergic<br />
alterations in SMA. In <strong>the</strong> uninjured hemisphere, we found neuronal <strong>an</strong>d glutamatergic<br />
alterations in M1, no biochemical alterations in PM, <strong>an</strong>d neuronal alterations in SMA. Although<br />
GM volumes were signific<strong>an</strong>tly decreased following stroke, no correlations were found between<br />
neurometabolite concentrations <strong>an</strong>d GM atrophy. The glutamate level in <strong>the</strong> injured hemisphere<br />
(M1, PM) was correlated with <strong>the</strong> clinical severity of arm motor impairment.<br />
Our findings provide evidence of long-lasting alterations of two cell types (neurons <strong>an</strong>d glia) <strong>an</strong>d<br />
dysfunctions of glutamatergic system in remote but interconnected cortical regions following a<br />
subcortical stroke. These results will assist <strong>the</strong> elucidation of neural mech<strong>an</strong>isms of functional<br />
recovery following stroke. Fur<strong>the</strong>r, this knowledge will help to develop pharmacological agents<br />
or procedures (i.e., neuropros<strong>the</strong>tic devices) targeting molecular level to facilitate <strong>the</strong> adaptive<br />
<strong>for</strong>ms of plasticity during recovery <strong>from</strong> stroke.<br />
Disclosures: C.M. Cirstea, None; W.M. Brooks, None; S.C. Craciunas, None; A.E. Popescu,<br />
None; L.J. Hutfles, None; C.R. Savage, None; R.J. Nudo, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.12/EE74<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NFNZ W &B Miller PhD Scholarship<br />
NFNZ Gr<strong>an</strong>t 3606666<br />
AMRF Gr<strong>an</strong>t 3606841<br />
Title: Priming <strong>the</strong> brain with <strong>the</strong>ta burst stimulation in chronic stroke patients
Authors: *S. J. ACKERLEY 1 , C. M. STINEAR 2 , P. A. BARBER 2 , W. D. BYBLOW 1 ;<br />
1 2<br />
Movement Neurosci. Lab., Dept. of Med. - Neurol., Univ. of Auckl<strong>an</strong>d, Auckl<strong>an</strong>d, New<br />
Zeal<strong>an</strong>d<br />
Abstract: This study evaluated <strong>the</strong> neurophysiological <strong>an</strong>d behavioural effects of a protocol of<br />
repetitive tr<strong>an</strong>scr<strong>an</strong>ial magnetic stimulation, <strong>the</strong>ta burst stimulation (TBS), delivered to <strong>the</strong><br />
primary motor cortex (M1) of chronic stroke patients prior to practice of a precision grip task.<br />
Ten chronic patients with subcortical stroke were recruited to this cross-over, sham-controlled,<br />
double-blinded study. Particip<strong>an</strong>ts received 600 pulses of TBS at 90% of contralesional M1<br />
active motor threshold, in 3 separate sessions. Intermittent TBS (iTBS) was delivered to<br />
ipsilesional M1, continuous TBS (cTBS) was delivered to contralesional M1, <strong>an</strong>d Sham TBS<br />
was delivered to ei<strong>the</strong>r M1. Be<strong>for</strong>e <strong>an</strong>d after TBS motor evoked potentials (MEPs) were<br />
recorded <strong>from</strong> first dorsal interosseous (FDI) bilaterally to assess ch<strong>an</strong>ge in corticomotor<br />
excitability. To qu<strong>an</strong>tify m<strong>an</strong>ual dexterity <strong>an</strong>d sensorimotor coordination, measures of grip <strong>an</strong>d<br />
lift <strong>for</strong>ces were derived using a custom grip-lift m<strong>an</strong>ipul<strong>an</strong>dum, be<strong>for</strong>e <strong>an</strong>d after TBS. Following<br />
<strong>the</strong> TBS protocol, particip<strong>an</strong>ts used <strong>the</strong> paretic upper limb to practice a precision grip task using<br />
a vari<strong>an</strong>t of <strong>the</strong> nine hole peg test <strong>for</strong> 16 minutes. Corticomotor excitability <strong>an</strong>d grip-lift data<br />
were obtained during <strong>an</strong>d after precision grip practice. All particip<strong>an</strong>ts completed <strong>the</strong> protocols<br />
with no adverse effects. Corticomotor excitability of <strong>the</strong> ipsilesional hemisphere was facilitated<br />
by iTBS. However, this facilitation was no longer evident after subsequent grip-lift task<br />
per<strong>for</strong>m<strong>an</strong>ce. There were no effects of iTBS on corticomotor excitability of <strong>the</strong> contralesional<br />
hemisphere. Nei<strong>the</strong>r cTBS nor Sham stimulation altered corticomotor excitability in ei<strong>the</strong>r<br />
hemisphere. Preload duration during <strong>the</strong> grip-lift task decreased after cTBS <strong>an</strong>d showed a trend<br />
to decrease after iTBS but not after Sham stimulation. The effects of real TBS persisted<br />
throughout subsequent precision grip practice. These results suggest that <strong>the</strong> effects of TBS on<br />
corticomotor excitability <strong>an</strong>d grip function are somewhat independent. Despite tr<strong>an</strong>sient (iTBS)<br />
or absent (cTBS) modulation of ipsilesional corticomotor excitability, both iTBS of ipsilesional<br />
M1 <strong>an</strong>d cTBS of contralesional M1 c<strong>an</strong> improve per<strong>for</strong>m<strong>an</strong>ce during subsequent practice of a<br />
precision grip task in chronic patients with subcortical stroke. Priming <strong>the</strong> brain with TBS<br />
<strong>the</strong>re<strong>for</strong>e c<strong>an</strong> open a <strong>the</strong>rapeutic window to promote use-dependent plasticity in <strong>the</strong>se patients.<br />
Disclosures: S.J. Ackerley, None; C.M. Stinear, None; P.A. Barber, None; W.D. Byblow,<br />
None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.13/EE75
Topic: D.17.d. Stroke, damage, or disease<br />
Support: PODSII, Foundation <strong>for</strong> Physical Therapy<br />
CA Physical Therapy Fund<br />
Title: Initial pl<strong>an</strong> <strong>an</strong>d compensatory adjustments of unconstrained reach actions after<br />
sensorimotor stroke<br />
Authors: *J. C. STEWART, J. GORDON, C. J. WINSTEIN;<br />
USC, Los Angeles, CA<br />
Abstract: Previous research has shown that individuals with stroke utilize both <strong>an</strong> initial pl<strong>an</strong><br />
<strong>an</strong>d compensatory adjustments when per<strong>for</strong>ming a single-joint action with <strong>the</strong> nonparetic arm<br />
(Fisher et al, 2000). It is unknown whe<strong>the</strong>r similar control is used when reaching with <strong>the</strong> paretic<br />
arm or under less constrained conditions. The purpose of this research was to determine whe<strong>the</strong>r<br />
individuals post-sensorimotor (SM) stroke use both <strong>an</strong> initial pl<strong>an</strong> <strong>an</strong>d compensatory adjustments<br />
<strong>for</strong> unconstrained reach actions to 3-D virtual targets. Six individuals post-SM stroke <strong>an</strong>d six<br />
age-matched, non-disabled adults reached quickly to 6 targets displayed in 2 directions (+45°, -<br />
45°) <strong>an</strong>d 3 dist<strong>an</strong>ces (8, 16, 24 cm) <strong>for</strong> a total of 120 trials with each arm. A deterministic<br />
multiple regression model (Gordon & Ghez, 1987) was used to determine <strong>the</strong> percent vari<strong>an</strong>ce of<br />
movement dist<strong>an</strong>ce explained by <strong>the</strong> initial pl<strong>an</strong> (initial peak acceleration) <strong>an</strong>d <strong>the</strong> percent<br />
explained by compensatory adjustments (adding target dist<strong>an</strong>ce). Me<strong>an</strong> endpoint error was<br />
larger, movement time longer, <strong>an</strong>d peak acceleration <strong>an</strong>d peak velocity lower <strong>for</strong> reaches to<br />
individual targets when reaching with <strong>the</strong> paretic <strong>an</strong>d nonparetic arms compared with <strong>the</strong><br />
matched arm in controls. Initial peak acceleration (pl<strong>an</strong>) varied in its ability to predict <strong>the</strong><br />
vari<strong>an</strong>ce in movement dist<strong>an</strong>ce in controls <strong>an</strong>d particip<strong>an</strong>ts with stroke when reaching with <strong>the</strong><br />
nonparetic arm (10-40%) but was <strong>an</strong> overall poor predictor when reaching with <strong>the</strong> paretic arm<br />
(3-12%). Adding target dist<strong>an</strong>ce (compensatory adjustments) to <strong>the</strong> model increased <strong>the</strong> vari<strong>an</strong>ce<br />
in movement dist<strong>an</strong>ce predicted when reaching with both arms but to a greater degree with <strong>the</strong><br />
nonparetic arm (total vari<strong>an</strong>ce 85-97%) compared with <strong>the</strong> paretic arm (65-80%). Adjustments<br />
between initial peak acceleration <strong>an</strong>d initial peak velocity accounted <strong>for</strong> a signific<strong>an</strong>t portion of<br />
<strong>the</strong> overall adjustments in controls <strong>an</strong>d in individuals with stroke reaching with <strong>the</strong> nonparetic<br />
arm (>60%) but not <strong>the</strong> paretic arm (
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.14/EE76<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: MJ Murdock Charitable Trust Foundation<br />
Title: Corticomotor excitation following robotic assisted movement post-stroke<br />
Authors: *C. T. LEONARD, S. LI, H. SIELER;<br />
Sch. of Physical Therapy <strong>an</strong>d Rehabil. Sci., Univ. Mont<strong>an</strong>a, Missoula, MT<br />
Abstract: Past studies have reported beneficial outcomes using robotic assisted movements of<br />
<strong>the</strong> upper extremity (UE) of stroke subjects. Mech<strong>an</strong>isms underlying <strong>the</strong>se improved outcomes<br />
remain unknown <strong>an</strong>d it remains open to question whe<strong>the</strong>r or not robotic assisted movement<br />
impart <strong>an</strong>y additional physiological or functional benefits over non-assisted repetitive voluntary<br />
movements. The primary purpose of this study was to assess corticomotor excitability following<br />
repetitive voluntary movements <strong>an</strong>d following robotic assisted movements. Specifically, we<br />
assessed ch<strong>an</strong>ges in TMS induced motor evoked potentials (MEPs) <strong>an</strong>d resting motor thresholds<br />
(rMTs) of <strong>the</strong> contralesional extensor carpi radialis (ECR) following non-fatiguing repetitive<br />
wrist extension movements. Methods: Eight chronic stroke patients with Fugl-Meyer UE scores<br />
r<strong>an</strong>ging <strong>from</strong> 27 to 51 have been tested. Subjects were positioned in <strong>the</strong> Portable Robotic Arm<br />
Movement Assistive Device (Patent Pending). The subjects voluntarily extended <strong>the</strong>ir wrist to<br />
maximal extension. This r<strong>an</strong>ge of motion was used as <strong>the</strong> threshold to trigger robotic assisted<br />
wrist extensions beyond this point (to subject com<strong>for</strong>t level). Protocol #1 involved 150<br />
repetitions of robot assisted wrist extensions. Protocol #2 involved 150 unassisted voluntary<br />
wrist extensions at <strong>the</strong> same cadence <strong>an</strong>d velocity as robotic assisted movements. Each protocol<br />
was r<strong>an</strong>domized <strong>an</strong>d took place 24 hours apart. Testing consisted of pre <strong>an</strong>d post MEP, (rMT),<br />
voluntary wrist extension r<strong>an</strong>ge of motion (ROM) <strong>an</strong>d maximal voluntary contraction (MVC)<br />
measurements. Results: Sufficient ECR MEPs at rest could only be obtained in 4 of <strong>the</strong> 8<br />
subjects. Two of <strong>the</strong> 4 had increases following voluntary movement (5.98+31.33%) whereas all<br />
subjects had reduced MEPs following robotic assisted movements (-39.91+2.57%). Voluntary<br />
wrist extension ROM increased in <strong>the</strong> robotic movement group only. No o<strong>the</strong>r ch<strong>an</strong>ges between<br />
<strong>the</strong> groups were discerned. Conclusions: Results are inconclusive but <strong>the</strong>se preliminary results<br />
would indicate that corticomotor excitation is not correlated with <strong>the</strong> robotic assisted wrist<br />
extension ROM gains in post-stroke subjects.<br />
Disclosures: C.T. Leonard, None; S. Li, None; H. Sieler, None.
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.15/EE77<br />
Topic: D.17.e. Cortical pl<strong>an</strong>ning <strong>an</strong>d execution<br />
Support: Sport <strong>an</strong>d Rehabilitation Engineering Program at EPFL<br />
Title: What apraxia tells us about <strong>the</strong> brain hemispheres’ contributions to hum<strong>an</strong> imitation<br />
Authors: *B. PETRESKA 1 , A. BILLARD 1 , J. HERMSDOERFER 2 , G. GOLDENBERG 3 ;<br />
1 LASA Lab. EPFL, Ecole Polytechnique Federale, Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d; 2 Clin.<br />
Neuropsychology Res. Group (EKN), Hosp. Muenchen-Bogenhausen, Munich, Germ<strong>an</strong>y;<br />
3 Neuropsychological Dept., Hosp. Muenchen-Bogenhausen, Munich, Germ<strong>an</strong>y<br />
Abstract: Praxic functions are frequently altered following brain lesion, giving rise to apraxia - a<br />
complex pattern of impairments that has proven difficult to assess or interpret. Here we revisit a<br />
study on imitation of me<strong>an</strong>ingless gestures in a patient with disconnected brain hemispheres. We<br />
per<strong>for</strong>med extensive statistical <strong>an</strong>alyses over several objective geometrical variables (e.g. h<strong>an</strong>d<br />
orientation) that revealed a clear dissociation in <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of imitating different goals. Our<br />
results show that <strong>the</strong> right hemisphere is perfectly capable of imitating a h<strong>an</strong>d posture with <strong>the</strong><br />
contralateral h<strong>an</strong>d, but not its position of contact with <strong>the</strong> face. We propose that <strong>the</strong> underlying<br />
deficit is incorrect coordination of <strong>the</strong> reproduction of multiple imitative goals. This accords well<br />
with a left hemisphere domin<strong>an</strong>ce <strong>for</strong> <strong>the</strong> body part coding of gestures. In addition, our results<br />
provide neuropsychological evidence in favor of <strong>the</strong> hypo<strong>the</strong>sis of goal-directed imitation.<br />
Finally, we found that only <strong>the</strong> stimuli with unnatural h<strong>an</strong>d postures were in<strong>for</strong>mative as <strong>the</strong>y<br />
were selectively impaired.<br />
Disclosures: B. Petreska, None; A. Billard, None; J. Hermsdoerfer, None; G. Goldenberg,<br />
None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.16/EE78<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH Gr<strong>an</strong>t 5R01HD039343-08<br />
Title: The effect of body position on upper extremity single <strong>an</strong>d multi-joint strength in<br />
individuals with chronic hemiparetic stroke<br />
Authors: *S. CHURCHILL 1 , D. KRAINAK 1,2 , M. GANDHI 1 , K. BURY 1 , E. PAVLOVICS 1 ,<br />
L. PEARSON 1 , M. D. ELLIS 1 , J. DEWALD 1,2,3 ;<br />
1 2 3<br />
Physical Therapy <strong>an</strong>d Hum<strong>an</strong> Movement Sci., Biomed. Engin., Physical Med. & Rehabil.,<br />
Northwestern Univ., Chicago, IL<br />
Abstract: Introduction: Individuals with moderate/severe chronic hemiparetic stroke have<br />
reduced reaching dist<strong>an</strong>ces associated with abnormal coupling of shoulder abduction <strong>an</strong>d elbow<br />
flexion. It has been suggested that <strong>the</strong> expression of this impairment is due to increased reli<strong>an</strong>ce<br />
on brainstem motor pathways following loss of corticofugal pathways due to stroke. Additional<br />
evidence <strong>for</strong> brainstem contributions, specifically <strong>from</strong> <strong>the</strong> vestibulospinal motor system, to <strong>the</strong><br />
voluntary control of <strong>the</strong> upper limb may be obtained by comparing <strong>the</strong> effect of body position on<br />
single <strong>an</strong>d dual task isometric strength in individuals with <strong>an</strong>d without stroke. We hypo<strong>the</strong>size<br />
that 1) <strong>the</strong>re will be a greater percent increase <strong>from</strong> supine to sit in individuals with stroke<br />
compared with <strong>the</strong> control group <strong>for</strong> maximum isometric elbow extension <strong>an</strong>d shoulder flexion<br />
strength, <strong>an</strong>d 2) individuals with stroke will produce greater elbow extension during submaximal<br />
shoulder abduction torque (50% of maximum) in sitting versus supine.<br />
Methods: Particip<strong>an</strong>ts include 11 subjects with hemiparesis due to chronic stroke (>1 year)<br />
without contractures or co-morbidities <strong>an</strong>d moderate to severe upper extremity impairment<br />
(average Fugl-Meyer 22.9/66). Ten adults without stroke or confounding comorbidity were used<br />
as a comparison group. Subjects per<strong>for</strong>med isometric maximum voluntary torques (MVT) in<br />
sitting <strong>an</strong>d supine recorded via 6-degrees-of-freedom load cell in six single tasks: shoulder<br />
abduction (SAB), shoulder adduction (SAD), shoulder flexion (SF), shoulder extension (SE),<br />
elbow flexion (EF), <strong>an</strong>d elbow extension (EE). Each particip<strong>an</strong>t also per<strong>for</strong>med a dual task<br />
maximizing EE torque while sustaining a submaximal (50%) SAB torque.<br />
Results: The percent increase MVT <strong>from</strong> supine to sitting was greater in individuals with stroke<br />
<strong>for</strong> SE <strong>an</strong>d EF, but not <strong>for</strong> o<strong>the</strong>r torque directions. Within group <strong>an</strong>alysis showed that both<br />
groups had greater SF <strong>an</strong>d EE MVT in sitting compared to supine. For <strong>the</strong> dual task, <strong>the</strong>re was<br />
no difference in maximum EE torque produced during 50% SAB across positions (sit/supine) <strong>for</strong><br />
both groups.<br />
Conclusion: Body position does increase arm extensor strength in sitting in both populations<br />
presumably due to <strong>an</strong> increase in vestibulospinal drive. Unique to <strong>the</strong> stroke population was <strong>the</strong><br />
increase in flexor activity measured in <strong>the</strong> paretic limb during sitting. However, body position<br />
does not appear to affect <strong>the</strong> ability of individuals with stroke to work outside <strong>the</strong> flexion pattern<br />
in a dual task. Future research of o<strong>the</strong>r dual tasks within <strong>the</strong> abnormal torque coupling patterns is<br />
necessary to fur<strong>the</strong>r explore this possibility.
Disclosures: S. Churchill, None; D. Krainak, Department of Biomedical Engineering,<br />
Northwestern University, O<strong>the</strong>r; M. G<strong>an</strong>dhi, None; K. Bury, None; E. Pavlovics, None; L.<br />
Pearson, None; M.D. Ellis, Northwestern University Department of Physical Therapy <strong>an</strong>d<br />
Hum<strong>an</strong> Movement Sciences, A. Employment (full or part-time); J. Dewald, Northwestern<br />
University Department of Physical Therapy <strong>an</strong>d Hum<strong>an</strong> Movement Sciences, A. Employment<br />
(full or part-time).<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.17/EE79<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH Gr<strong>an</strong>t K01HD047669<br />
NIH Gr<strong>an</strong>t T32HD007434<br />
Foundation <strong>for</strong> Physical Therapy<br />
Title: Moving bilaterally affects grip <strong>for</strong>ce <strong>an</strong>d motion during reach-grasp-lift movements early<br />
after stroke<br />
Authors: *S. L. DEJONG 1 , S. Y. SCHAEFER 2 , C. E. LANG 3 ;<br />
1 2<br />
Program in Physical Therapy, Program in Physical Therapy, Dept. of Biomed. Engin.,<br />
3<br />
Program in Physical Therapy, Program in Occup. Therapy, Dept. of Neurol., Washington Univ.,<br />
St Louis, MO<br />
Abstract: Previous studies have suggested that bilateral movement may improve per<strong>for</strong>m<strong>an</strong>ce in<br />
people with post stroke hemiparesis. We investigated grip <strong>for</strong>ce <strong>an</strong>d motion during unilateral<br />
versus bilateral reach-grasp-lift movements in people with hemiparesis three weeks after stroke<br />
<strong>an</strong>d in healthy adults, using 3D motion <strong>an</strong>alysis <strong>an</strong>d a vertical cylinder covered with a pressure<br />
mapping sensor. For palmar <strong>an</strong>d 3-finger grasp types, we measured duration of <strong>the</strong> reach phase,<br />
straightness of <strong>the</strong> reach path, <strong>an</strong>d <strong>the</strong> maximum dist<strong>an</strong>ce between <strong>the</strong> thumb <strong>an</strong>d index finger<br />
during <strong>the</strong> reach (peak aperture). Grip <strong>for</strong>ce variables included <strong>the</strong> time spent ramping up <strong>for</strong>ce<br />
prior to object lift-off, magnitude of <strong>the</strong> initial <strong>for</strong>ce peak, variability of <strong>for</strong>ce during <strong>the</strong> static<br />
phase of lift, <strong>an</strong>d time spent releasing <strong>for</strong>ce, relative to <strong>the</strong> time when <strong>the</strong> object returned to its<br />
starting position. Results showed that moving bilaterally affects grip <strong>for</strong>ce <strong>an</strong>d motion differently<br />
in people with hemiparesis th<strong>an</strong> in healthy adults. <strong>When</strong> reaching to grasp <strong>an</strong>d lift with <strong>the</strong> 3-
finger grasp type, <strong>the</strong> hemiparetic group showed faster but less direct arm motion when <strong>the</strong>y<br />
moved bilaterally instead of unilaterally. Lower initial <strong>for</strong>ce peaks <strong>an</strong>d a longer time to release<br />
<strong>for</strong>ce were also seen during bilateral movement, only <strong>for</strong> palmar grasping in <strong>the</strong> hemiparetic<br />
group. Results that approached signific<strong>an</strong>ce suggested more rapid <strong>for</strong>ce development in <strong>the</strong><br />
bilateral context, <strong>for</strong> both grasp types in <strong>the</strong> hemiparetic group. No signific<strong>an</strong>t effects of bilateral<br />
versus unilateral movement context were identified in <strong>the</strong> group of healthy adults. Implications<br />
<strong>for</strong> bilateral per<strong>for</strong>m<strong>an</strong>ce in people with hemiparesis may be ei<strong>the</strong>r positive (faster reach phase<br />
<strong>an</strong>d faster grip <strong>for</strong>ce development) or negative (less direct reach path, lower grip <strong>for</strong>ce, longer<br />
release time), <strong>an</strong>d may vary across grasp types. In addition to <strong>the</strong> above effects of bilateral<br />
movement context, expected effects of group <strong>an</strong>d grasp type were also evident. Compared to<br />
healthy adults, <strong>the</strong> hemiparetic group showed slower reaching, slower grip <strong>for</strong>ce development,<br />
<strong>an</strong>d more variable <strong>for</strong>ce during static lift. Compared to <strong>the</strong> 3-finger grasp, palmar grasp was<br />
associated with higher peak apertures, higher first <strong>for</strong>ce peaks <strong>an</strong>d higher variability during static<br />
lift. The findings of this study suggest that moving bilaterally may have mixed influence on grip<br />
<strong>for</strong>ce <strong>an</strong>d motion in people with hemiparesis.<br />
Disclosures: S.L. DeJong, None; S.Y. Schaefer, None; C.E. L<strong>an</strong>g, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.18/EE80<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NIH K01HD047669<br />
NIH T32HD007434<br />
Foundation <strong>for</strong> Physical Therapy<br />
Title: Contextual influences on reaching during reach-to-grasp following stroke<br />
Authors: *S. Y. SCHAEFER, S. L. DEJONG, C. E. LANG;<br />
Physical Therapy, Washington Univ., Saint Louis, MO<br />
Abstract: Daily activities such as brushing our teeth, opening a door, <strong>an</strong>d holding a glass are all<br />
tasks in which we use our upper extremities, but in different ways. Our movements are contextdependent,<br />
<strong>an</strong>d <strong>the</strong> ability to move our upper extremities in a variety of contexts is import<strong>an</strong>t to
daily life. Although studies in healthy adults have suggested <strong>the</strong> role of context in pl<strong>an</strong>ning <strong>an</strong>d<br />
controlling movement, it is not clear how context influences movement following stroke. The<br />
purpose of this study was to determine how movement context influences reaching per<strong>for</strong>m<strong>an</strong>ce<br />
during reach-to-grasp tasks following stroke. Specifically, we tested whe<strong>the</strong>r reach speed <strong>an</strong>d<br />
efficiency (reach path ratio) in hemiparetic patients 3 weeks post-stroke were differentially<br />
impaired when reaching to hold or lift <strong>an</strong> object using a precision (3-finger) grasp or a palmar<br />
grasp. We compared movements of <strong>the</strong> affected arm with those of healthy control subjects while<br />
reaching to <strong>an</strong> object of const<strong>an</strong>t size <strong>an</strong>d location. Reach speed <strong>for</strong> <strong>the</strong> hemiparetic group was<br />
lower th<strong>an</strong> <strong>for</strong> <strong>the</strong> control group, regardless of reach task, <strong>an</strong>d did not vary with grasp type.<br />
Reach speed did, however, ch<strong>an</strong>ge with grasp type in <strong>the</strong> control group, such that palmar<br />
grasping had faster reaches th<strong>an</strong> precision grasping. Although reach efficiency was not<br />
influenced by task, it was differentially affected by grasp type. In both palmar <strong>an</strong>d precision<br />
grasping, reach path ratios tended to be higher (less efficient) in <strong>the</strong> hemiparetic group th<strong>an</strong> in<br />
<strong>the</strong> control group; reaches within <strong>the</strong> control group, however, tended to be more efficient when<br />
reaching to grasp precisely. <strong>When</strong> examining ch<strong>an</strong>ges between 3 <strong>an</strong>d 12 weeks post-stroke, we<br />
found that <strong>the</strong> greatest improvement was <strong>the</strong> speed of reaching to lift <strong>an</strong> object with a palmar<br />
grasp. These results suggest that, even prior to grasping, arm movement is affected by how <strong>the</strong><br />
object will be grasped in healthy adults. It is possible that recovery of reaching ability post-stroke<br />
may be facilitated through different movement contexts that vary in speed <strong>an</strong>d accuracy<br />
requirements.<br />
Disclosures: S.Y. Schaefer, None; S.L. DeJong, None; C.E. L<strong>an</strong>g, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.19/EE81<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Title: Increased sensory feedback improves paretic limb end point control during targeted<br />
stepping post stroke<br />
Authors: *E. R. WALKER 1 , A. S. HYNGSTROM 2 , B. D. SCHMIT 1 ;<br />
1 Biomed. Engin., 2 Physical Therapy, Marquette Univ., Milwaukee, WI<br />
Abstract: Walking is a complex goal-oriented task that is modulated by reflexes <strong>an</strong>d sensory<br />
feedback. After stroke, motor <strong>an</strong>d sensory deficits, such as weakness <strong>an</strong>d decreased<br />
proprioception, c<strong>an</strong> lead to altered limb trajectories during walking. These altered trajectories
contribute to decreased end point control, affecting foot placement during walking. The purpose<br />
of this study was to evaluate whe<strong>the</strong>r increased sensory feedback <strong>from</strong> <strong>the</strong> paretic limb improves<br />
control of <strong>the</strong> paretic leg during a targeted stepping task. Five chronic stroke subjects<br />
participated in <strong>the</strong> study. During <strong>the</strong> targeted stepping task, subjects initiated <strong>the</strong> trial with <strong>the</strong>ir<br />
non-paretic limb <strong>an</strong>d stepped to one of five target locations projected onto <strong>the</strong> floor with <strong>the</strong>ir<br />
paretic limb. The five target locations were normalized to a percentage of <strong>the</strong> subject’s normal<br />
stride length. Subjects completed three blocks of 20 trials, <strong>an</strong>d during <strong>the</strong> second block, electrical<br />
stimulation was applied to <strong>the</strong> medial calc<strong>an</strong>eal nerve. The stimulation was delivered at a<br />
frequency of 30Hz, starting one second be<strong>for</strong>e movement initiation until <strong>the</strong> end of <strong>the</strong> trial. A<br />
six camera Vicon motion <strong>an</strong>alysis system <strong>an</strong>d <strong>for</strong>ce plates were used to record lower limb<br />
kinematics <strong>an</strong>d kinetics throughout <strong>the</strong> task. <strong>When</strong> electrical stimulation was applied to <strong>the</strong><br />
paretic limb, individuals followed a straighter path to <strong>the</strong> target <strong>an</strong>d decreased targeting error<br />
magnitude. The straighter trajectory of <strong>the</strong> paretic limb c<strong>an</strong> be attributed to decreased peak hip<br />
abduction during swing. In addition to decreased hip abduction, <strong>an</strong> increase in peak knee flexion<br />
was also observed during <strong>the</strong> stimulation block. However, no apparent ch<strong>an</strong>ges were observed in<br />
<strong>an</strong>kle kinematics. After stroke, increased hip abduction c<strong>an</strong> be used to compensate <strong>for</strong> decreased<br />
toe clear<strong>an</strong>ce during swing. The observed increased knee flexion may be used to maintain toe<br />
clear<strong>an</strong>ce due to <strong>the</strong> decreased hip adduction. Ch<strong>an</strong>ges in limb trajectory persisted when <strong>the</strong><br />
stimulation was removed, indicating a short-term adaptation. These preliminary results<br />
demonstrate <strong>an</strong> improvement in paretic limb control at <strong>the</strong> hip <strong>an</strong>d knee when increased sensory<br />
stimulation was applied to <strong>the</strong> paretic foot.<br />
Disclosures: E.R. Walker, None; A.S. Hyngstrom, None; B.D. Schmit, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.20/EE82<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: NHMRC (Australia) Peter Doherty Fellowship<br />
Title: Control of const<strong>an</strong>t low-level isometric <strong>for</strong>ce after stroke<br />
Authors: *P. A. MCNULTY;<br />
Prince Wales Med. Res. Inst., R<strong>an</strong>dwick, Australia
Abstract: M<strong>an</strong>y activities of daily living, r<strong>an</strong>ging <strong>from</strong> holding a telephone to carrying<br />
shopping, are critically dependent on stable low-level voluntary contractions with a const<strong>an</strong>t<br />
<strong>for</strong>ce output. Force steadiness has been measured previously after stroke, but only over short<br />
intervals such as 5-20s. We investigated <strong>for</strong>ce stability over a more functionally relev<strong>an</strong>t duration<br />
in hemiplegic stroke subjects. 10 subjects (between 1-13 years post stroke, me<strong>an</strong> 6.5 years; aged<br />
37-72) sat with <strong>the</strong> knee of <strong>the</strong> test leg extended to 120° <strong>an</strong>d <strong>the</strong> <strong>an</strong>kle at <strong>the</strong> mid-point of passive<br />
r<strong>an</strong>ge of motion. Dorsiflexion maximum voluntary contraction <strong>for</strong>ce was measured in this<br />
position during three brief (2-3 s) trials. Subjects <strong>the</strong>n produced ei<strong>the</strong>r 5 or 10% of <strong>the</strong>ir<br />
maximum <strong>an</strong>d once established, <strong>the</strong> target <strong>for</strong>ce was held <strong>for</strong> 6 minutes under different<br />
conditions including during a distraction task, <strong>an</strong>d with <strong>an</strong>d without visual feedback. Trials on<br />
each side were separated by 10 min rest, with <strong>the</strong> less affected side tested be<strong>for</strong>e <strong>the</strong> more<br />
affected side. Thus <strong>an</strong>y learning or training effect occurred on <strong>the</strong> less affected side. Surface<br />
EMG was recorded <strong>from</strong> tibialis <strong>an</strong>terior, peroneus longus <strong>an</strong>d triceps surae <strong>an</strong>d qu<strong>an</strong>tified <strong>for</strong><br />
each condition as <strong>the</strong> magnitude of <strong>the</strong> RMS processed signal. Force stability was measured as<br />
<strong>the</strong> me<strong>an</strong> square error <strong>from</strong> <strong>the</strong> target <strong>for</strong>ce. Signific<strong>an</strong>t differences in <strong>for</strong>ce stability were seen<br />
between sides (p
5R01HD047569-05<br />
Title: Ch<strong>an</strong>ges in cortical-muscular connectivity in frequency domain following stroke<br />
Authors: *J. YAO, J. P. DEWALD;<br />
Northwestern Univ., Chicago, IL<br />
Abstract: Introduction: M<strong>an</strong>y stroke survivors lose independent joint control in <strong>the</strong> affected<br />
limb. This study focused on <strong>the</strong> more prevalent abnormal coupling between shoulder abductors<br />
<strong>an</strong>d elbow flexors. This so-called flexor-synergy severely restricts <strong>the</strong> active r<strong>an</strong>ge of motion of<br />
<strong>the</strong> affected limb. The underlying neurological mech<strong>an</strong>isms responsible <strong>for</strong> its m<strong>an</strong>ifestation<br />
remain largely unknown, although <strong>an</strong> increased reli<strong>an</strong>ce on unaffected motor pathways following<br />
stroke has been implicated as a possible mech<strong>an</strong>ism. If it is true, associated ch<strong>an</strong>ges in Cortico-<br />
Muscular (C-M) connectivity should occur following stroke. In this study, we qu<strong>an</strong>tified ch<strong>an</strong>ges<br />
of C-M connectivity in frequency resolution, i.e., <strong>the</strong> ability to use distinct frequency<br />
components <strong>for</strong> C-M communication <strong>for</strong> muscles involved in shoulder or elbow joints.<br />
Methods: Four stroke <strong>an</strong>d 4 control subjects were recruited. Using visual feedback of shoulder<br />
abduction (SABD) torque, subjects were trained to generate single degree isometric SABD<br />
(sSABD) at 25% of <strong>the</strong>ir maximum voluntary torques. Three control subjects were fur<strong>the</strong>r<br />
trained <strong>for</strong> a matched SABD (mSABD) task by providing visual feedback showing both SABD<br />
<strong>an</strong>d EF torque levels generated by a matched stroke subject per<strong>for</strong>ming <strong>an</strong> sSABD task. Once<br />
trained, actual data collection was conducted without visual feedback. After reaching <strong>the</strong> target<br />
zone, subjects were asked to hold <strong>for</strong> 1 or 2 seconds. EEG, EMG <strong>an</strong>d torques during this holding<br />
phase was used to calculate <strong>the</strong> EEG-EMG coherence in 1s length windows. The frequency<br />
samples within <strong>the</strong> beta b<strong>an</strong>d that had signific<strong>an</strong>t values were used to qu<strong>an</strong>tify <strong>the</strong> histogram of<br />
EEG-EMG coherences in <strong>the</strong> frequency domain <strong>for</strong> each muscle. Subsequently, <strong>the</strong> overlap of<br />
<strong>the</strong> histogram between two muscles was qu<strong>an</strong>tified. An increased overlap indicates a decreased<br />
frequency resolution.<br />
Results: Our results showed that while control subjects use a narrowly distributed frequency<br />
b<strong>an</strong>d <strong>for</strong> C-M communication, stroke subjects use a signific<strong>an</strong>tly wider frequency b<strong>an</strong>d.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> overlap in <strong>the</strong> frequency b<strong>an</strong>ds between two muscles is signific<strong>an</strong>tly increased<br />
in stroke subjects. It is worthy to note that when control subjects per<strong>for</strong>ming <strong>the</strong> mSABD task as<br />
compared to <strong>the</strong> simple sSABD task, <strong>an</strong> even sharper frequency b<strong>an</strong>d was used.<br />
Discussions: The above results shown that <strong>the</strong>re is a loss of frequency resolution that is used <strong>for</strong><br />
C-M communication between different muscle pairs following stroke. The broadening of <strong>the</strong><br />
frequency distribution <strong>an</strong>d associated increase in overlap in C-M communication is attributed to<br />
<strong>an</strong> increase reli<strong>an</strong>ce on oligosynaptic cortico-bulbo-spinal pathways following stroke.<br />
Disclosures: J. Yao, None; J.P. Dewald, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.22/EE84<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: 5R01HD039343-07<br />
5R01HD047569-05<br />
Title: Reaching dist<strong>an</strong>ce c<strong>an</strong> be influenced by <strong>the</strong> asymmetric tonic neck reflex in chronic<br />
hemiparetic stroke survivors<br />
Authors: *S. LEE 1,2 , J. YAO 2 , A. ACOSTA 2 , J. DEWALD 2 ;<br />
1 Chicago, IL; 2 Northwestern Univ., Chicago, IL<br />
Abstract: In a previous study we showed that maximum isometric elbow flexion <strong>an</strong>d extension<br />
torques are affected by head rotation in moderately to severely impaired chronic stroke survivors.<br />
The effect of head rotation results in <strong>the</strong> expression of <strong>the</strong> asymmetric tonic neck reflex (ATNR)<br />
defined as <strong>an</strong> increase in elbow extension when <strong>the</strong> person rotates <strong>the</strong> head toward <strong>an</strong>d <strong>an</strong><br />
increase in elbow flexion when <strong>the</strong> head is rotated away <strong>from</strong> <strong>the</strong> paretic arm. The ATNR<br />
normally disappears in inf<strong>an</strong>cy but has been reported to reappear in patients with brain injury.<br />
We hypo<strong>the</strong>size that ATNR affects <strong>the</strong> reaching dist<strong>an</strong>ce of stroke survivors due to ch<strong>an</strong>ges in<br />
elbow extension torque generating ability as previously reported by our laboratory under static<br />
conditions. In order to investigate <strong>the</strong> effect of <strong>the</strong> ATNR during reaching movements, <strong>the</strong> Arm<br />
Coordination Training 3-D (ACT 3D ) device was used. The ACT 3D robot is able to simulate a<br />
virtual or haptic table, or provide different support levels. Three shoulder abduction loading<br />
conditions were tested in this study: table condition, 0%, <strong>an</strong>d 25% of maximum (Max) shoulder<br />
abduction (SABD) <strong>for</strong>ce. A total of 3 subjects with chronic hemiparetic stroke were studied <strong>an</strong>d<br />
per<strong>for</strong>med nine different experiment conditions. These consisted of three different head<br />
positions: away <strong>from</strong>, straight, <strong>an</strong>d toward <strong>the</strong> paretic arm, <strong>an</strong>d three different shoulder abduction<br />
<strong>for</strong>ce levels to investigate <strong>the</strong> effect of ATNR on reaching dist<strong>an</strong>ce. The effect of ATNR is<br />
reaching signific<strong>an</strong>ce at <strong>the</strong> 25% Max shoulder abduction condition (p=0.03). These preliminary<br />
observations demonstrate that when elbow extension is most compromised, as during 25% of<br />
max shoulder abduction, <strong>the</strong> effect of <strong>the</strong> ATNR on movement dist<strong>an</strong>ce is most apparent. The<br />
presence of ATNR in chronic stroke subjects suggests <strong>an</strong> enh<strong>an</strong>ced excitability of brainstem<br />
motor systems. In <strong>an</strong> ef<strong>for</strong>t to reduce <strong>the</strong> effect of impairment level on synergy- induced<br />
movement dist<strong>an</strong>ce, we pl<strong>an</strong> to determine <strong>the</strong> shoulder abduction loading that will result in 50%<br />
of <strong>the</strong> dist<strong>an</strong>ce <strong>from</strong> <strong>the</strong> home to <strong>the</strong> reaching target with <strong>the</strong> subject looking straight <strong>for</strong>ward.<br />
We believe that this will provide more conclusive evidence of <strong>the</strong> exact effects of ATNR on<br />
reaching abilities following stroke regardless of impairment level.
Disclosures: S. Lee, Jules Dewald, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Yao, None; A. Acosta,<br />
None; J. Dewald, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.23/EE85<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Title: Evidence of abnormal isometric trunk kinetics <strong>an</strong>d muscle activation patterns in chronic<br />
stroke subjects<br />
Authors: *S. PERLMUTTER, M. MAKHSOUS, F. LIN, M. BAJEMA, J. P. A. DEWALD;<br />
Northwestern Univ., Chicago, IL<br />
Abstract: Unlike <strong>the</strong> predomin<strong>an</strong>tly contralateral cortical control of <strong>the</strong> extremities, <strong>the</strong> trunk is<br />
thought to be bilaterally innervated by <strong>the</strong> brainstem as well as each of <strong>the</strong> cerebral cortices.<br />
Thus, it has <strong>the</strong>n been suggested that <strong>the</strong>se numerous bilateral projections may minimize trunk<br />
impairment in survivors of unilateral stroke. Yet, clinical measures show that trunk control poststroke<br />
<strong>an</strong>d <strong>the</strong> time course of recovery may not be different <strong>from</strong> what has been reported in <strong>the</strong><br />
paretic limbs. Thus, <strong>for</strong> <strong>the</strong> first time, this study identifies <strong>an</strong>d qu<strong>an</strong>tifies asymmetries in <strong>for</strong>ce<br />
<strong>an</strong>d torque generating abilities of <strong>the</strong> trunk, as well as associated EMG activity, in chronic stroke<br />
subjects.<br />
Using a custom-built device with a 6-DOF load cell, multi-directional isometric trunk control of<br />
stroke <strong>an</strong>d control subjects in a seated posture were evaluated. To date, one healthy control (62<br />
yrs) <strong>an</strong>d one chronic stroke subject (53 yrs, right hemisphere) have per<strong>for</strong>med maximum<br />
voluntary isometric <strong>for</strong>ces in sagittal <strong>an</strong>d coronal pl<strong>an</strong>es (flexion, extension, left <strong>an</strong>d right) while<br />
muscle activity was recorded <strong>from</strong> 14 muscles.<br />
Analysis of trunk kinetics showed that subjects’ produced larger <strong>for</strong>ces in <strong>the</strong> sagittal pl<strong>an</strong>e when<br />
compared to <strong>the</strong>ir respective coronal pl<strong>an</strong>e. Although, <strong>the</strong> control subject produced larger <strong>for</strong>ces<br />
th<strong>an</strong> <strong>the</strong> stroke subject in both pl<strong>an</strong>es with larger differences being seen in sagittal pl<strong>an</strong>e. The<br />
control produced less <strong>for</strong>ce <strong>an</strong>d torque in directions not related to <strong>the</strong> intended <strong>for</strong>ce direction,<br />
while <strong>the</strong> stroke subject generated larger axial trunk torques in all directions of <strong>for</strong>ce generation.<br />
Also, <strong>the</strong> stroke subject had asymmetries of muscle activity in all directions that differed <strong>from</strong><br />
<strong>the</strong> control. More specifically, large activity was seen in <strong>the</strong> ipsilateral-to-lesioned hemisphere<br />
(IL-H) trunk extensors of <strong>the</strong> stroke subject regardless of <strong>the</strong> intended direction of <strong>for</strong>ce<br />
generation.
These preliminary data suggests that, although <strong>the</strong> stroke subject generated less <strong>for</strong>ce th<strong>an</strong> <strong>the</strong><br />
control subject in all directions, larger <strong>for</strong>ces <strong>an</strong>d torques in untended directions of movement<br />
were observed. This suggests inefficient control as opposed to simply weakness of trunk<br />
musculature which may contribute to trunk discoordination. This is fur<strong>the</strong>r supported by <strong>the</strong> fact<br />
that <strong>the</strong>re were abnormal co-activations seen in <strong>the</strong> IL-H trunk extensors regardless of <strong>for</strong>ce<br />
direction. Thus, <strong>the</strong>se preliminary data suggest a deficit that affects <strong>the</strong> IL-H side of <strong>the</strong> trunk<br />
musculature. If <strong>the</strong>se findings remain consistent in a large sample size, <strong>the</strong>y may be explained by<br />
<strong>an</strong> increased reli<strong>an</strong>ce on bulbospinal pathways due to loss of corticofugal projections after<br />
unilateral stroke.<br />
Disclosures: S. Perlmutter, None; M. Makhsous, None; F. Lin, None; M. Bajema,<br />
None; J.P.A. Dewald, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.24/EE86<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: <strong>the</strong> Samsung Biomedical Research Institute gr<strong>an</strong>t (C-A7-407-1)<br />
02210)<br />
<strong>the</strong> Korea Science <strong>an</strong>d Engineering Foundation gr<strong>an</strong>t (M10644000022-06N4400-<br />
Title: Plasticity of <strong>the</strong> resting-state motor network during stroke recovery<br />
Authors: W. CHANG 1 , C.-H. PARK 1,2 , O. BANG 3 , S. KIM 4 , W.-K. YOO 6 , *Y.-H. KIM 1,5 ;<br />
1 Physical Med. <strong>an</strong>d Rehabil., 2 Samsung Biomed. Res., 3 Neurol., 4 Radiology, 5 Samsung Biomed.<br />
Res. Inst., Samsung Med. Cntr, Sungkyunkw<strong>an</strong> Univ., Seoul, Republic of Korea; 6 Physical Med.<br />
<strong>an</strong>d Rehabil., Hallym Univ. Sacred Heart Hosp., Any<strong>an</strong>g, Republic of Korea<br />
Abstract: Introduction<br />
Most fMRI studies investigating <strong>the</strong> reorg<strong>an</strong>ization of motor system were per<strong>for</strong>med by using<br />
task-based fMRI which has limitation in implicating to severely impaired stroke patients.<br />
Alternatively, we employed resting-state fMRI which exhibits spont<strong>an</strong>eous, low-frequency<br />
fluctuations of <strong>the</strong> fMRI signal in <strong>the</strong> absence of overt task per<strong>for</strong>m<strong>an</strong>ce. Our aim was to<br />
investigate <strong>the</strong> plastic ch<strong>an</strong>ges of <strong>the</strong> resting-state motor network in stroke patients during <strong>the</strong><br />
course of recovery.
Methods<br />
Twenty-nine stroke patients <strong>an</strong>d eleven healthy volunteers participated in this study. All patients<br />
underwent three times of resting-state fMRI sc<strong>an</strong>ning using 3.0T Philips Achieva sc<strong>an</strong>ner over<br />
three months after stroke: within 2 weeks, at 1 <strong>an</strong>d 3 months after stroke. During <strong>the</strong> sc<strong>an</strong>ning,<br />
subjects were instructed to keep <strong>the</strong>ir eyes closed <strong>an</strong>d remain motionless. Their motor function<br />
was assessed using motoricity index (MI) at each fMRI sc<strong>an</strong>ning <strong>an</strong>d at 6 month after stroke.<br />
After preprocessing of resting-state fMRI data, a motor network was obtained by correlating<br />
each voxel's time course with <strong>the</strong> reference time course of <strong>the</strong> ipsilesional primary motor cortex<br />
(M1). The motor network of <strong>the</strong> group of stroke patients at each time point was compared with<br />
those of <strong>the</strong> normal subjects. The motor network areas that showed signific<strong>an</strong>t correlation with<br />
long-term motor recovery were defined.<br />
Results<br />
Compared to <strong>the</strong> normal subjects, <strong>the</strong> stroke patients showed consistently higher functional<br />
connectivity of <strong>the</strong> ipsilesional M1 with <strong>the</strong> thalamus <strong>an</strong>d nonmotor areas such as <strong>the</strong> prefrontal<br />
cortex <strong>an</strong>d <strong>the</strong> inferior parietal cortex. The stroke patients also showed decreased functional<br />
connectivity of ipsilesional M1 with <strong>the</strong> contralesional M1 which me<strong>an</strong>t decreased laterality<br />
index of motor network connectivity. Functional connectivity of <strong>the</strong> <strong>the</strong> ipsilesional M1 with <strong>the</strong><br />
sensorimotor cortex, <strong>the</strong> secondary somatosensory cortex, <strong>the</strong> supplementary motor area, <strong>an</strong>d <strong>the</strong><br />
basal g<strong>an</strong>glia had a positive correlation with motor recovery as indexed by <strong>the</strong> total upper limb<br />
MI scores.<br />
ConclusionsOur study suggests that resting-state fMRI c<strong>an</strong> offer a distinctive approach <strong>for</strong><br />
identifying neural correlates of stroke recovery. (This study was supported by <strong>the</strong> Samsung<br />
Biomedical Research Institute gr<strong>an</strong>t (C-A7-407-1) <strong>an</strong>d <strong>the</strong> Korea Science <strong>an</strong>d Engineering<br />
Foundation gr<strong>an</strong>t (M10644000022-06N4400-02210).<br />
Disclosures: W. Ch<strong>an</strong>g, <strong>the</strong> Samsung Biomedical Research Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong> Korea<br />
Science <strong>an</strong>d Engineering Foundation gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); C. Park, <strong>the</strong><br />
Samsung Biomedical Research Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong> Korea Science <strong>an</strong>d Engineering<br />
Foundation gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); O. B<strong>an</strong>g, <strong>the</strong> Samsung Biomedical Research<br />
Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong> Korea Science <strong>an</strong>d Engineering Foundation gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); S. Kim, <strong>the</strong> Samsung Biomedical Research Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong> Korea Science <strong>an</strong>d<br />
Engineering Foundation gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); W. Yoo, <strong>the</strong> Samsung<br />
Biomedical Research Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong> Korea Science <strong>an</strong>d Engineering Foundation gr<strong>an</strong>t, B.<br />
Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as<br />
gr<strong>an</strong>ts already received); Y. Kim, <strong>the</strong> Samsung Biomedical Research Institute gr<strong>an</strong>t <strong>an</strong>d <strong>the</strong><br />
Korea Science <strong>an</strong>d Engineering Foundation gr<strong>an</strong>t, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.25/EE87<br />
Topic: D.17.i. Plasticity<br />
Title: Brain activation regions in <strong>the</strong> process of <strong>the</strong> functional recovery of hemiplegia patients<br />
with somatosensory deficit<br />
Authors: *T. MURAYAMA 1,2 , K. NUMATA 3 , T. KAWAKAMI 2 , T. TOSAKA 2 , N. OKA 4 , M.<br />
OGA 3 , J. TAKASUGI 1 , D. MATSUZAWA 1 , E. SHIMIZU 1 ;<br />
1 Chiba Univ., Chiba, Jap<strong>an</strong>; 2 Physical Therapy <strong>for</strong> Adult, Chiba Rehabil. Ctr., Chiba, Jap<strong>an</strong>;<br />
3 Ibaraki Prefectural Univ. of Hlth. Sci., Ibaraki, Jap<strong>an</strong>; 4 Chiba Ryogo Ctr., Chiba, Jap<strong>an</strong><br />
Abstract: Somatosensory deficits along with motor paralysis are common in <strong>the</strong> subcortical<br />
stroke patients. However, <strong>the</strong> process of functional recovery <strong>an</strong>d following brain activation<br />
regions after rehabilitation <strong>the</strong>rapy remain to be elucidated. In this study, we investigated <strong>the</strong><br />
brain activation pattern in <strong>the</strong> process of functional recovery after <strong>the</strong> constraint induced<br />
movement <strong>the</strong>rapy (CIMT) in hemiplegia patients with or without somatosensory deficits. Four<br />
subcortical stroke patients with mild right hemiplegia participated. Two of <strong>the</strong>m were suffering<br />
<strong>from</strong> severe somatosensory deficits due to thalamic lesion. Motor-sensory evaluation <strong>an</strong>d fMRI<br />
(auditory guided 1Hz paced right thumb-index finger tapping task, eyes closed) were per<strong>for</strong>med<br />
at 3 periods: pre-CIMT, post-CIMT <strong>an</strong>d 3 months after CIMT. Improvement in motor function<br />
was observed in all four subjects, but archived without recovery of somatosensory deficits in <strong>the</strong><br />
two patients. fMRI revealed activations in <strong>the</strong> contralateral primary sensorimotor cortex,<br />
supplementary motor area <strong>an</strong>d cerebellum at all periods in all four subjects. Ch<strong>an</strong>ges in <strong>the</strong><br />
cerebellum activation pattern were accomp<strong>an</strong>ied with motor recovery. Moreover, <strong>the</strong> activations<br />
in <strong>the</strong> inferior parietal lobe (BA40) <strong>an</strong>d ventral premotor cortex were observed at all periods in<br />
<strong>the</strong> patients with somatosensory deficits. It is suggested that those higher sensorimotor regions<br />
were compensatory involved in <strong>the</strong> motor execution <strong>an</strong>d <strong>the</strong> visual in<strong>for</strong>mation domin<strong>an</strong>tly<br />
contributed <strong>the</strong> motor function recovery in <strong>the</strong> patients with somatosensory deficits.<br />
Disclosures: T. Murayama, None; K. Numata, None; T. Kawakami, None; T. Tosaka,<br />
None; N. Oka, None; M. Oga, None; J. Takasugi, None; D. Matsuzawa, None; E. Shimizu,<br />
None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.26/EE88<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Title: The role of non-paretic quadriceps activation on paretic leg activity post-stroke<br />
Authors: *M. D. LEWEK, R. BRESLIN, L. HLAD, A. LANTON, J. ST. JOHN;<br />
Div. of Physical Therapy, Univ. of North Carolina At Chapel Hill, Chapel Hill, NC<br />
Abstract: Introduction: Muscle weakness <strong>an</strong>d inappropriate muscle activity are common<br />
following stroke. Paretic muscle activation may be influenced, in part, by volitional activation of<br />
<strong>the</strong> non-paretic limb. This pilot study extends our previous report to examine <strong>the</strong> role of nonparetic<br />
muscle activation on paretic thigh EMG activity <strong>an</strong>d knee joint torques during volitional<br />
<strong>an</strong>d reflexive tasks, respectively.<br />
Methods: Ten subjects with chronic stroke (time post-stroke: 29±22 months) sat on a Cybex to<br />
test knee extensor (KE) strength. The paretic sh<strong>an</strong>k was attached to <strong>the</strong> dynamometer <strong>an</strong>d <strong>the</strong><br />
non-paretic sh<strong>an</strong>k was rigidly attached to a load cell. EMG <strong>from</strong> <strong>the</strong> paretic quadriceps (VL,<br />
VM, <strong>an</strong>d RF) was recorded with independent KE torque data <strong>from</strong> each limb. For <strong>the</strong> volitional<br />
protocol, subjects per<strong>for</strong>med KE maximal voluntary isometric contractions (MVIC) under<br />
r<strong>an</strong>domly ordered conditions: (1) paretic leg only, (2) non-paretic leg only <strong>an</strong>d (3) both legs<br />
toge<strong>the</strong>r. Peak torque <strong>an</strong>d EMG were compared between conditions. For <strong>the</strong> reflex protocol, <strong>an</strong><br />
instrumented tendon tapper elicited a patellar tendon reflex of <strong>the</strong> paretic leg at a r<strong>an</strong>ge of<br />
tapping intensities while <strong>the</strong> non-paretic leg was (1) relaxed <strong>an</strong>d (2) maintaining 25% MVIC.<br />
The threshold to elicit a paretic quadriceps response was compared between conditions.<br />
Results: The sum of KE torques during <strong>the</strong> bilateral condition was less th<strong>an</strong> <strong>the</strong> sum of <strong>the</strong><br />
torques <strong>from</strong> <strong>the</strong> unilateral contractions (p=0.02). This was due to reduced torque in <strong>the</strong> nonparetic<br />
limb (p=0.02), but not <strong>the</strong> paretic limb (p=0.22), during <strong>the</strong> bilateral condition. During<br />
<strong>the</strong> bilateral condition, <strong>the</strong> paretic side generated greater relative, but less absolute torque th<strong>an</strong><br />
<strong>the</strong> non-paretic side (p=0.02). EMG of <strong>the</strong> paretic thigh was unaltered with activation of <strong>the</strong> nonparetic<br />
quadriceps (p>0.15). During <strong>the</strong> reflex test, <strong>the</strong> tendon tapping threshold of <strong>the</strong> paretic<br />
VL, <strong>an</strong>d VM muscles <strong>an</strong>d torque response decreased with non-paretic activity (p
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.27/EE89<br />
Topic: D.17.e. Cortical pl<strong>an</strong>ning <strong>an</strong>d execution<br />
Support: NIH Gr<strong>an</strong>t R01 MH077073<br />
Milton Fund<br />
Title: Procedural skill learning in Alzheimer's disease: Goal vs. action<br />
Authors: *D. PRESS, N. MOSHA, L. IGUCHI, D. COHEN, E. ROBERTSON;<br />
Dept Neurol., Beth Israel Deaconess Med. Ctr., Boston, MA<br />
Abstract: Background: One of <strong>the</strong> earliest signs of Alzheimer’s disease (AD) is a deficit in<br />
declarative memory, but procedural learning <strong>for</strong> motor skill is often preserved. Recent findings<br />
in procedural skill learning suggest that like declarative memory, procedural learning has<br />
different components. One aspect of a skill is action-based, tied to specific effectors, <strong>an</strong>d learned<br />
in body space. The o<strong>the</strong>r is goal-based, effector-independent <strong>an</strong>d learned in <strong>an</strong> extra-personal<br />
spatial framework. Each is subserved by distinct neural systems <strong>an</strong>d may be differentially<br />
impaired in AD.<br />
Methods: We measured procedural skill learning in patients with mild AD <strong>an</strong>d demographicallymatched<br />
controls using <strong>the</strong> Serial Reaction Time Task, a sequence learning task. After skill was<br />
acquired in <strong>the</strong>ir right, domin<strong>an</strong>t h<strong>an</strong>d, <strong>the</strong> particip<strong>an</strong>ts per<strong>for</strong>med <strong>the</strong> task in <strong>the</strong>ir contralateral<br />
h<strong>an</strong>d ei<strong>the</strong>r where <strong>the</strong> action, <strong>the</strong> sequence of finger movements, or where <strong>the</strong> goal, <strong>the</strong> sequence<br />
of stimuli on <strong>the</strong> screen, was maintained. Skill was defined as <strong>the</strong> shortening of reaction times<br />
during a block of a repeating 10-item sequence, as compared to a block of pseudo-r<strong>an</strong>dom trials<br />
that followed. Different 10-item sequences were used <strong>for</strong> <strong>the</strong> goal <strong>an</strong>d <strong>the</strong> action trials, with <strong>the</strong><br />
order of tasks counterbal<strong>an</strong>ced.<br />
Results: Preliminary results <strong>from</strong> Alzheimer’s particip<strong>an</strong>ts (n=7) <strong>an</strong>d controls (n=9) demonstrate<br />
that AD particip<strong>an</strong>ts do acquire a signific<strong>an</strong>t degree of skill in <strong>the</strong> SRTT (81 msec). Controls<br />
acquire similar skill (57 msec). While controls acquire roughly equal degrees of action-based<br />
skill (113 msec) <strong>an</strong>d goal-based skill (123 msec), AD particip<strong>an</strong>ts acquire more action- based<br />
skill (113 msec) th<strong>an</strong> goal-based skill (70 msec), albeit non-signific<strong>an</strong>tly.<br />
Conclusions: While most AD particip<strong>an</strong>ts with mild disease are capable of acquiring skill on a<br />
procedural, motor-sequence learning task, <strong>the</strong>ir profile of skill acquisition differs <strong>from</strong><br />
demographically-matched controls. AD particip<strong>an</strong>ts show a relative impairment in acquisition of
goal-based skill, with relative preservation of action-based skill. The findings support <strong>the</strong><br />
hypo<strong>the</strong>sis that different aspects of motor skills c<strong>an</strong> be separated <strong>an</strong>d differentially impaired by<br />
neurodegenerative disease.<br />
Disclosures: D. Press, None; N. Mosha, None; L. Iguchi, None; D. Cohen, None; E.<br />
Robertson, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.28/EE90<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: Department of Veter<strong>an</strong>s Affairs Rehabilitation Research <strong>an</strong>d Development Gr<strong>an</strong>t<br />
B4476R<br />
Title: Differentiating <strong>the</strong> effects of <strong>an</strong>terior versus posterior stroke in visuomotor adaptation<br />
Authors: *P. K. MUTHA 1,2 , R. L. SAINBURG 2 , K. Y. HAALAND 1,3 ;<br />
1 NM VA Healthcare Syst., Albuquerque, NM; 2 Kinesiology, Pennsylv<strong>an</strong>ia State Univ.,<br />
University Park, PA; 3 Psychiatry <strong>an</strong>d Neurol., Univ. of New Mexico, Albuquerque, NM<br />
Abstract: We recently showed signific<strong>an</strong>t differences in <strong>the</strong> m<strong>an</strong>ner in which left (LHD) <strong>an</strong>d<br />
right hemisphere damaged (RHD) patients adapted to a visuomotor rotation with <strong>the</strong>ir<br />
ipsilesional arm. While RHD patients learned <strong>the</strong> rotation by consistently ch<strong>an</strong>ging initial<br />
movement direction, LHD patients reduced movement error by making more effective online<br />
corrections. We now investigate whe<strong>the</strong>r damage to <strong>an</strong>terior or posterior regions within a<br />
hemisphere differentially affects adaptation. Previous research has highlighted <strong>the</strong> import<strong>an</strong>ce of<br />
posterior parietal regions <strong>for</strong> <strong>the</strong> recalibration of sensorimotor representations required during<br />
rotation learning. We <strong>the</strong>re<strong>for</strong>e expect that damage to posterior (POST-D), but not <strong>an</strong>terior brain<br />
regions (ANT-D) should impair adaptation. Twenty patients (11 ANT-D, 9 POST-D) <strong>an</strong>d 14<br />
healthy controls adapted to a 30 degrees counterclockwise rotation during a center-out reaching<br />
task per<strong>for</strong>med in eight directions. First, our comparison between LHD <strong>an</strong>d RHD groups showed<br />
trends similar to our previous report. We were thus able to replicate our findings with a different<br />
group of subjects with smaller, more focal lesions. Second, our data showed that when first<br />
exposed to <strong>the</strong> rotation, <strong>the</strong> ANT-D group showed large errors in movement accuracy. Over<br />
time, <strong>the</strong>se patients reduced final position errors, although <strong>the</strong> rate of adaptation was slower th<strong>an</strong><br />
that of healthy controls. Upon removal of <strong>the</strong> rotation, this group showed large after-effects in
final position. In contrast, <strong>the</strong> POST-D group showed smaller errors even during <strong>the</strong> first few<br />
trials of exposure to <strong>the</strong> rotation. These low errors were due to more efficient online corrections,<br />
as reflected by larger curvature of <strong>the</strong>ir movements. Moreover, this group showed reduced aftereffects<br />
when <strong>the</strong> rotation was removed, suggesting less effective sensorimotor remapping. These<br />
differences between <strong>the</strong> ANT-D <strong>an</strong>d POST-D groups were stronger in <strong>the</strong> RHD group. These<br />
findings indicate that <strong>an</strong>terior <strong>an</strong>d posterior brain regions play different, but specific roles in <strong>the</strong><br />
visuomotor adaptation process.<br />
Disclosures: P.K. Mutha, None; R.L. Sainburg, None; K.Y. Haal<strong>an</strong>d, None.<br />
Poster<br />
568. Stroke: Damage or Disease - Mech<strong>an</strong>isms of Abnormal Movement<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 568.29/EE91<br />
Topic: D.17.d. Stroke, damage, or disease<br />
Support: Medical Research Council<br />
Title: Reticulospinal <strong>an</strong>d ipsilateral corticospinal tract contributions to functional recovery after<br />
unilateral corticospinal lesion<br />
Authors: *B. ZAAIMI 1 , S. A. EDGLEY 2 , S. N. BAKER 1 ;<br />
1 Inst. Neurosci., Newcastle Univ., Newcastle upon Tyne, United Kingdom; 2 Dept. of Physiol.,<br />
Develop. <strong>an</strong>d Neuroscience,Cambridge Univ., Cambridge, United Kingdom<br />
Abstract: We recently reported (Riddle et al. <strong>2009</strong>, J Neurosci) inputs <strong>from</strong> <strong>the</strong> reticulospinal<br />
tract to motoneurones in primates. This included cells innervating h<strong>an</strong>d muscles, which was<br />
unexpected; control of <strong>the</strong> h<strong>an</strong>d is usually assigned to <strong>the</strong> corticospinal tract in higher primates.<br />
Our findings raised <strong>the</strong> ex<strong>citing</strong> possibility that <strong>the</strong> reticulospinal tract could subserve recovery<br />
of h<strong>an</strong>d use following corticospinal lesion. To test this idea, we made unilateral lesions of <strong>the</strong> left<br />
medullary pyramidal tract (PT) in three monkeys <strong>an</strong>d <strong>the</strong>n allowed recovery. Six months later we<br />
recorded intracellularly <strong>from</strong> right side cervical motoneurons under terminal <strong>an</strong>aes<strong>the</strong>sia. Cells<br />
were <strong>an</strong>tidromically identified by stimulation through cuff electrodes as projecting ei<strong>the</strong>r to<br />
<strong>for</strong>earm or h<strong>an</strong>d muscles. To activate central pathways, we impl<strong>an</strong>ted stimulating electrodes in<br />
<strong>the</strong> right medial longitudinal fasciculus (MLF, containing m<strong>an</strong>y reticulospinal axons) <strong>an</strong>d <strong>the</strong><br />
intact right PT. Motoneuron responses were classified as monosynaptic (segmental latency<br />
1ms, augmenting response to<br />
a train). Our preliminary <strong>an</strong>alysis measured 104 motoneuron responses. In <strong>for</strong>earm motoneurons,
15% responded to MLF stimuli with monosynaptic EPSPs (154±62µV, me<strong>an</strong> ± SEM); <strong>an</strong>d 40%<br />
with disynaptic EPSPs (308±48µV). For h<strong>an</strong>d motoneurons, 57% showed monosynaptic EPSPs<br />
to MLF (382±147µV), <strong>an</strong>d 45% responded disynaptically (281±77µV). Effects <strong>from</strong> stimulation<br />
of <strong>the</strong> intact (right) PT were very weak: 25% of h<strong>an</strong>d motoneurons had monosynaptic EPSPs<br />
(87±16µV); none had disynaptic EPSPs. 13% of <strong>for</strong>earm motoneurons had monosynaptic EPSPs<br />
(79±4µV) <strong>an</strong>d 3% had disynaptic EPSPs (65µV). Our preliminary results support <strong>the</strong> concept<br />
that streng<strong>the</strong>ning of reticulospinal connections may contribute to functional recovery following<br />
corticospinal tract lesion. By contrast, <strong>the</strong> contribution of <strong>the</strong> PT on <strong>the</strong> intact side appears<br />
minimal.<br />
Funded by <strong>the</strong> Medical Research Council.<br />
Keywords: reticulospinal, corticospinal, lesion, recovery, monkey<br />
Disclosures: B. Zaaimi, None; S.A. Edgley, None; S.N. Baker, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.1/EE92<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Training Program <strong>for</strong> Research M<strong>an</strong>agers in Integrated Arts <strong>an</strong>d Sciences, Hiroshima<br />
University<br />
Title: Social isolation stimulates hippocampal estradiol syn<strong>the</strong>sis<br />
Authors: *M. HATTORI, E. MUNETSUNA, K. TAKAHASHI, K. TAKEKAWA, A.<br />
ISHIDA, S. SAKATA, T. YAMAZAKI;<br />
Hiroshima Univ., Higashi-Hiroshima, Jap<strong>an</strong><br />
Abstract: Neurosteroids are syn<strong>the</strong>sized de novo <strong>from</strong> cholesterol in <strong>the</strong> central <strong>an</strong>d peripheral<br />
nervous systems through mech<strong>an</strong>isms that are independent of peripheral steroidogenic org<strong>an</strong>s.<br />
Neurosteroid syn<strong>the</strong>sis has been reported in various brain regions, including <strong>the</strong> hippocampus.<br />
Neurosteroids play import<strong>an</strong>t roles in brain function. For example, neurosteroid estradiol<br />
stimulates spine <strong>for</strong>mation. It has been demonstrated that estradiol plays import<strong>an</strong>t roles in<br />
processes such as synapse <strong>for</strong>mation <strong>an</strong>d neuroprotection in <strong>the</strong> hippocampus. Neurosteroid<br />
estradiol increases neuronal survival after different <strong>for</strong>ms of insults. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, it has<br />
also been demonstrated that neurosteroid allopregn<strong>an</strong>olone promotes proliferation of rodent<br />
neural progenitor cells.
Recently, <strong>the</strong> relationship between neurosteroidogenesis <strong>an</strong>d <strong>an</strong>imal housing condition has<br />
received increased attention. Social isolation decreased allopregn<strong>an</strong>olone level in <strong>the</strong> mouse<br />
olfactory bulb. This housing condition is reported to induce <strong>the</strong> decrease in 5α-reductase-1<br />
mRNA level in <strong>the</strong> mouse hippocampus. These data suggest that neurosteroidogenesis is affected<br />
by housing conditions. To date, however, most studies on <strong>the</strong> effects of social isolation on<br />
neurosteroidogenesis have focused on allopregn<strong>an</strong>olone syn<strong>the</strong>sis. No reports have appeared<br />
concerning <strong>the</strong> effects of social isolation on estradiol syn<strong>the</strong>sis, despite <strong>the</strong> fact that estradiol<br />
plays import<strong>an</strong>t roles in various brain regions such as hippocampus. The purpose of <strong>the</strong> present<br />
study was to <strong>an</strong>alyze <strong>the</strong> effects of social isolation on estradiol syn<strong>the</strong>sis in <strong>the</strong> rat hippocampus.<br />
[METHOD] Wistar rats on postnatal day 28 were r<strong>an</strong>domly divided into two groups: social<br />
isolation <strong>an</strong>d pair housed group. Socially isolated rats were housed individually in clear plastic<br />
cages while pair housed rats were housed two per cage in <strong>the</strong> same type of cage <strong>for</strong> 8 weeks.<br />
Hippocampus <strong>an</strong>d olfactory bulbs were freshly isolated <strong>from</strong> <strong>the</strong> whole brain of each rat, <strong>an</strong>d<br />
<strong>the</strong>n <strong>the</strong> mRNA contents of neurosteroidogenic enzymes in hippocampus <strong>an</strong>d olfactory bulbs<br />
were qu<strong>an</strong>tified by real-time RT-PCR.<br />
[RESULT] Social isolation activated <strong>the</strong> tr<strong>an</strong>scription of neurosteroidogenic molecules,<br />
including StAR <strong>an</strong>d P450arom. These two molecules are involved in <strong>the</strong> regulatory step <strong>for</strong><br />
steroidgenesis <strong>an</strong>d final step of estradiol syn<strong>the</strong>sis. In contrast, <strong>the</strong> mRNA levels were not<br />
affected in rat olfactory bulb. The hippocampal estradiol content was increased in accord<strong>an</strong>ce<br />
with <strong>the</strong> increased mRNA levels. These data suggest that social isolation may enh<strong>an</strong>ce de novo<br />
estradiol syn<strong>the</strong>sis in <strong>the</strong> hippocampus.<br />
Disclosures: M. Hattori, None; E. Munetsuna, None; K. Takahashi, None; K. Takekawa,<br />
None; A. Ishida, None; S. Sakata, None; T. Yamazaki, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.2/EE93<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Ministry of Education, Culture, Sports, Science <strong>an</strong>d Technology of Jap<strong>an</strong><br />
Ministry of Environment<br />
Title: Suppression of thyroid hormone-induced dendrite arborization of Purkinje cell by<br />
polybrominated diphenyl e<strong>the</strong>rs
Authors: *N. KOIBUCHI, K. IBHAZEHIEBO, N. SHIMOKAWA, T. IWASAKI;<br />
Dept. of Integrate Physiol., Gunma Univ. Grad. Sch. of Med., Maebashi/Gunma, Jap<strong>an</strong><br />
Abstract: Polybrominated dipheny e<strong>the</strong>rs (PBDEs) are a group of flame retard<strong>an</strong>t chemicals,<br />
which have been used in a variety of produces. PBDEs have become ubiquitous environmental<br />
contamin<strong>an</strong>ts <strong>an</strong>d have been detected in soil, air, sediments <strong>an</strong>d <strong>an</strong>imal tissues including hum<strong>an</strong>.<br />
Recent studies have shown that perinatal exposure to PBDE may alter spont<strong>an</strong>eous motor<br />
activity, <strong>an</strong>d disrupt learning <strong>an</strong>d memory. Although <strong>the</strong> molecular mech<strong>an</strong>isms of PBDE action<br />
have not yet been fully understood, studies have shown <strong>the</strong> possibilities that PBDE may act<br />
through <strong>the</strong> thyroid hormone system. To examine <strong>the</strong> mech<strong>an</strong>isms of PBDE action on thyroid<br />
hormone receptor (TR) -mediated tr<strong>an</strong>scription, we first per<strong>for</strong>med a tr<strong>an</strong>sient tr<strong>an</strong>sfection-based<br />
reporter assay. Several PBDE congeners suppressed TR-mediated tr<strong>an</strong>scription. The suppression<br />
was greatest with deca-BDE. This suppression is not mediated by competition of thyroid<br />
hormone binding to TR, but by partial dissociation of TR <strong>from</strong> thyroid hormone-response<br />
element of target gene. We fur<strong>the</strong>r confirmed <strong>the</strong> site of PBDE action within TR molecule using<br />
chimeric receptor generated <strong>from</strong> TR <strong>an</strong>d glucocorticoid receptor (GR). While all chimeric<br />
receptors harboring DNA-binding domain (DBD) of TR show suppression of tr<strong>an</strong>scription by<br />
deca-BDE regardless of <strong>the</strong> difference of lig<strong>an</strong>d binding domain, such suppression was not seen<br />
with receptors harboring DBD of GR, indicating that <strong>the</strong> site of PBDE action may be DBD of<br />
TR. Fur<strong>the</strong>rmore, PBDE signific<strong>an</strong>tly suppressed thyroid hormone-induced dendrite arborization<br />
of Purkinje cell in primary cerebellar culture derived <strong>from</strong> newborn rat. These results indicate<br />
that PBDE may disrupt thyroid hormone system by partial dissociation of TR <strong>from</strong> TRE, which<br />
may alter neuronal development.<br />
Disclosures: N. Koibuchi, Ministry of Environment, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); K.<br />
Ibhazehiebo, None; N. Shimokawa, None; T. Iwasaki, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.3/EE94<br />
Topic: B.08.c. LTP: Physiology <strong>an</strong>d behavior<br />
Support: HHMI Gr<strong>an</strong>t 52005137<br />
Title: The effects of P<strong>an</strong>ax ginseng extract <strong>an</strong>d constituent ginsenosides on long term synaptic<br />
plasticity in rat hippocampal slices
Authors: E. CHANG 1 , *J. T. KING 2 ;<br />
1 Program in Neurosci., 2 Program Neurosci, Pomona Col., Claremont, CA<br />
Abstract: Recently, much interest has been devoted to <strong>the</strong> study of various traditional Asi<strong>an</strong><br />
herbs using Western scientific techniques, such as electrophysiology <strong>an</strong>d qu<strong>an</strong>titative behavioral<br />
tasks. P<strong>an</strong>ax ginseng C.A. Meyer (PG), in particular, has been highlighted <strong>for</strong> its ameliorating<br />
effects on learning <strong>an</strong>d memory processes, including <strong>the</strong> induction of long-term potentiation<br />
(LTP). In <strong>the</strong> current study, we investigated <strong>the</strong> effects of <strong>an</strong> aqueous PG extract on LTP in <strong>the</strong><br />
CA1 subfield of rat hippocampal slices in vitro. Extracellular recordings of excitatory<br />
postsynaptic potentials were made following pretreatment with <strong>an</strong> aqueous PG extract or its<br />
constituent ginsenosides; high per<strong>for</strong>m<strong>an</strong>ce liquid chromatography electronspray ionization mass<br />
spectrometry (HPLC-ESI-MS) was utilized to identify <strong>an</strong>d qu<strong>an</strong>tify ginsenosides Rb1, Rb2, Rc,<br />
Rg1, <strong>an</strong>d Rg2 within <strong>the</strong> extract. Pretreatment with PG (12 or 4 ug/ml) signific<strong>an</strong>tly decreased<br />
LTP induction in a concentration dependent m<strong>an</strong>ner. The application of ginsenosides Rb1, Rb2,<br />
<strong>an</strong>d Rc also signific<strong>an</strong>tly decreased LTP, while <strong>the</strong> addition of ginsenosides Rg1 <strong>an</strong>d Rg2<br />
signific<strong>an</strong>tly increased LTP induction. These results suggest that PG alters LTP induction<br />
processes in vitro, though possibly in a different fashion th<strong>an</strong> in vivo. The ginsenosides likely<br />
contribute strongly to this effect, though <strong>the</strong>ir mech<strong>an</strong>ism of action remains unclear. The effect<br />
of both PG <strong>an</strong>d its constituent ginsenosides on LTP induction may underlie <strong>the</strong> ability of <strong>the</strong> herb<br />
to alter learning <strong>an</strong>d memory processes.<br />
Disclosures: E. Ch<strong>an</strong>g, None; J.T. King , None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.4/EE95<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Title: Long term ovariectomy elevates tr<strong>an</strong>slocator protein levels in <strong>the</strong> female rat brain:<br />
Implication <strong>for</strong> neurosteroidogenesis<br />
Authors: *S. HOROVITZ 1,2 , M. ARAD 2 , I. WEINER 2 , A. BIEGON 1,3 ;<br />
1 The Joseph Sagol Neurosci. Ctr., Ramat G<strong>an</strong>, Israel; 2 Dept. of Psychology, Tel Aviv Univ., Tel<br />
Aviv, Israel; 3 Med. Dept., Brookhaven Natl. Lab., Upton, NY<br />
Abstract: Background. The mammali<strong>an</strong> brain expresses numerous steroidogenic enzymes<br />
which constitute <strong>the</strong> molecular machinery required <strong>for</strong> local de novo sex steroid syn<strong>the</strong>sis, a
process known as neurosteroidogenesis. Cessation of ovari<strong>an</strong> function (menopause), <strong>the</strong> main<br />
source of sex steroids in females, is associated with vasomotor symptoms such as hot flashes <strong>an</strong>d<br />
psychological effects such as mood swings. Interestingly, most of <strong>the</strong>se symptoms are tr<strong>an</strong>sient<br />
by nature, taking 6 months to a few years to clear, despite continuously low peripheral levels of<br />
estrogen. This observation led us to hypo<strong>the</strong>size that chronic estrogen deprivation induces a slow<br />
compensatory increase in neurosteroidogenesis, mediated by <strong>the</strong> cholesterol tr<strong>an</strong>slocator protein<br />
(TSPO, PBR), <strong>the</strong> first <strong>an</strong>d rate-limiting step in <strong>the</strong> process. Here, we have tested whe<strong>the</strong>r<br />
chronic estrogen deprivation induced by ovariectomy (OVX) in rats would result in a<br />
compensatory increase in TSPO expression in <strong>the</strong> brain in a region- <strong>an</strong>d time-dependent m<strong>an</strong>ner.<br />
Methods. Animals underwent OVX/sham procedures <strong>an</strong>d were sacrificed after a short term (1<br />
week post OVX) or a long term (3 <strong>an</strong>d 6 months post OVX). Using autoradiography,<br />
qu<strong>an</strong>tification of TSPO binding levels was per<strong>for</strong>med on consecutive cryostat brain sections<br />
incubated with [ 3 H]PK11195, a TSPO lig<strong>an</strong>d. Statistical <strong>an</strong>alysis was done by 2 way ANOVA<br />
with repeated measures (surgery x time with region as <strong>the</strong> repeated measure) followed by<br />
appropriate posthoc comparisons. Results. [3H]PK11195 binding showed a time-dependent<br />
increase in <strong>the</strong> brains of OVX <strong>an</strong>imals (surgery x term interaction, p
Title: Retinoic acid stimulates estradiol <strong>an</strong>d testosterone syn<strong>the</strong>sis in rat hippocampal slice<br />
cultures<br />
Authors: *T. YAMAZAKI 1 , E. MUNETSUNA 1,2 , Y. HOJO 3 , M. HATTORI 1 , A. ISHIDA 1 , S.<br />
KAWATO 3 ;<br />
1 Integrated Arts <strong>an</strong>d Sci., Hiroshima Univ., Higashi-Hiroshima, Jap<strong>an</strong>; 2 Present address: Dept. of<br />
Biotechnology, Toyama Prefectural Univ., Toyama, Jap<strong>an</strong>; 3 The Univ. of Tokyo, Tokyo, Jap<strong>an</strong><br />
Abstract:<br />
The hippocampus is essentially involved in learning <strong>an</strong>d memory processes. Its functions are<br />
affected by various neuromodulators, including 17β-estradiol, testosterone <strong>an</strong>d retinoid. Brainsyn<strong>the</strong>sized<br />
steroid hormones act as autocrine <strong>an</strong>d paracrine modulators. The regulatory<br />
mech<strong>an</strong>ism underlying brain steroidogenesis has not been fully elucidated. Although <strong>the</strong> function<br />
of <strong>the</strong> retinoic acids <strong>an</strong>d sex steroids in <strong>the</strong> hippocampus are similar, cross-talk between thier<br />
signaling pathways has not been focused on. Syn<strong>the</strong>sis of sex steroids in <strong>the</strong> gonads is stimulated<br />
by retinoic acids. There<strong>for</strong>e, we examined <strong>the</strong> effects of retinoic acids on estradiol <strong>an</strong>d<br />
testosterone biosyn<strong>the</strong>sis in <strong>the</strong> rat hippocampus.<br />
We used cultured hippocampal slices <strong>from</strong> 10-12-day-old male rats to investigate de novo<br />
steroidogenesis. The inf<strong>an</strong>t rat hippocampus possesses mRNAs <strong>for</strong> steroidogenic enzymes <strong>an</strong>d<br />
retinoid receptors. Slices were used after 24 hours of pre-culture to obtain maximal steroidogenic<br />
activity, because steroidogenesis in cultured slices decreases by longer pre-incubation. mRNA<br />
contetns in <strong>the</strong> slices were qu<strong>an</strong>tified by real-time RT-PCR. Protein content of P45017α was<br />
determined by Western blotting. The levels of steroids were <strong>an</strong>alyzed by RIA or EIA after<br />
purification by HPLC <strong>an</strong>d/or mini-column.<br />
The mRNA levels <strong>for</strong> P45017α, P450arom <strong>an</strong>d estrogen receptor β in <strong>the</strong> slices were increased by<br />
treatment with 9-cis-retinoic acid, but not by all-tr<strong>an</strong>s-isomer. The magnitude of stimulation <strong>an</strong>d<br />
<strong>the</strong> shape of <strong>the</strong> dose-response curve <strong>for</strong> <strong>the</strong> mRNA level <strong>for</strong> P45017α were similar to those <strong>for</strong><br />
cellular retinoid binding protein type-2, tr<strong>an</strong>scription of which is activated by retinoid X receptor<br />
signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in <strong>the</strong> protein content of P45017α,<br />
<strong>an</strong>d a 2-fold increase in de novo syn<strong>the</strong>sis of 17β-estradiol <strong>an</strong>d testosterone. These steroids may<br />
be syn<strong>the</strong>sized <strong>from</strong> a steroid precursor(s), such as pregnenolone or o<strong>the</strong>r steroids, or <strong>from</strong><br />
cholesterol, as so-called neurosteroids. The stimulation of estradiol <strong>an</strong>d testosterone syn<strong>the</strong>sis by<br />
9-cis-retinoic acid might be caused by activation of P45017α tr<strong>an</strong>scription via retinoid X receptor<br />
signaling.<br />
Disclosures: T. Yamazaki, None; E. Munetsuna, None; Y. Hojo, None; M. Hattori, None; A.<br />
Ishida, None; S. Kawato, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.6/EE97<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Bioin<strong>for</strong>matics JST Jap<strong>an</strong><br />
CREST Jap<strong>an</strong><br />
Title: Rapid modulation of spines <strong>an</strong>d LTD/LTP by estrogen <strong>an</strong>d <strong>an</strong>drogen in rat hippocampus:<br />
Neuro-intracrinology<br />
Authors: *S. KAWATO 1 , H. MUKAI 1 , Y. HOJO 1 , Y. HATANAKA 1 , S. HIGO 1 , G.<br />
MURAKAMI 2 , Y. OOISHI 1 , T. KIMOTO 1 , T. KOMINAMI 1 ;<br />
1 2<br />
Dept Biophys & Life Sci, Grad Sch. Arts & Sci., Univ. of Tokyo, Tokyo, Jap<strong>an</strong>; Rockefeller<br />
Univ., New York, NY<br />
Abstract: Estradiol (E2)-induced rapid modulation (within 2 h) was <strong>an</strong>alyzed about spinogenesis<br />
<strong>an</strong>d LTD/ LTP in adult male rat hippocampal slices. Spine <strong>an</strong>alysis was per<strong>for</strong>med by dyeinjected<br />
single pyramidal neurons in <strong>the</strong> hippocampal slices. E2 at 1-10 nM rapidly increased <strong>the</strong><br />
density of spines. in CA1 pyramidal neurons, but in CA3 estradiol decreased <strong>the</strong> density of<br />
(spine-like) thorns which are postsynaptic pairs of mossy fiber terminals. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, both<br />
testosterone (T) <strong>an</strong>d dihydrotetosterone (DHT) increased <strong>the</strong> density of CA1 spines as well as<br />
CA3 thorns. MAP kinase was involved in <strong>the</strong>se processes. Multielectrode <strong>an</strong>alysis showed that<br />
LTD was signific<strong>an</strong>tly enh<strong>an</strong>ced by 1- 10 nM estradiol in CA1 <strong>an</strong>d CA3. ERalpha agonist but<br />
not ERbeta agonist, induced <strong>the</strong> same effect as that of estradiol in both spinogenesis <strong>an</strong>d LTD.<br />
These rapid responses were driven via ERalpha that was found at synapses of glutamatergic<br />
neurons. Mass-spectrometric <strong>an</strong>alysis demonstrated that even after castration to deplete<br />
circulating T, <strong>the</strong> male hippocampal E2 level was not decreased, indicating that E2, syn<strong>the</strong>sized<br />
within <strong>the</strong> hippocampus, plays <strong>an</strong> essential role <strong>for</strong> rapid synaptic plasticity. Note that castration<br />
decreased T <strong>from</strong> 18 nM to 3 nM in <strong>the</strong> hippocampus.<br />
Disclosures: S. Kawato, None; H. Mukai, None; Y. Hojo, None; Y. Hat<strong>an</strong>aka, None; S.<br />
Higo, None; G. Murakami, None; Y. Ooishi, None; T. Kimoto, None; T. Kominami, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 569.7/EE98<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: R37 AG06647<br />
PO1 AG16765<br />
Title: Synaptic estrogen receptor alpha (ER-α) is present in prefrontal cortex <strong>an</strong>d correlated with<br />
cognitive per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: *A.-J. WANG 1 , Y. HARA 2 , W. LOU 4 , W. G. M. JANSSEN 2 , P. RAPP 5 , J. H.<br />
MORRISON 3 ;<br />
1 Mount Sinai Sch. Med., New York, NY; 2 Neurosci., Mount Sinai Sch. Med., NewYork, NY;<br />
3 Neurosci., Mount Sinai Sch. Med., New York, NY; 4 Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada;<br />
5 Neurocognitive Aging Section, Natl. instution on aging, Baltimore, MD<br />
Abstract: Long-term cyclic estrogen (E) protects against age-related impairment of cognitive<br />
function mediated by <strong>the</strong> dorsolateral prefrontal cortex (dlPFC) in ovariectomized (OVX) female<br />
rhesus monkeys. Improved cognitive function is accomp<strong>an</strong>ied by <strong>an</strong> increase in spine density in<br />
layer III dlPFC pyramidal neurons (Hao, et.al 2007). The ability of E to rescue spines <strong>an</strong>d<br />
dlPFC-dependent cognitive function suggests that <strong>the</strong> molecular mech<strong>an</strong>ism that responds to E<br />
remains intact in aged <strong>an</strong>imals. To test this hypo<strong>the</strong>sis, we examined synaptic distribution of<br />
estrogen receptor alpha (ER-α) in twelve young <strong>an</strong>d fourteen aged female rhesus monkeys with<br />
or without E treatment (Y+Veh, Y+E, A+Veh, A+E). These <strong>an</strong>imals were all tested on Delayed<br />
Response (DR), a cognitive task dependent on dlPFC. We <strong>the</strong>n per<strong>for</strong>med postembeding<br />
immunogold electron microscopy to determine <strong>the</strong> distribution of ER-α in axospinous (AS)<br />
synapses in area 46 of dlPFC. Approximately 50% of AS synapses were labeled with ER-α<br />
regardless of age or treatment. The abund<strong>an</strong>ce of ER-α as reflected by numbers of gold particles<br />
in presynaptic regions was twice that of postsynaptic regions in all groups. There was a positive<br />
correlation between % ER-α labeling <strong>an</strong>d DR accuracy (P=0.021), in Y+Veh <strong>an</strong>d A+Veh but not<br />
in E treated groups. Fur<strong>the</strong>rmore, <strong>the</strong> pre-synaptic ER-α labeling had a positive correlation with<br />
DR accuracy (P=0.004) Thus, synaptic ER-α is clearly retained in dlPFC of aged female rhesus<br />
monkeys, <strong>an</strong>d OVX monkeys may rely on locally syn<strong>the</strong>sized E <strong>an</strong>d presynaptic ER-α to sustain<br />
per<strong>for</strong>m<strong>an</strong>ce dlPFC-dependent tasks.<br />
Disclosures: A. W<strong>an</strong>g , None; Y. Hara, None; W. Lou, None; W.G.M. J<strong>an</strong>ssen, None; P.<br />
Rapp, None; J.H. Morrison, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.8/EE99<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: NIH Gr<strong>an</strong>t MH065990 (RSC)<br />
Title: Hetereogeneity of dose <strong>an</strong>d time effects of estradiol benzoate (EB) on neuron-specific<br />
protein (NeuN) <strong>an</strong>d phosphorylated cyclic AMP response element-binding protein (pCREB) in<br />
<strong>the</strong> hippocampus of ovariectomized rats<br />
Authors: *B. W. BAKKUM 1,2 , L. FAN 2 , R. S. COHEN 2 ;<br />
1 2<br />
Illinois Coll Optometry, Chicago, IL; Dept. of Anat. <strong>an</strong>d Cell Biol., Univ. of Illinois at<br />
Chicago, Chicago, IL<br />
Abstract: Estrogen effects in <strong>the</strong> hippocampus may depend on several factors, including<br />
hormone dose <strong>an</strong>d length of treatment. Because of <strong>the</strong> potential effects of estrogen on cell <strong>an</strong>d<br />
synaptic growth <strong>an</strong>d survival via CREB-related gene products, we examined <strong>the</strong> effects of high<br />
(10µg <strong>for</strong> 14 days) or moderate (2.5µg <strong>for</strong> 4 or 14 days) regimens of EB on me<strong>an</strong> numbers of<br />
NeuN- <strong>an</strong>d pCREB-immunolabled neuronal <strong>an</strong>d nuclear numbers, respectively, <strong>an</strong>d brain region<br />
volume defined by NeuN <strong>an</strong>d pCREB in <strong>the</strong> CA1, CA2 <strong>an</strong>d CA3 regions, dorsal (DDG) <strong>an</strong>d<br />
ventral (VDG) dentate gyrus, <strong>an</strong>d hilus of <strong>the</strong> hippocampus in young, adult, Sprague Dawley,<br />
ovariectomized rats by stereology with StereoInvestigator (MicroBrightField). <strong>When</strong> <strong>an</strong>alyzing<br />
<strong>the</strong> extent of <strong>the</strong> entire hippocampus, all regions showed a signific<strong>an</strong>t increase in <strong>the</strong><br />
me<strong>an</strong> number of NeuN- <strong>an</strong>d pCREB-immunolabeled neuronal <strong>an</strong>d nuclear numbers,<br />
respectively, <strong>an</strong>d <strong>an</strong> increase volume defined by NeuN staining or pCREB labeling with all EB<br />
regimens compared to <strong>the</strong> vehicle, sesame oil (SO). We also <strong>an</strong>alyzed <strong>the</strong> same parameters in <strong>the</strong><br />
a<strong>for</strong>ementioned regions (except <strong>the</strong> hilus) of <strong>the</strong> rostral hippocampus. In terms of <strong>the</strong><br />
me<strong>an</strong> number of NeuN-immunolabeled neuronal numbers, <strong>the</strong> CA1 <strong>an</strong>d CA2, but not CA3,<br />
regions <strong>an</strong>d DDG <strong>an</strong>d VDG displayed a signific<strong>an</strong>t increase in this parameter compared to SO<br />
with all regimens. There was a signific<strong>an</strong>t increase in me<strong>an</strong> pCREB-labeled nuclear numbers<br />
with all EB regimens compared to SO only in CA1. Only <strong>the</strong> moderate EB regimens resulted in a<br />
signific<strong>an</strong>t increase in <strong>the</strong> me<strong>an</strong> pCREB-immunolabeled nuclear numbers over SO in <strong>the</strong> CA3,<br />
DDG <strong>an</strong>d VDG; <strong>the</strong> high regimen showed values that were not different <strong>from</strong> those of SO. For<br />
volume defined by NeuN immunolabeling, <strong>the</strong> CA3, but not CA1 or CA2, region displayed a<br />
signific<strong>an</strong>t increase in volume with <strong>the</strong> moderate regimens only. The DDG <strong>an</strong>d VDG showed a<br />
signific<strong>an</strong>t effect with <strong>the</strong> two moderate EB regimens; a signific<strong>an</strong>t effect was also seen with <strong>the</strong><br />
high regimen in <strong>the</strong> rostral VDG, but not DDG, with <strong>the</strong> high EB regimen. For volume defined<br />
by pCREB immunolabeling, no effect was seen with <strong>an</strong>y of <strong>the</strong> regimes in CA1. All regimens<br />
signific<strong>an</strong>tly increased me<strong>an</strong> volume in <strong>the</strong> CA2; in <strong>the</strong> CA3 only <strong>the</strong> moderate regimes elicited<br />
this effect. Both <strong>the</strong> moderate <strong>an</strong>d high EB regimens were effective in signific<strong>an</strong>tly increasing<br />
<strong>the</strong> DDG me<strong>an</strong> volume compared to SO, although <strong>for</strong> <strong>the</strong> rostral VDG, only <strong>the</strong> two moderate<br />
regimens produced this effect. Responsiveness to different regimens of estrogen appears to<br />
depend on <strong>the</strong> specific subregion of <strong>the</strong> hippocampus.
Disclosures: B.W. Bakkum, None; L. F<strong>an</strong>, None; R.S. Cohen, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.10/EE101<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Neurological Foundation of New Zeal<strong>an</strong>d<br />
Department of Physiology, University of Otago<br />
Title: Ameliorative effects of non-classical estrogen action on basal <strong>for</strong>ebrain cholinergic<br />
neurons<br />
Authors: *Z. KOSZEGI, E. TOLOD-KEMP, I. M. ABRAHAM;<br />
Ctr. <strong>for</strong> Neuroendocrinology, Dept. of Physiol., Univ. of Otago, Dunedin, New Zeal<strong>an</strong>d<br />
Abstract: Estrogen alters <strong>the</strong> function of basal <strong>for</strong>ebrain cholinergic (BFC) neurons <strong>an</strong>d is<br />
thought to exert ameliorative effects on <strong>the</strong>se cells. Typically, estrogen alters gene tr<strong>an</strong>scription<br />
via estrogen-receptor mediated “classical” genomic mech<strong>an</strong>isms. However, estrogen also exerts<br />
rapid, non-classical effects via intracellular signaling systems. In our experiment, we have<br />
examined <strong>the</strong> potential ameliorative properties of non-classical estrogen actions on BFC neurons<br />
using <strong>an</strong> in vivo neurodegenerative <strong>an</strong>imal model. N-methyl-D-aspartate (NMDA), a glutamate<br />
<strong>an</strong>alogue was injected into <strong>the</strong> subst<strong>an</strong>tia innominata (SI) of ovariectomized female mice in order<br />
to elicit cholinergic cell death. One hour after <strong>the</strong> NMDA injection, <strong>an</strong>imals received a single<br />
injection of 17-beta-estradiol (E2), a non-classical estrogen pathway activator, estren, or vehicle.<br />
In <strong>the</strong> E2 treated group some <strong>an</strong>imals were pre-treated with specific inhibitors <strong>for</strong> protein kinase<br />
A (PKA) or mitogen-activated protein kinase (MAPK) signaling pathways. The survival rates of<br />
BFC neurons were assessed by identifying <strong>the</strong> cholinergic cell bodies in <strong>the</strong> SI <strong>an</strong>d fibers in <strong>the</strong><br />
somatosensory cortex using computerized qu<strong>an</strong>titative immunohistochemistry <strong>an</strong>d<br />
histochemistry, respectively. NMDA injection results in 50% cholinergic fiber loss in <strong>the</strong><br />
somatosensory cortex <strong>an</strong>d <strong>an</strong> 80% decrease in cell bodies of BFC neurons in <strong>the</strong> SI.<br />
Administration of E2 or estren after NMDA-injection signific<strong>an</strong>tly (p
estore synaptic cholinergic fibers in <strong>the</strong> cerebral cortex after <strong>the</strong> loss of subcortical cholinergic<br />
input without rescuing BFC neurons against excitotoxic cell death. The experiment with estren<br />
<strong>an</strong>d signaling pathway inhibitors suggest that non-classical estrogen actions are involved in this<br />
ameliorative mech<strong>an</strong>ism.<br />
Disclosures: Z. Koszegi, None; E. Tolod-Kemp, None; I.M. Abraham, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.11/EE102<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Karo Bio<br />
NIMH<br />
Title: Strain differences in dose-dependent effects of estradiol <strong>for</strong> <strong>an</strong>xiety, cognitive, sexual <strong>an</strong>d<br />
depressive behaviors in mice<br />
Authors: *D. M. OSBORNE 1 , A. A. WALF 2 , C. A. FRYE 3 ;<br />
2 Psychol, 3 Psychol, Bio, Ctr. Life & Neurosci. Res., 1 Univ. Alb<strong>an</strong>y, Alb<strong>an</strong>y, NY<br />
Abstract: Estradiol (E2) c<strong>an</strong> alter <strong>an</strong>xiety, cognitive, sex, <strong>an</strong>d/or depressive behaviors. However,<br />
when compared to basic <strong>an</strong>imal research indicating that E2-based <strong>the</strong>rapy should provide<br />
symptom relief associated with reproductive senescence, clinical studies present incongruent<br />
findings regarding E2’s effectiveness. Large clinical trials, such as <strong>the</strong> Women’s Health<br />
Initiative, where few beneficial effects of E2 were found, offer a prime example of <strong>the</strong> need to<br />
fur<strong>the</strong>r investigate <strong>the</strong> source of <strong>the</strong>se discrep<strong>an</strong>cies in what has been shown <strong>for</strong> E2’s effects. Of<br />
interest in <strong>the</strong> present study was <strong>the</strong> effects of individual/strain differences <strong>for</strong> responses to E2 <strong>for</strong><br />
its functional effects. To fur<strong>the</strong>r underst<strong>an</strong>d E2’s effects, we examined two different strains of<br />
c57bl/6 mice (c57Bl/6 <strong>an</strong>d in house/bred c57Bl/6/UA). In experiment 1, mice were administered<br />
E2 (0.0, 0.5, 0.10. 0.15 mg/kg) <strong>an</strong>d tested in <strong>an</strong>xiety (emergence, elevated plus maze), cognitive<br />
(object recognition, Morris water maze), sex, <strong>an</strong>d depressive (<strong>for</strong>ced swim task) tests. In<br />
experiment 2, mice were administered pharmacological controls (diazepam, scopolamine, or<br />
fluoxetine) <strong>an</strong>d tested in <strong>the</strong> same battery. Results show varying effects of E2 depending on strain<br />
<strong>an</strong>d task. c57bl/6 mice had greater per<strong>for</strong>m<strong>an</strong>ce in object recognition <strong>an</strong>d sex testing task, while<br />
c57bl/6/UA mice <strong>an</strong>d decreased <strong>an</strong>xiety-like <strong>an</strong>d depressive behaviors. These differences were
specific to E2. In support, pharmacological controls elicited similar results <strong>from</strong> both strains.<br />
Fur<strong>the</strong>rmore, radioimmunoassay results confirm no signific<strong>an</strong>t differences between central <strong>an</strong>d<br />
plasma E2 levels between <strong>the</strong> two strains. These results in two similar mouse strains suggest that<br />
individual <strong>an</strong>d epigenetic differences may contribute to divergent effects of E2 that have been<br />
reported.<br />
Disclosures: D.M. Osborne, None; A.A. Walf, None; C.A. Frye, NIMH, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); NSF, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); DOD, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.12/EE103<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Title: Synaptogenesis <strong>an</strong>d LTP are impaired in cyclic <strong>an</strong>d in ovariectomized mice after<br />
inhibition of aromatase<br />
Authors: *L. FESTER, L. ZHOU, R. VIERK, R. BENDER, D. DUDZINSKI, G. M. RUNE;<br />
Univ. Med. Ctr. Hamburg-Eppendorf, Inst. of Anat. I, Hamburg, Germ<strong>an</strong>y<br />
Abstract: Inhibition of estrogen syn<strong>the</strong>sis by aromatase inhibitors has become a favoured<br />
<strong>the</strong>rapy <strong>for</strong> breast c<strong>an</strong>cer in postmenopausal women. Estrogen is, however, import<strong>an</strong>t <strong>for</strong> synapse<br />
<strong>for</strong>mation in <strong>the</strong> hippocampus. Inhibition of aromatase induces spine synapse loss in org<strong>an</strong>otypic<br />
hippocampal slice cultures. We <strong>the</strong>re<strong>for</strong>e, studied <strong>the</strong> effect of systemic treatment with <strong>the</strong> potent<br />
aromatase inhibitor letrozole, <strong>for</strong> periods of seven days <strong>an</strong>d four weeks, on spine <strong>for</strong>mation <strong>an</strong>d<br />
synaptic proteins in <strong>the</strong> hippocampi of female mice. In cyclic, letrozole-treated females <strong>an</strong>d in<br />
ovariectomized, letrozole-treated females, <strong>the</strong> number of spine synapses was signific<strong>an</strong>tly<br />
reduced in <strong>the</strong> hippocampus but not in <strong>the</strong> prefrontal or cerebellar cortex. Consequently, <strong>the</strong><br />
expression of NMDA receptors NR1 <strong>an</strong>d NR2b were downregulated after treatment with<br />
letrozole <strong>an</strong>d long-term potentiation (LTP) was dramatically impaired. In cyclic <strong>an</strong>imals <strong>the</strong><br />
expression of <strong>the</strong> synaptic proteins synaptophysin <strong>an</strong>d spinophilin was downregulated in<br />
response to letrozole. In ovariectomized <strong>an</strong>imals, however, protein expression was<br />
downregulated after seven days of treatment, whereas <strong>the</strong> expression was upregulated after four<br />
weeks of treatment. Our results indicate that systemic inhibition of aromatase in mice affects
structural synaptic plasticity <strong>an</strong>d LTP in <strong>the</strong> hippocampus. This may be <strong>an</strong> underlying cause of<br />
cognitive deficits in postmenopausal women treated with aromatase inhibitors.<br />
Disclosures: L. Fester, None; L. Zhou, None; R. Vierk, None; R. Bender, None; D.<br />
Dudzinski, None; G.M. Rune, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.13/EE104<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: NIMH<br />
NSF<br />
DOD<br />
Title: Conjugated equine estrogens <strong>an</strong>d/or medroxprogesterone acetate decrease <strong>an</strong>xiety-like<br />
behavior of middle-aged rats, depending upon status of ovari<strong>an</strong> function<br />
Authors: *K. L. EDINGER 1 , A. A. WALF 2 , J. J. PARIS 2 , D. M. OSBORNE 2 , C. A. FRYE 3 ;<br />
1 2 3<br />
SUNY Alb<strong>an</strong>y, Alb<strong>an</strong>y, NY; Psychol, Psychol, Bio, Ctr. Life & Neurosci., Univ. Alb<strong>an</strong>y,<br />
Alb<strong>an</strong>y, NY<br />
Abstract: Ovari<strong>an</strong> hormones c<strong>an</strong> influence nervous system function in young adults. Ch<strong>an</strong>ges in<br />
levels of estradiol <strong>an</strong>d progesterone that occur with <strong>the</strong> tr<strong>an</strong>sition to reproductive senescence may<br />
influence affective responding throughout <strong>the</strong> lifesp<strong>an</strong>. Syn<strong>the</strong>tic hormones may also be a<br />
treatment option <strong>for</strong> some women during menopause, when symptoms associated with ovari<strong>an</strong><br />
steroid decline are m<strong>an</strong>ifest. The effects of commonly-prescribed hormone-replacement<br />
<strong>the</strong>rapies, such as conjugated equine estrogen (CEE) <strong>an</strong>d/or medroxyprogesterone acetate<br />
(MPA), <strong>for</strong> brain function are understudied. We have been investigating <strong>the</strong>se factors in <strong>an</strong>imal<br />
models of reproductive senescence, in which a ch<strong>an</strong>ge in affective behavior <strong>an</strong>d neuroendocrine<br />
function are apparent. We have shown that acute administration of CEE to middle-aged rats c<strong>an</strong><br />
improve cognitive per<strong>for</strong>m<strong>an</strong>ce, reduce <strong>an</strong>xiety, <strong>an</strong>d increase prosocial behavior, but whe<strong>the</strong>r<br />
MPA <strong>an</strong>d/or reproductive status would influence <strong>the</strong>se processes was of interest. In <strong>the</strong> present<br />
study, we compared <strong>the</strong> effects of CEE (0.625 mg/k) <strong>an</strong>d/or MPA (4 mg/kg) <strong>for</strong> affective<br />
processes of middle-aged (12-14 months old) female rats that were reproductively-competent or
not (reproductively-senescent). We hypo<strong>the</strong>sized that CEE <strong>an</strong>d/or MPA would increase <strong>an</strong>ti<strong>an</strong>xiety-like<br />
behavior in <strong>the</strong> open field <strong>an</strong>d elevated plus maze tasks, particularly in<br />
reproductively-competent rats. Our hypo<strong>the</strong>sis that CEE <strong>an</strong>d/or MPA would increase <strong>an</strong>ti<strong>an</strong>xiety-like<br />
behavior, depending upon reproductive senescence status, was partially supported.<br />
Although CEE <strong>an</strong>d CEE+MPA increased entries in <strong>the</strong> center of <strong>the</strong> open field, compared to<br />
vehicle or MPA, only CEE+MPA increased time on <strong>the</strong> open arms compared to vehicle, CEE, or<br />
MPA, <strong>an</strong> effect which was most pronounced among reproductively-competent rats. These data<br />
demonstrate that <strong>the</strong> typically-prescribed hormone <strong>the</strong>rapies, CEE <strong>an</strong>d MPA, have <strong>an</strong>ti-<strong>an</strong>xiety<br />
effects when administered to middle-aged female rats, but <strong>the</strong> nature of <strong>the</strong> effect depends upon<br />
individual reproductive senescence status. Indeed, middle-aged rats c<strong>an</strong> respond favorably to<br />
HRT, but <strong>the</strong> nature of <strong>the</strong> response may be influenced by ch<strong>an</strong>ges in ovari<strong>an</strong> function with<br />
aging.<br />
Disclosures: K.L. Edinger, None; A.A. Walf, None; J.J. Paris, None; D.M. Osborne, None;<br />
C.A. Frye, NIMH, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); NSF, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
DOD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as<br />
well as gr<strong>an</strong>ts already received).<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.14/EE105<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: DOD<br />
NIMH<br />
NSF<br />
Title: Trophic effects of estradiol <strong>an</strong>d selective estrogen receptor modulators <strong>for</strong> hippocampusmediated<br />
behavior <strong>an</strong>d proliferation in reproductive tissues<br />
Authors: *A. A. WALF 1 , J. C. RUSCONI 2 , C. A. FRYE 1 ;<br />
1 Dept Psychol, Univ. Alb<strong>an</strong>y, Alb<strong>an</strong>y, NY; 2 Taconic Inc, Rensselaer, NY
Abstract: Estradiol (E2), secreted by <strong>the</strong> ovaries, c<strong>an</strong> profoundly influence physiology <strong>an</strong>d<br />
behavior throughout <strong>the</strong> lifesp<strong>an</strong>. Much focus has been on whe<strong>the</strong>r E2 <strong>the</strong>rapies are beneficial to<br />
treating <strong>the</strong> sequelae of menopause associated with ch<strong>an</strong>ges in E2 because <strong>the</strong>re are clear<br />
unw<strong>an</strong>ted side effects of E2 <strong>the</strong>rapies, including increasing risk <strong>for</strong> reproductive c<strong>an</strong>cers.<br />
Differences in <strong>the</strong> favorable effects of E2 <strong>the</strong>rapies may depend in part upon <strong>the</strong>ir actions at ERs.<br />
The ER iso<strong>for</strong>ms, ERalpha <strong>an</strong>d ERbeta,) are differentially distributed in <strong>the</strong> body <strong>an</strong>d brain.<br />
Generally, ERalpha is widely expressed throughout most E2-sensitive tissues, whereas ERbeta<br />
expression is more circumspect. Indeed, increased expression of ERalpha in <strong>the</strong> breast <strong>an</strong>d uterus<br />
may account <strong>for</strong> negative trophic effects of E2 in <strong>the</strong>se tissues. The role of E2 to increase risk <strong>for</strong><br />
breast c<strong>an</strong>cer is widely recognized. Differential expression of ERalpha <strong>an</strong>d ERbeta may underlie<br />
how some effects of estrogens c<strong>an</strong> be beneficial or not. We utilized <strong>an</strong> <strong>an</strong>imal model to<br />
investigate potential mech<strong>an</strong>isms <strong>for</strong> E2’s beneficial effects in <strong>the</strong> brain on behavioral processes<br />
<strong>an</strong>d negative proliferative effects in <strong>the</strong> body (on tumors, uterine weights). Ovariectomized rats<br />
were administered E2 (0.09 mg/kg), <strong>an</strong> ERalpha-selective estrogen receptor modulator (SERM;<br />
PPT; 0.09 mg/kg), <strong>an</strong>d <strong>an</strong> ERbeta-SERM (DPN; 0.09 mg/kg), or placebo vehicle, 44-48 hours<br />
be<strong>for</strong>e testing in affective tasks that are mediated by <strong>the</strong> hippocampus (e.g. elevated plus maze,<br />
light-dark tr<strong>an</strong>sition, <strong>for</strong>ced swim test). Some rats in each hormone condition were administered<br />
<strong>the</strong> chemical carcinogen (DMBA; 12.5 mg), or inert vegetable oil vehicle, by gavage. Tumor<br />
burden <strong>an</strong>d uterine weight were <strong>an</strong>alyzed. Our hypo<strong>the</strong>sis, that <strong>the</strong>re may be some specificity in<br />
<strong>the</strong> mech<strong>an</strong>isms of E2 <strong>for</strong> trophic effects in <strong>the</strong> hippocampus <strong>an</strong>d periphery of rats, was<br />
supported. We found that E2 <strong>an</strong>d DPN, but not PPT, decreased <strong>an</strong>xiety-like <strong>an</strong>d depression-like<br />
behavior. In support, E2 <strong>an</strong>d DPN, compared to vehicle, increased <strong>the</strong> time spent on <strong>the</strong> open<br />
arms of <strong>the</strong> plus maze <strong>an</strong>d <strong>the</strong> time spent on <strong>the</strong> light side of <strong>the</strong> light-dark tr<strong>an</strong>sition chamber,<br />
<strong>an</strong>d decreased <strong>the</strong> time spent immobile in <strong>the</strong> <strong>for</strong>ced swim test. Rats administered DMBA,<br />
compared to no carcinogen exposure, had increased tumor burden. E2 <strong>an</strong>d PPT increased tumor<br />
burden, compared to vehicle administration. E2 <strong>an</strong>d PPT increased uterine weights compared to<br />
vehicle. Thus, <strong>the</strong>se data suggest that <strong>the</strong>re may be ER subtype-specificity <strong>for</strong> estrogens’ trophic<br />
effects in <strong>the</strong> brain <strong>an</strong>d body.<br />
Disclosures: A.A. Walf, None; J.C. Rusconi, None; C.A. Frye, NIMH, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); NSF, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); DOD, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.15/EE106
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: DOD<br />
NIMH<br />
NSF<br />
Title: Histone modifications may underlie some trophic effects of estradiol <strong>an</strong>d selective<br />
estrogen receptor modulators <strong>for</strong> tumorigenesis, uterine proliferation, <strong>an</strong>d hippocampal processes<br />
in ovariectomized rats<br />
Authors: *J. RUSCONI 1 , A. A. WALF 2 , C. A. FRYE 3 ;<br />
1 2 3<br />
Taconic, Inc, Rensselaer, NY; Psychol, Psychol, Bio, Ctr. Life & Neurosci. Res., Univ.<br />
Alb<strong>an</strong>y, Alb<strong>an</strong>y, NY<br />
Abstract: The ovari<strong>an</strong> steroid, estradiol (E2), has pleiotrophic effects <strong>for</strong> processes in <strong>the</strong> central<br />
nervous system <strong>an</strong>d in <strong>the</strong> periphery. Although <strong>the</strong>re is some indication of beneficial trophic<br />
effects of E2 in <strong>the</strong> brain, its potential to enh<strong>an</strong>ce tumorigenic processes may outweigh <strong>the</strong>se<br />
beneficial trophic effects in <strong>the</strong> brain. Key to underst<strong>an</strong>ding whe<strong>the</strong>r E2’s trophic effects may be<br />
beneficial, or not, may lie in <strong>the</strong> cellular mech<strong>an</strong>isms of <strong>the</strong>se effects. E2 has actions at estrogen<br />
receptors (ERs), of which two iso<strong>for</strong>ms have been best characterized (ERalpha <strong>an</strong>d ERbeta).<br />
Research suggests that <strong>the</strong> beneficial effects of E2 in <strong>the</strong> hippocampus may be due to actions at<br />
ERbeta, whereas proliferative effects in <strong>the</strong> body may be due to actions at ERalpha. The<br />
downstream mech<strong>an</strong>isms of ERalpha <strong>an</strong>d ERbeta <strong>for</strong> <strong>the</strong>se effects on proliferation are of interest.<br />
Histone modifications (acetylation, phosphorylation), downstream of ERs, may underlie<br />
divergent trophic effects in <strong>the</strong> body <strong>an</strong>d brain. Histone deacetylases are c<strong>an</strong>didates <strong>for</strong> c<strong>an</strong>cer<br />
treatments owing to <strong>the</strong>ir ability to promote expression of genes involved in cell differentiation<br />
<strong>an</strong>d death, <strong>an</strong>d cell cycle arrest. We <strong>an</strong>alyzed ch<strong>an</strong>ges in histone 3 acetylation <strong>an</strong>d<br />
phosphorylation in hippocampus, carcinogen-induced tumors, <strong>an</strong>d uteri of ovariectomized rats<br />
administered E2 or selective estrogen receptor modualtors (SERMs) that act at ERalpha or<br />
ERbeta with western blotting. These experiments have suggested that <strong>the</strong>re are differences in<br />
histone 3 acetylation/phosphorylation in <strong>the</strong> uterus, tumors, <strong>an</strong>d hippocampus of rats<br />
administered E2, PPT (ERalpha-SERM), DPN (ERbeta-SERM) or placebo. In <strong>the</strong> hippocampus,<br />
acetylated histone 3 was increased by E2 <strong>an</strong>d decreased by DPN, compared to vehicle. The<br />
phosphor-acetylated <strong>for</strong>m of histone 3 was reduced by E2, PPT, <strong>an</strong>d DPN similarly. A different<br />
pattern emerged in tumor <strong>an</strong>d uterine tissues, which was sensitive to <strong>the</strong> effects of carcinogen<br />
exposure. In excised tumors, both acetylated <strong>an</strong>d phospho-acetylated histone 3 was increased by<br />
E2 <strong>an</strong>d SERMs, effects that were attenuated by carcinogen. In <strong>the</strong> uterus, E2 did not alter, <strong>an</strong>d<br />
PPT <strong>an</strong>d DPN increased expression of acetylated histone 3. Phospho-acetylated histone 3 was<br />
decreased by E2 <strong>an</strong>d PPT in <strong>the</strong> uterus, but increased by DPN, compared to vehicle. In <strong>the</strong> uterus,<br />
carcinogen exposure reversed <strong>the</strong>se effects. These data suggest that differences in trophic effects<br />
in <strong>the</strong> body <strong>an</strong>d brain may be related to histone 3 modifications. Indeed, fur<strong>the</strong>r investigation of<br />
<strong>the</strong> effects of E2 on cell cycle ch<strong>an</strong>ges to underlie differential trophic effects on reproductive <strong>an</strong>d<br />
neural tissue is subst<strong>an</strong>tiated.
Disclosures: J. Rusconi, None; A.A. Walf, None; C.A. Frye, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.16/EE107<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Pacific Alzheimer Research Foundation<br />
Title: The ability of estrogens to upregulate cell proliferation in <strong>the</strong> hippocampus of older<br />
females is dependent on reproductive experience<br />
Authors: *C. K. BARHA 1 , L. A. M. GALEA 2 ;<br />
2 Psychology, 1 Univ. British Columbi, V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: Estrogen has been implicated as a possible <strong>the</strong>rapeutic agent <strong>for</strong> improving cognition<br />
in postmenopausal women <strong>an</strong>d has been linked to neurodegenerative disorders such as<br />
Alzheimer’s disease. Despite <strong>the</strong> m<strong>an</strong>y different <strong>for</strong>ms of estrogen most research has focused on<br />
estradiol. Estradiol is <strong>the</strong> more common estrogen in young women, while estrone is more<br />
common in older women. The most commonly prescribed hormone replacement <strong>the</strong>rapy (HRT)<br />
consists primarily of estrone, <strong>an</strong>d does not have as m<strong>an</strong>y cognitive-enh<strong>an</strong>cing benefits as HRTs<br />
that consist primarily of estradiol. In addition, treatment with estrone upregulates ERα whereas<br />
treatment with estradiol upregulates ERβ in <strong>the</strong> rodent hippocampus. Fur<strong>the</strong>rmore, <strong>the</strong>re are two<br />
naturally occurring isomers of estradiol, 17β-estradiol <strong>an</strong>d 17α-estradiol. Although 17α-estradiol<br />
is a putative lig<strong>an</strong>d <strong>for</strong> <strong>the</strong> estrogen membr<strong>an</strong>e receptor, most research conducted with estradiol<br />
has used 17β-estradiol. We have previously shown that all of <strong>the</strong>se estrogens promote<br />
hippocampal neurogenesis in a dose-dependent m<strong>an</strong>ner in young, virgin ovariectomized female<br />
rats. The aim of <strong>the</strong> present study was to determine <strong>the</strong> acute effects of 17β-estradiol, 17αestradiol,<br />
<strong>an</strong>d estrone on hippocampal neurogenesis in aged ovariectomized female rats <strong>an</strong>d to<br />
determine whe<strong>the</strong>r effects are dependent on previous reproductive experience. Middle-aged 13 to<br />
14 month old retired breeder female rats <strong>an</strong>d aged-matched virgin female rats were injected<br />
subcut<strong>an</strong>eously with vehicle (sesame oil), or 10µg dose of 17β-estradiol, 17α-estradiol, or<br />
estrone. Rats were <strong>the</strong>n injected with bromodeoxyuridine, a thymidine <strong>an</strong>alog that incorporates<br />
itself into <strong>the</strong> DNA of cells during <strong>the</strong> syn<strong>the</strong>sis phase of <strong>the</strong> cell cycle, 30 min after hormone<br />
injections <strong>an</strong>d were perfused 24 hours later. We have found that 17β-estradiol, 17α-estradiol, <strong>an</strong>d<br />
estrone increase cell proliferation in retired breeder female rats compared to ovariectomized<br />
female controls <strong>an</strong>d sham operated controls, with 17α-estradiol having <strong>the</strong> most potent effect.
Interestingly, none of <strong>the</strong>se estrogens had <strong>an</strong> effect on cell proliferation in virgin ovariectomized<br />
female rats. There<strong>for</strong>e, previous reproductive experience may make <strong>the</strong> older brain more<br />
responsive to estrogens later in life. Findings <strong>from</strong> this study will adv<strong>an</strong>ce our underst<strong>an</strong>ding of<br />
how different <strong>for</strong>ms of estrogen mediate hippocampal neurogenesis in aged rats, which may<br />
ultimately lead to <strong>the</strong> development of new <strong>the</strong>rapeutic adv<strong>an</strong>ces in <strong>the</strong> treatment of symptoms<br />
associated with menopause in women.<br />
Disclosures: C.K. Barha, None; L.A.M. Galea, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.17/EE108<br />
Topic: B.08.i. Structural plasticity<br />
Support: Mahidol University<br />
Title: Estrogen induces activity-regulated cytoskeleton associated protein expression <strong>an</strong>d<br />
synaptic plasticity via ER- dependent pathways in SH-SY5Y cells<br />
Authors: *S. CHONGTHAMMAKUN 1 , S. CHAMNIANSAWAT 2 ;<br />
1 Dept Anat. <strong>an</strong>d Ctr. <strong>for</strong> Neurosci., Fac. of Sci., B<strong>an</strong>gkok, Thail<strong>an</strong>d; 2 Dept Anat. <strong>an</strong>d Ctr. <strong>for</strong><br />
Neurosci., Fac. of Sci., B<strong>an</strong>gkok 10400, Thail<strong>an</strong>d<br />
Abstract: Synaptic plasticity is a main cellular response underlying memory consolidation. It<br />
requires novel protein syn<strong>the</strong>sis to support structural ch<strong>an</strong>ges, <strong>an</strong>d could depend on rapid<br />
induction of gene tr<strong>an</strong>scription <strong>an</strong>d subsequent protein tr<strong>an</strong>slation. The activity-regulated<br />
cytoskeleton associated protein (Arc) is one of <strong>the</strong> immediate-early genes in activity-dependent<br />
synaptic plasticity that have been implicated in <strong>the</strong> mainten<strong>an</strong>ce of long-term memory. Since<br />
estrogen promote synaptic plasticity <strong>an</strong>d Arc is a marker protein underlying mech<strong>an</strong>ism of<br />
synaptic plasticity, estrogen-enh<strong>an</strong>ced synaptic plasticity probably involves expression of Arc.<br />
The aims of this study were to investigate <strong>the</strong> effects of estrogen on Arc <strong>an</strong>d synaptic protein<br />
expression in SH-SY5Y cells. The signaling mech<strong>an</strong>isms underlying estrogenic effect on Arc<br />
expression were also investigated. SH-SY5Y cells were exposed to 10 -8 M estrogen, ERα agonist<br />
(2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) or ERβ agonist (4,4',4''-(4-Propyl-[1H]-pyrazole-<br />
1,3,5-triyl) trisphenol (PPT). The expressions of Arc, PSD-95, <strong>an</strong>d synaptophysin (SYP) were<br />
determined using qRT-PCR, Western blot <strong>an</strong>d immunocytochemistry techniques. To investigate<br />
<strong>the</strong> involved signaling pathways, SH-SY5Y cells were preincubated with 200 nM ICI182,780,
<strong>the</strong> estrogen receptor (ER) <strong>an</strong>tagonist; 200 µM Wortm<strong>an</strong>nin, <strong>the</strong> PI−3K inhibitor; 1 µM U0126,<br />
<strong>the</strong> MAPK inhibitor; 1 µM Y27632, <strong>the</strong> Rho-associated kinase (ROCK) inhibitor; or 200 µM<br />
GFX, <strong>the</strong> protein kinase C (PKC) inhibitor, <strong>for</strong> 1 h prior to incubation with 10 -8 M estrogen. The<br />
results revealed that estrogen increased Arc mRNA <strong>an</strong>d protein in different time dependent<br />
m<strong>an</strong>ner. Estrogen-induced Arc expression is MAPK- <strong>an</strong>d PI-3K-dependent pathways but not<br />
ROCK- <strong>an</strong>d PKC-dependent pathways. In addition, estrogen-induced Arc expression is mediated<br />
via membr<strong>an</strong>e ERβ but not ERα. However, DPN but not PPT, markedly induced Arc expression<br />
that mimics <strong>the</strong> action of estrogen. In conclusion, <strong>the</strong> present study demonstrates <strong>the</strong> nongenomic<br />
effects of estrogen on Arc expression, which mediated via ERβ-, MAPK-, <strong>an</strong>d PI-3Kdependent<br />
pathways. However, <strong>the</strong> estrogen-induced synaptic markers, PSD-95 <strong>an</strong>d SYP<br />
expression, indicated <strong>the</strong> classical genomic estrogenic effects on synaptic plasticity. Toge<strong>the</strong>r,<br />
<strong>the</strong>se results demonstrate <strong>the</strong> crosstalk mech<strong>an</strong>ism between non-genomic <strong>an</strong>d genomic estrogenic<br />
actions.<br />
Disclosures: S. Chongthammakun, None; S. Chamni<strong>an</strong>sawat, None.<br />
Poster<br />
569. Steroids <strong>an</strong>d Plasticity I<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 569.18/EE109<br />
Topic: E.01.d. Steroids <strong>an</strong>d plasticity<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>from</strong> <strong>the</strong> Ministry of Education, Culture, Sports, Science <strong>an</strong>d Technology<br />
of Jap<strong>an</strong> 20510061<br />
Title: Dynamics of <strong>the</strong> interaction of GR or TR with <strong>the</strong> general coactivator SYT<br />
Authors: *M. B. LONDONO-LAZARO 1,2 , T. IWASAKI 1 , K. IBHAZEHIEBO 1 , F.<br />
LIZCANO 2 , N. SHIMOKAWA 1 , N. KOIBUCHI 1 ;<br />
1 2<br />
Dept. of Integrative Physiol., Gunma Univ. Grad. Sch. of Med., Maebashi / Gunma, Jap<strong>an</strong>; La<br />
Sab<strong>an</strong>a Univ., Bogota, Colombia<br />
Abstract: Nuclear hormone receptors (NR) regulate tr<strong>an</strong>scription by interacting with<br />
coactivators <strong>an</strong>d corepressors. After synovial sarcoma tr<strong>an</strong>slocation (SYT) was identified as<br />
component of <strong>the</strong> fusion protein that appears to be fundamental in hum<strong>an</strong> synovial sarcomas<br />
pathogenesis, we <strong>an</strong>d o<strong>the</strong>r groups characterized SYT as a general tr<strong>an</strong>scriptional coactivator.<br />
However, its physiological roles remain unknown.<br />
To underst<strong>an</strong>d <strong>the</strong> role of SYT in nuclear receptors mediated tr<strong>an</strong>scription we <strong>an</strong>alyzed <strong>the</strong>
spatio-temporal subcellular distribution of pEGFP fused SYT in <strong>the</strong> presence of unlig<strong>an</strong>ded <strong>an</strong>d<br />
lig<strong>an</strong>ded glucocorticoid receptor (GR) <strong>an</strong>d thyroid hormone receptor (TR)β1 by using time-lapse<br />
imaging of living cells.<br />
We found a progressive <strong>an</strong>d signific<strong>an</strong>t increase in <strong>the</strong> nuclear fluorescence intensity in a<br />
punctate pattern in <strong>the</strong> cells efficiently cotr<strong>an</strong>sfected with pEGFP-SYT <strong>an</strong>d ei<strong>the</strong>r pcDNA3-GR<br />
or pcDNA3-TRβ1 starting at 5 minutes of dexametasone or T3 treatment (10 -7 M) <strong>an</strong>d lasting <strong>for</strong><br />
more th<strong>an</strong> 40 min. Double recording of pEGFP <strong>an</strong>d mCherry-fused proteins are currently being<br />
done. Additionally, direct binding of pEGFP-SYT-S to both receptors in <strong>the</strong> absence <strong>an</strong>d<br />
presence of <strong>the</strong> lig<strong>an</strong>ds was evidenced by immunoprecipitation in CHO cells. Experiments to<br />
identify <strong>the</strong> domains involved in <strong>the</strong>se interactions are now undergoing. The coactivation<br />
efficiency of <strong>the</strong> pEGFP-SYT-S chimera was evaluated with luciferase reporter assays.<br />
In conclusion, here we show that with lig<strong>an</strong>ded nuclear receptors <strong>the</strong>re is clear SYT<br />
redistribution within <strong>the</strong> nucleus in a punctate pattern <strong>an</strong>d that SYT directly interacts with <strong>the</strong><br />
glucocorticoid <strong>an</strong>d <strong>the</strong> thyroid hormone receptors.<br />
The present findings provide visual evidence of <strong>the</strong> dynamic ch<strong>an</strong>ge in <strong>the</strong> subcellular<br />
localization of NR coactivators according to <strong>the</strong> NR state of activation.<br />
Disclosures: M.B. Londono-Lazaro, None; T. Iwasaki, T.Iwasaki was previously working in<br />
Eli Lilly & Comp<strong>an</strong>y, A. Employment (full or part-time); K. Ibhazehiebo, None; F. Lizc<strong>an</strong>o,<br />
None; N. Shimokawa, None; N. Koibuchi, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.1/EE110<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIMH047538<br />
Title: Prenatal immune stress attenuates juvenile social play behavior in male but not in female<br />
rats<br />
Authors: *P. V. TAYLOR, G. J. DE VRIES;<br />
Umass Amherst, Amherst, MA<br />
Abstract: Prenatal immune stress is a risk factor <strong>for</strong> behavioral disorders such as<br />
schizophrenia <strong>an</strong>d autism, suggesting that immune stress may interfere<br />
with development of <strong>the</strong> neural circuitry driving social behavior. This
study explores <strong>the</strong> behavioral <strong>an</strong>d physiological m<strong>an</strong>ifestations of<br />
prenatal immune stress in rats. Lipopolysaccharide (LPS), a component of<br />
<strong>the</strong> cell wall of gram-negative bacteria, or saline was administered to<br />
pregn<strong>an</strong>t rats on embryonic day 15. Play behaviors of pups were<br />
video-taped <strong>from</strong> 22-45 days of age. Animals were tested in <strong>the</strong> home cage<br />
with all littermates present or in age- <strong>an</strong>d sex-matched pairs after one<br />
hour of isolation. Males showed more play behavior th<strong>an</strong> females in <strong>an</strong>y<br />
setting at all ages. LPS tended to reduce play behavior in <strong>the</strong> home<br />
cage, but differences did not reach signific<strong>an</strong>ce (p=0.06). However, LPS<br />
signific<strong>an</strong>tly decreased <strong>the</strong> frequency of play in male offspring in<br />
treatment-matched pairs at all ages tested. Although <strong>the</strong>re was no<br />
difference in <strong>the</strong> frequency of play behaviors between LPS- <strong>an</strong>d<br />
saline-treated rats in mixed treatment pairs, LPS-treated rats initiated<br />
play signific<strong>an</strong>tly less often th<strong>an</strong> saline-treated rats. This suggests<br />
that LPS may not interfere with <strong>the</strong> ability to respond to social play<br />
but alters <strong>the</strong> tendency to initiate play. LPS did not signific<strong>an</strong>tly<br />
influence <strong>the</strong> frequency of play behaviors in female rats. This sex<br />
difference may be due to <strong>the</strong> physiological response of <strong>the</strong> fetal immune<br />
system to LPS, <strong>the</strong> neural circuitry upon which mediators of <strong>the</strong> immune<br />
response act, or both. The stronger effects of LPS on males th<strong>an</strong> on<br />
females suggest that LPS treatment may be helpful in underst<strong>an</strong>ding <strong>the</strong><br />
etiology of behavioral disorders that affect males more th<strong>an</strong> females,<br />
such as autism. Preliminary data suggest that LPS also reduces <strong>the</strong> vasopressin expression in <strong>the</strong><br />
medial amygdala, which may contribute to <strong>the</strong> effects of LPS on social play.<br />
Disclosures: P.V. Taylor, None; G.J. de Vries, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.2/EE111<br />
Topic: E.02.b. Behavioral effects<br />
Support: PICT2006, ANPCyT<br />
University of Buenos Aires
Title: Maternal care <strong>an</strong>d perinatal inflammation contributions to postnatal programming of<br />
<strong>an</strong>xiety <strong>an</strong>d depression-related behavior<br />
Authors: *A. M. DEPINO 1,2,3 , L. LUCCHINA 1,2 , F. PITOSSI 1,2 ;<br />
1 Leloir Inst. Fndn., Buenos Aires, Argentina; 2 CONICET, Buenos Aires, Argentina; 3 DFBMyC,<br />
FCEyN, Univ. of Buenos Aires, Buenos Aires, Argentina<br />
Abstract: The perinatal development of <strong>the</strong> nervous system is influenced by different external<br />
<strong>an</strong>d internal stimuli. Previous data show that maternal care <strong>an</strong>d perinatal inflammation c<strong>an</strong><br />
induce long-term ch<strong>an</strong>ges in <strong>an</strong>xiety <strong>an</strong>d depression-related behavior. Our hypo<strong>the</strong>sis is that both<br />
events act through interacting biological pathways during specific temporal windows to program<br />
adult behavior. Our aim is to study how <strong>the</strong>se stimuli program adult behavior <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>re<br />
is <strong>an</strong> interaction between maternal care <strong>an</strong>d inflammatory stimuli programming. To this aim, we<br />
used two protocols of maternal care variation in mice [H<strong>an</strong>dling/Maternal Separation, <strong>an</strong>d F1<br />
offspring -C57BL/6J (C57) x Balb/c (Balb)] <strong>an</strong>d injected <strong>the</strong> bacterial endotoxin<br />
lipopolysaccharide (LPS) at ei<strong>the</strong>r of two previously reported sensitive development ages<br />
[gestational day 9 (GD9) or postnatal day 3 (PD3)].<br />
The <strong>an</strong>alysis of maternal behavior revealed that pups subjected to Maternal Separation (both<br />
Balb <strong>an</strong>d C57 strains) <strong>an</strong>d <strong>the</strong> F1 pups of C57 dams received more maternal attention. The<br />
<strong>an</strong>alysis of <strong>the</strong> adult offspring revealed that h<strong>an</strong>dled C57 males spent more time in <strong>the</strong> open arms<br />
of elevated-plus maze (EPM) <strong>an</strong>d in <strong>the</strong> illuminated side of <strong>the</strong> Light/Dark box. Maternally<br />
separated mice showed reduced immobility in both <strong>the</strong> tail suspension <strong>an</strong>d <strong>the</strong> Porsolt tests.<br />
Control Balb adult mice showed increased locomotion in <strong>the</strong> EPM <strong>an</strong>d reduced immobility in <strong>the</strong><br />
Porsolt test, compared to both m<strong>an</strong>ipulated groups. The <strong>an</strong>alysis of <strong>the</strong> F1 revealed that mice of<br />
<strong>the</strong> C57 pedigree showed increased <strong>an</strong>xiety-related behavior in <strong>the</strong> open field (OF) <strong>an</strong>d EPM,<br />
<strong>an</strong>d increased depression-related behavior in <strong>the</strong> Porsolt test.<br />
Postnatal (PD3) inflammatory stimulation had no long-term effects on C57 or Balb mice. LPS at<br />
PD3 reduced exploration in <strong>the</strong> OF in F1 females, regardless of pedigree. Conversely, prenatal<br />
LPS (GD9) led to increased <strong>an</strong>xiety <strong>an</strong>d depression-related behavior in C57 male mice.<br />
We failed to observe a strong interaction between maternal care <strong>an</strong>d inflammatory stimuli in <strong>the</strong><br />
programming of adult <strong>an</strong>xiety <strong>an</strong>d depression-related behavior in <strong>an</strong>y of <strong>the</strong> experimental groups.<br />
In summary, we characterized a mouse model of postnatal programming of <strong>an</strong>xiety <strong>an</strong>d<br />
depression-related behavior in which we c<strong>an</strong> study <strong>the</strong> molecular mech<strong>an</strong>isms involved. Our<br />
behavioral results suggest that maternal care <strong>an</strong>d inflammatory stimuli affect perinatal<br />
programming of adult behavior presumably through different molecular <strong>an</strong>d cellular<br />
mech<strong>an</strong>isms.<br />
Disclosures: A.M. Depino, None; L. Lucchina, None; F. Pitossi, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.3/EE112<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIH gr<strong>an</strong>t NS 19327<br />
Isis gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> <strong>Society</strong> <strong>for</strong> Women’s Health Research<br />
Title: Peripubertal immunogenic stress reduces <strong>the</strong> effects of ovari<strong>an</strong> steroids on <strong>an</strong>xiety-like<br />
behavior in adult female mice<br />
Authors: *K. M. OLESEN, N. ISMAIL, P. GARAS, R. ROGAN, N. BOUCHENTOUF-<br />
IDRISS, S. SERVATTALAB, C. DIGLORIA, J. D. BLAUSTEIN;<br />
Univ. Massachusetts-Amherst, Amherst, MA<br />
Abstract: Brain reorg<strong>an</strong>ization during <strong>the</strong> peripubertal period contributes to <strong>the</strong> development of<br />
adult neural <strong>an</strong>d behavioral responses to gonadal steroids. Exposure to shipping or <strong>an</strong> immune<br />
stressor at 6 weeks of age reduces sexual receptivity in response to ovari<strong>an</strong> steroids in adult<br />
female mice. We hypo<strong>the</strong>sized that peripubertal immunogenic stress would also reduce <strong>the</strong><br />
responsiveness of <strong>an</strong>xiety-like behavior to ovari<strong>an</strong> steroids. Anxiety-like behavior varies over <strong>the</strong><br />
estrous cycle, <strong>an</strong>d ovariectomy increases, <strong>an</strong>d acute hormone replacement decreases <strong>an</strong>xiety-like<br />
behavior. To determine if peripubertal immunogenic stress impacts <strong>the</strong> sensitivity of <strong>an</strong>xiety-like<br />
behavior to ovari<strong>an</strong> steroids, we examined <strong>an</strong>xiety-like behavior in response to estradiol<br />
benzoate (E) <strong>an</strong>d progesterone (P) in adult female mice exposed to a bacterial endotoxin,<br />
lipopolysaccharide (LPS) during <strong>the</strong> peripubertal period. Because h<strong>an</strong>dling c<strong>an</strong> alter immune<br />
response to LPS, we also examined <strong>the</strong> effects of h<strong>an</strong>dling on LPS-induced ch<strong>an</strong>ges in <strong>an</strong>xietylike<br />
behavior. Female C57Bl/6 mice were shipped <strong>from</strong> <strong>the</strong> supplier at 3 weeks old. Beginning<br />
upon arrival, h<strong>an</strong>dled mice were allowed to walk on <strong>the</strong> experimenters’ h<strong>an</strong>ds <strong>for</strong> ~2 min/day, 3<br />
times/week <strong>for</strong> <strong>the</strong> duration of <strong>the</strong> study. Non-h<strong>an</strong>dled <strong>an</strong>imals were not exposed to contact<br />
beyond weekly cage ch<strong>an</strong>ges. At 6 weeks of age, mice received injections of 1.5mg/kg LPS (ip)<br />
or saline <strong>an</strong>d were ovariectomized one week later. One week after ovariectomy, mice were<br />
primed with ovari<strong>an</strong> steroids <strong>an</strong>d tested <strong>for</strong> <strong>an</strong>xiety-like behavior in <strong>the</strong> light-dark box, elevated<br />
plus maze, <strong>an</strong>d marble burying tests. Overall, E+P reduced <strong>an</strong>xiety-like behaviors. In nonh<strong>an</strong>dled<br />
mice, E+P increased <strong>the</strong> amount of time spent on <strong>the</strong> light side of <strong>the</strong> light-dark box <strong>an</strong>d<br />
tended to increase <strong>the</strong> amount of time spent on <strong>the</strong> open arm of <strong>the</strong> elevated plus maze in saline-,<br />
but not LPS-treated, mice. H<strong>an</strong>dling did not alter <strong>the</strong> amount of time spent in <strong>the</strong> light in <strong>the</strong><br />
light-dark box, but did increase <strong>the</strong> amount of time spent on <strong>the</strong> open arm of <strong>the</strong> elevated plus<br />
maze in saline-, but not LPS-treated mice. Surprisingly, in <strong>the</strong> marble burying paradigm, E+P did<br />
not alter <strong>the</strong> number of marbles buried by non-h<strong>an</strong>dled <strong>an</strong>imals; however, E+P reduced <strong>the</strong><br />
number of marbles buried by saline-, but not LPS-treated, h<strong>an</strong>dled females. These data suggest<br />
that a peripubertal immunogenic stressor reduces <strong>the</strong> responsiveness of <strong>an</strong>xiety-like behavior to<br />
ovari<strong>an</strong> steroids in adult mice, as it reduces sexual receptivity in response to ovari<strong>an</strong> steroids.
Disclosures: K.M. Olesen, None; N. Ismail, None; P. Garas, None; R. Rog<strong>an</strong>, None; N.<br />
Bouchentouf-Idriss, None; S. Servattalab, None; C. Digloria, None; J.D. Blaustein, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.4/EE113<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIH NS19327<br />
Isis gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Study of Women’s Health Research<br />
Title: Chronic estradiol treatment increases, not decreases, depression-like symptoms in adult<br />
female mice treated with lipopolysaccharide during <strong>the</strong> peripubertal period<br />
Authors: *N. ISMAIL, K. M. OLESEN, P. GARAS, R. ROGAN, S. SERVETTALAB, J. D.<br />
BLAUSTEIN;<br />
Psychology, Univ. of Massachusetts, Amherst, MA<br />
Abstract: A single injection of <strong>the</strong> bacterial endotoxin, lipopolysaccharide (LPS; 1.5mg/kg)<br />
during <strong>the</strong> peripubertal period defeminized sexual behavior in response to estradiol <strong>an</strong>d<br />
progesterone <strong>an</strong>d altered neural expression of estrogen <strong>an</strong>d progestin receptors. These findings<br />
suggest that peripubertal LPS treatment reduces <strong>the</strong> sensitivity to gonadal steroid hormones in<br />
adulthood. Since estradiol <strong>an</strong>d progesterone influence m<strong>an</strong>y non-reproductive behaviors, we<br />
were interested in investigating <strong>the</strong> effect of peripubertal LPS on non-sexual behaviors. Estradiol<br />
decreases state-directed expression of depression-like behavior in adult females. There<strong>for</strong>e, we<br />
hypo<strong>the</strong>sized that estradiol would be less effective at reducing depression-like behavior in female<br />
mice treated peripubertally with LPS. Six-week old female mice were injected with ei<strong>the</strong>r saline<br />
or LPS. Two weeks later, <strong>the</strong>y were ovariectomized <strong>an</strong>d subcut<strong>an</strong>eously impl<strong>an</strong>ted with ei<strong>the</strong>r <strong>an</strong><br />
oil- or estradiol-filled Silastic capsule. Following one week of recovery, behavioral testing <strong>for</strong><br />
depression-like symptoms beg<strong>an</strong>. The duration of immobility during <strong>the</strong> tail suspension <strong>an</strong>d<br />
<strong>for</strong>ced swim tests was used as a behavioral measure of learned helplessness, <strong>an</strong> import<strong>an</strong>t<br />
symptom of <strong>the</strong> depression state. Chronic estradiol decreased depression-like behavior (duration<br />
of immobility) in saline-treated females. However, in LPS-treated females, chronic estradiol<br />
strikingly increased <strong>the</strong> duration of immobility. No difference in locomotion was found between<br />
<strong>the</strong> groups in <strong>an</strong> open field or in locomotor activity chambers. These findings illustrate that<br />
although estradiol decreases depression-like behavior (learned helplessness), it increases this
ehavior in mice treated with LPS. These results suggest a complex interplay between<br />
peripubertal immune challenge <strong>an</strong>d chronic estradiol on depression-like symptoms in adulthood.<br />
Disclosures: N. Ismail, None; K.M. Olesen, None; P. Garas, None; R. Rog<strong>an</strong>, None; S.<br />
Servettalab, None; J.D. Blaustein, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.5/EE114<br />
Topic: E.02.b. Behavioral effects<br />
Title: Spinal TNF-α neutralization reduces hyperalgesia, attenuates peripheral inflammation <strong>an</strong>d<br />
suppresses autonomic responses to inflammation<br />
Authors: M. K. BOETTGER 1 , K. WEBER 1 , D. GROSSMANN 1 , M. GAJDA 2 , R. BRÄUER 2 ,<br />
R. BAUER 3 , K.-J. BÄR 4,6 , S. SCHULZ 7 , A. VOSS 7 , C. GEIS 5 , *A. EBERSBERGER 8 , H.-G.<br />
SCHAIBLE 1 ;<br />
1 Inst. of Physiol. I, 2 Inst. of Pathology / Immunopathology, 3 Dept. of Pathophysiology, Inst. of<br />
Mol. Cell Biol., 4 Dept. of Psychiatry, Univ. Hosp., Jena, Germ<strong>an</strong>y; 5 Dept. of Neurol., Univ.<br />
Hosp., Würzburg, Germ<strong>an</strong>y; 6 Dept. of Psychiatry, Psycho<strong>the</strong>rapy <strong>an</strong>d Psychosomatic Med., Ruhr<br />
Univ., Bocum, Germ<strong>an</strong>y; 7 Dept. of Med. Engin. <strong>an</strong>d Biotech., Univ. of Applied Sci., Jena,<br />
Germ<strong>an</strong>y; 8 Univ. Jena, D-07740 Jena, Germ<strong>an</strong>y<br />
Abstract: Systemic neutralization of TNF-α has been shown to reduce pain-related behavior, but<br />
only to a minor degree <strong>the</strong> inflammatory response in <strong>an</strong>imal models of arthritis. For <strong>the</strong>se effects,<br />
a neuronal target has been proposed.<br />
Here, we aimed to elucidate <strong>the</strong> import<strong>an</strong>ce of spinal TNF-α on inflammation mech<strong>an</strong>isms.<br />
There<strong>for</strong>e, <strong>an</strong>tigen-induced arthritis was induced in female Lewis rats that were ei<strong>the</strong>r treated<br />
with saline (n=22), intraperitoneal et<strong>an</strong>ercept (n=20) or intra<strong>the</strong>cal et<strong>an</strong>ercept (n=20) <strong>an</strong>d<br />
parameters of pain-related behavior, inflammation <strong>an</strong>d cardiac autonomic response were<br />
obtained.<br />
In comparison to peripheral application, spinal TNF-α neutralization exhibited even stronger<br />
<strong>an</strong>tinociceptive <strong>an</strong>d <strong>an</strong>ti-inflammatory effects in both <strong>the</strong> acute (day 3) <strong>an</strong>d chronic phase (day<br />
21). In particular, primary mech<strong>an</strong>ical hyperalgesia was grossly attenuated <strong>an</strong>d histopathological<br />
signs of inflammation <strong>an</strong>d joint destruction were signific<strong>an</strong>tly reduced. Serum concentrations of<br />
et<strong>an</strong>ercept in <strong>an</strong>imals treated intra<strong>the</strong>cally were far below those of intraperitoneally treated<br />
<strong>an</strong>imals, thus making a peripheral mode of action unlikely.
Intriguingly, <strong>the</strong> autonomic response to inflammation that could be seen in saline-treated controls<br />
was absent during spinal TNF-α neutralization. Here, heart rate as well as linear <strong>an</strong>d non-linear<br />
parameters of heart rate variability indicating autonomic modulation were shifted towards a<br />
sympa<strong>the</strong>tically dominated state in saline treated <strong>an</strong>imals, but remained unch<strong>an</strong>ged after<br />
induction of inflammation as compared to baseline in <strong>an</strong>imals that received intra<strong>the</strong>cal<br />
et<strong>an</strong>ercept.<br />
Overall, spinal TNF-α appears to drive peripheral inflammation <strong>an</strong>d neutralization of <strong>the</strong> latter<br />
exhibits beneficial effects on inflammation <strong>an</strong>d related symptoms. In particular, spinal TNF-α<br />
seems to be import<strong>an</strong>t <strong>for</strong> <strong>the</strong> development of central sensitization <strong>an</strong>d hyperalgesia. Whe<strong>the</strong>r <strong>the</strong><br />
altered autonomic modulation reflects <strong>an</strong> afferent mech<strong>an</strong>ism due to diminished inflammation or<br />
might actually contribute to <strong>the</strong> beneficial effects c<strong>an</strong> not be <strong>an</strong>swered <strong>from</strong> <strong>the</strong>se data <strong>an</strong>d<br />
should be investigated in greater detail in future studies.<br />
Disclosures: M.K. Boettger, None; K. Weber, None; D. Grossm<strong>an</strong>n, None; M. Gajda,<br />
None; R. Bräuer, None; R. Bauer, None; K. Bär, None; S. Schulz, None; A. Voss, None; C.<br />
Geis, None; A. Ebersberger, None; H. Schaible, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.6/EE115<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIMH Intramural Research Program<br />
Title: Effects of subacute IFN-alpha treatment on diurnal rhythm in monkeys<br />
Authors: K. CHISHOLM 1 , D. DELANEY 1 , P. L. NOBLE 1 , E. E. NELSON 2 , A. H. MILLER 3 ,<br />
*J. T. WINSLOW 1 ;<br />
1 NIMH IRP Primate Res. Core, 2 NIMH Mood <strong>an</strong>d Anxiety Disorders Program, NIH, Be<strong>the</strong>sda,<br />
MD; 3 Dept. of Psychiatry <strong>an</strong>d Behavioral Sci., Emory Univ. Sch. of Medicine, Winship C<strong>an</strong>cer<br />
Inst., Atl<strong>an</strong>ta, GA<br />
Abstract: Considerable evidence indicates that circadi<strong>an</strong> <strong>an</strong>d sleep disturb<strong>an</strong>ces are import<strong>an</strong>t in<br />
<strong>the</strong> pathophysiology of hum<strong>an</strong> mood disorders. Recent findings also suggest that <strong>the</strong> c<strong>an</strong>certreatment<br />
drug Interferon-alpha (IFNa) produces a depressive-like syndrome in hum<strong>an</strong>s <strong>an</strong>d<br />
<strong>an</strong>imals including non-hum<strong>an</strong> primates. We have recently determined that adverse rearing<br />
experiences, a putative risk factor <strong>for</strong> <strong>the</strong> emergence of hum<strong>an</strong> mood <strong>an</strong>d <strong>an</strong>xiety disorders,
produces alterations in both diurnal plasma cortisol levels as well as activity patterns determined<br />
by automated activity monitors in adolescent rhesus monkeys. We are currently examining<br />
whe<strong>the</strong>r four weeks of daily administration of (20 MIU/m(2), S.C., 5 days/wk) hum<strong>an</strong><br />
recombin<strong>an</strong>t IFNa to healthy adult rhesus monkeys (a dose previously associated with depressive<br />
symptoms) may also alter <strong>the</strong>ir diurnal activity patterns using automated activity monitors.<br />
Additional measures of <strong>an</strong>hedonia <strong>an</strong>d spont<strong>an</strong>eous home-cage behavior are also examined.<br />
Initial findings suggest that chronic treatment produces a 50% reduction in overall activity within<br />
2-3 weeks of treatment in all <strong>an</strong>imals tested. Ongoing <strong>an</strong>alysis examines whe<strong>the</strong>r <strong>an</strong>d when<br />
treatment might alter diurnal patterns of activity <strong>an</strong>d whe<strong>the</strong>r <strong>the</strong>se might be comparable to those<br />
measured in patients with mood disorders. These findings will likely in<strong>for</strong>m a relationship<br />
between altered diurnal rhythm <strong>an</strong>d IFNa induced mood-disorders.<br />
Disclosures: K. Chisholm, None; D. Del<strong>an</strong>ey, None; P.L. Noble, None; E.E. Nelson, None;<br />
A.H. Miller, National Institute of Mental Health Gr<strong>an</strong>t Nos. MH069124 <strong>an</strong>d M01-RR00039;<br />
GlaxoSmithKline; Schering Plough, Centocor, J<strong>an</strong>ssen, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
GlaxoSmithKline, D. Speakers Bureau/Honoraria (speakers bureau, symposia, <strong>an</strong>d expert<br />
witness); Schering Plough, AstraZeneca, F. Consult<strong>an</strong>t/Advisory Board; J.T. Winslow, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.7/EE116<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIH Gr<strong>an</strong>t MH086296-01<br />
Title: Increased stress-induced plasma IL-6 responses in male depressed patients with increased<br />
early life stress: Role of catecholamines<br />
Authors: *A. I. LIATIS, A. H. MILLER, T. M. MLETZKO, C. M. HEIM, R. M. BONSALL,<br />
T. W. W. PACE;<br />
Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: Previous work in our laboratory has shown that male patients with major depression<br />
<strong>an</strong>d increased early life stress (ELS) exhibit <strong>an</strong> exaggerated inflammatory response to stress<br />
compared to male controls. This exaggerated stress-induced inflammatory response may be<br />
mediated, in part, by increased stress-induced catecholamines in <strong>the</strong>se patients. There<strong>for</strong>e, we
measured plasma norepinephrine (NE) <strong>an</strong>d interleukin (IL)-6 be<strong>for</strong>e, during, <strong>an</strong>d after challenge<br />
with <strong>the</strong> Trier Social Stress Test (a public speaking <strong>an</strong>d mental arithmetic task) in male patients<br />
with major depression <strong>an</strong>d increased ELS (n = 14) <strong>an</strong>d non-depressed male controls (n = 13).<br />
Despite signific<strong>an</strong>tly greater stress-induced plasma IL-6 responses, patients with major<br />
depression <strong>an</strong>d increased ELS displayed NE responses to stress that were similar to controls.<br />
Interestingly, however, when NE responses were divided into those above <strong>an</strong>d below <strong>the</strong> medi<strong>an</strong>,<br />
depressed patients with NE responses below <strong>the</strong> medi<strong>an</strong> exhibited signific<strong>an</strong>tly greater stress<br />
induced plasma IL-6 responses compared to controls with NE responses below <strong>the</strong> medi<strong>an</strong>. IL-6<br />
responses in patients <strong>an</strong>d controls with NE responses above <strong>the</strong> medi<strong>an</strong> were comparable, <strong>an</strong>d<br />
not different th<strong>an</strong> IL-6 responses in depressed patients with NE responses below <strong>the</strong> medi<strong>an</strong>.<br />
These preliminary data indicate <strong>an</strong> increased sensitivity to NE in male depressed patients with<br />
increased ELS. Because <strong>the</strong>se depressed patients also exhibited higher baseline IL-6<br />
concentration (suggesting chronic inflammation), <strong>the</strong> increased sensitivity to NE may be<br />
mediated by upregulation of <strong>the</strong> alpha-1 adrenergic receptor <strong>an</strong>d down regulation of beta-2<br />
adrenergic receptors. Alpha-1 adrenoreceptor upregulation <strong>an</strong>d beta-2 adrenoreceptor down<br />
regulation has been found in patients with chronic inflammatory disorders <strong>an</strong>d chronic stress, <strong>an</strong>d<br />
has been associated with <strong>an</strong> increased inflammatory response to catecholamines.<br />
Disclosures: A.I. Liatis, None; A.H. Miller, None; T.M. Mletzko, None; C.M. Heim,<br />
None; R.M. Bonsall, None; T.W.W. Pace, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.8/EE117<br />
Topic: E.02.b. Behavioral effects<br />
Support: NSF IOS-05-54514<br />
NSF DBI-320988<br />
NIMH 067782<br />
Title: Brain mast cells affect neurogenesis to impact emotionality<br />
Authors: *K. M. NAUTIYAL 1 , A. IYER 2 , R. SILVER 1,2 ;<br />
1 Psyc, Columbia Univ., New York, NY; 2 Barnard Col., New York, NY
Abstract: Given mast cell location in hippocampal areas of <strong>the</strong> brain, <strong>the</strong>ir neurotrophic factor<br />
content <strong>an</strong>d impact on behavior, we hypo<strong>the</strong>size that <strong>the</strong>se immune cells function as endogenous<br />
“<strong>an</strong>ti-depress<strong>an</strong>t reservoirs” <strong>the</strong>reby modulating hippocampal neurogenesis <strong>an</strong>d influencing<br />
emotionality. Mast cells are constitutively active <strong>an</strong>d syn<strong>the</strong>size, store, <strong>an</strong>d release neuroactive<br />
mediators including tr<strong>an</strong>smitters <strong>an</strong>d trophic factors. Their numbers <strong>an</strong>d activational state<br />
fluctuate with hormone, stress <strong>an</strong>d arousal status. Mast cell deficient mice display increased<br />
<strong>an</strong>xiety-like behavior <strong>an</strong>d physiology. Here, we examined mast cell influence on <strong>the</strong> number <strong>an</strong>d<br />
phenotype of mitotic cells in neurogenenic niches in <strong>the</strong> adult brain by comparing mast cell<br />
deficient “sash” mice <strong>an</strong>d <strong>the</strong>ir heterozygous <strong>an</strong>d homozygous littermate controls.<br />
Immunohistochemical (IHC) detection of 5-bromo-2-deoxyuridine (BrdU) incorporation was<br />
used to assay cell proliferation <strong>an</strong>d survival (28d later). Effects on neuronal proliferation were<br />
also confirmed by <strong>the</strong> IHC detection of Ki67, a protein expressed in cells undergoing mitosis.<br />
The phenotype of <strong>the</strong> proliferating cells was assessed at early stages by labeling <strong>for</strong> doublecortin,<br />
a protein expressed in immature neurons. The phenotype of <strong>the</strong> surviving cells 28d later was also<br />
determined by double labeling <strong>for</strong> neuronal specific nuclei protein (NeuN) <strong>an</strong>d BrdU. Sash mice<br />
have ~30% less cell proliferation <strong>an</strong>d ~40% fewer surviving cells compared to littermate<br />
controls. Import<strong>an</strong>tly, <strong>the</strong>re was no effect of mast cell deficiency on cell proliferation in <strong>the</strong><br />
subventricular zone or cell survival in <strong>the</strong> olfactory bulb - loci lacking resident mast cells.<br />
Doublecortin staining is reduced <strong>an</strong>d <strong>the</strong>re are fewer BrdU-labeled NeuN cells in sash mice<br />
compared to littermate controls, confirming <strong>an</strong> effect on neurons. Evidence suggests that this<br />
reduction in neurogenesis is not <strong>an</strong> indirect effect of <strong>the</strong> stress phenotype in sash mice, but ra<strong>the</strong>r<br />
due to a lack of brain mast cells. Measures of hippocampal levels of brain derived neurotrophic<br />
factor (BDNF) <strong>an</strong>d nerve growth factor (NGF) in sash <strong>an</strong>d control mice will help determine mast<br />
cell contribution of neurotrophic factors. The results to date support a role <strong>for</strong> brain mast cells in<br />
<strong>the</strong> mainten<strong>an</strong>ce of neurogenesis, suggesting a mech<strong>an</strong>ism whereby mast cells impact<br />
emotionality.<br />
Disclosures: K.M. Nautiyal, None; A. Iyer, None; R. Silver, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.9/EE118<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIH AG 027697.<br />
Title: Effects of systemic IL-1 beta on <strong>an</strong>xiety-like behavior <strong>an</strong>d MCP-1 expression in <strong>the</strong> brain
Authors: E. T. GILBREATH 1 , J. L. NUNEZ 2 , P. S. MOHANKUMAR 1 , *S. M.<br />
MOHANKUMAR 3 ;<br />
1 Pathobiology <strong>an</strong>d Diagnos. Investigation, 2 Neurosci., Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI;<br />
3 Pharmacol. <strong>an</strong>d Toxicology, East L<strong>an</strong>sing, MI<br />
Abstract: Interleukin-1 beta (IL-1β), a cytokine, has recently been implicated in major<br />
depression <strong>an</strong>d <strong>an</strong>xiety. We hypo<strong>the</strong>sized that IL-1-induced ch<strong>an</strong>ges in <strong>the</strong> chemokine, MCP-1<br />
<strong>an</strong>d monoamine levels may play a role in IL-1-induced ch<strong>an</strong>ges in behavior. To test this, adult<br />
male Sprague-Dawley rats were treated with <strong>the</strong> vehicle or 5µg of IL-1 β, i.p. An hour later, <strong>the</strong><br />
<strong>an</strong>imals were subjected to behavioral tests such as <strong>the</strong> Morris water maze, radial arm maze, <strong>an</strong>d<br />
open field task. The following day, <strong>an</strong>imals were subjected to <strong>the</strong> same i.p. treatment <strong>an</strong>d<br />
sacrificed <strong>an</strong> hour later. The brains were frozen, sectioned <strong>an</strong>d <strong>the</strong> central amygdala, prefrontal<br />
cortex, hippocampus, basal g<strong>an</strong>glia, thalamus <strong>an</strong>d paraventricular nucleus were microdissected<br />
<strong>an</strong>d <strong>an</strong>alyzed <strong>for</strong> MCP-1 levels using a commercial ELISA kit. Acute IL-1 treatment produced<br />
<strong>an</strong>xiety-like behavior as evidenced by increased time spent in <strong>the</strong> open arms of <strong>the</strong> radial maze.<br />
IL-1 treatment also increased MCP-1 levels in brain regions that are associated with <strong>the</strong><br />
regulation of emotion <strong>an</strong>d stress. Signific<strong>an</strong>t increases in MCP-1 levels (Me<strong>an</strong>±SE; pg/µg<br />
protein) were observed in <strong>the</strong> amygdala (65.68±3.6 vs 21.98±3.5), basal g<strong>an</strong>glia (81.4±18.6 vs<br />
27.5±5.6), hippocampus (55.6±9.6 vs.24.4±1.6) <strong>an</strong>d prefrontal cortex (86.5±11.7 vs. 33.01±4.4)<br />
in IL-treated <strong>an</strong>imals compared to controls (p
Abstract: The etiology of depression is a matter of intense debate. For <strong>the</strong> last two decades, a<br />
<strong>the</strong>ory suggesting that enh<strong>an</strong>ced production of pro-inflammatory cytokines might underlie<br />
depression has been widely studied. This hypo<strong>the</strong>sis is supported by studies showing <strong>an</strong><br />
increased production of pro-inflammatory cytokines (such as IL-6, IL-1β, IFN-γ <strong>an</strong>d TNF) in<br />
depressed patients. Moreover, immuno<strong>the</strong>rapy with pro-inflammatory cytokines, has been<br />
associated with depressive symptoms. The fact that <strong>the</strong>se symptoms almost immediately<br />
disappear after termination of cytokine administration supports a causal role <strong>for</strong> cytokines in <strong>the</strong><br />
mediation of depression. Increased production of pro-inflammatory cytokines occurs naturally as<br />
part of <strong>the</strong> immune response to infection. In <strong>an</strong>imal models, acute infection or administration of<br />
LPS, which are both associated with high levels of pro-inflammatory cytokines during a short<br />
period of time, cause what is called sickness behaviour. This syndrome is characterised by a<br />
pattern of behavioural alterations that share similarities with those of depressed individuals.<br />
However, chronic infections (with moderate levels of pro-inflammatory cytokines during a long<br />
period of time) are very frequent <strong>an</strong>d in<strong>for</strong>mation is lacking of <strong>the</strong>ir possible role in depression.<br />
To investigate <strong>the</strong> role of chronic infection in mood disorders we chose <strong>the</strong> mouse model of<br />
Mycobacterium avium infection since it is a very well characterized model , <strong>an</strong>d also because<br />
mycobacterial infections are among <strong>the</strong> major health threats worldwide. Here we show that<br />
although <strong>the</strong> levels of at least two of <strong>the</strong> most import<strong>an</strong>t pro-inflammatory cytokines, IFN-γ <strong>an</strong>d<br />
TNF, are increased during <strong>the</strong> course of infection, C57BL/6 female mice chronically infected<br />
with M. avium do not display obvious signs of <strong>an</strong>xious or depressive-like behaviour. Additional<br />
studies are underway to investigate <strong>the</strong> reason why a prolonged production of moderate levels of<br />
pro-inflammatory cytokines does not induce depressive or <strong>an</strong>xious symptomatology.<br />
Disclosures: S. Roque, None; C. Nobrega, None; C. Nunes-Alves, None; N. Sousa, None; J.<br />
Palha, None; M. Correia-Neves, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.11/EE120<br />
Topic: E.02.b. Behavioral effects<br />
Support: NIH Gr<strong>an</strong>t MH068834<br />
Title: Immunotoxic lesion of hypothalamic noradrenergic/adrenergic input ameliorates <strong>the</strong><br />
effects of peripheral LPS challenge on sickness behavior <strong>an</strong>d associated brain c-Fos expression
Authors: *R. P. GAYKEMA, G. C. THACKER, N. J. SHAPIRO, L. E. GOEHLER;<br />
Ctr. <strong>for</strong> <strong>the</strong> Study of Complementary <strong>an</strong>d Alternative Therapies, Univ. Virginia Sch. of Nursing,<br />
Charlottesville, VA<br />
Abstract: Caudal medullary catecholamine neurons that innervate <strong>the</strong> hypothalamus play a<br />
major role in <strong>the</strong> activation of paraventricular neurons that drive pituitary adrenocorticotropin<br />
<strong>an</strong>d adrenal corticosteroid release in response to peripheral pro-inflammatory challenges with<br />
interleukin-1 or lipopolysaccharide (LPS). Pro-inflammatory challenges also lead to marked<br />
behavioral ch<strong>an</strong>ges, including fatigue, loss of social interest, <strong>an</strong>orexia, somnolence, but <strong>the</strong><br />
precise neuronal mech<strong>an</strong>isms that underlie sickness behavior remain elusive. We reasoned that<br />
<strong>the</strong> medulla-hypothalamic catecholaminergic pathway may also contribute to <strong>the</strong> behavioral<br />
m<strong>an</strong>ifestations in illness. To investigate such possible role, we applied a targeted lesion approach<br />
in rats to determine whe<strong>the</strong>r or not caudal brainstem catecholaminergic neurons that innervate<br />
<strong>the</strong> hypothalamus are also necessary <strong>for</strong> <strong>the</strong> expression of sickness behavior. Anti-dopamine beta<br />
hydroxylase <strong>an</strong>tibodies conjugated to saporin (DSAP), when injected into a target region,<br />
selectively poisons <strong>an</strong>d destroy noradrenergic/adrenergic neurons that innervate <strong>the</strong> target. DSAP<br />
was micro-injected bilaterally into <strong>the</strong> hypothalamic paraventricular nucleus (PVN), whereas<br />
control rats received unconjugated saporin (SAP controls). Fourteen days later <strong>the</strong> <strong>an</strong>imals were<br />
injected intraperitoneally with ei<strong>the</strong>r LPS or saline, <strong>an</strong>d 2h later were submitted to <strong>the</strong> open field<br />
to record <strong>the</strong>ir exploratory behavior, 1h after which <strong>the</strong> rats were sacrificed <strong>for</strong> brain<br />
immunohistochemical <strong>an</strong>alyses. LPS-treated SAP control rats showed drastic reduction in<br />
exploratory behavior (reduced locomotion dist<strong>an</strong>ce <strong>an</strong>d velocity). Prior DSAP microinjections<br />
largely reversed <strong>the</strong> LPS-induced reduction in locomotor behavior. The brains of <strong>the</strong>se DSAP<br />
rats showed a dramatic loss of noradrenergic innervation of <strong>the</strong> PVN but also in o<strong>the</strong>r parts of <strong>the</strong><br />
medial, tuberal <strong>an</strong>d tuberomammilary regions of <strong>the</strong> hypothalamus. The behavioral resilience to<br />
LPS coincided with diminished LPS-related c-Fos staining in <strong>the</strong> PVN, <strong>an</strong>d increased c-Fos<br />
staining in <strong>the</strong> lateral <strong>an</strong>d tuberomammillary regions related to behavior <strong>an</strong>d/or arousal. In<br />
summary, our findings support <strong>the</strong> hypo<strong>the</strong>sis that hypothalamic catecholaminergic projections<br />
originating in <strong>the</strong> lower brainstem play a critical role in <strong>the</strong> expression of sickness behavior in<br />
<strong>the</strong> context of novelty-induced exploratory activity, but we c<strong>an</strong>not determine with precision in<br />
which part of <strong>the</strong> hypothalamus <strong>the</strong> noradrenergic/adrenergic input contributes to <strong>the</strong> expression<br />
of sickness behavior due to extensive collateralization of <strong>the</strong> ascending projections throughout<br />
<strong>the</strong> hypothalamus.<br />
Disclosures: R.P. Gaykema, None; G.C. Thacker, None; N.J. Shapiro, None; L.E. Goehler,<br />
None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 570.12/EE121<br />
Topic: E.02.b. Behavioral effects<br />
Support: 21st COE program 'Base to Overcome Fatigue'<br />
CREST<br />
Title: Interleukin-1β expression in <strong>the</strong> brain is involved in poly I:C-induced immunological<br />
fatigue-like behavior in rats<br />
Authors: *M. YAMATO 1,3 , K. OKUYAMA 1 , J. GUANGHUA 1,3 , A. EGUCHI 1 , Y.<br />
WATANABE 2,3 , Y. KATAOKA 1,3 ;<br />
1 Cell. Function Imaging Lab., 2 Mol. Probe Dynamics Lab., RIKEN Ctr. For Mol. Imaging Sci.,<br />
Hyogo, Jap<strong>an</strong>; 3 Physiol., Osaka City Univ. Grad. Sch. of Med., Osaka, Jap<strong>an</strong><br />
Abstract: Intraperitoneal (i.p) injection of polyriboinosinic:polyribocytidylic acid (poly I:C),<br />
double-str<strong>an</strong>d RNA, is known to mimic viral infection, <strong>an</strong>d induces immunological fatigue-like<br />
behavior including tr<strong>an</strong>sient fever <strong>an</strong>d <strong>the</strong> suppression of locomotor activity in rats. In <strong>the</strong> last<br />
<strong>an</strong>nual meeting of <strong>the</strong> <strong>Society</strong> <strong>for</strong> Neuroscience, we reported that Interleukin (IL)-1β <strong>an</strong>d IL-6<br />
mRNAs were expressed in <strong>the</strong> cerebral cortex, hippocampus, <strong>an</strong>d hypothalamus in <strong>the</strong> <strong>an</strong>imals,<br />
<strong>an</strong>d that peripheral injection of rat recombin<strong>an</strong>t (rr) IL-1β induced IL-1β mRNA expression in<br />
<strong>the</strong> brain <strong>an</strong>d mimicked poly I:C-induced fatigue-like behavior. In <strong>the</strong> present report, we<br />
demonstrate that intracerebroventricular (i.c.v) injection of rrIL-1β suppressed locomotor activity<br />
in normal rats, <strong>an</strong>d that i.c.v infusion of IL-1 receptor <strong>an</strong>tagonist signific<strong>an</strong>tly attenuated poly<br />
I:C-induced fatigue-like behavior. These observations suggest that brain IL-1β is a key mediator<br />
<strong>for</strong> <strong>the</strong> induction of immunological fatigue in viral infection.<br />
This study was supported by <strong>the</strong> funds <strong>from</strong> COE program <strong>an</strong>d CREST.<br />
Disclosures: M. Yamato, None; K. Okuyama, None; J. Gu<strong>an</strong>ghua, None; A. Eguchi,<br />
None; Y. Wat<strong>an</strong>abe, None; Y. Kataoka, None.<br />
Poster<br />
570. Neuroimmunology: Behavioral Effects<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 570.13/EE122<br />
Topic: E.02.b. Behavioral effects
Support: NSERC<br />
C<strong>an</strong>adi<strong>an</strong> Foundation <strong>for</strong> Innovation<br />
Title: Role of class I immune molecules in regulating sickness behaviour<br />
Authors: A. SANKAR, R. N. MACKENZIE, *J. A. FOSTER;<br />
Psych & Behav Neurosci, Brain Body Inst, McMaster Univ., Hamilton, ON, C<strong>an</strong>ada<br />
Abstract: The role <strong>for</strong> class I MHC proteins is growing past <strong>the</strong> immune realm. In particular, <strong>the</strong><br />
discovery of class I MHC proteins in neurons <strong>an</strong>d <strong>the</strong>ir possible role as cellular indicators of<br />
stress highlights <strong>the</strong> import<strong>an</strong>ce of this molecule in immune-brain signalling. In response to<br />
immune challenge, <strong>the</strong> CNS mounts <strong>an</strong> innate immune response; previous work has shown that<br />
neuronal expression of class I MHC contributes to <strong>the</strong> neuronal component of this response. In<br />
order to fur<strong>the</strong>r eludicate <strong>the</strong> role of class I MHC to <strong>the</strong> CNS innate immune response, our group<br />
per<strong>for</strong>med behavioural testing on functional class I MHC knockout mice (constitutive β2microglobulin<br />
(B2M) <strong>an</strong>d tr<strong>an</strong>sporter associated with <strong>an</strong>tigen processing (TAP) double knockout<br />
mice). Firstly, we examined basal locomotor activity in <strong>the</strong> open field of knockout <strong>an</strong>d wild-type<br />
mice. Secondly, we examined sickness behaviour following i.p injection with endotoxin (LPS).<br />
Our experiments testing basal behaviour yielded signific<strong>an</strong>t differences in locomotor activity<br />
between female B2M-/-/TAP-/- <strong>an</strong>d B6 mice while no differences were observed in males. <strong>When</strong><br />
sickness behaviour was <strong>an</strong>alyzed, more differences emerged. LPS-injected B2M-/-TAP-/- mice<br />
showed no acute sickness response (first 30 mins) compared to SAL-injected B2M-/-TAP-/-.<br />
LPS-injected B2M-/-TAP-/- mice showed reduced locomotor activity later in <strong>the</strong> test suggesting<br />
a delayed or reduced response to immune challenge. The results of our experiment point to<br />
aberr<strong>an</strong>t signalling of <strong>the</strong> immune stress signal in <strong>the</strong> B2M-/-/TAP-/- group. Molecular <strong>an</strong>alysis<br />
of CNS <strong>an</strong>d peripheral innate immune signalling pathways are underway.<br />
Disclosures: A. S<strong>an</strong>kar, None; R.N. MacKenzie, None; J.A. Foster, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.1/EE123<br />
Topic: E.03.b. Parental behavior<br />
Support: 2008 NARSAD Young Investigator Award to M. Pereira
Title: Adenosine-dopamine receptor interaction in <strong>the</strong> regulation of maternal responsiveness<br />
across postpartum<br />
Authors: *M. PEREIRA 1 , A. M. FARRAR 2 , J. HOCKEMEYER 3 , C. E. MÜLLER 3 , J. D.<br />
SALAMONE 4 , J. I. MORRELL 2 ;<br />
2 Ctr. Mol & Behav Neurosci, 1 Rutgers Univ., Newark, NJ; 3 Pharmazeutische Chemie, Univ.<br />
Bonn, Pharmazeutisches Institut,, Bonn, Germ<strong>an</strong>y; 4 Dept. of Psychology, Univ. of Connecticut,<br />
Storrs, CT<br />
Abstract: Mesolimbic dopamine (DA), particularly in <strong>the</strong> nucleus accumbens, import<strong>an</strong>tly<br />
regulates activational aspects of maternal behavior across <strong>the</strong> postpartum period. DA <strong>an</strong>tagonism<br />
<strong>an</strong>d accumbens DA depletions interfere with early postpartum maternal motivation by selectively<br />
affecting most <strong>for</strong>ms of active maternal behaviors (retrieval <strong>an</strong>d grouping of <strong>the</strong> pups at <strong>the</strong> nest<br />
site, pup licking <strong>an</strong>d nest building), while leaving nursing behavior (a motorically inactive state)<br />
relatively intact. Considerable evidence indicates that <strong>the</strong>re is a functional interaction between<br />
D2 DA <strong>an</strong>d A2A adenosine receptors in striatal areas, including <strong>the</strong> nucleus accumbens. For<br />
inst<strong>an</strong>ce, adenosine A2A <strong>an</strong>tagonism has been shown to reverse <strong>the</strong> disruptive effects of DA<br />
<strong>an</strong>tagonism on behavioral output <strong>an</strong>d ef<strong>for</strong>t-related decision-making using instrumental tasks that<br />
involve food-seeking behaviors. The first set of experiments was conducted to determine if<br />
adenosine A2A receptor <strong>an</strong>tagonism could reverse <strong>the</strong> effects of DA receptor <strong>an</strong>tagonism on<br />
early postpartum maternal responsiveness. The adenosine A2A receptor <strong>an</strong>tagonist MSX-3 (0.5,<br />
0.75, 1.0 <strong>an</strong>d 2.0 mg/kg, IP) was investigated <strong>for</strong> its ability to reverse <strong>the</strong> effects of <strong>the</strong> D2 DA<br />
receptor <strong>an</strong>tagonist haloperidol (0.1 mg/kg, IP) on <strong>the</strong> maternal behavior of early postpartum<br />
female rats. Haloperidol severely impaired <strong>the</strong> expression of active maternal components, while<br />
MSX-3, when administered in <strong>the</strong> absence of haloperidol, did not affect early postpartum<br />
maternal responding. Co-administration of MSX-3 (0.5-2.0 mg/kg IP) with haloperidol produced<br />
a dose-related attenuation of <strong>the</strong> haloperidol-induced behavioral deficits in early postpartum<br />
females. As deactivation of <strong>the</strong> mesolimbic DA system appears to be involved in <strong>the</strong> natural<br />
decline of maternal responsiveness characteristic of late postpartum, a fur<strong>the</strong>r experiment<br />
examined <strong>the</strong> effects of MSX-3 alone on <strong>the</strong> maternal behavior of late postpartum females. As<br />
hypo<strong>the</strong>sized, all doses of MSX-3 (0.5-2.0 mg/kg) facilitated <strong>the</strong> expression of maternal behavior<br />
in late postpartum females. However, it was observed that <strong>the</strong> high dose of MSX-3 also induced<br />
oral stereotypy in late postpartum females. Results show that adenosine <strong>an</strong>d DA systems interact<br />
to regulate maternal responsiveness across <strong>the</strong> postpartum period. This research may potentially<br />
contribute to <strong>the</strong> development of strategies <strong>for</strong> treatments of psychiatric disorders during <strong>the</strong><br />
postpartum period, with particular emphasis in maintaining or restoring mo<strong>the</strong>r-inf<strong>an</strong>t bonding.<br />
Disclosures: M. Pereira, None; A.M. Farrar, None; J. Hockemeyer, None; C.E. Müller,<br />
None; J.D. Salamone, None; J.I. Morrell, None.<br />
Poster<br />
571. Parental Behavior
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.2/EE124<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH Gr<strong>an</strong>t RO1HD039668<br />
Title: Diazepam reduces latencies to behave maternally in naïve primigravid rats<br />
Authors: *P. E. MANN, J. O. LEE, P. GERWIN;<br />
Dept Biomed Sci., Tufts Univ., North Grafton, MA<br />
Abstract: <strong>When</strong> virgin (naïve) female rats are exposed to foster pups <strong>the</strong>y not only fail to<br />
display maternal behavior, <strong>the</strong>y actively avoid or c<strong>an</strong>nibalize <strong>the</strong> pups. This observation <strong>an</strong>d<br />
o<strong>the</strong>r evidence has lead to <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> initial onset of maternal behavior is a conflict<br />
between approach <strong>an</strong>d avoid<strong>an</strong>ce, with <strong>the</strong> avoid<strong>an</strong>ce due to <strong>the</strong> possible aversive quality of <strong>the</strong><br />
pups. The involvement of <strong>the</strong> GABAergic system in <strong>the</strong> resolution of this conflict has not been<br />
well-studied. The objective of <strong>the</strong> present study was to determine whe<strong>the</strong>r subcut<strong>an</strong>eous<br />
injections of diazepam, a benzodiazepine receptor agonist, would stimulate <strong>the</strong> onset of maternal<br />
behavior in rats during <strong>an</strong> initial pregn<strong>an</strong>cy. Groups of nulliparous (virgin) Sprague-Dawley rats<br />
were time-mated. Starting on day 14 of gestation until parturition, females received once daily<br />
injections of ei<strong>the</strong>r diazepam (0.1, 0.5, or 2.5 mg/kg; Hospira) or vehicle (40% propylene glycol,<br />
10% eth<strong>an</strong>ol in water). On day 15 of gestation, subjects were tested on <strong>the</strong> elevated plus maze<br />
(EPM; between 0900-1200 h) to evaluate <strong>the</strong> effects of diazepam on that task. Maternal behavior<br />
testing beg<strong>an</strong> following EPM exposure <strong>an</strong>d continued once daily until parturition. Rats treated<br />
with <strong>the</strong> 0.5 mg/kg dose of diazepam had signific<strong>an</strong>tly shorter latencies (medi<strong>an</strong> days) to display<br />
maternal behavior compared to all o<strong>the</strong>r groups: 0.5 mg/kg (5 days) vs. vehicle (8 days); 0.5<br />
mg/kg (5 days) vs. 0.1 mg/kg (8 days); 0.5 mg/kg (5 days) vs. 2.5 mg/kg (8 days), all P’s
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.3/EE125<br />
Topic: E.03.b. Parental behavior<br />
Support: NIMH<br />
CIHR<br />
Title: Brain derived neurotrophic factor mediates variations in maternal behavior<br />
Authors: *D. K. SHAHROKH, T. ZHANG, M. J. MEANEY;<br />
McGill, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: The aim of <strong>the</strong> current study was to investigate <strong>the</strong> role of mesolimbic brain derived<br />
neurotrophic factor (BDNF) in maternal behavior. Naturally occurring variations in maternal<br />
licking/grooming (LG) of pups influence neural development, <strong>an</strong>d are tr<strong>an</strong>smitted <strong>from</strong> mo<strong>the</strong>r<br />
to female offspring. Thus, <strong>the</strong> female offspring of high LG mo<strong>the</strong>rs will in turn be high LG dams<br />
as adults. BDNF, a prominent CREB-regulated gene, mediates m<strong>an</strong>y enduring ch<strong>an</strong>ges in<br />
neuroplasticity <strong>an</strong>d synaptogenesis. In order to assess <strong>the</strong> role of BDNF in maternal behavior,<br />
BDNF levels <strong>an</strong>d expression were measured across various brain regions of lactating dams. The<br />
effect of a temporary knock-down of BDNF on maternal behavior was assessed using <strong>an</strong> <strong>an</strong>ti-<br />
BNDF <strong>an</strong>tibody. Anti-BDNF was infused into <strong>the</strong> nucleus accumbens (nAcc) shell of lactating<br />
female rats twice a day <strong>for</strong> <strong>the</strong> first four days of post-partum. The results revealed that <strong>the</strong><br />
infusions disrupted maternal behavior such that <strong>the</strong> licking/grooming behavior of High LG dams<br />
resembled that of <strong>the</strong> Low LG dams. To check <strong>for</strong> differences in BDNF expression, BDNF <strong>an</strong>d<br />
TrkB (BDNF receptor) mRNA levels were measured using in situ hybridization. The results<br />
revealed <strong>an</strong> increase in BDNF expression at <strong>the</strong> level of mRNA in <strong>the</strong> hippocampus <strong>an</strong>d ventral<br />
tegmental area (VTA) of high LG mo<strong>the</strong>rs on post partum day 4 (PP4): Both regions project to<br />
<strong>the</strong> nAcc. The <strong>an</strong>alysis <strong>for</strong> TrkB mRNA levels revealed no signific<strong>an</strong>t difference between high<br />
<strong>an</strong>d low LG dams. In addition, ELISA results revealed <strong>an</strong> increase in BDNF protein levels in <strong>the</strong><br />
nAcc <strong>an</strong>d medial preoptic area (MPOA) of high LG dams compared to low LG dams on PP4. In<br />
addition, a Chromatin Immunoprecipitation (ChIP) was per<strong>for</strong>med on <strong>the</strong> lysine 9 risidue of<br />
histone 3 (H3K9) <strong>an</strong>d <strong>the</strong> promoter region of <strong>the</strong> BDNF exon IV, which has been indicated in<br />
mediating drug addiction <strong>an</strong>d depression. High LG dams on PP4 were found to express more<br />
acetylated H3K9 <strong>an</strong>d hence higher levels of BDNF expression compared to low LG dams. To<br />
fur<strong>the</strong>r consolidate <strong>the</strong> findings, a chip was used to look <strong>for</strong> differential binding of MeCP2<br />
(methyl binding protein commonly associated with tr<strong>an</strong>scriptional repression) to <strong>the</strong> BDNF exon<br />
IV promoter. Results revealed that Low LG dams had <strong>an</strong> increase MeCP2 binding to Exon IV<br />
compared to High LG dams which supports <strong>the</strong> H3K9 data. Bisulfite mapping was used to assess<br />
differences in methylation in <strong>the</strong> exon IV promoter region of High compared to Low LG dams<br />
on PP4. Results revealed no differences in nei<strong>the</strong>r site specific nor overall methylation between<br />
<strong>the</strong> two groups. In summary, <strong>the</strong>se findings suggest that variations in maternal care are
associated with differences in both <strong>the</strong> regulation <strong>an</strong>d expression of BDNF in <strong>the</strong> mesolimbic<br />
system.<br />
Disclosures: D.K. Shahrokh , None; T. Zh<strong>an</strong>g, None; M.J. Me<strong>an</strong>ey, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.4/EE126<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH Gr<strong>an</strong>t R01 DA019946<br />
Title: Vasopressin V1a receptors modulate maternal BOLD fMRI response to a novel male<br />
intruder<br />
Authors: *M. K. CAFFREY, B. C. NEPHEW, M. FEBO;<br />
Nor<strong>the</strong>astern Univ., Boston, MA<br />
Abstract: Vasopressin V1a receptors in <strong>the</strong> rat brain have been studied <strong>for</strong> <strong>the</strong>ir role in<br />
modulating aggression <strong>an</strong>d <strong>an</strong>xiety. Maternal aggression is a robust <strong>for</strong>m of aggression displayed<br />
by lactating females when presented with a novel male intruder. In <strong>the</strong> current study, bloodoxygen-level-dependent<br />
(BOLD) functional MRI was used to test whe<strong>the</strong>r V1a receptors<br />
modulate neural processing in <strong>the</strong> maternal brain when dams are exposed to a male intruder.<br />
Primiparous females were given <strong>an</strong> intracerebroventricular (ICV) injection of vehicle or V1a<br />
receptor <strong>an</strong>tagonist ([deamino-Pen 1 , O-Me-Tyr, Arg 8 ]-Vasopressin, 125 ng/10 µL) 90-120<br />
minutes be<strong>for</strong>e imaging. During fMRI, fully awake dams were presented with <strong>the</strong>ir own pups<br />
<strong>an</strong>d a male intruder using a specialized chamber that contained separate compartments <strong>for</strong> <strong>the</strong><br />
pups <strong>an</strong>d <strong>the</strong> intruder. The results indicate that multiple primary sensory processing areas are<br />
modulated by V1a receptor blockade. Dams treated with V1a <strong>an</strong>tagonist showed signific<strong>an</strong>tly<br />
greater BOLD responses in <strong>the</strong> <strong>an</strong>terior olfactory nucleus, infralimbic prefrontal cortex,<br />
gustatory cortex, subst<strong>an</strong>tia innominata, <strong>an</strong>d somatosensory cortex when presented with a novel<br />
male intruder. BOLD responses were reduced in <strong>the</strong> cortical amygdala <strong>an</strong>d ventromedial<br />
hypothalamus. These results indicate that vasopressin, acting through V1a receptors, modulates<br />
<strong>the</strong> neural response of <strong>the</strong> maternal rat brain in <strong>the</strong> presence of a novel male intruder.<br />
Disclosures: M.K. Caffrey, None; B.C. Nephew, None; M. Febo, None.
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.5/EE127<br />
Topic: E.03.b. Parental behavior<br />
Support: NSERC Gr<strong>an</strong>t<br />
Title: Oxytocin in <strong>the</strong> nucleus accumbens <strong>an</strong>d maternal memory in postpartum rats<br />
Authors: T. M. D'CUNHA 1 , S. J. KING 2 , F. LÉVY 3 , *A. S. FLEMING 1 ;<br />
1 Univ. Toronto Mississauga, Mississauga, ON, C<strong>an</strong>ada; 2 Inst. of Neurosci., Carleton Univ.,<br />
Ottawa, ON, C<strong>an</strong>ada; 3 Lab. de Comportement, Neurobiologie et Adaptation, Inra-Cnrs-<br />
Université de Tours-Haras Nationaux, Nouzilly, Fr<strong>an</strong>ce<br />
Abstract: Female rats with prior maternal experience display a shorter onset of maternal<br />
responsiveness toward <strong>the</strong>ir pups compared to those with no prior experience. This phenomenon<br />
is called <strong>the</strong> ‘maternal experience effect’ or ‘maternal memory’, <strong>an</strong>d is critically dependent on<br />
<strong>the</strong> nucleus accumbens (NA) shell. Recent evidence has shown that oxytocin (OT) receptors<br />
present in <strong>the</strong> NA are import<strong>an</strong>t <strong>for</strong> <strong>the</strong> <strong>for</strong>mation of social bonds, <strong>an</strong>d that blocking <strong>the</strong>se<br />
receptors has been shown to delay <strong>the</strong> onset of maternal responsiveness. This study investigated<br />
<strong>the</strong> roles of OT receptors on maternal memory. In Experiment 1, postpartum female rats given<br />
one hour of maternal experience were infused at <strong>the</strong> end of <strong>the</strong> experience with ei<strong>the</strong>r a high or<br />
low dose of <strong>an</strong> OT <strong>an</strong>tagonist, (d(CH2)5 Tyr (Me)2, Orn 8)-AVT, into <strong>the</strong> NA shell <strong>an</strong>d <strong>the</strong>n<br />
tested <strong>for</strong> maternal behaviour after a 10-day pup isolation period. Females showed a signific<strong>an</strong>tly<br />
longer latency to exhibit full maternal behaviour after <strong>the</strong> pup isolation compared to females that<br />
received a vehicle control. In Experiment 2, postpartum female rats were infused with ei<strong>the</strong>r a<br />
high or low dose of OT into <strong>the</strong> NA shell immediately after a 15 minute maternal experience <strong>an</strong>d<br />
tested <strong>for</strong> maternal behaviour after a 10-day pup isolation period. A trend emerged indicating<br />
that females infused with a high dose of OT displayed a shorter latency to become maternal<br />
compared to females treated with a vehicle control. These findings taken toge<strong>the</strong>r suggest that<br />
OT receptors in <strong>the</strong> NA shell play a role in <strong>the</strong> consolidation of maternal memory.<br />
Disclosures: T.M. D'Cunha, None; S.J. King, None; F. Lévy, None; A.S. Fleming, None.<br />
Poster
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.6/EE128<br />
Topic: E.03.b. Parental behavior<br />
Title: Regulatory function of oxytocin receptor in maternal behavior of mouse<br />
Authors: *K. SATO 1 , A. YAMASHITA 1 , D. OSADA 1 , M. YOSHIDA 1 , Y. AOYAGI 1 , H.<br />
MIZUKAMI 2 , K. OZAWA 2 , K. NISHIMORI 1 ;<br />
1 2<br />
Grad. Sch. of Agr. Science, Tohoku Univ., Tohoku Univ., Sendai, Jap<strong>an</strong>; Jichi Med. Univ.,<br />
Tochigi, Jap<strong>an</strong><br />
Abstract: Oxytocin (OXT) was known to have hormonal function, <strong>an</strong>d recently has been<br />
considered as a neuromodulator. Previously, we generated oxytocin receptor (OXTR) knockout<br />
mice, <strong>an</strong>d reported that OXTR had essential role in regulation of social behaviors (i.e. maternal<br />
behavior, affiliative behavior, social recognition, aggression <strong>an</strong>d so on)[1]. In order to identify<br />
<strong>an</strong>d characterize <strong>the</strong> neurons expressing OXTR in <strong>the</strong> brain, next we generated OXTR-Venus<br />
knockin mice, <strong>an</strong>d found that OXTR was expressed in various nuclear regions, suspected to<br />
control social behaviors [2].<br />
Recently, we developed adeno-associated viral vector expressing OXTR (AAV-Oxtr). It is <strong>an</strong><br />
ideal tool to reveal <strong>the</strong> region-specific function of OXTR in <strong>the</strong> brain, by stereotaxic injection to<br />
various nuclear regions in OXTR knockout mice showing impairment of maternal behaviors.<br />
With this experiment, we found rescued phenotype in retrieving behavior, one of <strong>the</strong> typical<br />
maternal behaviors (n = 3).<br />
On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, we <strong>an</strong>alyzed alteration of OXTR expression in <strong>the</strong> OXT expressing neuron<br />
with OXTR-Venus female mice, when maternal behavior was induced after parturition. In <strong>the</strong><br />
result, <strong>the</strong> number of Venus-positive cells, indicating expression of OXTR, increased<br />
signific<strong>an</strong>tly in <strong>the</strong> medial preoptic area (mPOA), known to be <strong>an</strong> essential region <strong>for</strong> maternal<br />
behavior, <strong>an</strong>d in <strong>the</strong> supraoptic nucleus (SON), where OXT releasing cells are restrictedly<br />
distributed, with parturition <strong>an</strong>d induction of maternal behavior. The increase of Venus-positive<br />
cells in mPOA <strong>an</strong>d SON of <strong>the</strong> virgin female mice after induction of maternal behavior was also<br />
detected.<br />
References:<br />
1.Takay<strong>an</strong>agi Y et al. (2005) Proc Natl Acad Sci USA 102:16096<br />
2.Yoshida M et al. (<strong>2009</strong>) J Neurosci 29:2259<br />
Disclosures: K. Sato, None; A. Yamashita, None; D. Osada, None; M. Yoshida, None; Y.<br />
Aoyagi, None; H. Mizukami, None; K. Ozawa, None; K. Nishimori, None.
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.7/EE129<br />
Topic: E.03.b. Parental behavior<br />
Support: CIHR<br />
NSERC<br />
Title: The effects of early maternal separation on accumbal dopamine in adult female rats as a<br />
function of parity <strong>an</strong>d hormonal priming<br />
Authors: *V. M. AFONSO, S. J. KING, A. S. FLEMING;<br />
Univ. of Toronto At Mississauga, Mississauga, ON, C<strong>an</strong>ada<br />
Abstract: Mo<strong>the</strong>rless rats, raised through artificial rearing (AR), show altered maternal<br />
responses to pup-stimuli that c<strong>an</strong> be partially reversed with varying amounts of pre-we<strong>an</strong>ing<br />
tactile stimulation. Previously in mo<strong>the</strong>r reared rats, dopamine (DA) responses to pup-stimuli<br />
varied with maternal responsiveness (Afonso et al., <strong>2009</strong>). To fur<strong>the</strong>r investigate accumbal DA<br />
mediation of maternal behavior, <strong>the</strong> present study examined DA responses in <strong>the</strong> NACsh, to pup-<br />
<strong>an</strong>d food- stimuli in adult AR female rats with varying amounts of pre-we<strong>an</strong>ing tactile<br />
stimulation (ARmin; ARmax). In Experiment 1, samples were collected ei<strong>the</strong>r <strong>from</strong> postpartum<br />
day 1 dams or cycling females. In Experiment 2, samples were collected <strong>from</strong> ovariectomized<br />
females given ei<strong>the</strong>r parturition-like hormone or sham treatments. For in vivo procedure, after<br />
baseline collection in <strong>the</strong> dialysis chamber, pup- <strong>an</strong>d <strong>the</strong>n food- stimuli were individually<br />
presented to females. Prior to stimulus presentation, postpartum <strong>an</strong>d hormone-treated AR<br />
females had decreased basal DA concentrations compared to <strong>the</strong>ir controls. In response to pupstimuli,<br />
only postpartum <strong>an</strong>d hormone-treated AR females had increased DA levels compared to<br />
basal concentrations. However, unlike our previous study with maternally reared postpartum <strong>an</strong>d<br />
hormone-treated females in which DA response remained elevated <strong>for</strong> <strong>the</strong> duration of <strong>the</strong> pupstimuli<br />
availablility, AR mo<strong>the</strong>r rats did not have robust sustained elevated DA responses. These<br />
DA responses were affected by pre-we<strong>an</strong>ing tactile stimulation. In response to food-stimuli, all<br />
AR females had similar increased DA responses.<br />
Disclosures: V.M. Afonso, None; S.J. King, None; A.S. Fleming, None.<br />
Poster
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.8/EE130<br />
Topic: E.03.b. Parental behavior<br />
Support: FAPESP<br />
CNPq - Brazil<br />
Title: Serotonin receptors mRNA qu<strong>an</strong>tification in <strong>the</strong> hypothalamus of female inbred mice<br />
differing in maternal per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: *S. CHIAVEGATTO 1,2 , G. AMBAR 3 , B. SAUCE 4 , A. C. PERIPATO 4 ;<br />
1 Pharmacol., Biomed. Sci. Institute; Univ. of Sao Paulo, Sao Paulo, Brazil; 2 INCT of<br />
Developmental Psychiatry, CNPq, Brazil; 3 Psychiatry, Inst. of Psychiatry Univ. of Sao Paulo<br />
Med. Sch., Sao Paulo, Brazil; 4 Genet. <strong>an</strong>d Evolution, Biol. Sci. <strong>an</strong>d Hlth. Center; Federal Univ.<br />
Sao Carlos, SP, Brazil<br />
Abstract: Maternal per<strong>for</strong>m<strong>an</strong>ce is a complex behavioral trait that is essential in early stages in<br />
mammals. Recently, <strong>an</strong> import<strong>an</strong>t role <strong>for</strong> central serotonin (5-HT) system in maternal behavior<br />
was suggested using mice deficient <strong>for</strong> a 5-HT tr<strong>an</strong>scription factor (Lerch-H<strong>an</strong>er et al., 2008).<br />
We previously reported that female mice <strong>from</strong> LG/J <strong>an</strong>d SM/J inbred strains exhibit abnormal<br />
maternal per<strong>for</strong>m<strong>an</strong>ce <strong>for</strong> offspring survival (60% <strong>an</strong>d 26% of litter loss, respectively) (Peripato<br />
et al., 2002). In <strong>the</strong> present study, we investigated hypothalamic 5-HT receptor tr<strong>an</strong>scription in<br />
females <strong>from</strong> <strong>the</strong>se both strains <strong>an</strong>d fur<strong>the</strong>r characterize <strong>the</strong>ir difference in maternal behavior.<br />
Different sets of LG/J <strong>an</strong>d SM/J female mice were used to investigate maternal behaviors (nest<br />
building, milk provision, aggressive behavior; n=24-29 each strain) or several 5-HT receptors<br />
gene expression by qRT-PCR (whole hypothalamic area was obtained at <strong>the</strong> 2 nd day postpartum;<br />
n=13 each strain).<br />
LG/J <strong>an</strong>d SM/J females built pre <strong>an</strong>d postpartum nests to assembly <strong>the</strong>ir offspring, however only<br />
<strong>the</strong> SM/J dams showed a more elaborated pre (p
tr<strong>an</strong>scripts in <strong>the</strong> hypothalamus, when compared to females with better maternal per<strong>for</strong>m<strong>an</strong>ce.<br />
These correlations may suggest <strong>the</strong> participation of <strong>the</strong> specific subtypes 2A, 5B, <strong>an</strong>d 7 <strong>from</strong><br />
hypothalamic postsynaptic 5-HT receptors in maternal behaviors <strong>an</strong>d warr<strong>an</strong>t additional<br />
investigation.<br />
Disclosures: S. Chiavegatto, None; G. Ambar, None; B. Sauce, None; A.C. Peripato, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.9/EE131<br />
Topic: E.03.b. Parental behavior<br />
Support: NIMH Gr<strong>an</strong>t MH41447<br />
Title: Ch<strong>an</strong>ges in prolactin-induced JAK-STAT signaling in <strong>the</strong> ring dove brain as a function of<br />
reproductive state <strong>an</strong>d in response to exogenous prolactin administration<br />
Authors: *J. D. BUNTIN, L. BUNTIN;<br />
Univ. Wisconsin, Milwaukee, WI<br />
Abstract: Prolactin stimulates several physiological <strong>an</strong>d behavioral ch<strong>an</strong>ges associated with<br />
parental care in birds, but <strong>the</strong> cellular mech<strong>an</strong>isms that mediate <strong>the</strong>se actions are poorly<br />
understood. In mammals, <strong>the</strong> J<strong>an</strong>us Kinase (JAK)-signal tr<strong>an</strong>sducer <strong>an</strong>d activator of tr<strong>an</strong>scription<br />
(STAT) signaling pathway mediates m<strong>an</strong>y of <strong>the</strong> actions of prolactin in several target tissues,<br />
including <strong>the</strong> brain. The activation of JAK2 that occurs following prolactin binding to its<br />
receptor is required <strong>for</strong> phosphorylation of STAT proteins, which in turn act as tr<strong>an</strong>scription<br />
factors to modify gene expression. We examined ch<strong>an</strong>ges in phosphorylated STAT5 (pSTAT5)<br />
expression in discrete diencephalic brain regions by immunocytochemistry in untreated nonbreeding<br />
female ring doves, non-breeding females that received chronic infusions of ovine<br />
prolactin (80µg/day) or vehicle <strong>for</strong> seven days, <strong>an</strong>d breeding females sampled during <strong>the</strong> prelaying,<br />
early incubation, <strong>an</strong>d early post-hatching phases of <strong>the</strong> reproductive cycle. Prolactintreated<br />
females showed strong pSTAT5 immunostaining in several brain areas, including <strong>the</strong><br />
preoptic area, lateral hypothalamus, paraventricular nucleus, tuberal hypothalamus, <strong>an</strong>d lateral<br />
bed nucleus of <strong>the</strong> stria terminalis. In contrast, pSTAT5 immunostaining was virtually absent in<br />
<strong>the</strong>se regions in vehicle treated doves. A similar <strong>an</strong>atomical distribution of pSTAT5immunostained<br />
cells were observed in females sampled during <strong>the</strong> breeding cycle. Breeding<br />
females sampled during <strong>the</strong> early post-hatching period when peak levels of plasma prolactin are
attained had over 10 times <strong>the</strong> number of pSTAT5-immunostaining cells in <strong>the</strong> POA as nonbreeding<br />
females <strong>an</strong>d over twice <strong>the</strong> number seen in females sampled during <strong>the</strong> prelaying <strong>an</strong>d<br />
early incubation phases. A similar pattern of differences was observed when pSTAT5 staining in<br />
o<strong>the</strong>r brain regions was compared across breeding stages. These results suggest that that JAK-<br />
STAT signaling pathway, <strong>an</strong>d STAT5 in particular, is strongly activated by prolactin during <strong>the</strong><br />
ring dove breeding cycle.<br />
Disclosures: J.D. Buntin, None; L. Buntin, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.10/EE132<br />
Topic: E.03.b. Parental behavior<br />
Title: Pelvic signaling influences postpartum maternal care in prairie voles<br />
Authors: *U. L. HAYES 1 , A. ORTIZ 2 ;<br />
1 2<br />
Ctr. Neuroendocrine Studies, Univ. Massachusetts, Amherst, MA; Dept. of Psychology, Univ.<br />
of Massachusetts, Amherst, MA<br />
Abstract: Female prairie voles (Microtus ochrogaster) show a dramatic shift in <strong>the</strong>ir behavioral<br />
reactions to pups at parturition. As virgins, female prairie voles are inf<strong>an</strong>ticidal (i.e., attack<br />
young). They continue to attack pups during pregn<strong>an</strong>cy but are fully maternal once pups are<br />
born. However, caesare<strong>an</strong> delivery blocks this ch<strong>an</strong>ge in behavior, <strong>an</strong>d females remain<br />
aggressive toward pups. We hypo<strong>the</strong>size that sensory feedback triggered by distension of <strong>the</strong><br />
cervix during delivery induces maternal behaviors. To test this hypo<strong>the</strong>sis, maternal<br />
responsiveness was determined at term (Day 22) in inf<strong>an</strong>ticidal <strong>an</strong>d maternal females that had <strong>the</strong><br />
pelvic nerves innervating <strong>the</strong> cervix severed on Day 19-20 of pregn<strong>an</strong>cy.<br />
Severing <strong>the</strong> pelvic nerve during pregn<strong>an</strong>cy blocked <strong>the</strong> induction of maternal behaviors<br />
normally seen at delivery. Females that were inf<strong>an</strong>ticidal or maternal during pregn<strong>an</strong>cy remained<br />
inf<strong>an</strong>ticidal or maternal, respectively, when tested at term. However, inf<strong>an</strong>ticidal females became<br />
maternal if <strong>the</strong> pelvic nerves were intact. These findings demonstrate that sensory output via <strong>the</strong><br />
pelvic nerve during parturition triggers maternal behaviors in inf<strong>an</strong>ticidal female prairie voles.<br />
Our preliminary data in hum<strong>an</strong>s also indicate that blocked pelvic signaling at delivery is<br />
associated with increased postpartum depression symptomatology, a major predictor of <strong>the</strong><br />
quality of maternal care in women.
Disclosures: U.L. Hayes, None; A. Ortiz, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.11/EE133<br />
Topic: E.03.b. Parental behavior<br />
Support: NSERC C<strong>an</strong>ada Discovery Gr<strong>an</strong>t to ACWW<br />
Title: Synaptic ultrastructural plasticity following <strong>the</strong> onset of maternal behaviour<br />
Authors: *A. C. WEEKS 1 , J. BLOOMFIELD 1 , Y. HENDERSON 1 , C. MAHEDO 2 , J. C.<br />
LEBOUTILLIER 2 , M. J. SAARI 1 ;<br />
1 Nipissing Univ., North Bay, ON, ON, C<strong>an</strong>ada; 2 Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Past research has shown that <strong>the</strong> hypothalamus <strong>an</strong>d limbic system play a critical role<br />
in <strong>the</strong> expression of maternal behaviour. Less is known about <strong>the</strong> potential role that synaptic<br />
ch<strong>an</strong>ge plays in <strong>the</strong> onset <strong>an</strong>d expression of maternal behaviour. This experiment examined<br />
synaptic ch<strong>an</strong>ges in c<strong>an</strong>didate brain structures following <strong>the</strong> onset of maternal behaviour. Four<br />
experimental groups included: first time mo<strong>the</strong>rs with pups, first time mo<strong>the</strong>rs with pups<br />
removed immediately following birth, control virgins <strong>an</strong>d virgins exposed to pups. Synapses<br />
were qu<strong>an</strong>tified 24 hours after <strong>the</strong> onset of maternal behaviour in <strong>the</strong> relev<strong>an</strong>t groups. Control<br />
females were time yoked to rats that expressed maternal behaviour. Synaptic measures included:<br />
<strong>the</strong> total number of synapses per neuron, <strong>the</strong> number of excitatory <strong>an</strong>d inhibitory contacts,<br />
synaptic curvature type, <strong>the</strong> presence of synaptic per<strong>for</strong>ations, <strong>an</strong>d <strong>the</strong> maximum length of <strong>the</strong><br />
synapses. Synapses were found to ch<strong>an</strong>ge in <strong>the</strong> maternal groups relative to controls.<br />
Disclosures: A.C. Weeks, None; J. Bloomfield, None; Y. Henderson, None; C. Mahedo,<br />
None; J.C. LeBoutillier, None; M.J. Saari, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.12/EE134<br />
Topic: E.03.b. Parental behavior<br />
Support: NZ Lottery Health Gr<strong>an</strong>t<br />
HS & JC Anderson Charitable Trust<br />
Title: Low prolactin during early pregn<strong>an</strong>cy causes postpartum <strong>an</strong>xiety <strong>an</strong>d impaired maternal<br />
behavior: Role of neurogenesis?<br />
Authors: C. M. LARSEN 1 , *D. R. GRATTAN 2 ;<br />
1 2<br />
Ctr. <strong>for</strong> Neuroendocrinology, Anat. <strong>an</strong>d Structural Biol., Univ. of Otago, Dunedin, New<br />
Zeal<strong>an</strong>d<br />
Abstract: An increase in neurogenesis occurs in <strong>the</strong> subventricular zone (SVZ) of female mice<br />
during early pregn<strong>an</strong>cy, stimulated by elevated prolactin secretion that occurs at this time. The<br />
functional consequences of enh<strong>an</strong>ced neurogenesis during pregn<strong>an</strong>cy are unclear. To test <strong>the</strong><br />
hypo<strong>the</strong>sis that neurogenesis may be import<strong>an</strong>t <strong>for</strong> behavioural ch<strong>an</strong>ges occurring in <strong>the</strong><br />
peripartum period, we aimed to suppress <strong>the</strong> prolactin-induced increase of neurogenesis on day 7<br />
of pregn<strong>an</strong>cy, ei<strong>the</strong>r using low doses of bromocriptine, carefully titrated to avoid causing<br />
pregn<strong>an</strong>cy termination, or <strong>the</strong> mitotic inhibitor, methylazoxymeth<strong>an</strong>ol (MAM). Groups of<br />
bromocriptine- or vehicle-treated <strong>an</strong>imals were injected with bromodeoxyuridine, <strong>an</strong>d <strong>the</strong>n killed<br />
<strong>for</strong> assessment of neurogenesis in <strong>the</strong> SVZ. O<strong>the</strong>r groups were allowed to maintain pregn<strong>an</strong>cy,<br />
<strong>an</strong>d <strong>the</strong>n were assessed on day 2 postpartum <strong>for</strong> <strong>an</strong>xiety on <strong>an</strong> elevated plus maze or maternal<br />
behaviour in a pup retrieval paradigm. Bromocriptine-induced suppression of prolactin<br />
signific<strong>an</strong>tly increased <strong>an</strong>xiety postpartum <strong>an</strong>d impaired maternal behaviour. Daily prolactin<br />
injections in early pregn<strong>an</strong>cy to replace prolactin in bromocriptine-treated mice, restored <strong>the</strong><br />
normal increase in neurogenesis, prevented <strong>the</strong> increased postpartum <strong>an</strong>xiety <strong>an</strong>d restored normal<br />
maternal behaviour. Injections of MAM also specifically suppressed neurogenesis during early<br />
pregn<strong>an</strong>cy, but without altering prolactin secretion. While this treatment dramatically impaired<br />
maternal behaviour, it did not affect postpartum <strong>an</strong>xiety. These data demonstrate that elevated<br />
prolactin during early pregn<strong>an</strong>cy is required <strong>for</strong> suppression of <strong>an</strong>xiety in <strong>the</strong> post partum period<br />
<strong>an</strong>d normal expression of maternal behaviour. While <strong>the</strong> effect on maternal behaviour appears to<br />
be mediated by enh<strong>an</strong>ced neurogenesis in <strong>the</strong> SVZ, <strong>the</strong> prolactin-induced suppression of<br />
postpartum <strong>an</strong>xiety is not mediated by neurogenesis. These data provide new insights into central<br />
nervous system functions of prolactin <strong>an</strong>d prolactin-induced neurogenesis in <strong>the</strong> maternal brain<br />
during pregn<strong>an</strong>cy.<br />
Disclosures: C.M. Larsen, None; D.R. Gratt<strong>an</strong>, None.<br />
Poster
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.13/EE135<br />
Topic: E.03.b. Parental behavior<br />
Support: UNM gr<strong>an</strong>t RAC0803<br />
Title: Good stress regulator begets good stress regulator: Predicting offspring stress regulation<br />
<strong>from</strong> maternal stress regulation<br />
Authors: *A. C. TANG 1 , Z. YANG 1 , R. D. ROMEO 2,3 , A. CHEN 1 , A. PLAKIO 1 , D.<br />
DELVECCHIO 1 , V. NGUYEN 1 , Y. ZHANG 1 , J. YOUNGBLOOD 1 , A. GONZALES 1 , B. S.<br />
MCEWEN 3 ;<br />
1 Univ. New Mexico, Albuquerque, NM; 2 Dept. of Psychology Neurosci. <strong>an</strong>d Behavior Program,<br />
Barnard Col., New York, NY; 3 Lab. of Neuroendocrinology, Rockefeller Univ., New York, NY<br />
Abstract: Early life experiences, particularly traumatic experiences, c<strong>an</strong> have a long lasting<br />
impact on social, cognitive, <strong>an</strong>d emotional development. However it is not <strong>the</strong> case that those<br />
who experience negative events will necessarily develop psychopathology in <strong>the</strong>ir adult life. It<br />
has been suggested that individual differences in stress regulation play <strong>an</strong> import<strong>an</strong>t role in<br />
determining how a given stressful event is experienced differentially by each individual. Here we<br />
show that maternal basal <strong>an</strong>d evoked corticosterone (CORT) levels measured after we<strong>an</strong>ing c<strong>an</strong><br />
serve as a trait-like marker to predict individual differences in her adult offspring’s basal <strong>an</strong>d<br />
evoked stress response, as well as measures of emotionality in <strong>an</strong> open field. Maternal basal <strong>an</strong>d<br />
1-min swim-stress evoked CORT levels were measured on postnatal day (PND) 28 <strong>an</strong>d 29<br />
(we<strong>an</strong>ing occurred on PND 21) <strong>an</strong>d her adult offspring’s emotionality <strong>an</strong>d basal <strong>an</strong>d evoked<br />
CORT were measured at ~ 60 days <strong>an</strong>d 13 months of age respectively. We found that (1)<br />
mo<strong>the</strong>rs with low basal <strong>an</strong>d high evoked CORT levels have adult offspring who were less fearful<br />
in <strong>an</strong> open field [maternal basal (MB): r=0.522, p=0.032; Maternal evoked (ME): r=-0.605,<br />
p=0.013]; (2) <strong>the</strong> maternal stress profile c<strong>an</strong> predict her offspring’s stress profile at late<br />
adulthood with mo<strong>the</strong>rs of low basal CORT levels having adult offspring with low basal CORT<br />
levels, mo<strong>the</strong>rs of high evoked CORT having adult offspring with high evoked CORT, <strong>an</strong>d low<br />
basal CORT levels being associated with higher evoked CORT levels [MB & offspring basal<br />
(OB): r=0.605, p=0.013; ME & OE: r=0.809, p=0.001; MB & OE: r=-0.672, p=0.009; ME &<br />
OB: r=-0.803, p=0.001]. These findings suggest that maternal individual differences in <strong>the</strong>ir<br />
regulation of HPA function, specifically <strong>the</strong>ir ability to maintain a low basal CORT level <strong>an</strong>d a<br />
complementary ability to mount a large CORT response on dem<strong>an</strong>d, may be one of <strong>the</strong><br />
contributing factors <strong>for</strong> <strong>the</strong> development of offspring HPA regulation. As <strong>the</strong> amount of maternal<br />
care received during inf<strong>an</strong>cy has been suggested to be a key variable in shaping <strong>the</strong> development<br />
of <strong>the</strong> HPA axis, we also measured postnatal maternal care during PND 1-12 using <strong>an</strong> average<br />
round-<strong>the</strong>-clock measure similar to that used by Me<strong>an</strong>ey <strong>an</strong>d colleagues. We found no evidence
that this average round-<strong>the</strong>-clock maternal care measure predicts adult offpsring’s basal <strong>an</strong>d<br />
evoked CORT levels or open-field emotionality measure. Toge<strong>the</strong>r, <strong>the</strong>se findings indicate that<br />
maternal regulation of her own stress response system may be <strong>an</strong> import<strong>an</strong>t player in <strong>the</strong> creation<br />
of individual differences in offspring stress regulation <strong>an</strong>d coping.<br />
Disclosures: A.C. T<strong>an</strong>g, None; Z. Y<strong>an</strong>g, None; R.D. Romeo, None; A. Chen, None; A.<br />
Plakio, None; D. Delvecchio, None; V. Nguyen, None; Y. Zh<strong>an</strong>g, None; J. Youngblood,<br />
None; A. Gonzales, None; B.S. McEwen, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.14/EE136<br />
Topic: E.03.b. Parental behavior<br />
Title: Autoradiographic <strong>an</strong>alysis of [3H]muscimol <strong>an</strong>d [3H]flunitrazepam binding in <strong>the</strong> brains<br />
of female <strong>an</strong>d male rats<br />
Authors: *S. M. MILLER 1 , J. S. LONSTEIN 2 ;<br />
1 Psychology, 2 Psychology & Neurosci., Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Postpartum female rats show reduced <strong>an</strong>xiety compared with diestrous virgins. While<br />
<strong>the</strong> neural mediation of this phenomenon is not well understood, one import<strong>an</strong>t influence appears<br />
to be increased neurotr<strong>an</strong>smission at <strong>the</strong> GABAA receptor. For inst<strong>an</strong>ce, postpartum females that<br />
are allowed access to <strong>the</strong>ir pups experience reduced <strong>an</strong>xiety <strong>an</strong>d <strong>an</strong> increase in cerebrospinal<br />
fluid concentrations of GABA when compared to females that are separated <strong>from</strong> <strong>the</strong>ir litters.<br />
Fur<strong>the</strong>rmore, inhibiting this receptor increases postpartum females’ freezing in response to <strong>an</strong><br />
acoustic stimulus <strong>an</strong>d also reduces <strong>the</strong>ir punished drinking (H<strong>an</strong>sen et al., 1985; H<strong>an</strong>sen, 1990),<br />
indicating increased <strong>an</strong>xiety. Our laboratory has recently found that <strong>an</strong>tagonizing <strong>the</strong> GABA <strong>an</strong>d<br />
benzodiazepine sites on <strong>the</strong> GABAA receptor increases <strong>an</strong>xiety-related behaviors in <strong>the</strong> elevated<br />
plus-maze <strong>an</strong>d light-dark box, often specifically in postpartum females <strong>an</strong>d not diestrous virgins.<br />
These findings imply that one mech<strong>an</strong>ism <strong>for</strong> reducing <strong>an</strong>xiety during <strong>the</strong> postpartum period<br />
could be a naturally occurring difference in binding affinity of lig<strong>an</strong>ds to <strong>the</strong> GABAA receptor,<br />
including <strong>the</strong> GABA <strong>an</strong>d benzodiazepine sites, in neural sites involved in <strong>an</strong>xiety. To investigate<br />
this possibility, we are per<strong>for</strong>ming autoradiography utilizing [3H]muscimol <strong>an</strong>d<br />
[3H]flunitrazepam to compare <strong>the</strong> GABA <strong>an</strong>d benzodiazepine binding sites in female <strong>an</strong>d male<br />
rats in brain regions including <strong>the</strong> central amygdala, bed nucleus of <strong>the</strong> stria terminalis,<br />
hippocampus, periaqueductal gray, <strong>an</strong>d <strong>the</strong> <strong>an</strong>terior cingulate, prelimbic, <strong>an</strong>d infralimbic
cortices. Female rats were sacrificed as diestrous virgins, on day 10 of pregn<strong>an</strong>cy, or on day 7<br />
postpartum, while sexually naïve male rats were sacrificed between 75 <strong>an</strong>d 95 days of age. We<br />
predict that postpartum females will show signific<strong>an</strong>tly greater GABA <strong>an</strong>d benzodiazepine<br />
binding in m<strong>an</strong>y of <strong>the</strong> brain regions examined compared to all o<strong>the</strong>r groups. We are also using<br />
[3H]TBOB to determine differences among <strong>the</strong>se same groups in binding affinity to <strong>the</strong><br />
picrotoxin site on <strong>the</strong> GABAA receptor. Results <strong>from</strong> <strong>the</strong>se studies will contribute to our<br />
underst<strong>an</strong>ding of how tonic upregulation of GABA systems contributes to postpartum ch<strong>an</strong>ges in<br />
<strong>an</strong>xiety, sex differences in <strong>an</strong>xiety, as well as o<strong>the</strong>r emotional states <strong>an</strong>d behaviors that differ<br />
across reproductive states <strong>an</strong>d between <strong>the</strong> sexes.<br />
Disclosures: S.M. Miller, None; J.S. Lonstein, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.15/EE137<br />
Topic: E.03.b. Parental behavior<br />
Title: Maternal resilience: Neurobiological <strong>an</strong>d behavioral responses following neurotoxin<br />
exposure<br />
Authors: *A. FRANSSEN 1 , C. L. FRANSSEN 2 , A. M. RZUCIDLO 2 , S. A. BENKOVIC, Jr. 3 ,<br />
E. A. SHEA 2 , A. BARANOVA 1 , C. H. KINSLEY 1 , K. G. LAMBERT 2 ;<br />
1 2<br />
Psychology, Univ. of Richmond, Richmond, VA; Psychology, R<strong>an</strong>dolph-Macon Col., Ashl<strong>an</strong>d,<br />
VA; 3 NIOSH, CDC, Morg<strong>an</strong>town, WV<br />
Abstract: Previous work <strong>from</strong> our labs <strong>an</strong>d o<strong>the</strong>rs has demonstrated that <strong>the</strong> hormones <strong>an</strong>d<br />
experience of pregn<strong>an</strong>cy <strong>an</strong>d lactation enh<strong>an</strong>ce hippocampal plasticity <strong>an</strong>d spatial learning<br />
abilities in <strong>the</strong> maternal rat. We hypo<strong>the</strong>sized that <strong>the</strong>se maternal brains may have acquired some<br />
protection <strong>from</strong> neuronal damage. A recent study has shown that lactating rats have less cell<br />
damage th<strong>an</strong> diestrus rats induced by KA excitotoxicity in CA1, CA3, <strong>an</strong>d <strong>the</strong> dentate gyrus of<br />
<strong>the</strong> hippocampus (V<strong>an</strong>oye-Carlo et al., 2008). As hippocampal plasticity <strong>an</strong>d enh<strong>an</strong>ced learning<br />
abilities extend well beyond <strong>the</strong> time of pregn<strong>an</strong>cy <strong>an</strong>d lactation, we hypo<strong>the</strong>sized that<br />
neuroprotective effects may also extend beyond time of we<strong>an</strong>ing, <strong>an</strong>d that cellular<br />
neuroprotection may tr<strong>an</strong>slate to preserving learned behaviors. In this study, we compared<br />
multiparous rats (those having 2 pregn<strong>an</strong>cy <strong>an</strong>d lactation experiences, with <strong>the</strong> most recent<br />
we<strong>an</strong>ing ~1 month prior to <strong>the</strong> start of this study) with age-matched virgin females in response to<br />
KA injections. We found that seizure activity was not different between those with <strong>an</strong>d without
maternal experience. Our preliminary <strong>an</strong>alysis indicates no maternal effect on glial cell<br />
activation in <strong>the</strong> hippocampus, as assessed with GFAP immunoreactivity, however we do find<br />
that maternal rats have less neurodegeneration, assessed with FluoroJade-B, in <strong>the</strong> CA2 <strong>an</strong>d CA3<br />
regions of <strong>the</strong> hippocampus. Cell damage is currently being assessed with Nissl staining.<br />
Behaviorally, maternal <strong>an</strong>imals seem to better preserve <strong>the</strong> memory <strong>for</strong> a recently acquired task<br />
(Dry-l<strong>an</strong>d version of <strong>the</strong> Morris water maze; DLM) through <strong>the</strong> experience of KA exposure,<br />
have a better recall on <strong>an</strong> object recognition task, <strong>an</strong>d exhibit bolder responses in <strong>the</strong>ir approach<br />
to a novel object following KA injection. These data suggest that <strong>the</strong> experience of mo<strong>the</strong>rhood<br />
provides some long-term protection <strong>from</strong> neurotoxic insult.<br />
Disclosures: A. Fr<strong>an</strong>ssen, None; C.L. Fr<strong>an</strong>ssen, None; A.M. Rzucidlo, None; S.A. Benkovic,<br />
None; E.A. Shea, None; A. Bar<strong>an</strong>ova, None; C.H. Kinsley, None; K.G. Lambert, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.16/EE138<br />
Topic: E.03.b. Parental behavior<br />
Support: NSF Gr<strong>an</strong>t #0723341<br />
Title: Hippocampal plasticity associated with paternal responsivity in two Peromyscus species<br />
Authors: *C. L. FRANSSEN 1 , S. KARNSER 1 , E. TU 1 , M. M. HYER 1 , A. FRANSSEN 2 , C.<br />
KINSLEY 2 , K. G. LAMBERT 1 ;<br />
1 R<strong>an</strong>dolph-Macon Col., Ashl<strong>an</strong>d, VA; 2 Univ. of Richmond, Richmond, VA<br />
Abstract: Maternal experience alters both <strong>the</strong> brain <strong>an</strong>d behavior of <strong>the</strong> rat, benefiting mo<strong>the</strong>r<br />
<strong>an</strong>d inf<strong>an</strong>t. Mounting <strong>an</strong>d converging evidence suggests a wide variety of subst<strong>an</strong>tial <strong>an</strong>d<br />
enduring effects on <strong>the</strong> neurobiology <strong>an</strong>d behavior of parous <strong>an</strong>imals. For example, signific<strong>an</strong>t<br />
restructuring in <strong>the</strong> hippocampus <strong>an</strong>d amygdala, including dendritic spine density ch<strong>an</strong>ges,<br />
seems to underlie <strong>the</strong>se maternal rats’ enh<strong>an</strong>ced abilities, including spatial learning<br />
improvements (Kinsley et al., 1999; Rasia-Filho et al., 2004; Lambert & Kinsley, 2008). Rats,<br />
like over 90% of mammali<strong>an</strong> species, are a polygamous, uni-parental <strong>an</strong>imal. In <strong>the</strong> current<br />
study, however, we were interested in <strong>the</strong> generalization of parenting-induced neurobiological<br />
ch<strong>an</strong>ges in <strong>an</strong>imals representing alternative parenting strategies, specifically paternal males in biparental<br />
species. Thus, we compared <strong>the</strong> monogamous, bi-parental Cali<strong>for</strong>nia Mouse<br />
(Peromyscus cali<strong>for</strong>nicus) <strong>an</strong>d <strong>the</strong> congeneric, polygamous, non-paternal Deer Mouse
(Peromyscus m<strong>an</strong>iculatus). In this study, we show that P. cali<strong>for</strong>nicus males, both biological<br />
fa<strong>the</strong>rs <strong>an</strong>d pup-exposed virgins, quickly acquire <strong>an</strong>d maintain a suite of paternal behaviors.<br />
Following <strong>an</strong> assessment of <strong>the</strong>se paternal behavioral responses, nestin-immunoreactivity (IR)<br />
was utilized to explore <strong>the</strong> neuroplasticity associated with <strong>the</strong> development of novel paternal<br />
behaviors. Specifically, nestin-IR, <strong>an</strong> intermediate filament indicative of cellular growth in <strong>the</strong><br />
brain, was assessed throughout <strong>the</strong> hippocampus of adult males of each species with variable<br />
paternal experiences (males with families, pup-exposed virgins, <strong>an</strong>d virgins with no pup<br />
exposure). Results indicated <strong>an</strong> enh<strong>an</strong>cement of nestin immunoreactivity in CA2 <strong>an</strong>d CA3<br />
regions of <strong>the</strong> hippocampus in P. cali<strong>for</strong>nicus compared to <strong>the</strong>ir P. m<strong>an</strong>iculatis counterparts.<br />
Analysis of glial contributions to <strong>the</strong> neuroplasticity, assessed with GFAP-IR, in <strong>the</strong>se brains is<br />
currently being conducted. In sum, as observed in maternal <strong>an</strong>imals, <strong>the</strong> emergence of paternal<br />
responses is likely associated with modifications of existing neural circuitry in relev<strong>an</strong>t brain<br />
areas.<br />
Disclosures: C.L. Fr<strong>an</strong>ssen, None; S. Karnser, None; E. Tu, None; M.M. Hyer, None; A.<br />
Fr<strong>an</strong>ssen, None; C. Kinsley, None; K.G. Lambert, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.17/EE139<br />
Topic: E.03.b. Parental behavior<br />
Support: NSF Gr<strong>an</strong>t 0723341<br />
Title: The paternal neurobiological circuit: Explorations in two congeneric Peromyscus species<br />
Authors: *K. G. LAMBERT 1 , C. L. FRANSSEN 2 , L. HAINLEY 2 , S. KARSNER 2 , E. TU 2 , M.<br />
HYER 2 , A. CROCKETT 2 , A. BARANOVA 3 , T. FERGUSON 3 , C. KINSLEY 3 ;<br />
1 R<strong>an</strong>dolph Macon Col., Ashl<strong>an</strong>d, VA; 2 Psychology, R<strong>an</strong>dolph-Macon Col., Ashl<strong>an</strong>d, VA;<br />
3 Psychology, Univ. of Richmond, Richmond, VA<br />
Abstract: Research in our laboratories has revealed parenting-induced neurobiological<br />
alterations in rodents leading to adaptive responses directed toward <strong>the</strong> offspring (Kinsley et al.,<br />
1999). Hippocampal modifications such as increased dendritic density have been shown to<br />
enh<strong>an</strong>ce valuable “off <strong>the</strong> nest” responses such as <strong>for</strong>aging <strong>an</strong>d efficient emotional processing<br />
(Lambert & Kinsley, 2008). Of interest in <strong>the</strong> current study was <strong>the</strong> generalization of parentinginduced<br />
alterations in males <strong>from</strong> two closely related species in <strong>the</strong> genus Peromyscus exhibiting
divergent paternal responses. P. cali<strong>for</strong>nicus males are both monogamous <strong>an</strong>d bi-parental,<br />
exhibiting all of <strong>the</strong> parenting responses displayed by maternal mice except lactation; P.<br />
m<strong>an</strong>iculatus, on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, is a non-paternal species. Age-matched biological fa<strong>the</strong>rs, pupexposed<br />
virgins <strong>an</strong>d pup-naïve virgins of each species (n=6 <strong>for</strong> each group, total n=36) were<br />
used in <strong>the</strong> current study. Assessments of paternal responses in biological fa<strong>the</strong>rs <strong>an</strong>d pupexposed<br />
males confirmed that <strong>the</strong> P. cali<strong>for</strong>nicus males exhibited signific<strong>an</strong>tly more nurturing<br />
responses (e.g., crouching over pups, grooming pups) <strong>an</strong>d signific<strong>an</strong>tly fewer <strong>an</strong>xiogenic<br />
responses (e.g., stretch attend, freezing) in <strong>the</strong> presence of pups th<strong>an</strong> <strong>the</strong> P. m<strong>an</strong>iculatus males.<br />
Following a 24 hr separation <strong>an</strong>d subsequent “reunion” with <strong>the</strong>ir family (<strong>for</strong> biological dads),<br />
with alien pups (<strong>for</strong> pup-exposed virgins) or cage mate bro<strong>the</strong>rs (<strong>for</strong> pup-naïve virgins), <strong>an</strong>imals<br />
were <strong>an</strong>es<strong>the</strong>tized <strong>for</strong> histological processing to determine c-fos activation (i.e., neuronal<br />
activation) in response to exposure to <strong>the</strong> relev<strong>an</strong>t social stimulus. Results indicated altered c-fos<br />
activation in several brain areas: <strong>the</strong> cingulate cortex, lateral septum, paraventricular nucleus of<br />
<strong>the</strong> hypothalamus (PVN), medial amygdala <strong>an</strong>d medial preoptic area (MPOA). The convergence<br />
of varying signific<strong>an</strong>t effects observed in this study suggest <strong>the</strong> presence of dual systems in <strong>the</strong><br />
demonstration of paternal responsiveness: decreased activation of <strong>an</strong>xiety/fear brain areas (e.g.,<br />
<strong>the</strong> medial amygdala, PVN) <strong>an</strong>d enh<strong>an</strong>ced activation of brain areas involved in nurturing<br />
responses (e.g., <strong>the</strong> MPOA). Supported by NSF #0723341.<br />
Disclosures: K.G. Lambert, None; C.L. Fr<strong>an</strong>ssen, None; L. Hainley, None; S. Karsner,<br />
None; E. Tu, None; M. Hyer, None; A. Crockett, None; A. Bar<strong>an</strong>ova, None; T. Ferguson,<br />
None; C. Kinsley, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.18/EE140<br />
Topic: E.03.b. Parental behavior<br />
Support: ABS research gr<strong>an</strong>t to E.D.G.<br />
NSF IOS-0620042 to C.A.M.<br />
Title: Epigenetic tr<strong>an</strong>smission of paternal behavior in <strong>the</strong> monogamous <strong>an</strong>d biparental Cali<strong>for</strong>nia<br />
mouse, Peromyscus cali<strong>for</strong>nicus<br />
Authors: E. D. GLEASON 1 , *C. A. MARLER 2 ;<br />
1 Psychology, Univ. of Wisconsin-Madison, Madison, WI; 2 Univ. Wisconsin, Madison, WI
Abstract: In <strong>the</strong> monogamous <strong>an</strong>d biparental Cali<strong>for</strong>nia mouse (Peromyscus cali<strong>for</strong>nicus),<br />
paternal care is critical <strong>for</strong> maximal offspring survival. Moreover, <strong>an</strong>imals <strong>for</strong>m pair bonds <strong>an</strong>d<br />
do not engage in extrapair matings. We hypo<strong>the</strong>sized that similar to female house mice <strong>an</strong>d rats,<br />
paternal behavior could be passed on to future generations via epigenetic mech<strong>an</strong>isms. Previous<br />
studies have shown that testosterone supports paternal behavior in Cali<strong>for</strong>nia mice, <strong>an</strong>d<br />
castration reduces <strong>the</strong> amount of huddling <strong>an</strong>d grooming (HG) that a male contributes to<br />
offspring. We castrated a group of male Cali<strong>for</strong>nia mice, <strong>an</strong>d allowed <strong>the</strong>m to raise young<br />
without <strong>an</strong>y fur<strong>the</strong>r intervention. Sons of <strong>the</strong>se castrated males (F1 males) were <strong>the</strong>n paired with<br />
females in adulthood, observed during courtship interactions, <strong>an</strong>d subsequently with <strong>the</strong>ir own<br />
offspring. Preliminary review of <strong>the</strong> data indicates that sons of low HG fa<strong>the</strong>rs also huddle <strong>an</strong>d<br />
groom <strong>the</strong>ir young at lower levels th<strong>an</strong> <strong>the</strong> sons of sham-operated fa<strong>the</strong>rs. In addition, sons of<br />
castrates also appear to respond to a paternal behavior challenge by spending less time with <strong>the</strong><br />
pups, <strong>an</strong>d leaving <strong>the</strong> nest more frequently. These data support <strong>the</strong> hypo<strong>the</strong>sis that paternal<br />
behavior, like maternal behavior, c<strong>an</strong> be tr<strong>an</strong>sferred to future generations via epigenetic<br />
mech<strong>an</strong>isms, <strong>an</strong>d suggests that in a biparental species both parents may be contributing to<br />
offspring behavioral development.<br />
Disclosures: E.D. Gleason, None; C.A. Marler, None.<br />
Press Conferences<br />
PC-05. Gender Research: New Male Insights<br />
Location: Time: Sunday, October 18, <strong>2009</strong>, 2:30 pm - 3:30 pm<br />
Program#/Poster#: Poster Board Number:<br />
Topic: E.03.b. Parental behavior<br />
Support: ABS research gr<strong>an</strong>t to E.D.G.<br />
NSF IOS-0620042 to C.A.M.<br />
Title: Epigenetic tr<strong>an</strong>smission of paternal behavior in <strong>the</strong> monogamous <strong>an</strong>d biparental Cali<strong>for</strong>nia<br />
mouse, Peromyscus cali<strong>for</strong>nicus<br />
Authors: E. D. GLEASON 1 , *C. A. MARLER 2 ;<br />
1 Psychology, Univ. of Wisconsin-Madison, Madison, WI; 2 Univ. Wisconsin, Madison, WI<br />
Abstract: In <strong>the</strong> monogamous <strong>an</strong>d biparental Cali<strong>for</strong>nia mouse (Peromyscus cali<strong>for</strong>nicus),<br />
paternal care is critical <strong>for</strong> maximal offspring survival. Moreover, <strong>an</strong>imals <strong>for</strong>m pair bonds <strong>an</strong>d<br />
do not engage in extrapair matings. We hypo<strong>the</strong>sized that similar to female house mice <strong>an</strong>d rats,<br />
paternal behavior could be passed on to future generations via epigenetic mech<strong>an</strong>isms. Previous<br />
studies have shown that testosterone supports paternal behavior in Cali<strong>for</strong>nia mice, <strong>an</strong>d
castration reduces <strong>the</strong> amount of huddling <strong>an</strong>d grooming (HG) that a male contributes to<br />
offspring. We castrated a group of male Cali<strong>for</strong>nia mice, <strong>an</strong>d allowed <strong>the</strong>m to raise young<br />
without <strong>an</strong>y fur<strong>the</strong>r intervention. Sons of <strong>the</strong>se castrated males (F1 males) were <strong>the</strong>n paired with<br />
females in adulthood, observed during courtship interactions, <strong>an</strong>d subsequently with <strong>the</strong>ir own<br />
offspring. Preliminary review of <strong>the</strong> data indicates that sons of low HG fa<strong>the</strong>rs also huddle <strong>an</strong>d<br />
groom <strong>the</strong>ir young at lower levels th<strong>an</strong> <strong>the</strong> sons of sham-operated fa<strong>the</strong>rs. In addition, sons of<br />
castrates also appear to respond to a paternal behavior challenge by spending less time with <strong>the</strong><br />
pups, <strong>an</strong>d leaving <strong>the</strong> nest more frequently. These data support <strong>the</strong> hypo<strong>the</strong>sis that paternal<br />
behavior, like maternal behavior, c<strong>an</strong> be tr<strong>an</strong>sferred to future generations via epigenetic<br />
mech<strong>an</strong>isms, <strong>an</strong>d suggests that in a biparental species both parents may be contributing to<br />
offspring behavioral development.<br />
Disclosures: E.D. Gleason, None; C.A. Marler, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.19/FF1<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH Gr<strong>an</strong>t R01DA013190<br />
NIH Gr<strong>an</strong>t P01DA022446<br />
Title: The association between maternal cocaine use during pregn<strong>an</strong>cy <strong>an</strong>d mo<strong>the</strong>r-inf<strong>an</strong>t play<br />
interactions in <strong>the</strong> first year of life<br />
Authors: R. EIDEN 1 , P. SCHUETZE 2 , *J. M. JOHNS, Ph.D. 3 ;<br />
1 Res. Inst. on Addictions, Univ. at Buffalo, SUNY, Buffalo, NY; 2 Psychology, Buffalo State<br />
Col., Buffalo, NY; 3 Univ. North Carolina, Chapel Hill, NC<br />
Abstract: The study examined <strong>the</strong> association between maternal cocaine use during pregn<strong>an</strong>cy<br />
<strong>an</strong>d mo<strong>the</strong>r-inf<strong>an</strong>t play interactions at 7 <strong>an</strong>d 13 months of child age. Maternal cocaine use is<br />
associated with more negative parenting behavior <strong>an</strong>d higher risk <strong>for</strong> negative birth outcomes<br />
(e.g., low birthweight). However, only a h<strong>an</strong>dful of studies have examined mo<strong>the</strong>r-inf<strong>an</strong>t play<br />
interactions as a function of maternal cocaine use <strong>an</strong>d inf<strong>an</strong>t risk characteristics at birth.<br />
Particip<strong>an</strong>ts consisted of 167 mo<strong>the</strong>r-inf<strong>an</strong>t dyads recruited at delivery (87 cocaine exposed, 80<br />
non-cocaine exposed). The poster includes data collected at 7 <strong>an</strong>d 13 months of inf<strong>an</strong>t ages.
MANOVA with cocaine group status as <strong>the</strong> independent variable, maternal negative affect <strong>an</strong>d<br />
sensitivity at 7 months as <strong>the</strong> dependent measures, <strong>an</strong>d frequency of o<strong>the</strong>r subst<strong>an</strong>ce use during<br />
pregn<strong>an</strong>cy (alcohol, cigarettes, <strong>an</strong>d mariju<strong>an</strong>a) as covariates yielded a signific<strong>an</strong>t multivariate<br />
effect on maternal behavior, F (2, 157) = 4.00, p < .05, ηp 2 = .05. Mo<strong>the</strong>rs in <strong>the</strong> cocaine group<br />
exhibited higher negative affect toward <strong>the</strong>ir inf<strong>an</strong>t during play interactions at 7 months.<br />
MANOVA with <strong>the</strong> five measures of maternal behavior at 13 months (sensitivity, connectedness,<br />
negative affect, intrusiveness, <strong>an</strong>d positive involvement) as <strong>the</strong> dependent measures indicated a<br />
signific<strong>an</strong>t multivariate effect of cocaine group status on maternal behavior at 13 months, F (5,<br />
156) = 2.78, p < .05, ηp 2 = .08. Cocaine using mo<strong>the</strong>rs were less sensitive, displayed lower<br />
positive involvement, <strong>an</strong>d higher negative affect during play interactions with <strong>the</strong>ir 13 month old<br />
inf<strong>an</strong>ts. O<strong>the</strong>r subst<strong>an</strong>ce use was not associated with maternal or inf<strong>an</strong>t behavior. Cocaine<br />
exposed inf<strong>an</strong>ts had lower birthweight, gestational age, <strong>an</strong>d higher obstetrical complications at<br />
delivery. Lower birthweight was associated with lower child responsiveness <strong>an</strong>d lower child<br />
positive affect during play at 7 months. Mo<strong>the</strong>rs of inf<strong>an</strong>ts with lower birthweight were more<br />
intrusive (r = -.20, p < .05) <strong>an</strong>d less connected (r = -.16, p < .05) during play at 13 months.<br />
Results indicate that maternal cocaine use during pregn<strong>an</strong>cy is a signific<strong>an</strong>t predictor of maternal<br />
behavior. Inf<strong>an</strong>t birth outcomes have independent associations with maternal <strong>an</strong>d child behavior.<br />
Disclosures: R. Eiden, None; P. Schuetze, None; J.M. Johns , None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.20/FF2<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH Gr<strong>an</strong>t MH072935<br />
Title: Alloparenting in male prairie voles releases oxytocin <strong>an</strong>d elevates heart rate<br />
Authors: *W. KENKEL, J. PAREDES, A. J. GRIPPO, H. P. NAZARLOO, S. W. PORGES, C.<br />
S. CARTER;<br />
Dept. of Psychiatry, Univ. Illinois At Chicago, Chicago, IL<br />
Abstract: ALLOPARENTING IN MALE PRAIRIE VOLES RELEASES OXYTOCIN AND<br />
ELEVATES HEART RATE<br />
Social monogamy in mammals is characterized by pair bond <strong>for</strong>mation <strong>an</strong>d biparental care of<br />
offspring. Both of <strong>the</strong>se features c<strong>an</strong> be found in prairie voles (Microtus ochrogaster) <strong>an</strong>d may
e related. In this study, sexually naïve male prairie voles (aged 60-90 days) were placed in a<br />
novel cage with ei<strong>the</strong>r <strong>an</strong> unrelated pup (aged 1-3 days), pup-sized wooden dowel, or no<br />
stimulus. After 20 minutes, <strong>the</strong> stimulus was removed <strong>an</strong>d a time course of plasma peptides<br />
(oxytocin, vasopressin <strong>an</strong>d corticosterone) was assayed at 10, 20 <strong>an</strong>d 60 minutes. In <strong>the</strong> 60<br />
minute group, immunohistochemistry was carried out in order to examine <strong>the</strong> activation (as<br />
measured by c-fos) of oxytocin <strong>an</strong>d vasopressin positive neurons in <strong>the</strong> hypothalamus. In a<br />
parallel study, a similar cohort of males was impl<strong>an</strong>ted with radio telemetric tr<strong>an</strong>smitters <strong>for</strong> <strong>the</strong><br />
purpose of recording electrocardiographic parameters. Males exposed to pups were found to<br />
display a short-term (10 min time point) elevation in oxytocin as well as lower corticosterone<br />
levels at <strong>the</strong> same 10 minute time point. Pup exposure also elicited <strong>an</strong> increase in heart-rate<br />
which was sustained throughout <strong>the</strong> 20 minute testing period in a m<strong>an</strong>ner uniquely different <strong>from</strong><br />
exposure to a dowel or novel adult female. Fur<strong>the</strong>r, this increase in heart-rate did not habituate<br />
with repeated pup exposures, nor was it influenced by locomotor activity, which decreased<br />
during pup exposure relative to dowels. These findings are discussed with respect to previously<br />
found behavioral consequences of pup exposure <strong>an</strong>d additionally as a novel combination of<br />
elevated heart-rate <strong>an</strong>d diminished glucocorticoid levels in response to a stimulus.<br />
Disclosures: W. Kenkel, None; J. Paredes, None; A.J. Grippo, None; H.P. Nazarloo,<br />
None; S.W. Porges, None; C.S. Carter, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.21/FF3<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH Gr<strong>an</strong>t 1P01DA022446-02<br />
Title: Gestational cocaine affects maternal motivation <strong>an</strong>d pup preference: Possible dependence<br />
on pup vocalizations<br />
Authors: *S. WILLIAMS 1 , E. T. COX 1 , C. F. HEATON 1 , N. N. DESAI 1 , E. E. FAY 2 , M. S.<br />
MCMURRAY 1 , J. M. JOHNS 1 ;<br />
1 2<br />
Dept Psych, UNC-Chapel Hill, Chapel Hill, NC; Univ. of Med. <strong>an</strong>d Denistry of New Jersey,<br />
Newark, NJ<br />
Abstract: Background: Approximately 5% of pregn<strong>an</strong>t women report cocaine use despite<br />
widespread publicity of neurological <strong>an</strong>d behavioral abnormalities in prenatally exposed
offspring. Children of mo<strong>the</strong>rs who abuse cocaine are more likely to suffer <strong>from</strong> maternal<br />
neglect <strong>an</strong>d child abuse. Animal models indicate that cocaine treatment results in neglect of<br />
offspring <strong>an</strong>d that prenatally exposed offspring are less able to elicit care <strong>from</strong> all mo<strong>the</strong>rs. The<br />
early postpartum period represents a time of rapid adjustment <strong>for</strong> first-time mo<strong>the</strong>rs, <strong>an</strong>d<br />
motivational state driven by offspring-produced cues likely plays a role in maternal response.<br />
Objective: To determine whe<strong>the</strong>r cocaine treatment during pregn<strong>an</strong>cy alters maternal preference<br />
<strong>for</strong> prenatally cocaine exposed or control pups, <strong>an</strong>d whe<strong>the</strong>r ultrasonic vocalizations (USVs)<br />
<strong>from</strong> pups are associated with differential retrieval behavior. Methods: Pregn<strong>an</strong>t Sprague-<br />
Dawley females were r<strong>an</strong>domly assigned to untreated (UN) or chronic cocaine (CC) treatment<br />
groups. CC dams were given bi-daily subcut<strong>an</strong>eous injections of cocaine HCL (15 mg/kg)<br />
throughout gestation <strong>an</strong>d not after. All dams were tested <strong>for</strong> pup retrieval <strong>an</strong>d preference on<br />
postpartum days (PPD) 1, (choice of <strong>the</strong>ir own pups versus a neutral stimulus), 3 <strong>an</strong>d 5 (choice<br />
between UN or CC pups). USVs (42-46 kHz) were detected using a bat detector <strong>an</strong>d UltraVox<br />
software <strong>for</strong> 5 minutes immediately prior to retrieval testing. Results: CC treatment resulted in:<br />
PPD 1 - a signific<strong>an</strong>tly increased latency to retrieve all pups (p< 0.05), retrieval of fewer pups<br />
(p< 0.05), <strong>an</strong>d fewer maternal behaviors were displayed (lick (p< 0.01), nurse (p< 0.01), touch<br />
(p< 0.01),); PPD 3- <strong>an</strong> increased latency <strong>for</strong> retrieval <strong>an</strong>d signific<strong>an</strong>tly decreased levels of<br />
maternal behaviors (lick (p< 0.01), nurse (p< 0.01), touch (p< 0.01)); PPD 5- continuing deficits<br />
in maternal behaviors (lick (p< 0.01), nurse (p< 0.01), touch (p< 0.01),). On PPD 5, UN dams<br />
chose UN pups over CC-exposed pups. CC-exposed pups displayed fewer USVs across all test<br />
days compared to UN pups (p< 0.05). Conclusions: CC treatment during gestation alters<br />
maternal behavior <strong>an</strong>d response to pups cues while UN pup-produced cue differences are<br />
preferentially responded to by UN dams indicating cues may alter motivational state of dams.<br />
Disclosures: S. Williams, None; E.T. Cox, None; C.F. Heaton, None; N.N. Desai, None; E.E.<br />
Fay, None; M.S. McMurray, None; J.M. Johns, None.<br />
Poster<br />
571. Parental Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 571.22/FF4<br />
Topic: E.03.b. Parental behavior<br />
Support: NIH DA014025<br />
Title: Tr<strong>an</strong>sient inactivation of <strong>the</strong> venteral tegmental area disrupts <strong>the</strong> expression of place<br />
preference <strong>for</strong> a natural but not a drug stimulus
Authors: *K. M. SEIP 1 , J. I. MORRELL 2 ;<br />
1 2<br />
Lab. of <strong>the</strong> Biol. of Addictive Dis., The Rockefeller Univ., New York City, NY; CMBN,<br />
Rutgers Univ., Newark, NJ<br />
Abstract: The ventral tegmental area (VTA) is critically involved in motivated behavior,<br />
including <strong>the</strong> seeking out of stimuli attributed with strong positive incentive value. We used a<br />
conditioned place preference (CPP) paradigm to examine whe<strong>the</strong>r intact VTA function is<br />
required <strong>for</strong> a subject to express stimulus-seeking behavior directed toward a unique<br />
environment (i.e., a CPP chamber) that has been paired with a natural or a pharmacological<br />
stimulus. Prior to <strong>the</strong> start of <strong>the</strong> CPP paradigm, virgin <strong>an</strong>d postpartum female rats were<br />
impl<strong>an</strong>ted with bilateral c<strong>an</strong>nulae directed ei<strong>the</strong>r at <strong>the</strong> VTA or control regions. Subjects <strong>the</strong>n<br />
learned to associate one CPP chamber with a stimulus with strong incentive value <strong>an</strong>d a distinct<br />
chamber with a neutral stimulus: <strong>for</strong> virgin females, chambers were paired with injections of<br />
cocaine <strong>an</strong>d saline, while, <strong>for</strong> postpartum females, chambers were paired with young offspring<br />
(pups) or remained neutral. Immediately be<strong>for</strong>e CPP testing, subjects received a bilateral<br />
microinfusion of bupivacaine (a NA+ ch<strong>an</strong>nel blocker <strong>an</strong>d local <strong>an</strong>es<strong>the</strong>tic) into <strong>the</strong> VTA to<br />
tr<strong>an</strong>siently inactivate <strong>the</strong> region <strong>an</strong>d were promptly tested <strong>for</strong> <strong>the</strong>ir conditioned preference.<br />
Subjects were also tested following saline microinfusion, using a counterbal<strong>an</strong>ced design.<br />
Postpartum females’ preference <strong>for</strong> <strong>the</strong> pup-paired chamber was eliminated by VTA inactivation<br />
but was very strong following saline microinfusion (P
Title: Cocaine treatment prior to pregn<strong>an</strong>cy affects subsequent offspring care <strong>an</strong>d maternal<br />
aggression during early, but not late lactation<br />
Authors: *B. NEPHEW, M. FEBO;<br />
Nor<strong>the</strong>astern Univ., Boston, MA<br />
Abstract: Although m<strong>an</strong>y studies have investigated <strong>the</strong> effects of gestational <strong>an</strong>d lactational<br />
cocaine use on maternal behavior, few studies have examined <strong>the</strong> effects of prior adult cocaine<br />
use on maternal behavior. In <strong>the</strong> current study, intraperitoneal cocaine or saline was administered<br />
to adult female rats <strong>for</strong> 10 days, treatments were stopped <strong>for</strong> 4 days, <strong>an</strong>d <strong>the</strong> females were <strong>the</strong>n<br />
mated. Litter size <strong>an</strong>d weight was recorded on day 2 of lactation, <strong>an</strong>d maternal care, maternal<br />
aggression, <strong>an</strong>d <strong>an</strong>xiety levels were assessed on days 2, 9, <strong>an</strong>d 16 of lactation. Although <strong>the</strong>re<br />
was no effect of cocaine on me<strong>an</strong> pup weight on day 2, mo<strong>the</strong>rs previously treated with cocaine<br />
spent more time caring <strong>for</strong> <strong>the</strong> pups (increased pup retrieval, pup grooming, <strong>an</strong>d crouching over<br />
<strong>the</strong> pups by <strong>the</strong> mo<strong>the</strong>r) <strong>an</strong>d were more aggressive towards a male intruder compared to saline<br />
treated control <strong>an</strong>imals at this time point. These effects seem to be specific to maternal behavior,<br />
as o<strong>the</strong>r behaviors (self-grooming, locomotor activity) were mostly unaffected by prior cocaine<br />
treatment. Data <strong>from</strong> light/dark box tests suggest that <strong>an</strong>xiety levels were also similar between<br />
cocaine <strong>an</strong>d saline treated mo<strong>the</strong>rs. There were no signific<strong>an</strong>t effects of prior cocaine treatment<br />
on lactation day 16. It appears that prior cocaine use may have lasting effects on offspring care<br />
<strong>an</strong>d maternal aggression, specifically during early lactation. The present findings provide fur<strong>the</strong>r<br />
support <strong>for</strong> <strong>the</strong> hypo<strong>the</strong>sis that maternal motivation to exhibit maternal behavior was enh<strong>an</strong>ced<br />
by repeated cocaine exposure <strong>an</strong>d withdrawal, possibly due to cross sensitization between<br />
cocaine <strong>an</strong>d <strong>the</strong> natural reward of maternal behavior.<br />
Disclosures: B. Nephew, None; M. Febo, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.1/FF6<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH Gr<strong>an</strong>t 5R01HL081823<br />
Title: Effect of chronic hypoxia on reactive oxygen species in <strong>the</strong> nucleus of <strong>the</strong> solitary tract<br />
(NTS)
Authors: *D. POPA, E. BREEN, L. DUGAN, F. POWELL;<br />
UCSD, La Jolla, CA<br />
Abstract: Recent work has implicated reactive oxygen species (ROS) as possible signals <strong>for</strong><br />
plasticity in chemoreflexes that control ventilation. We hypo<strong>the</strong>sized that ROS contribute to <strong>the</strong><br />
time-dependent ch<strong>an</strong>ges that occur in ventilation, <strong>an</strong>d its modulation by O2 <strong>an</strong>d CO2, during<br />
ventilatory acclimatization to hypoxia (VAH). We studied 3 groups of adult male C57BL/6 mice:<br />
CN = chronic normoxia (controls), 21% O2; AH = acute hypoxia, 10% O2 <strong>for</strong> 6 hours; CH =<br />
chronic hypoxia, 10% O2 <strong>for</strong> 7 days. To measure ROS in respiratory centers of <strong>the</strong> medulla, we<br />
injected dihydroethidium (DHE, 54 mg/kg ip) <strong>an</strong>d kept <strong>the</strong> mice in 21% (CN) or 10% O2 (AH<br />
<strong>an</strong>d CH) <strong>for</strong> 6 hours be<strong>for</strong>e sacrifice <strong>an</strong>d perfusion fixation (4% para<strong>for</strong>maldehyde). The<br />
brainstems were removed <strong>an</strong>d sectioned (30um) at <strong>the</strong> level of obex (rostral NTS) <strong>an</strong>d 60um<br />
caudally (caudal NTS). These sites are import<strong>an</strong>t <strong>for</strong> ventilatory control because <strong>the</strong>y contain <strong>the</strong><br />
primary synapse <strong>from</strong> carotid body chemoreceptors <strong>an</strong>d exhibit CO2-sensitivity. We qu<strong>an</strong>tified<br />
<strong>the</strong> intensity of <strong>the</strong> DHE signal with confocal fluorescent microscopy (excitation Λ = 543nm,<br />
emission Λ = 590-690nm; Image J <strong>an</strong>alysis). Images <strong>from</strong> mice with no injections or only<br />
vehicle injected showed no measurable DHE signal. The average DHE staining per neuron (+<br />
SEM) in caudal <strong>an</strong>d rostral NTS sections, normalized to <strong>the</strong> value in CN values (= 1.000) were:<br />
Group Caudal NTS Rostral NTS<br />
AH 0.615 +/- 0.060 (n=2) 0.568 +/- 0.127 (n=2)<br />
CH 0.792 +/- 0.058 (n=3) 0.994 +/- 0.077 (n=2)<br />
These preliminary results show ROS decrease in AH but return to baseline levels with CH,<br />
which is consistent with our hypo<strong>the</strong>sis that ROS signal neural plasticity during VAH.<br />
Additional experiments are being done to determine <strong>the</strong> effects of CH on <strong>the</strong> number <strong>an</strong>d types<br />
of neurons producing DHE, <strong>the</strong> source <strong>an</strong>d type of ROS produced in neurons, <strong>an</strong>d <strong>the</strong><br />
physiological signific<strong>an</strong>ce of such ROS <strong>for</strong> VAH.<br />
Disclosures: D. Popa, None; E. Breen, None; L. Dug<strong>an</strong>, None; F. Powell, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.2/FF7<br />
Topic: E.04.b. Respiratory regulation
Support: NIH Gr<strong>an</strong>t NS057778<br />
Title: Spinal ERK inhibition halts <strong>the</strong> progression of phrenic long-term facilitation following<br />
acute intermittent hypoxia in rats<br />
Authors: *N. L. NICHOLS, G. S. MITCHELL;<br />
Dept Comp Bio, Univ. Wisconsin, Madison, WI<br />
Abstract: Acute intermittent hypoxia (AIH; 3, 5-minute episodes of 11% O2, 5 min intervals)<br />
induces a <strong>for</strong>m of respiratory plasticity known as phrenic long-term facilitation (pLTF). pLTF<br />
requires spinal serotonin receptor activation <strong>an</strong>d new syn<strong>the</strong>sis of brain-derived neurotrophic<br />
factor (BDNF; Baker-Herm<strong>an</strong> et al., 2004). BDNF subsequently activates its high affinity<br />
receptor, <strong>the</strong> receptor tyrosine kinase TrkB. Serotonin <strong>an</strong>d TrkB receptor activation are necessary<br />
<strong>for</strong> pLTF induction, but not mainten<strong>an</strong>ce. Activation of <strong>the</strong> ERK MAP kinase pathway<br />
(presumably via TrkB activation) is also necessary <strong>for</strong> pLTF, although it is not known if it is<br />
critical <strong>for</strong> induction versus mainten<strong>an</strong>ce of pLTF once established. To test <strong>the</strong> hypo<strong>the</strong>sis that<br />
ERK MAP kinase activation is necessary to maintain pLTF, we injected <strong>the</strong> MEK inhibitor<br />
UO126 (100 µM; 12 µl) into <strong>the</strong> intra<strong>the</strong>cal space of <strong>the</strong> cervical spinal cord (~C4) after pLTF<br />
had been induced with AIH. Integrated phrenic <strong>an</strong>d hypoglossal (XII) nerve burst amplitudes<br />
were monitored in <strong>an</strong>es<strong>the</strong>tized, vagotomized, paralyzed <strong>an</strong>d ventilated rats <strong>an</strong>d UO126 or<br />
vehicle (DMSO + saline) were administered 30 minutes after pLTF had been induced with AIH.<br />
In vehicle treated rats, both phrenic (n = 7) <strong>an</strong>d XII (n = 12) nerve burst amplitudes increased<br />
progressively <strong>from</strong> baseline, reaching values of 87 ± 14% (phrenic) <strong>an</strong>d 79 ± 18% (XII) above<br />
baseline at 90 minutes post-AIH. In contrast, although pLTF developed <strong>for</strong> <strong>the</strong> first 30 minutes<br />
following AIH in UO126-treated rats (34 ± 11%, n = 8), fur<strong>the</strong>r development of pLTF was not<br />
observed at 60 (32 ± 11%) or 90 minutes (35 ± 9%) post-AIH. In <strong>the</strong>se same rats, XII LTF (n =<br />
9) increased <strong>from</strong> 40 ± 10% above baseline (30 minutes post-AIH) to 62 ± 25% (60 minutes) <strong>an</strong>d<br />
112 ± 36% (90 minutes). Thus, localized injections of UO126 halted fur<strong>the</strong>r progression of<br />
pLTF, but not LTF in XII motor output. In summary, ERK activation is necessary <strong>for</strong> <strong>the</strong><br />
development of pLTF, but is not necessary <strong>for</strong> pLTF mainten<strong>an</strong>ce in <strong>the</strong> time frame observed<br />
since UO126 halted fur<strong>the</strong>r development, but did not reverse AIH-induced pLTF.<br />
Disclosures: N.L. Nichols, None; G.S. Mitchell, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.3/FF8
Topic: E.04.b. Respiratory regulation<br />
Support: NIH 1R01HD052682-01A1 (DDF)<br />
NIH 1R01NS054025 (PJR)<br />
Title: Phrenic motoneuron discharge during hypoxia-induced short term potentiation<br />
Authors: *K.-Z. LEE 1 , P. J. REIER 2 , D. D. FULLER 1 ;<br />
1 2<br />
Physical Therapy, Univ. of Florida, Gainesville, FL; Neurosci., Univ. of Florida, McKnight<br />
Brain Inst., Gainesville, FL<br />
Abstract: Hypoxia-induced short term potentiation (STP) of respiratory motor output represents<br />
a progressive increase in activity during hypoxia <strong>an</strong>d a slow return in activity following hypoxia.<br />
We hypo<strong>the</strong>sized that STP would be differentially expressed between physiologically defined<br />
phrenic motoneurons (PhrMN). Single fiber recordings were used to characterize PhrMN<br />
discharge in <strong>an</strong>es<strong>the</strong>tized, vagotomized <strong>an</strong>d ventilated rats. PhrMNs were divided into early-<br />
(Early-I) or late-inspiratory (Late-I) according to discharge onset relative commencement of <strong>the</strong><br />
contralateral phrenic burst during normocapnic baseline. Discharge frequency of both Early-I<br />
<strong>an</strong>d Late-I PhrMNs were abruptly increased during hypoxia (FIO2=0.12-0.14, 3-min). Both<br />
PhrMN types showed a gradual enh<strong>an</strong>cement in frequency over <strong>the</strong> remainder of hypoxia.<br />
However, <strong>the</strong> discharge duration <strong>an</strong>d total spikes/breath of Early-I PhrMNs were attenuated<br />
during hypoxia whereas Late-I PhrMNs maintained const<strong>an</strong>t discharge duration <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e<br />
increased <strong>the</strong> number of spikes/breath. A population of previously inactive (i.e. silent) PhrMNs<br />
was recruited 48±8 sec after hypoxia onset. These PhrMNs had a Late-I onset <strong>an</strong>d <strong>the</strong> majority<br />
(8/9) ceased bursting promptly upon termination of hypoxia. In contrast, both Early-I <strong>an</strong>d Late-I<br />
PhrMNs showed post-hypoxia STP as reflected by greater discharge frequencies <strong>an</strong>d<br />
spikes/breath during <strong>the</strong> post-hypoxic period (P < 0.01 vs. baseline). We conclude that 1)<br />
induction of STP reflects activation of Early-I, Late-I, <strong>an</strong>d recruited Late-I (i.e. silent) PhrMNs,<br />
<strong>an</strong>d 2) STP following hypoxia primarily reflects persistent increases in <strong>the</strong> discharge of Early-I<br />
<strong>an</strong>d Late-I PhrMNs. Long-lasting ch<strong>an</strong>ges in silent PhrMN activity may require repeated<br />
activation such would occur during intermittent hypoxia.<br />
Disclosures: K. Lee, None; P.J. Reier, None; D.D. Fuller, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.4/FF9
Topic: E.04.b. Respiratory regulation<br />
Support: NIH HL69064<br />
Title: Phrenic motor facilitation following phrenic inactivity requires spinal atypical protein<br />
kinase C activity<br />
Authors: *T. BAKER-HERMAN;<br />
Comparative Biosci., Univ. of Wisconsin, Madison, WI<br />
Abstract: Phrenic inactivity elicits mech<strong>an</strong>isms of compensatory plasticity that enh<strong>an</strong>ce phrenic<br />
motor output once activity is restored. We hypo<strong>the</strong>sized that atypical protein kinase C (PKC)<br />
plays a criticial role in <strong>the</strong> induction of this <strong>for</strong>m of homeostatic plasticity. Anes<strong>the</strong>tized,<br />
vagotomized <strong>an</strong>d pump-ventilated rats were exposed to 30 min of hypocapnia (5-10 mmHg<br />
below baseline) to reversibly deprive phrenic motor neurons of rhythmic respiratory activity.<br />
Rats pre-treated with intra<strong>the</strong>cal vehicle or control peptide prior to activity deprivation<br />
signific<strong>an</strong>tly increased phrenic motor output once baseline conditions were restored, <strong>an</strong> effect<br />
that lasted at least 60 min (45% <strong>an</strong>d 50% baseline, respectively). Rats receiving intra<strong>the</strong>cal ZIP<br />
(myristoylated PKC zeta pseudosubstrate), <strong>an</strong> inhibitor to atypical PKC io<strong>for</strong>ms zeta <strong>an</strong>d iota,<br />
had no signific<strong>an</strong>t phrenic motor facilitation (7% baseline). In contrast, PKC zeta/iota inhibition<br />
did not block phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH;<br />
81% baseline), suggesting that pLTF <strong>an</strong>d homeostatic plasticity of phrenic motor output are<br />
distinct mech<strong>an</strong>isms. These data suggest a critical role <strong>for</strong> atypical PKC activity in compensatory<br />
increases in phrenic motor output following acute activity deprivation.<br />
Disclosures: T. Baker-Herm<strong>an</strong>, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.5/FF10<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH Gr<strong>an</strong>t AG18760<br />
Title: Testosterone restores respiratory long term facilitation in old male rats
Authors: N. R. NELSON 1 , *M. BEHAN 2 ;<br />
1 2<br />
Comparative Biosci., Univ. of Wisconsin, Madison, WI; Univ. Wisconsin Sch. Vet Med.,<br />
Madison, WI<br />
Abstract: Previous studies in our laboratory have shown that gonadectomy in young male rats (3<br />
months) eliminates long term facilitation of hypoglossal <strong>an</strong>d phrenic motor output following<br />
intermittent hypoxia (LTF). LTF c<strong>an</strong> be restored in gonadectomized rats by administration of<br />
testosterone in a <strong>for</strong>m that is converted to estrogen in <strong>the</strong> brain. Additionally, circulating<br />
testosterone levels in <strong>the</strong>se <strong>an</strong>imals are positively correlated with hypoglossal LTF. Aging has a<br />
negative effect on respiratory LTF. By middle age (12 months), intact male rats no longer exhibit<br />
hypoglossal LTF, <strong>an</strong>d phrenic LTF is signific<strong>an</strong>tly reduced. This reduction in respiratory LTF<br />
persists into old age. We asked <strong>the</strong> question: c<strong>an</strong> testosterone supplementation in old male rats<br />
restore respiratory LTF? Male Fischer 344 rats (>20 months) were impl<strong>an</strong>ted with Silastic tubes<br />
containing testosterone. One week later rats were <strong>an</strong>es<strong>the</strong>tized, vagotomized, artificially<br />
ventilated, <strong>an</strong>d <strong>the</strong> activity of hypoglossal <strong>an</strong>d phrenic nerves recorded during 3, 5-minute<br />
episodes of hypoxia <strong>an</strong>d <strong>for</strong> 60 minutes <strong>the</strong>reafter to measure LTF. By comparison with control<br />
rats (intact <strong>an</strong>d sham-operated), <strong>the</strong>re was a signific<strong>an</strong>t increase in both hypoglossal <strong>an</strong>d phrenic<br />
LTF in testosterone-treated rats, with levels approaching that of normal young rats. Serum<br />
testosterone levels were measured <strong>an</strong>d correlated with <strong>the</strong> degree of recovery of respiratory LTF.<br />
These data suggest that a progressive decline in testosterone-mediated respiratory plasticity with<br />
increasing age may alter <strong>the</strong> ability of respiratory motoneurons to maintain upper airway tone,<br />
especially in response to intermittent hypoxia. We speculate that in old male rats testosterone is<br />
converted to estrogen in <strong>the</strong> brain by aromatase, <strong>an</strong>d exerts its effect by me<strong>an</strong>s of estrogen<br />
receptors in respiratory control circuits.<br />
Disclosures: N.R. Nelson, None; M. Beh<strong>an</strong> , None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.6/FF11<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH HL-90554<br />
NIH HL-76537<br />
NIH HL-86493
Title: Sensory plasticity of <strong>the</strong> carotid body requires activation of NADPH oxidase<br />
Authors: *Y.-J. PENG 1 , J. NANDURI 1 , G. YUAN 1 , N. WANG 1 , E. DENERIS 2 , S.<br />
PENDYALA 1 , V. NATARAJAN 1 , G. KUMAR 1 , N. R. PRABHAKAR 1 ;<br />
1 Dept. of Med., Univ. of Chicago, Chicago, IL; 2 Case Western Reserve Univ., Clevel<strong>an</strong>d, OH<br />
Abstract: Repetitive hypoxia induces long-lasting increase in sensory discharge of <strong>the</strong> carotid<br />
bodies or sensory long-term facilitation (sLTF) in rodents exposed to chronic intermittent<br />
hypoxia (CIH). In <strong>the</strong> present study, we tested <strong>the</strong> role of NADPH oxidase (NOX) in CIHevoked<br />
sLTF. Experiments were per<strong>for</strong>med on ex vivo carotid bodies <strong>from</strong> rats <strong>an</strong>d mice exposed<br />
ei<strong>the</strong>r to 10d of CIH or normoxia. CIH-evoked sLTF was absent in mice deficient in NOX2. CIH<br />
increases NOX activity via 5-HT dependent mech<strong>an</strong>ism(s). Studies with ROS scavengers<br />
revealed that H2O2 generated <strong>from</strong> O2 ·- contributes to CIH-evoked sLTF. In <strong>the</strong> presence of H2O2,<br />
acute repetitive hypoxia elicited sLTF of carotid bodies <strong>from</strong> normoxic control rats <strong>an</strong>d mice,<br />
similar to that seen in CIH-treated <strong>an</strong>imals. These observations suggest that CIH results in 5-HTdependent<br />
NOX2 activation <strong>an</strong>d ROS generation, which in turn induce sensory plasticity of <strong>the</strong><br />
carotid body.<br />
Disclosures: Y. Peng, None; J. n<strong>an</strong>duri, None; G. Yu<strong>an</strong>, None; N. W<strong>an</strong>g, None; E. Deneris,<br />
None; S. Pendyala, None; V. Nataraj<strong>an</strong>, None; G. Kumar, None; N.R. Prabhakar, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.7/FF12<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH Gr<strong>an</strong>t HL072849<br />
Title: Suppression of excitatory tr<strong>an</strong>smission in Kölliker-Fuse nucleus reverses initial shortening<br />
of inspiration during hypoxia <strong>an</strong>d attenuates post-hypoxic frequency decline<br />
Authors: *G. SONG, C. TIN, C.-S. POON;<br />
Div. Hlth. Sci. & Technol., MIT, Cambridge, MA<br />
Abstract: The acute respiratory response to brief hypoxic challenge was tested in 10 ureth<strong>an</strong>e<strong>an</strong>es<strong>the</strong>tized,<br />
vagotomized, paralyzed <strong>an</strong>d ventilated adult male SD rats be<strong>for</strong>e <strong>an</strong>d after<br />
unilateral microinjections of GABAA receptor agonist muscimol (10 mM, 20-50 nl) or glutamate
eceptor <strong>an</strong>tagonists AP-5 <strong>an</strong>d/or CNQX (25 mM, 20-50 nl) at <strong>the</strong> pontine Kölliker-Fuse (KF)<br />
nucleus. Microinjections were made at sites within <strong>the</strong> intermediate KF nucleus where electrical<br />
stimulation provoked strong phrenic inspiratory inhibition <strong>an</strong>d excitation of vagal efferent postinspiratory<br />
discharge. Unilateral microinjection of <strong>an</strong>y of <strong>the</strong>se agents signific<strong>an</strong>tly leng<strong>the</strong>ned<br />
inspiratory duration (TI) by 48.8±9.64% (me<strong>an</strong>±SE, P
Title: Molecular mech<strong>an</strong>isms involved in <strong>the</strong> postnatal loss of hypoxic sensitivity in<br />
adrenomedullary chromaffin cells: Role of opioid receptor stimulation<br />
Authors: S. SALMAN 1 , J. BUTTIGIEG 3 , *C. A. NURSE 2 ;<br />
1 Biol., 2 McMaster Univ., Hamilton, ON, C<strong>an</strong>ada; 3 Toronto Western Hosp., Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Prior to preg<strong>an</strong>glionic innervation, adrenomedullary chromaffin cells (AMCs) respond<br />
directly to hypoxic stress at birth by evoking catecholamine (CAT) secretion. CAT secretion is a<br />
crucial physiological response during <strong>the</strong> tr<strong>an</strong>sition to extrauterine life because it regulates<br />
cardiac conduction <strong>an</strong>d prepares <strong>the</strong> lungs <strong>for</strong> air breathing. This non-neurogenic response to<br />
hypoxia is suppressed postnatally along a time course that parallels <strong>the</strong> development of<br />
spl<strong>an</strong>chnic innervation <strong>an</strong>d is mediated by K + ch<strong>an</strong>nel inhibition leading to membr<strong>an</strong>e<br />
depolarization <strong>an</strong>d voltage-gated Ca 2+ entry. Opioid peptides released <strong>from</strong> spl<strong>an</strong>chnic nerve<br />
terminals toge<strong>the</strong>r with acetylcholine (ACh) have been proposed to play a role in <strong>the</strong> suppression<br />
of <strong>the</strong> direct hypoxic response of AMCs. Recent studies <strong>from</strong> our laboratory indicated that<br />
chronic ACh receptor stimulation with nicotine in vivo <strong>an</strong>d in vitro caused a loss of hypoxic<br />
sensitivity in primary neonatal AMCs <strong>an</strong>d immortalized chromaffin (MAH) cells. This effect<br />
was attributed to upregulation of KATP ch<strong>an</strong>nels which are actually activated during hypoxia,<br />
causing membr<strong>an</strong>e hyperpolarization. Here, we tested whe<strong>the</strong>r opioid receptor stimulation also<br />
leads to loss of hypoxic sensitivity in chromaffin cells. Chronic stimulation of neonatal AMCs<br />
with µ- <strong>an</strong>d δ-opioid agonists (2 µM) in vitro led to suppression of hypoxic sensitivity.<br />
Moreover, Qu<strong>an</strong>titative Real Time-PCR <strong>an</strong>d western blot <strong>an</strong>alysis indicated that chronic<br />
exposure of MAH cells to µ- <strong>an</strong>d δ-opioid receptor agonists <strong>for</strong> ~7 days in culture resulted in<br />
KATP ch<strong>an</strong>nel upregulation. Fur<strong>the</strong>rmore, <strong>the</strong>re was <strong>an</strong> upregulation of cystathionine beta<br />
synthase (CBS), <strong>the</strong> enzyme responsible <strong>for</strong> <strong>the</strong> production of hydrogen sulfide (H2S), a<br />
gasotr<strong>an</strong>smitter proposed to be involved in O2-sensing in certain cell types. All of <strong>the</strong> above<br />
effects of opioid agonists were inhibited by <strong>the</strong> general <strong>an</strong>tagonist, naloxone (2 µM). Because<br />
accumulating evidence points to <strong>the</strong> KATP ch<strong>an</strong>nel as a target of H2S, we propose that<br />
upregulation of KATP ch<strong>an</strong>nels <strong>an</strong>d <strong>the</strong>ir activation by endogenous H2S production during chronic<br />
opioid receptor stimulation may contribute to <strong>the</strong> suppression of hypoxic sensitivity in<br />
chromaffin cells. Given <strong>the</strong> link between prenatal exposure to opiates <strong>an</strong>d <strong>the</strong> higher risk of<br />
Sudden Inf<strong>an</strong>t Death Syndrome (SIDS), this study suggests that opioid receptor-mediated loss of<br />
hypoxic sensitivity in adrenomedullary chromaffin cells may be a contributing factor.<br />
Disclosures: S. Salm<strong>an</strong>, None; J. Buttigieg, None; C.A. Nurse, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 572.9/FF14<br />
Topic: E.04.b. Respiratory regulation<br />
Support: CIHR Gr<strong>an</strong>t 480237<br />
Title: Long-term facilitation of genioglossus muscle tone induced by obstructive apneas requires<br />
TrkB receptor activation at <strong>the</strong> hypoglossal motor pool<br />
Authors: *A. TADJALLI 1 , J. PEEVER 1,2 , J. DUFFIN 2 ;<br />
1 Cell <strong>an</strong>d Systems Biol., 2 Physiol., Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada<br />
Abstract: The respiratory neural network is flexible <strong>an</strong>d c<strong>an</strong> undergo neuronal plasticity. Recent<br />
work suggests that neurotrophins <strong>an</strong>d <strong>the</strong>ir high-affinity receptor tyrosine kinase B (TrkB) are<br />
involved in mediating plasticity of respiratory motor output elicited by intermittent hypoxia.<br />
Previously we showed that repeated obstructive apneas induce long-term facilitation (LTF) of<br />
genioglossus motor outflow in rats. This study aimed to determine whe<strong>the</strong>r TrkB receptor<br />
activation is required <strong>for</strong> respiratory LTF induced by repeated obstructive apneas. Experiments<br />
were per<strong>for</strong>med on <strong>an</strong>es<strong>the</strong>tized, tracheostomized, spont<strong>an</strong>eously breathing adult rats. A<br />
microdialysis probe was placed into <strong>the</strong> hypoglossal motor pool through which ei<strong>the</strong>r K252a (a<br />
TrkB receptor inhibitor; 10 uM in 0.9% saline) or 0.9% saline (control) was perfused into <strong>the</strong><br />
motor pool. Genioglossus muscle EMG activity served as <strong>an</strong> index of respiratory motor outflow.<br />
Apneas (10, 15-second apneas each separated by 1-minute) were induced by obstructing tracheal<br />
airflow using a specially-constructed device. The following two hypo<strong>the</strong>ses were tested. 1)<br />
Perfusion of saline (n=7) alone into <strong>the</strong> hypoglossal motor pool would have no effect on <strong>the</strong><br />
expression of apnea-induced LTF. 2) Perfusion of a TrkB receptor inhibitor (n=6) into <strong>the</strong><br />
hypoglossal motor pool prior (i.e. 20 minute prior) to repeated apneas would prevent LTF of<br />
genioglossus muscle tone. We found that saline perfusion into <strong>the</strong> hypoglossal motor pool did<br />
not affect <strong>the</strong> expression of apnea-induced respiratory LTF. Indeed we found that airway<br />
obstructions caused a signific<strong>an</strong>t long-term enh<strong>an</strong>cement of genioglossus muscle tone, increasing<br />
it by 72 ± 14% above baseline levels at 60 minutes following repeated apneas (RM ANOVA,<br />
p0.05). LTF of genioglossus muscle tone requires a functional<br />
neurotrophic signaling cascade. Triggering LTF by pharmacological mech<strong>an</strong>isms could be a<br />
potentially useful strategy <strong>for</strong> improving airway patency in obstructive sleep apnea patients.<br />
Disclosures: A. Tadjalli, None; J. Peever, None; J. Duffin, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.10/FF15<br />
Topic: E.04.b. Respiratory regulation<br />
Support: Heart <strong>an</strong>d Stroke Foundation of Ontario<br />
Natural Sciences <strong>an</strong>d Engineering Research Council of C<strong>an</strong>ada<br />
Title: Immunocytochemical <strong>an</strong>d functional characterization of rat aortic body chemoreceptors:<br />
Role of neurotr<strong>an</strong>smitters<br />
Authors: *N. PISKURIC, C. VOLLMER, M. ZHANG, C. NURSE;<br />
Biol., McMaster Univ., Hamilton, ON, C<strong>an</strong>ada<br />
Abstract: Distributed diffusely in <strong>the</strong> region of <strong>the</strong> aortic arch are clusters of catecholaminergic<br />
glomus cells (parag<strong>an</strong>glia) collectively referred to as aortic bodies (ABs). These parag<strong>an</strong>glia are<br />
putative peripheral arterial chemoreceptors, presumed to sense blood O2 levels <strong>an</strong>d elicit<br />
cardiovascular reflexes during low O2 (hypoxia). Though ABs are thought to function similarly<br />
to <strong>the</strong> well-characterized carotid body (CB) chemoreceptors, studies at <strong>the</strong> cellular level are<br />
lacking. To address this, <strong>the</strong> present study combines confocal immunofluorescence with<br />
functional assays in a novel AB cell culture model generated <strong>from</strong> juvenile rats. In whole mounts<br />
of <strong>the</strong> left vagus <strong>an</strong>d recurrent laryngeal nerves, AB glomus cells showed positive<br />
immunoreactivity <strong>for</strong> tyrosine hydroxylase, vesicular acetylcholine (ACh) tr<strong>an</strong>sporter <strong>an</strong>d<br />
serotonin (5-HT). Fur<strong>the</strong>rmore, nerve terminals surrounding AB glomus cells were<br />
immunopositive <strong>for</strong> several purinoceptor subunits, including P2X2, P2X3 <strong>an</strong>d P2X7, suggesting<br />
that, similar to <strong>the</strong> CB, <strong>the</strong>y may be activated by ATP released <strong>from</strong> AB glomus cells.<br />
Interestingly, <strong>the</strong>se purinoceptor subunits were also found in neuronal cell bodies that occurred at<br />
<strong>the</strong> periphery of AB parag<strong>an</strong>glia, raising <strong>the</strong> possibility of a local circuit involved in afferent<br />
signalling. Dissociation of <strong>the</strong> AB region of <strong>the</strong> vagus <strong>an</strong>d recurrent laryngeal nerves yielded<br />
viable cultures containing both AB glomus cells <strong>an</strong>d <strong>the</strong> local circuit neurons, which appeared to<br />
<strong>for</strong>m connections as revealed by immunocytochemistry. Indeed, in Ca2+ imaging experiments,<br />
some cultured neurons responded to chemostimul<strong>an</strong>ts, such as hypoxia, consistent with <strong>the</strong><br />
presence of functional synapses between neurons <strong>an</strong>d AB glomus cells in culture. Moreover,<br />
similar to <strong>the</strong> responses of CB afferent neurons, co-cultured AB neurons were excited by applied<br />
ACh <strong>an</strong>d ATP. There<strong>for</strong>e, this novel culture preparation appears <strong>an</strong> attractive one <strong>for</strong> <strong>the</strong><br />
elucidation of AB chemosensing mech<strong>an</strong>isms.<br />
Disclosures: N. Piskuric, None; C. Vollmer, None; M. Zh<strong>an</strong>g, None; C. Nurse, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.11/FF16<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH Gr<strong>an</strong>t HL085108<br />
Title: Role of serotonin receptor type 2A (5-HTR2A) in <strong>the</strong> nucleus of <strong>the</strong> solitary tract (nTS)<br />
after ten days of chronic intermittent hypoxia<br />
Authors: *J. R. AUSTGEN 1,2 , H. DANTZLER 2 , D. D. KLINE 1,2 ;<br />
1 Biomed. Sci., 2 Dalton Cardiovasc. Res. Ctr., Univ. of Missouri, Columbia, MO<br />
Abstract: The arterial chemoreflex is <strong>an</strong> import<strong>an</strong>t autonomic regulator of blood oxygen <strong>an</strong>d<br />
carbon dioxide levels. Alterations in <strong>the</strong>se levels activate <strong>the</strong> reflex, which induces ch<strong>an</strong>ges in<br />
breathing patterns <strong>an</strong>d sympa<strong>the</strong>tic nerve output. It is hypo<strong>the</strong>sized that <strong>the</strong> neurotr<strong>an</strong>smitter<br />
serotonin (5-HT) influences <strong>the</strong> primary synapse between chemosensory <strong>an</strong>d visceral afferents,<br />
<strong>an</strong>d second order nTS neurons. We have shown that 5-HT <strong>an</strong>d 5-HTR2 receptors are import<strong>an</strong>t<br />
in modulating synaptic tr<strong>an</strong>smission <strong>an</strong>d firing properties of nTS neurons under normal<br />
conditions. Chronic obstructive sleep apnea (OSA) alters <strong>the</strong> chemoreflex circuit, resulting in<br />
sustained hypertension <strong>an</strong>d breathing abnormalities. The pathway <strong>an</strong>d mech<strong>an</strong>ism by which sleep<br />
apnea induces sustained hypertension has not been fully elucidated. We have previously<br />
demonstrated that chronic intermittent hypoxia (CIH), a model of OSA, alters synaptic<br />
tr<strong>an</strong>smission <strong>an</strong>d cellular activity in <strong>the</strong> nTS. To determine <strong>the</strong> role of 5-HT within <strong>the</strong> nTS in<br />
CIH-induced cardiorespiratory <strong>an</strong>d synaptic alterations, rats (Sprague-Dawley, 3-4 weeks) were<br />
exposed to 10 days of CIH. nTS neurons were recorded using <strong>the</strong> patch-clamp technique in<br />
horizontal slices (275-285 µm). Spont<strong>an</strong>eous <strong>an</strong>d solitary tract evoked excitatory postsynaptic<br />
currents (s/eEPSC) <strong>an</strong>d current-induced action potential discharges (APD) were recorded in <strong>the</strong><br />
presence <strong>an</strong>d absence of 5-HT pharmacological agents. Bath application of <strong>the</strong> specific 5-<br />
HTR2A agonist TCB-2 (10 µM, n=5) resulted in a signific<strong>an</strong>t (p
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.12/FF17<br />
Topic: E.04.b. Respiratory regulation<br />
Support: FAPESP Gr<strong>an</strong>t 06/60174-9<br />
CNPq<br />
Title: Role of <strong>the</strong> commissural nucleus of <strong>the</strong> solitary tract on cardiorespiratory responses to<br />
chemoreflex activation<br />
Authors: *A. T. TAKAKURA 1 , M. T. FAVERO 2 , P. M. DE PAULA 2 , J. V. MENANI 2 , E.<br />
COLOMBARI 3 , T. S. MOREIRA 4 ;<br />
2 Physiol. <strong>an</strong>d Pathology, 1 Sao Paulo State Univ., Araraquara, Brazil; 3 Physiol., Federal Univ. of<br />
Sao Paulo, Sao Paulo, Brazil; 4 Physiol. <strong>an</strong>d Biophysics, Univ. of Sao Paulo, Sao Paulo, Brazil<br />
Abstract: A large variety of sensory afferents converges to <strong>the</strong> nucleus of <strong>the</strong> solitary tract<br />
(NTS) <strong>an</strong>d influences <strong>the</strong> activity of ventrolateral medullary neurons involved in cardiovascular<br />
<strong>an</strong>d respiratory control. Here, we investigated <strong>the</strong> effects of inhibition of <strong>the</strong> commissural NTS<br />
(commNTS) on cardiovascular <strong>an</strong>d respiratory responses to central or peripheral chemoreceptor<br />
activation in conscious <strong>an</strong>d <strong>an</strong>es<strong>the</strong>tized rats. Male Holtzm<strong>an</strong> rats (280-300 g) were used. In<br />
conscious rats, inhibition of <strong>the</strong> commNTS with <strong>the</strong> GABA-A agonist muscimol (120 pmol/60<br />
nl) reduced <strong>the</strong> increase in me<strong>an</strong> arterial pressure (MAP) (+17±4, vs. saline: +36±6 mmHg) <strong>an</strong>d<br />
ventilation (549±32, vs. saline: 1217±49 ml/min/kg) produced by activation of peripheral<br />
chemoreflex with brief period of hypoxia (10% O2, 15-20 s). However, inhibition of <strong>the</strong><br />
commNTS did not ch<strong>an</strong>ge <strong>the</strong> increase in ventilation produced by activation of central<br />
chemoreceptors with increased levels of CO2 (10%) (1234±52, vs. saline: 1187±26 ml/min/kg).<br />
In ureth<strong>an</strong>e <strong>an</strong>es<strong>the</strong>tized rats, <strong>the</strong> injection of muscimol into <strong>the</strong> commNTS eliminated <strong>the</strong><br />
increase in MAP (+3±2, vs. saline: +17±5 mmHg) <strong>an</strong>d phrenic nerve discharge (PND) <strong>an</strong>d<br />
reduced spl<strong>an</strong>chnic sympa<strong>the</strong>tic nerve discharge (sSND) (+93±15, vs. saline: +283±26%)<br />
produced by hypoxia. Muscimol into <strong>the</strong> commNTS did not affect <strong>the</strong> ch<strong>an</strong>ge on PND <strong>an</strong>d sSND<br />
produced by <strong>the</strong> activation of central chemoreceptors with 10% CO2. In conclusion, <strong>the</strong> integrity<br />
of commNTS is import<strong>an</strong>t <strong>for</strong> <strong>the</strong> cardiovascular <strong>an</strong>d respiratory responses elicited by hypoxia<br />
(dependent on peripheral chemoreceptors), but not hypercapnia (dependent on central<br />
chemoreceptors).
Disclosures: A.T. Takakura, None; M.T. Favero, None; P.M. De Paula, None; J.V. Men<strong>an</strong>i,<br />
None; E. Colombari, None; T.S. Moreira, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.13/FF18<br />
Topic: E.04.b. Respiratory regulation<br />
Support: NIH Gr<strong>an</strong>t PO1 HD36379<br />
First C<strong>an</strong>dle/SIDS Alli<strong>an</strong>ce SP0030<br />
Title: Arousal habituation in response to acute intermittent hypoxia is modulated by GABAergic<br />
mech<strong>an</strong>isms<br />
Authors: R. W. SCHNEIDER 1 , C. M. TOBIA 2 , *R. A. DARNALL 3 ;<br />
1 Physiol., 2 Physiol, 3 Ped & Physiol, Dartmouth Med. Sch., Leb<strong>an</strong>on, NH<br />
Abstract: Arousal <strong>from</strong> sleep is <strong>an</strong> essential first line defense in response to hypoxia, whe<strong>the</strong>r<br />
<strong>from</strong> apnea, suffocation, or rebreathing. We have recently shown that inf<strong>an</strong>t rat pups (P15)<br />
exposed to repeated brief episodes of hypoxia take progressively longer <strong>an</strong>d longer to wake up<br />
with each successive exposure. The mech<strong>an</strong>isms underlying this arousal ‘habituation’ are<br />
unknown. The medullary raphe contains neurons that modulate breathing <strong>an</strong>d m<strong>an</strong>y autonomic<br />
functions including <strong>the</strong>rmoregulation <strong>an</strong>d sleep <strong>an</strong>d arousal, <strong>an</strong>d may play a role in arousal<br />
habituation. Since this region receives abund<strong>an</strong>t GABAergic innervation, we hypo<strong>the</strong>sized that<br />
inhibiting raphe neurons with muscimol, a GABAA receptor agonist, would blunt arousals <strong>an</strong>d<br />
enh<strong>an</strong>ce arousal habituation <strong>an</strong>d that blocking GABAA receptors with bicuculline would enh<strong>an</strong>ce<br />
arousal <strong>an</strong>d attenuate habituation. A total of 27 P15 rat pups were briefly <strong>an</strong>es<strong>the</strong>tized<br />
(isoflur<strong>an</strong>e) <strong>an</strong>d microinjections with ei<strong>the</strong>r saline, muscimol (100-500 pmol), or bicuculline (50<br />
pmol) were made into <strong>the</strong> medullary raphe near <strong>the</strong> ventral surface. After recovery <strong>from</strong><br />
<strong>an</strong>es<strong>the</strong>sia (~40 min) <strong>the</strong>y were exposed to 4 repeated episodes of 10% oxygen (3 min hypoxia/6<br />
minutes normoxia) at <strong>the</strong>rmoneutrality while chamber oxygen concentration (ChO2), heart rate<br />
(HR), body temperature (Tbody), <strong>an</strong>d oxyhemoglobin saturation (SpO2) were continuously<br />
recorded. The time to arousal <strong>from</strong> <strong>the</strong> onset of hypoxia progressively increased in <strong>the</strong> saline<br />
injected pups, not different <strong>from</strong> our previous observations in non-injected pups. Compared to<br />
controls, <strong>the</strong> overall time to arousal was greater after muscimol administration (P=0.004) <strong>an</strong>d<br />
less after bicuculline administration (P=0.034). Arousal habituation (slope over 4 trials) was also
enh<strong>an</strong>ced after muscimol (P=0.028) <strong>an</strong>d attenuated or abolished after bicuculline (P=0.06).<br />
These results were mirrored by ch<strong>an</strong>ges in ChO2 <strong>an</strong>d SpO2: compared to controls, pups aroused<br />
at lower ChO2 <strong>an</strong>d SpO2 after muscimol (P=0.015) <strong>an</strong>d at higher ChO2 <strong>an</strong>d SpO2 after<br />
bicuculline (P=0.023). HR increased in all groups during hypoxia but was greatest in muscimol<br />
injected pups. Tbody was also greater in bicuculline injected pups consistent with <strong>the</strong> role of<br />
neurons in <strong>the</strong> medullary raphe in <strong>the</strong>rmoregulatory brown fat activation <strong>an</strong>d vasoconstriction.<br />
These results indicate that structures in <strong>the</strong> medullary raphe contribute to <strong>the</strong> phenomenon of<br />
arousal habituation <strong>an</strong>d that <strong>the</strong>se are in turn modulated by GABAergic inputs. We speculate that<br />
this arousal ‘habituation’ may contribute to death in Sudden Inf<strong>an</strong>t Death syndrome (SIDS)<br />
inf<strong>an</strong>ts who often experience repeated periods of apnea <strong>an</strong>d bradycardia in <strong>the</strong> days <strong>an</strong>d weeks<br />
be<strong>for</strong>e death.<br />
Disclosures: R.W. Schneider, None; C.M. Tobia, None; R.A. Darnall, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.14/FF19<br />
Topic: E.04.b. Respiratory regulation<br />
Support: HL73474<br />
HL 80208<br />
Title: To what extent are second order neurons targeted by carotid body afferents to <strong>the</strong> nucleus<br />
of <strong>the</strong> solitary tract (NTS), also projection neurons to respiratory compartments in <strong>the</strong> brainstem?<br />
Authors: *G. F. ALHEID, W. JIAO, D. R. MCCRIMMON;<br />
Physiol., Northwestern Univ. Feinberg Sch. of Med., Chicago, IL<br />
Abstract: We are currently re-examining tr<strong>an</strong>s-NTS pathways to respiratory targets in <strong>the</strong><br />
ventral respiratory column of <strong>the</strong> brainstem. The nucleus of <strong>the</strong> solitary tract is crucial to central<br />
reflexes controlling respiration, driven by topographically org<strong>an</strong>ized projections to <strong>the</strong> NTS <strong>from</strong><br />
peripheral respiratory related afferents. To underst<strong>an</strong>d <strong>the</strong> relative contribution of intrinsic vs.<br />
extrinsic projecting NTS relay neurons to processing chemororeceptor afferents, we are<br />
examining terminal appositions of <strong>the</strong> carotid sinus nerve (CSN) on NTS neurons retrogradely<br />
labeled <strong>from</strong> physiologically defined regions of <strong>the</strong> ventral respiratory column of <strong>the</strong> medulla<br />
(VRC) or <strong>from</strong> respiratory related areas of <strong>the</strong> pons. Crystals of <strong>the</strong> red fluorescent carbocy<strong>an</strong>ine
dye “DiI” into <strong>the</strong> carotid body were used to label <strong>the</strong> peripheral chemoafferents of <strong>the</strong> CSN<br />
targeting <strong>the</strong> NTS; in <strong>the</strong> same <strong>an</strong>imals injections of fluorescent retrograde tracers (FluoroGold,<br />
Fast Blue) were made at medullary sites where respiratory activity was recorded <strong>from</strong> <strong>the</strong> same<br />
electrode, or in medullary or pontine regions where respiratory activity was evoked by n<strong>an</strong>oliter<br />
injections of <strong>the</strong> excitatory amino acid DL-homocysteic acid. DiI terminal appositions on <strong>the</strong><br />
soma <strong>an</strong>d proximal dendrites of retrogradely labeled NTS neuron were evaluated both<br />
qualitatively <strong>an</strong>d qu<strong>an</strong>titatively. As control, terminal appositions were additionally evaluated on<br />
non-retrogradely labeled neurons visualized by immunolabeling <strong>for</strong> MAP2, a microtubule<br />
associated protein detected in neuronal soma <strong>an</strong>d dendrites but not in axons. Our preliminary<br />
data (based on scoring apparent appositions in confocal z-stacks through sample areas containing<br />
both retrogradely labeled neurons - <strong>from</strong> <strong>the</strong> VRC - <strong>an</strong>d CSN varicosities) indicate that (<strong>for</strong> <strong>the</strong><br />
targeted areas) 75% of probable terminal varicosities were judged to make contacts with NTS<br />
neurons. Of <strong>the</strong>se, 88% of <strong>the</strong> apparent terminal-neuron appositions were on dendrites (labeled<br />
with MAP2). Of all neurons counted ~ 4.7% were retrogradely labeled; ~ 30% of <strong>the</strong> apparent<br />
somatic terminations, however, were on <strong>the</strong> retrogradely labeled neurons. This data are<br />
consistent with a signific<strong>an</strong>t role of <strong>for</strong> both intrinsic CSN relays within <strong>the</strong> NTS <strong>an</strong>d <strong>for</strong> 2nd<br />
order CSN relay neurons projecting directly to brainstem respiratory related areas.<br />
Disclosures: G.F. Alheid, None; W. Jiao, None; D.R. McCrimmon, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.15/FF20<br />
Topic: E.04.b. Respiratory regulation<br />
Support: HL63175<br />
Title: Evaluation of <strong>the</strong> effects of different classes of <strong>an</strong>es<strong>the</strong>tics on <strong>the</strong> incidence <strong>an</strong>d pattern of<br />
hypoxia-induced gasping in adult rat in vivo<br />
Authors: I. M. REID, K. MATHEWS, V. KURUVILLA, K. ONO, *I. C. SOLOMON;<br />
Physiol. & Biophysics, SUNY Stony Brook, Stony Brook, NY<br />
Abstract: Although <strong>the</strong>re is increasing interest in identifying <strong>the</strong> sites <strong>an</strong>d mech<strong>an</strong>isms<br />
mediating gasping in response to severe hypoxia, <strong>the</strong>re is little data <strong>from</strong> <strong>the</strong> in vivo <strong>an</strong>es<strong>the</strong>tized<br />
adult rat. We previously suggested that this sparsity of data may be related to <strong>the</strong> use of <strong>the</strong> longlasting<br />
sedative-hypnotic ureth<strong>an</strong>e (U) as <strong>the</strong> <strong>an</strong>es<strong>the</strong>sic agent, <strong>an</strong>d went on to demonstrate that
hypoxic gasping was readily induced in barbiturate-<strong>an</strong>es<strong>the</strong>tized rats. The barbiturate <strong>an</strong>es<strong>the</strong>tics<br />
assessed were <strong>the</strong> long-lasting barbiturate inactin (IN), which was evaluated ei<strong>the</strong>r alone or in<br />
combination with <strong>the</strong> dissociative ketamine (IN-K) <strong>an</strong>d <strong>the</strong> short-acting barbiturate sodium<br />
pentobarbital (P). Here, we have continued to conduct experiments to systematically investigate<br />
<strong>the</strong> influence of different classes of <strong>an</strong>es<strong>the</strong>tics on <strong>the</strong> incidence of gasping <strong>an</strong>d its characteristics<br />
to determine whe<strong>the</strong>r <strong>the</strong> <strong>an</strong>es<strong>the</strong>tized adult in vivo rat might serve as a viable <strong>an</strong>imal model <strong>for</strong><br />
studies investigating mech<strong>an</strong>isms <strong>for</strong> generation of hypoxia-induced gasping. In addition to U<br />
<strong>an</strong>d <strong>the</strong> barbiturate <strong>an</strong>es<strong>the</strong>tics identified above, we have also examined <strong>the</strong> effects of IN in<br />
combination with <strong>the</strong> thiazole xylazine (IN-X, n=3), ketamine in combination with xyzaline (K-<br />
X, n=5), <strong>the</strong> nonbarbiturate sedative propofol (PRO, n=1), <strong>an</strong>d <strong>the</strong> inhal<strong>an</strong>t <strong>an</strong>es<strong>the</strong>tic isoflur<strong>an</strong>e<br />
(ISO, n=9). Our data, thus far, demonstrate that (1) IN-, IN-K-, IN-X-, P-, <strong>an</strong>d K-X-<strong>an</strong>es<strong>the</strong>tized<br />
rats exhibit longer basal inspiratory burst durations (TI) th<strong>an</strong> U- <strong>an</strong>d ISO-<strong>an</strong>es<strong>the</strong>tized rats (~360-<br />
520 ms vs ~320 ms, P200% vs ~6% over baseline amplitude, P
Authors: J.-H. ZHANG 1,2 , S. J. FUNG1 1,2 , M. XI 1,2 , S. SAMPOGNA 1,2 , *J. YAMUY 2 , M. H.<br />
CHASE 1,2,3 ;<br />
1 WebSciences Intl., Los Angeles, CA; 2 Dept. of Res., VA Greater LA Healthcare Syste, Los<br />
Angeles, CA; 3 Dept. of Physiol., UCLA Sch. of Med., Los Angeles, CA<br />
Abstract: Obstructive sleep apnea <strong>an</strong>d o<strong>the</strong>r sleep-related breathing disorders are accomp<strong>an</strong>ied<br />
by a number of pathologies that involve disruption of <strong>the</strong> cardiovascular <strong>an</strong>d respiratory systems<br />
<strong>an</strong>d various behavioral states, including sleep <strong>an</strong>d wakefulness, among o<strong>the</strong>rs. Consequently, we<br />
were interested in determining, in <strong>an</strong> <strong>an</strong>imal model of sleep apnea, whe<strong>the</strong>r neuronal<br />
degeneration, due to apoptosis, occurs in regions of <strong>the</strong> pons <strong>an</strong>d medulla that are responsible <strong>for</strong><br />
controlling a variety of systems <strong>an</strong>d behaviors. Adult guinea pigs, which were divided into<br />
control (non-apneic) <strong>an</strong>d experimental (apneic) groups, were <strong>an</strong>es<strong>the</strong>tized with α-chloralose <strong>an</strong>d<br />
immobilized with Flaxedil. Apnea was induced by ventilatory arrest in order to desaturate <strong>the</strong><br />
oxyhemoglobin to 75% SpO2. A sequence of apnea, followed by ventilation with recovery to<br />
>95% SpO2, was repeated <strong>for</strong> a period of 3 hours. At <strong>the</strong> end of <strong>the</strong> period of recurrent apnea,<br />
<strong>the</strong> <strong>an</strong>imals were sacrificed <strong>an</strong>d brain sections were immunostained with a mouse monoclonal<br />
<strong>an</strong>tibody raised against single-str<strong>an</strong>ded DNA (ssDNA), which was used as a marker <strong>for</strong> <strong>the</strong><br />
presence of apoptosis. Light microscopic <strong>an</strong>alysis revealed that neurons labeled by <strong>the</strong> <strong>an</strong>tissDNA<br />
<strong>an</strong>tibody (i.e., apoptotic cells) were present principally in brainstem regions involved in<br />
<strong>the</strong> control of respiration (e.g., <strong>the</strong> parafacial respiratory group <strong>an</strong>d <strong>the</strong> ventral respiratory group),<br />
cardiovascular functions (e.g., <strong>the</strong> nucleus ambiguus, <strong>the</strong> nucleus tractus solitarius <strong>an</strong>d <strong>the</strong> dorsal<br />
motor nucleus of <strong>the</strong> vagus) as well as REM sleep (<strong>the</strong> nucleus pontis oralis) <strong>an</strong>d wakefulness<br />
(e.g., <strong>the</strong> dorsal raphe <strong>an</strong>d locus coeruleus). Apoptosis was not present in <strong>the</strong> pons <strong>an</strong>d medulla<br />
of control <strong>an</strong>imals. We suggest that neuronal degeneration, due to apoptosis, occurs in critical<br />
areas of <strong>the</strong> pons <strong>an</strong>d medulla that are responsible <strong>for</strong> m<strong>an</strong>y of <strong>the</strong> comorbities that are<br />
experienced by patients with sleep-disordered breathing pathologies.<br />
Disclosures: J. Zh<strong>an</strong>g, None; S.J. Fung1, None; M. Xi, None; S. Sampogna, None; J.<br />
Yamuy, None; M.H. Chase, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.17/FF22<br />
Topic: E.09.d. Hypoxia/Hyperoxia<br />
Support: CNPq (Brazil)
CAPES (Brazil)<br />
Title: Control of carbohydrate metabolism in <strong>an</strong> <strong>an</strong>oxia-toler<strong>an</strong>t nervous system during <strong>an</strong>oxia<br />
<strong>an</strong>d reoxygenation<br />
Authors: *L. S. DE FRAGA, R. S. M. DA SILVA, D. M. ZANCAN;<br />
Physiol., Federal Univ. of Rio Gr<strong>an</strong>de Do Sul, Porto Alegre, Brazil<br />
Abstract: Toler<strong>an</strong>ce to lack of oxygen is necessary <strong>for</strong> m<strong>an</strong>y org<strong>an</strong>isms, because reduced<br />
oxygen availability is a common situation affecting different habitats. Since lack of oxygen is<br />
one of <strong>the</strong> major causes of damage to sensitive tissues, like brain <strong>an</strong>d heart, <strong>an</strong>oxia-toler<strong>an</strong>t<br />
org<strong>an</strong>isms have become import<strong>an</strong>t models to underst<strong>an</strong>d protective mech<strong>an</strong>isms which prevent<br />
cellular damage during <strong>the</strong> absence of oxygen. As glycogen is <strong>the</strong> main fermentable fuel<br />
supporting energy production during oxygen tension reduction, underst<strong>an</strong>ding <strong>the</strong> glycogen<br />
metabolism c<strong>an</strong> provide import<strong>an</strong>t insights about processes involved in <strong>an</strong>oxia survival. Thus,<br />
<strong>the</strong> aim of <strong>the</strong> present study was to look at possible modifications of carbohydrate metabolism in<br />
<strong>the</strong> central nervous system (CNS) of <strong>the</strong> <strong>an</strong>oxia-toler<strong>an</strong>t l<strong>an</strong>d snail Megalobulimus abbreviatus<br />
during experimental <strong>an</strong>oxia exposure (3h e 12h) <strong>an</strong>d subsequent reoxygenation (15h of aerobic<br />
recovery after 3h of <strong>an</strong>oxia). Glucose uptake, glycogen syn<strong>the</strong>sis <strong>from</strong> glucose <strong>an</strong>d <strong>the</strong> activity of<br />
<strong>the</strong> glycogen metabolism key-enzymes glycogen synthase (GS) <strong>an</strong>d phosphorylase (GP) were<br />
<strong>an</strong>alyzed. <strong>When</strong> exposed to <strong>an</strong>oxia, nervous g<strong>an</strong>glia of <strong>the</strong> snail achieve sustained glucose uptake<br />
<strong>an</strong>d glycogen syn<strong>the</strong>sis levels, which seems import<strong>an</strong>t to maintain neural homeostasis. However,<br />
<strong>the</strong> activities of GS <strong>an</strong>d GP were reduced, indicating a possible CNS metabolic depression.<br />
During <strong>the</strong> aerobic recovery period <strong>the</strong> enzyme activities returned to basal values. Whereas no<br />
modifications were achieved by <strong>an</strong>oxia in <strong>the</strong> glucose uptake <strong>an</strong>d glycogen syn<strong>the</strong>sis, it seems<br />
that <strong>the</strong> snail CNS displays metabolic arrest since both GP <strong>an</strong>d GS enzymes experience a striking<br />
reduction in <strong>the</strong> CNS of <strong>an</strong>oxic <strong>an</strong>imals. Undoubtedly, more studies must be carried out in order<br />
to demonstrate possible metabolic differences between <strong>the</strong> glial <strong>an</strong>d neuronal snail cells <strong>an</strong>d<br />
clarify <strong>the</strong> mech<strong>an</strong>isms acting to maintain homeostasis in M. abbreviatus CNS during <strong>an</strong>oxia<br />
exposure <strong>an</strong>d reoxygenation.<br />
Disclosures: L.S. De Fraga, None; R.S.M. da Silva, None; D.M. Z<strong>an</strong>c<strong>an</strong>, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.18/FF23<br />
Topic: E.09.d. Hypoxia/Hyperoxia
Support: NSERC Gr<strong>an</strong>t<br />
Department of Biological Sciences, University of Calgary<br />
Title: Serotonin enh<strong>an</strong>ces survival of Helisoma trivolvis embryos exposed to long-term <strong>an</strong>oxia<br />
Authors: *R. B. SHARTAU, J. I. GOLDBERG;<br />
Biol. Sci., Univ. of Calgary, Calgary, AB, C<strong>an</strong>ada<br />
Abstract: Embryos of <strong>the</strong> pond snail, Helisoma trivolvis, develop two bilateral serotonergic<br />
neurons named embryonic neuron C1 (ENC1) that innervate ciliary b<strong>an</strong>ds <strong>an</strong>d stimulate ciliary<br />
beating. These are sensorimotor neurons that detect environmental hypoxia <strong>an</strong>d increase <strong>the</strong> rate<br />
of cilia-driven embryonic rotation. This behavior is postulated to be <strong>an</strong> adaptation to periods of<br />
hypoxia by increasing oxygen availability through <strong>the</strong> mixing of <strong>the</strong> capsular fluid. We<br />
hypo<strong>the</strong>size that <strong>the</strong> stimulation of ciliary-driven rotation by serotonin enh<strong>an</strong>ces <strong>the</strong> survival of<br />
embryos exposed to long-term hypoxic challenges. To address this, we examined <strong>the</strong> effect of<br />
serotonin during long-term hypoxia on embryonic behavior <strong>an</strong>d survival. Control <strong>an</strong>d serotonintreated<br />
embryos (100 mM) were subjected to different levels of hypoxia <strong>for</strong> 24-48 hours. The<br />
oxygen levels used were normoxia (PO2 = 140 torr), hypoxia (PO2 = 11 torr) <strong>an</strong>d <strong>an</strong>oxia (PO2 =<br />
0 torr). Gas mixtures were bubbled into a sealed petri dish containing a single egg mass in<br />
artificial pond water in <strong>the</strong> presence or absence of serotonin. Rotational behavior was recorded<br />
using time-lapse digital videomicroscopy at hourly intervals <strong>an</strong>d played back to measure <strong>the</strong><br />
embryonic rotation rate <strong>an</strong>d document mortality. Embryos subjected to normoxia <strong>for</strong> 24 hours<br />
maintained a const<strong>an</strong>t rotation rate. Serotonin induced <strong>an</strong> increase in rotation rate that was<br />
sustained <strong>for</strong> <strong>the</strong> entire recording period. Hypoxia caused both control <strong>an</strong>d serotonin-treated<br />
embryos to undergo a tr<strong>an</strong>sient increase in rotation rate that persisted <strong>for</strong> around 3 hours, with<br />
serotonin-treated embryos having <strong>an</strong> elevated rotation rate over controls. Exposure to <strong>an</strong>oxia also<br />
induced a tr<strong>an</strong>sient increase in rotation rate in both groups. After <strong>the</strong> increase, rotation steadily<br />
slowed in control embryos until it stopped by 13 hours. In contrast, rotation persisted in<br />
serotonin-treated embryos <strong>for</strong> up to 40 hours. In <strong>an</strong>oxia, embryonic death occurred shortly<br />
following <strong>the</strong> cessation of rotation. Control embryos reached 50% mortality at 9 hours of <strong>an</strong>oxia,<br />
whereas mortality was delayed in serotonin-treated embryos, which reached 50% mortality at 24<br />
hours of <strong>an</strong>oxia. The serotonin <strong>an</strong>tagonist mi<strong>an</strong>serin (50 mM) partially blocked <strong>the</strong> rotational<br />
response to long-term serotonin treatment, but not <strong>the</strong> delay in mortality. The ability of serotonin<br />
to prolong survival in <strong>an</strong>oxia may be due to a rotation-induced distribution of <strong>the</strong> limited O2<br />
present in <strong>the</strong> embryo’s vicinity, or a serotonin-activated metabolic pathway that liberates <strong>an</strong><br />
alternative energy source.<br />
Disclosures: R.B. Shartau, None; J.I. Goldberg, None.<br />
Poster<br />
572. Carotid Bodies, Hypoxia, <strong>an</strong>d Chronic Hypoxia
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 572.19/FF24<br />
Topic: E.09.d. Hypoxia/Hyperoxia<br />
Support: HL-90554<br />
HL-89616<br />
Title: Evidence <strong>for</strong> <strong>the</strong> involvement of reactive oxygen species mediated alterations of protein<br />
phosphatase 2A <strong>an</strong>d protein kinases in activation of tyrosine hydroxylase by intermittent hypoxia<br />
Authors: G. RAGHURAMAN, Y.-J. PENG, N. R. PRABHAKAR, *G. K. KUMAR;<br />
Med., Univ. of Chicago, Chicago, IL<br />
Abstract: Intermittent hypoxia (IH) associated with recurrent apneas leads to autonomic<br />
abnormalities resulting in cardio-respiratory morbidities. Brain stem catecholaminergic neurons<br />
have been implicated in <strong>the</strong> regulation of cardio-respiratory systems. In this report we examined<br />
<strong>the</strong> effect of two patterns of IH on <strong>the</strong> activity of tyrosine hydroxylase (TH), <strong>the</strong> rate-limiting<br />
enzyme in catecholamine biosyn<strong>the</strong>sis, in <strong>the</strong> rat brain stem. Also we investigated whe<strong>the</strong>r IH<br />
affects <strong>the</strong> activity <strong>an</strong>d expression of protein phosphatases (PP) <strong>an</strong>d protein kinases which are<br />
known to regulate TH activity. Rats exposed to ei<strong>the</strong>r IH15s (15 s, 5% O2; 5 min, 21%O2) or IH90s<br />
(90 s each of 10% O2 & 21%O2) <strong>for</strong> 10 days were used. IH15s but not IH90s caused a robust<br />
increase in TH activity, dopamine (DA) level <strong>an</strong>d TH phosphorylation at Ser-31 <strong>an</strong>d Ser-40 in<br />
<strong>the</strong> medulla but not in <strong>the</strong> pons. Likewise IH15s but not IH90s decreased activity <strong>an</strong>d expression of<br />
PP2A <strong>an</strong>d increased <strong>the</strong> activity of multiple protein kinases. In vitro dephosphorylation with<br />
PP2A nearly abolished IH15s-induced increase in TH activity. IH15s increased generation of<br />
reactive oxygen species (ROS) in brain stem medullary regions which was nearly 3-fold higher<br />
th<strong>an</strong> that evoked by IH90s. Anti-oxid<strong>an</strong>ts prevented IH15s-induced down-regulation of PP2A <strong>an</strong>d<br />
increases in multiple protein kinase activity with subsequent reversal of serine phosphorylation<br />
of TH, TH activity <strong>an</strong>d DA to control levels. These findings demonstrate that IH in a pattern<br />
specific m<strong>an</strong>ner activates TH involving ROS-mediated sustained increase in TH phosphorylation<br />
via down regulation of PP2A <strong>an</strong>d up regulation of protein kinases (supported by HL-90554 <strong>an</strong>d<br />
HL-89616).<br />
Disclosures: G. Raghuram<strong>an</strong>, None; Y. Peng, None; N.R. Prabhakar, None; G.K. Kumar,<br />
None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.1/FF25<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: NIH Fogarty International Center gr<strong>an</strong>t TW/HD-00668 to P. Michael Conn<br />
MH62677 to CLB<br />
U54 18185<br />
RR00163<br />
Title: The role of stress sensitivity <strong>an</strong>d <strong>the</strong> effect of citalopram treatment on corticotropin<br />
releasing hormone (CRH) <strong>an</strong>d urocortin I (UCN) fiber density in <strong>the</strong> midbrain raphe region of<br />
macaques<br />
Authors: K. V. WEISSHEIMER, S. M. HEROD, J. L. CAMERON, *C. L. BETHEA;<br />
Oregon Natl. Primate Res. Ctr., Beaverton, OR<br />
Abstract: Stress-induced amenorrhea occurs in a percentage of women <strong>an</strong>d monkeys when <strong>the</strong>y<br />
experience moderate combined psychosocial <strong>an</strong>d metabolic stresses. We have shown that when<br />
female cynomolgus monkeys experience a moderate psychosocial stress (move to a new room)<br />
<strong>an</strong>d diet <strong>for</strong> a 2-mo period, highly stress resilient monkeys (HSR) continue to ovulate whereas<br />
stress sensitive monkeys (SS) become amenorrheic <strong>an</strong>d <strong>an</strong>ovulatory. We have previously<br />
reported that SS monkeys have deficits in global serotonin release <strong>an</strong>d serotonin-related gene<br />
expression in <strong>the</strong> raphe nucleus. Administration of <strong>the</strong> SSRI, citalopram, increased estrogen <strong>an</strong>d<br />
progesterone production in SS monkeys. However, <strong>the</strong> deficits in expression of serotonin-related<br />
genes in <strong>the</strong> dorsal raphe nucleus were not corrected by citalopram. In this study, we questioned<br />
whe<strong>the</strong>r <strong>the</strong>re was a difference in CRH <strong>an</strong>d UCN fiber staining in <strong>the</strong> midbrain raphe region<br />
between HSR <strong>an</strong>d SS monkeys treated with placebo or citalopram. Monkeys characterized as<br />
HSR or SS were administered placebo (P) or s-citalopram (CIT) <strong>for</strong> 8 weeks at doses that<br />
normalized ovari<strong>an</strong> steroid secretion in <strong>the</strong> SS <strong>an</strong>imals <strong>an</strong>d that maintained blood CIT levels in a<br />
<strong>the</strong>rapeutic r<strong>an</strong>ge (n= 3 or 4/group). Sections of <strong>the</strong> DRN were immunostained with ei<strong>the</strong>r<br />
<strong>an</strong>tibody against hum<strong>an</strong> CRH (courtesy of Dr. Wiley Vale; 4 sections/<strong>an</strong>imal) or against UCN1<br />
(Sigma, U4757; 3 sections/<strong>an</strong>imal). A montage of <strong>the</strong> DRN was created with a Mari<strong>an</strong>as<br />
Stereology Workstation. The immunostained CRH- <strong>an</strong>d UCN-positive pixel area was qu<strong>an</strong>tified<br />
using a Slidebook 4.2 <strong>an</strong>d <strong>an</strong>alyzed with two-way ANOVA. Results were expressed as percent<br />
positive pixel area (immunostained pixel area/total pixel area). Citalopram signific<strong>an</strong>tly<br />
decreased <strong>the</strong> CRH fiber density in SS <strong>an</strong>imals, but not in HSR <strong>an</strong>imals. In HSR+P, HSR+CIT,<br />
SS+P <strong>an</strong>d SS+CIT respectively, <strong>the</strong> percent CRH positive pixel area equaled 6.7±0.68%,<br />
5.8±0.17%, 7.5±1.30% <strong>an</strong>d 4.3±0.21% (p=0.639 <strong>for</strong> stress sensitivity; p=0.029 <strong>for</strong> treatment).<br />
SS+P monkeys had signific<strong>an</strong>tly lower UCN fiber density compared to HSR+P monkeys.
However, CIT treatment did not alter <strong>the</strong> UCN fiber density. In HSR+P, HSR+CIT, SS+P <strong>an</strong>d<br />
SS+CIT respectively, <strong>the</strong> percent UCN positive pixel area was 1.5±0.31%, 1.7±0.49%,<br />
0.8±0.16% <strong>an</strong>d 1.0±0.20% (p=0.047 <strong>for</strong> stress sensitivity; p=0.495 <strong>for</strong> treatment). In summary,<br />
<strong>the</strong> action of CIT in SS individuals involves a reduction in CRH tr<strong>an</strong>sport to <strong>the</strong> dorsal raphe<br />
nucleus. In addition, SS individuals have lower tr<strong>an</strong>sport of UCN1 to <strong>the</strong> dorsal raphe th<strong>an</strong> HSR<br />
individuals.<br />
Supported by NIH Fogarty International Center gr<strong>an</strong>t TW/HD-00668 to P. Michael Conn,<br />
MH62677 to CLB, U54 18185 <strong>an</strong>d RR00163.<br />
Disclosures: K.V. Weissheimer, None; S.M. Herod, None; J.L. Cameron, None; C.L.<br />
Be<strong>the</strong>a, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.2/FF26<br />
Topic: E.05.d. Stress-modulated pathways<br />
Title: The role of Urocortin peptides in regulating <strong>the</strong> central stress response: Evidence <strong>from</strong> a<br />
novel Urocortin triple knockout mouse model<br />
Authors: *M. M. TSOORY 1 , A. NEUFELD 1 , D. GETSELTER 1 , W. VALE 2 , A. CHEN 1 ;<br />
1 2<br />
Weizm<strong>an</strong>n Inst. of Sci., Rehovot, Israel; Clayton Fndn. Labs. <strong>for</strong> Peptide Biol., The Salk Inst.<br />
<strong>for</strong> Biol. Studies, La Jolla, CA<br />
Abstract: The corticotropin releasing factor (CRF) -peptide family includes, in addition to CRF,<br />
<strong>the</strong> three Urocortin (Ucn) peptides (Ucn1, 2 <strong>an</strong>d 3). These peptides are involved in <strong>the</strong><br />
integration of <strong>the</strong> neuroendocrine, autonomic <strong>an</strong>d behavioral responses to stressors through<br />
selective activation of two receptors, CRF receptor type 1 (CRF-R1) <strong>an</strong>d CRF-R2, both widely<br />
expressed in stress-related brain nuclei. CRF has relatively lower affinity <strong>for</strong> CRF-R2 compared<br />
to its affinity <strong>for</strong> CRF-R1; Ucn1 has equal affinities <strong>for</strong> both receptors; <strong>an</strong>d Ucn2 <strong>an</strong>d Ucn3<br />
appear to be selective <strong>for</strong> CRF-R2. Opposing roles in regulating <strong>the</strong> central stress response were<br />
suggested <strong>for</strong> <strong>the</strong> two receptors systems; <strong>the</strong> CRF-R1 system was found critical <strong>for</strong> initiating<br />
stress responses, while <strong>the</strong> CRF-R2 system appears to be primarily <strong>for</strong> reestablishing<br />
homeostasis.<br />
This study evaluated <strong>the</strong> role of Ucn1, 2 <strong>an</strong>d 3 in regulating <strong>the</strong> central stress response by<br />
utilizing a novel knockout mice model that we generated, lacking all three known Urocortins<br />
(tripleUcnKO). We compared <strong>an</strong>xiety indices of tripleUcnKO mice with those of wild-type mice
(WT) obtained <strong>from</strong> <strong>the</strong> same colony, under basal conditions, both immediately <strong>an</strong>d 24 hours<br />
following exposure to <strong>an</strong> acute stressor.<br />
Under basal conditions <strong>an</strong>d immediately following exposure to acute stress, tripleUcnKO mice<br />
did not differ <strong>from</strong> WT mice in most <strong>an</strong>xiety indices of <strong>the</strong> open field <strong>an</strong>d dark/light tr<strong>an</strong>sfer<br />
tests. However, 24 hrs following <strong>the</strong> exposure to stress, tripleUcnKO mice exhibited increased<br />
levels of <strong>an</strong>xiety compared to WT mice. In addition, tripleUcnKO mice exhibited <strong>an</strong> altered<br />
corticosterone response profile following restraint stress.<br />
Collectively, our results suggest <strong>an</strong> import<strong>an</strong>t role <strong>for</strong> endogenous CRF-R2 lig<strong>an</strong>ds in mediating<br />
coping mech<strong>an</strong>isms following stressful experience, <strong>an</strong>d support <strong>the</strong> tripleUcnKO mouse line as a<br />
useful stress sensitive mouse model. Fur<strong>the</strong>r elucidation of <strong>the</strong> role of central urocortins in<br />
mediating <strong>the</strong> central stress response may provide new insights that could be used <strong>for</strong> <strong>the</strong><br />
development of novel <strong>the</strong>rapeutic tools <strong>for</strong> stress-related psychopathologies.<br />
Disclosures: M.M. Tsoory, None; A. Neufeld, None; D. Getselter, None; W. Vale, None; A.<br />
Chen, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.3/FF27<br />
Topic: E.01.c. Neuroendocrine Regulation: O<strong>the</strong>r<br />
Support: NWO gr<strong>an</strong>t 864.05.008.<br />
Title: Ucn1-null mice indicate a role of urocortin-1 in depression-associated behavior<br />
Authors: *L. STERRENBURG 1 , E. W. ROUBOS 1 , L. SANTBERGEN 1 , A. NEUFELD 2 , D.<br />
GALILI 2 , W. W. VALE 3 , A. CHEN 2 , T. KOZICZ 1 , A. PEETERS 1,4 ;<br />
1 Cell. <strong>an</strong>imal physiology, Radboud Univ., Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Neurobio., Weizm<strong>an</strong>n<br />
institute of science, Rehovot, Israel; 3 Peptide biology lab, Salk institute, La Jolla, CA; 4 Schering-<br />
Plough, Oss, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: The main production site of <strong>the</strong> corticotropin-releasing factor-related neuropeptide,<br />
urocortin-1 (Ucn1) is <strong>the</strong> mammali<strong>an</strong> non-preg<strong>an</strong>glionic Edinger-Westphal nucleus. Because <strong>the</strong><br />
Ucn1-producing neurons in this nucleus are activated by various acute <strong>an</strong>d chronic stressors, <strong>the</strong>y<br />
are assumed to be involved in <strong>the</strong> stress response. Fur<strong>the</strong>rmore, dysregulation of <strong>the</strong> nucleus has<br />
been implicated in <strong>the</strong> pathogenesis of major depression, which was recently supported by our<br />
finding of a dramatically high Ucn1 content of its neurons in suicide victims.
In this study we have used a battery of four tests <strong>for</strong> depression-related behavior <strong>an</strong>d compared<br />
<strong>the</strong> test results of wild type mice with those of ucn1-null mice. The Porsolt swim test revealed a<br />
subtle but distinct reducing effect of ucn1 deletion on immobility at <strong>the</strong> second day. This effect is<br />
interpreted as reduced memory retrieval, indicating that under severe stressful conditions Ucn1<br />
may promote <strong>the</strong> retrieval of aversive memories <strong>an</strong>d, hence, contribute to <strong>the</strong> pathogenesis of<br />
major depression. O<strong>the</strong>r aspects of depression-related behavior, like <strong>an</strong>xiety, <strong>an</strong>hedonia <strong>an</strong>d<br />
memory function under less stressful conditions, were measured in <strong>the</strong> marble burying test, sugar<br />
water preference test <strong>an</strong>d novel object recognition test. These aspects were not affected in ucn1null<br />
mice. We propose that a disturb<strong>an</strong>ce of Ucn1 in <strong>the</strong> non-preg<strong>an</strong>glionic Edinger-Westphal<br />
nucleus affects a specific aspect of depression <strong>an</strong>d is part of multiple brain center dysfunction<br />
that accounts <strong>for</strong> <strong>the</strong> full phenotype of major depression.<br />
Disclosures: L. Sterrenburg, None; E.W. Roubos, None; L. S<strong>an</strong>tbergen, None; A. Neufeld,<br />
None; D. Galili, None; W.W. Vale, None; A. Chen, None; T. Kozicz, None; A. Peeters, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.4/FF28<br />
Topic: E.05.d. Stress-modulated pathways<br />
Title: Involvement of c<strong>an</strong>nabinoids in functioning of <strong>the</strong> non-preg<strong>an</strong>glionic Edinger-Westphal<br />
urocortin 1 system<br />
Authors: *N. DERKS 1 , O. PINTÉR 2 , B. MULDERS 3 , E. HRABOVSZKY 4 , E. ROUBOS 3 , D.<br />
ZELENA 2 , T. KOZICZ 3 ;<br />
1 Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Lab. Stress <strong>an</strong>d Behavior, Inst. of Exptl. Medicine, HAS, Budapest,<br />
Hungary; 3 Radboud Univ. Nijmegen, Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 Lab. of Endocrine Neurobiology,<br />
Inst. of Exptl. Medicine, HAS, Budapest, Hungary<br />
Abstract: The brain produces endogenous c<strong>an</strong>nabinoids: <strong>an</strong><strong>an</strong>damide <strong>an</strong>d 2-arachidonoyl<br />
glycerol. Receptors <strong>for</strong> <strong>the</strong>se c<strong>an</strong>nabinoids, <strong>the</strong> c<strong>an</strong>nabinoid receptor 1 <strong>an</strong>d 2 (CB1/CB2), are G<br />
protein-coupled receptors present in brain areas involved in stress adaptation response <strong>an</strong>d<br />
depressive-like behavior, like <strong>the</strong> hypothalamic paraventricular nucleus (PVN) <strong>an</strong>d <strong>the</strong> lateral<br />
septum. Indeed, endogenous c<strong>an</strong>nabinoids c<strong>an</strong> influence <strong>the</strong> stress response <strong>an</strong>d mediate <strong>an</strong>xietyrelated<br />
responses. Urocortin 1 (Ucn1), a peptide produced in <strong>the</strong> non-preg<strong>an</strong>glionic Edinger-<br />
Westphal nucleus (npEW), seems to play <strong>an</strong> import<strong>an</strong>t role in stress adaptation <strong>an</strong>d <strong>the</strong><br />
development of depression. We hypo<strong>the</strong>sized that endogenous c<strong>an</strong>nabinoid system would control
<strong>the</strong> activity of npEW-Ucn1 system during stress adaptation. To test this hypo<strong>the</strong>sis, we <strong>an</strong>alyzed<br />
if CB1 receptor is expressed in npEW neurons, compared Ucn1 expression <strong>from</strong> wildtype <strong>an</strong>d<br />
CB1 knockout mice (KO), in males <strong>an</strong>d females <strong>an</strong>d in control, acute stress as well as chronic<br />
mild variable stress (CMS) situations. We confirmed <strong>the</strong> expression of CB1 mRNA in npEW,<br />
<strong>an</strong>d found 34% lower amount of Ucn1 mRNA in male CB1 knockouts th<strong>an</strong> in male wildtypes. In<br />
addition, we found a 55% higher Ucn1 expression in wildtype mice after chronic but not after<br />
acute stress, <strong>an</strong>d in knockout mice a 56% higher expression after acute but not after chronic<br />
stress. Immunocytochemistry could not reveal differences in <strong>the</strong> amount of Ucn1 neurons <strong>an</strong>d <strong>the</strong><br />
specific signal density of <strong>the</strong> immunosignal. At <strong>the</strong> same time basal <strong>an</strong>d stress induced<br />
adrenocorticotropin (ACTH) levels were higher in CB1 KO mice with lower ACTH <strong>an</strong>d higher<br />
corticosterone plasma levels in females. The CB1 KO mice were more <strong>an</strong>xious on <strong>the</strong> elevated<br />
plus maze <strong>an</strong>d <strong>the</strong> <strong>an</strong>xiolytic effect of CMS was equally present in both genotypes. Females<br />
developed less depressive-like behavior in <strong>the</strong> <strong>for</strong>ced swim test without <strong>an</strong>y effect of genotype or<br />
stress. From <strong>the</strong>se data we c<strong>an</strong> conclude that <strong>the</strong> c<strong>an</strong>nabinoid system influences Ucn1 dynamics<br />
in <strong>the</strong> npEW, which c<strong>an</strong> be strongly involved in <strong>the</strong> modulation of stress adaptation <strong>an</strong>d<br />
accomp<strong>an</strong>ied <strong>an</strong>xiety level.<br />
Disclosures: N. Derks, None; O. Pintér, None; B. Mulders, None; E. Hrabovszky, None; E.<br />
Roubos, None; D. Zelena, None; T. Kozicz, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.5/FF29<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: CIHR Gr<strong>an</strong>t MOP-81124<br />
NSERC Studenship to JFP<br />
Title: Downregulation of enkephalin expression in <strong>the</strong> ventral striatum correlates with stressinduced<br />
<strong>an</strong>hedonia<br />
Authors: *J.-F. POULIN, S. LAFOREST, G. DROLET;<br />
Neurosciences, Univ. Laval/CRCHUL, Québec, QC, C<strong>an</strong>ada<br />
Abstract: The impact of stress on hum<strong>an</strong> health c<strong>an</strong>not be underestimated <strong>an</strong>d numerous <strong>an</strong>imal<br />
models have demonstrated <strong>the</strong> deleterious effects of chronic stress. The endogenous opioid
enkephalin is a prime c<strong>an</strong>didate <strong>for</strong> providing <strong>an</strong> interface between chronic stress <strong>an</strong>d its ensuing<br />
behavioral deficits. We submitted rats to 14 days of restraint stress (60 minutes per day) <strong>an</strong>d<br />
measured <strong>the</strong> physiological <strong>an</strong>d behavioral impact of this stress regimen. Stressed <strong>an</strong>imals had a<br />
reduced weight gain (29% decreased) <strong>an</strong>d <strong>an</strong> elevated basal level of plasma corticosterone (48%<br />
increased) compared to control. There was also a marked reduction in sucrose preference in 25%<br />
of stressed <strong>an</strong>imals, but no effect of stress was observed on <strong>an</strong>xiety-like behaviors as measured in<br />
<strong>the</strong> elevated-plus maze four days after <strong>the</strong> last restraint. In <strong>an</strong>hedonic (vulnerable) <strong>an</strong>imals, in<br />
situ hybridization revealed a downregulation of enkephalin expression in <strong>the</strong> caudal core <strong>an</strong>d<br />
shell portions of <strong>the</strong> nucleus accumbens, which was not present in <strong>the</strong> resilient <strong>an</strong>imals.<br />
Fur<strong>the</strong>rmore, in this region proenkephalin mRNA density correlated with individual sucrose<br />
preference. There was no difference between vulnerable <strong>an</strong>d resilient individuals in dynorphin<br />
expression in <strong>an</strong>y part of <strong>the</strong> striatum. The tr<strong>an</strong>scription factor delta FosB may be implicated in<br />
<strong>the</strong> downregulation of enkephalin as we found a reduction in <strong>the</strong> expression of delta FosB in <strong>the</strong><br />
caudal accumbens in vulnerable individuals as well as a reduction of double-labeled neurons <strong>for</strong><br />
delta FosB <strong>an</strong>d enkephalin mRNA. These results support a functional role of enkephalin release<br />
in <strong>the</strong> nucleus accumbens in individual vulnerability to chronic stress-induced <strong>an</strong>hedonia.<br />
Disclosures: J. Poulin, None; S. La<strong>for</strong>est, None; G. Drolet, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.6/FF30<br />
Topic: E.05.d. Stress-modulated pathways<br />
Title: Social status alters behavioral <strong>an</strong>d cellular responses to social stress<br />
Authors: *K. E. MCINTYRE, D. W. CURRY, M. A. COOPER;<br />
Univ. Tennessee, Knoxville, TN<br />
Abstract: We investigated <strong>the</strong> effect of social status on stress-induced behavioral responses <strong>an</strong>d<br />
neural activation using a conditioned defeat model in Syri<strong>an</strong> hamsters (Mesocricetus auratus).<br />
Conditioned defeat is a model in which normal territorial aggression is replaced by increased<br />
submissive <strong>an</strong>d defensive behavior following social defeat. We paired weight-matched hamsters<br />
in daily 5-min aggressive encounters <strong>for</strong> two weeks <strong>an</strong>d identified domin<strong>an</strong>ts <strong>an</strong>d subordinates.<br />
We also included controls, which were exposed to <strong>an</strong> empty cage every day <strong>for</strong> two weeks.<br />
Twenty-four hours after <strong>the</strong> final pairing or empty cage exposure, half of <strong>the</strong> subjects were<br />
socially defeated in 3, 5-min encounters at 10-min intervals. The o<strong>the</strong>r subjects were not socially
defeated <strong>an</strong>d instead exposed to empty cages. Twenty-four hours after social defeat, <strong>an</strong>imals<br />
received conditioned defeat testing which involved a 5-min social interaction test with a nonaggressive<br />
intruder. In Experiment 1, we collected brains after conditioned defeat testing <strong>for</strong> c-<br />
Fos immunohistochemistry. In Experiment 2, we collected brains after social defeat <strong>for</strong> c-Fos<br />
immunohistochemistry. We found that <strong>for</strong>mer domin<strong>an</strong>ts showed less submissive <strong>an</strong>d defensive<br />
behavior at conditioned defeat testing th<strong>an</strong> both <strong>for</strong>mer subordinates <strong>an</strong>d controls. Defeated<br />
<strong>an</strong>imals had increased c-Fos immunoreactivity after social defeat compared to non-defeated<br />
<strong>an</strong>imals in <strong>the</strong> medial prefrontal cortex, medial amygdala, lateral amygdala, basolateral<br />
amygdala, <strong>an</strong>d ventromedial hypothalamus. Additionally, among defeated <strong>an</strong>imals, <strong>for</strong>mer<br />
domin<strong>an</strong>ts showed increased c-Fos immunoreactivity after social defeat compared to <strong>for</strong>mer<br />
subordinates in <strong>the</strong> medial prefrontal cortex <strong>an</strong>d ventromedial hypothalamus. Also, after social<br />
defeat <strong>for</strong>mer domin<strong>an</strong>ts had increased c-Fos immunoreactivity in <strong>the</strong> medial amygdala<br />
compared to both <strong>for</strong>mer subordinates <strong>an</strong>d controls. These results indicate that <strong>for</strong>mer domin<strong>an</strong>t<br />
<strong>an</strong>imals show increased neural activation following social defeat in several brain regions<br />
including <strong>the</strong> medial prefrontal cortex, ventromedial hypothalamus, <strong>an</strong>d medial amygdala. Our<br />
findings suggest that increased neural activation in select brain regions in domin<strong>an</strong>t <strong>an</strong>imals may<br />
account, in part, <strong>for</strong> <strong>the</strong>ir resist<strong>an</strong>ce to social defeat.<br />
Disclosures: K.E. McIntyre, None; D.W. Curry, None; M.A. Cooper, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.7/FF31<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: CIHR<br />
FRSQ<br />
Title: Interhemispheric regulation of <strong>the</strong> medial prefrontal glutamate response to stress in rats:<br />
Involvement of dopamine, norepinephrine <strong>an</strong>d GABA<br />
Authors: *D. A. LUPINSKY, L. MOQUIN, A. P. GRATTON;<br />
McGill Univ/Douglas, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: In addition to potently activating medial prefrontal cortex dopamine (DA)<br />
tr<strong>an</strong>smission, stress will also elicit TTX-sensitive increases in PFC glutamate (GLUT) levels.
The PFC receives GLUT-containing inputs <strong>from</strong> several regions (eg. hippocampus, amygdala,<br />
<strong>an</strong>d thalamus). However, some PFC pyramidal GLUT neurons are known to project to <strong>the</strong><br />
opposite PFC. Signific<strong>an</strong>t hemispheric asymmetries in PFC DA function as well as evidence that<br />
neuroendocrine <strong>an</strong>d autonomic responses to stress are differentially altered by left vs right PFC<br />
lesions suggest a possible involvement of such callosal projection neurons. In support of this<br />
idea, we have reported evidence suggesting that <strong>the</strong> PFC GLUT stress response is at least partly<br />
due to activation of callosal projection neurons originating in <strong>the</strong> opposite PFC; <strong>the</strong> PFC GLUT<br />
stress response was completely abolished both by ibotenate-induced lesions <strong>an</strong>d local mGLU2/3<br />
receptor activation in <strong>the</strong> opposite PFC. In <strong>the</strong> present study, we used microdialysis to investigate<br />
<strong>the</strong> role of DA, NE <strong>an</strong>d GABA on <strong>the</strong> interhemispheric regulation of <strong>the</strong> PFC GLUT stress<br />
response. We report here that local D1 receptor blockade in <strong>the</strong> left PFC dose-dependently<br />
enh<strong>an</strong>ces <strong>the</strong> GLUT response to a 20 min episode of tail-pinch stress in <strong>the</strong> right PFC but that <strong>the</strong><br />
same treatment applied to <strong>the</strong> right PFC produces a comparatively weaker enh<strong>an</strong>cement of <strong>the</strong><br />
left PFC GLUT stress response. In addition, whereas <strong>the</strong> right PFC GLUT stress response was<br />
partially inhibited by local left PFC α1-adrenoreceptor blockade it was potentiated by local<br />
GABAB receptor activation in <strong>the</strong> left PFC. These findings provide fur<strong>the</strong>r evidence that stress<br />
stimulates PFC GLUT tr<strong>an</strong>smission in one hemisphere at least partly as a consequence of<br />
activating or disinhibiting callosal projection neurons in <strong>the</strong> opposite hemisphere. Our data<br />
indicate that stress-induced activation of <strong>the</strong>se neurons is modulated by DA, NE <strong>an</strong>d GABA.<br />
They also suggest that <strong>the</strong> D1 receptor-mediated inhibitory influence over this interhemispheric<br />
communication is asymmetrical in that <strong>the</strong> DA input to <strong>the</strong> left PFC exerts a relatively stronger<br />
inhibitory influence on GLUT tr<strong>an</strong>smission in <strong>the</strong> right PFC th<strong>an</strong> <strong>the</strong> right PFC does over left<br />
PFC GLUT. The present findings also raise <strong>the</strong> possibility that disruption of PFC<br />
interhemispheric GLUT communication might contribute to maladaptive stress responsivity<br />
associated with abnormal lateralization of cortically-mediated function. Supported by CIHR<br />
(AG) <strong>an</strong>d FRSQ (DAL).<br />
Disclosures: D.A. Lupinsky, None; L. Moquin, None; A.P. Gratton, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.8/FF32<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: NIH R01 #HL025449
Title: Pre-locus coeruleus neurons are activated by sodium deprivation, express FoxP2, <strong>an</strong>d<br />
project to <strong>the</strong> diencephalon<br />
Authors: *J. C. GEERLING 1 , J.-W. SHIN 2 , A. LOEWY 3 ;<br />
1 Dept Anat & Neurobiol, Washington Univ., Saint Louis, MO; 2 Anat & Neurobiol, Washington<br />
Univ. Sch. of Med., Satin Louis, MO; 3 Anat & Neurobiol, Washington Univ. Sch. of Med., Saint<br />
Louis, MO<br />
Abstract: Immediately rostral to <strong>the</strong> locus coeruleus (LC) in <strong>the</strong> dorsolateral pons lies a cluster<br />
of neurons referred to as <strong>the</strong> pre-locus coeruleus (pre-LC). We identified <strong>the</strong> pre-LC based on<br />
two observations in rats. First, it receives a dense axon terminal field <strong>from</strong> <strong>the</strong> nucleus of <strong>the</strong><br />
solitary tract. Second, it contains a prominent cluster of c-Fos-positive (presumptively activated)<br />
neurons after a rat is deprived of dietary sodium <strong>for</strong> a week or more. Here, we elaborate<br />
additional properties of this unique brainstem nucleus. First, sodium deprivation-activated<br />
neurons in <strong>the</strong> pre-LC (<strong>an</strong>d in <strong>the</strong> neighboring parabrachial nucleus) constituitively express <strong>the</strong><br />
tr<strong>an</strong>scription factor FoxP2. Similar to o<strong>the</strong>r FoxP2+ cells in this region, none of <strong>the</strong>se neurons<br />
contain tyrosine hydroxlyase immunoreactivity, showing that <strong>the</strong> pre-LC is entirely separate<br />
<strong>from</strong> <strong>the</strong> LC. Second, although a small number of pontine FoxP2+ neurons outside <strong>the</strong> pre-LC<br />
express choline acetyltr<strong>an</strong>sferase, <strong>the</strong>se neurons do not, distinguishing <strong>the</strong>m <strong>from</strong> <strong>the</strong> laterodorsal<br />
tegmental nucleus (LDT), <strong>an</strong>o<strong>the</strong>r well-known group in this region of <strong>the</strong> brainstem. Third,<br />
<strong>an</strong>terograde (PHAL) <strong>an</strong>d retrograde (CTb) neuronal tracing techniques were used to determine<br />
<strong>the</strong> efferent projections of pre-LC neurons. In <strong>the</strong> retrograde tracing experiments, FoxP2 was<br />
used as a surrogate marker <strong>for</strong> its sodium deprivation-activated neurons. These experiments<br />
revealed major projections to multiple target sites in <strong>the</strong> diencephalon, including most<br />
prominently <strong>the</strong> paraventricular thalamic nucleus (PVT), paraventricular hypothalamic nucleus<br />
(PVH), dorsomedial, peri<strong>for</strong>nical, <strong>an</strong>d lateral hypothalamus (DMH, PeF, LHA), <strong>an</strong>d <strong>the</strong><br />
parasubthalamic nucleus (PSTN). O<strong>the</strong>r, more moderate projection targets included <strong>the</strong> bed<br />
nucleus of <strong>the</strong> stria terminals (BST) <strong>an</strong>d caudal nucleus accumbens (NAc), subparafasicular<br />
thalamus (SPF) <strong>an</strong>d a caudal region of <strong>the</strong> midline thalamus near <strong>the</strong> intermediodorsal (IMD) <strong>an</strong>d<br />
centromedi<strong>an</strong> (CM) nuclei. Minor target sites included <strong>the</strong> ventrolateral periaqueductal gray<br />
matter (PAG), central linear raphe nucleus (CLi), retrorubral field (RRF), <strong>an</strong>d medullary reticular<br />
<strong>for</strong>mation. Of note, virtually none of <strong>the</strong> FoxP2+ neurons in <strong>the</strong> pre-LC project to <strong>the</strong> central<br />
nucleus of <strong>the</strong> amygdala (CeA), insular cortex (IC), or lateral septum (LS) despite signific<strong>an</strong>t<br />
projections to <strong>the</strong>se sites <strong>from</strong> non-FoxP2 neurons in <strong>the</strong> medial parabrachial nucleus (CeA <strong>an</strong>d<br />
IC) <strong>an</strong>d LDT (LS). In summary, <strong>the</strong> pre-LC contains FoxP2-expressing neurons that are<br />
activated by dietary sodium deprivation <strong>an</strong>d which direct <strong>the</strong> bulk of <strong>the</strong>ir output to diencephalic<br />
nuclei implicated in behavioral homeostasis <strong>an</strong>d interoceptive stress responses.<br />
Disclosures: J.C. Geerling, None; J. Shin, None; A. Loewy, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.9/FF33<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: AstraZeneca<br />
Title: Caloric restriction reprogramming of stress <strong>an</strong>d reward neurocircuitry increases<br />
vulnerability to stress-induced binge eating<br />
Authors: *D. E. PANKEVICH 1 , G. SMAGIN 2 , T. L. BALE 1 ;<br />
1 Animal Biol, Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA; 2 AstraZeneca, Wilmington, DE<br />
Abstract: Despite dire health consequences, caloric overconsumption continues to produce<br />
increasing rates of overweight <strong>an</strong>d obese phenotypes. The high failure rate of behavioral<br />
treatments <strong>an</strong>d repeated or “yo-yo” dieting is associated with <strong>an</strong> increased risk <strong>for</strong> weight regain<br />
that is above <strong>the</strong> pre-diet weight. We have previously reported that three weeks of moderate<br />
caloric restriction produced <strong>an</strong> increased behavioral stress state associated with long-term<br />
ch<strong>an</strong>ges in corticotrophin releasing factor (CRF) expression driven by altered methylation of <strong>the</strong><br />
CRF promoter that was specific to <strong>the</strong> bed nucleus of <strong>the</strong> stria terminalis (BNST). Fur<strong>the</strong>r<br />
examination of pathways involved in stress, feeding <strong>an</strong>d reward signaling found that exposure to<br />
a high fat diet following caloric restriction resulted in elevations in <strong>the</strong> orexegenic hormones<br />
mel<strong>an</strong>in-concentrating hormone (MCH) <strong>an</strong>d orexin, indicating a potential mech<strong>an</strong>ism <strong>for</strong> greater<br />
weight gain following caloric restriction. We hypo<strong>the</strong>sized that stress neurocircuitry<br />
reprogramming <strong>an</strong>d its consequent effects on stress-sensitivity would increase <strong>the</strong> intake of<br />
palatable, calorically dense foods under stressful conditions <strong>an</strong>d that this effect would be<br />
alleviated with <strong>an</strong> orexegenic <strong>an</strong>tagonist. To examine this, <strong>the</strong> effects of stress on binge-eating<br />
responses were measured in <strong>for</strong>merly restricted mice. We found that chronic variable stress<br />
increased binge consumption of a high fat diet in previously calorically restricted mice compared<br />
to controls. Administration of a MCH-receptor 1 (MCHR1) <strong>an</strong>tagonist reduced overall caloric<br />
intake in previously calorically restricted mice. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that<br />
moderate caloric restriction increases stress-sensitivity <strong>the</strong>reby promoting binge-like eating<br />
which may be partially alleviated with orexegenic <strong>an</strong>tagonists. Overall, <strong>the</strong> stress associated with<br />
caloric restriction results in reprogramming of stress, feeding <strong>an</strong>d reward pathways that may<br />
establish vulnerability to future insults leading to increased high fat food intake <strong>an</strong>d long-term<br />
overweight <strong>an</strong>d obesity.<br />
Disclosures: D.E. P<strong>an</strong>kevich, None; G. Smagin, None; T.L. Bale, None.<br />
Poster
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.10/FF34<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: HSFO<br />
Title: Co-expression of orexin-B <strong>an</strong>d corticotrophin-releasing factor in <strong>the</strong> paraventricular<br />
nucleus of <strong>the</strong> hypothalamus <strong>an</strong>d limbic structures<br />
Authors: *W. IQBAL, J. M. MOREAU, J. CIRIELLO;<br />
Physiol. <strong>an</strong>d Pharmacology, Schulich Sch. of Med. <strong>an</strong>d Dent., Univ. of Western Ontario,<br />
London, ON, C<strong>an</strong>ada<br />
Abstract: Orexin-A <strong>an</strong>d -B (OX-A <strong>an</strong>d OX-B, respectively; hypocretins) are neuropeptides<br />
involved in homeostatic mech<strong>an</strong>isms controlling food intake, energy expenditure, blood pressure<br />
<strong>an</strong>d wakefulness. OX expressing neurons have previously been characterized in <strong>the</strong> lateral<br />
hypothalamus <strong>an</strong>d have been shown to have extensive projections throughout <strong>the</strong> neuraxis. In<br />
addition, a specific subgroup of OX-B immunopositive neurons have also been identified in <strong>the</strong><br />
central nucleus of <strong>the</strong> amygdala (ACe) <strong>an</strong>d bed nucleus of <strong>the</strong> stria terminalis (BST).<br />
Corticotropin-releasing factor (CRF) is a neuropeptide that mediates <strong>the</strong> response to stress<br />
through activation of <strong>the</strong> hypothalamic-pituitary-adrenal axis. CRF is expressed not only in <strong>the</strong><br />
paraventricular nucleus of <strong>the</strong> hypothalamus (PVH), but also in ACe <strong>an</strong>d BST. This study<br />
investigated whe<strong>the</strong>r OX-B <strong>an</strong>d CRF were co-localized within <strong>the</strong>se <strong>for</strong>ebrain regions in female<br />
Spraque Dawley rats on a high fat (45 kcal fat) diet. Intact, estrogen treated ovariectomized<br />
(OVX), <strong>an</strong>d OVX only female rats were placed ei<strong>the</strong>r on a normal or high fat diet. After 2<br />
months on <strong>the</strong> diets <strong>the</strong> <strong>an</strong>imals were given <strong>an</strong> intracerebroventricular injection of colchicine <strong>an</strong>d<br />
<strong>the</strong> <strong>for</strong>ebrain was processed 24 hrs later <strong>for</strong> OX-B <strong>an</strong>d CRF immunoreactivity. In all female rats,<br />
overlapping regions of OX-B <strong>an</strong>d CRF labelled neurons were found in <strong>the</strong> ACe <strong>an</strong>d BST.<br />
However, in <strong>the</strong> OVX only female given a high fat diet, a distinct group of OX-B labelled<br />
neurons appeared in <strong>the</strong> paravocellular PVH. The distribution of OX-B neurons in <strong>the</strong> medial<br />
parvocellular component of PVH overlapped that of CRF neurons. As OX has been shown to<br />
function as a neuromodulatory hormone that stimulates <strong>the</strong> ACTH-mediated response to stress,<br />
<strong>the</strong>se findings suggest that in females without estrogen <strong>an</strong>d on a high fat diet OX-B may function<br />
to modulate CRF release in neurons that co-express both peptides.<br />
Supported by HSFO<br />
Disclosures: W. Iqbal, None; J.M. Moreau, None; J. Ciriello, HSFO, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received).
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.11/FF35<br />
Topic: E.05.d. Stress-modulated pathways<br />
Title: The orexin system is critical <strong>for</strong> a subset of adaptive responses to acute hypercapnia<br />
Authors: *P. L. JOHNSON 1 , S. D. FITZ 1 , S. LIGHTMAN 2 , C. A. LOWRY 3 , A. SHEKHAR 1 ;<br />
1 Psychiatry, Indi<strong>an</strong>a Univ. Med. Sch., Indi<strong>an</strong>apolis, IN; 2 Henry Wellcome Labs. <strong>for</strong> Integrative<br />
Neurosci. <strong>an</strong>d Endrocrinol., Univ. of Bristol, Bristol, United Kingdom; 3 Integrative Physiol.,<br />
Univ. of Colorado, Boulder, CO<br />
Abstract: Acute hypercapnia (elevated arterial CO2) is a life-threatening condition that rapidly<br />
mobilizes adaptive ch<strong>an</strong>ges in breathing, behavioral arousal, <strong>an</strong>d aversive p<strong>an</strong>ic responses in<br />
order to restore homeostasis. Recent evidence has demonstrated that hypothalamic orexin (also<br />
known as hypocretin) neurons are highly sensitive to local ch<strong>an</strong>ges in CO2/H + through acidinduced<br />
closure of leak-like K+ ch<strong>an</strong>nels (Williams et al., 2007), <strong>an</strong>d mice lacking prepro-orexin<br />
have blunted respiratory responses to hypercapnia (Deng et al., 2007). Central orexin neurons are<br />
also crucial in maintaining waking states <strong>an</strong>d vigil<strong>an</strong>ce. There<strong>for</strong>e, a dysfunctional orexin<br />
system, implicated in some sleep-related disorders, could contribute to <strong>the</strong> lack of arousal in<br />
response to hypercapnia [e.g., sleep apnea, sudden inf<strong>an</strong>t death syndrome <strong>an</strong>d Ondine’s curse].<br />
There<strong>for</strong>e, <strong>the</strong> present study attempted to determine orexin’s role in physiological responses to 5<br />
min exposure to 20% hypercapnic, normoxic gas. Exposing conscious rats to hypercapnic, but<br />
not atmospheric air elicited: increased me<strong>an</strong> arterial blood pressure (MAP); decreased heart rate<br />
(HR) responses; increased <strong>an</strong>xiety behavior (as measured by open field test); hypo<strong>the</strong>rmia; <strong>an</strong>d<br />
selectively increased cellular response (as measured by nuclear c-Fos induction) in orexin<br />
neurons in <strong>the</strong> dorsomedial/peri<strong>for</strong>nical, but not lateral, hypothalamus. In a subsequent<br />
experiment, systemically pre-treating rats with <strong>an</strong> orexin1 receptor <strong>an</strong>tagonist (30mg/kg<br />
SB334867 i.p.) prior to CO2 exposure attenuated hypercapnia-induced pressor, locomotor <strong>an</strong>d<br />
hypo<strong>the</strong>rmia responses, without altering reductions in HR. Taken toge<strong>the</strong>r our results suggest<br />
that adaptive responses to hypercapnia are complex <strong>an</strong>d that orexin system plays a critical role in<br />
regulating a specific subset of adaptive responses to hypercapnia.<br />
Disclosures: P.L. Johnson, None; S.D. Fitz, None; S. Lightm<strong>an</strong>, None; C.A. Lowry, None; A.<br />
Shekhar, None.
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.12/FF36<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: NIH Gr<strong>an</strong>t R01 MH078194<br />
Title: Inhibition of hypocretin neurons by local injections of N/OFQ into <strong>the</strong> lateral<br />
hypothalamus modulates stress-induced <strong>an</strong>algesia in rats<br />
Authors: *D. GERASHCHENKO 1 , X. S. XIE 2 ;<br />
1 SRI Intl., Menlo Park, CA; 2 AfaSci, Inc., Burlingame, CA<br />
Abstract: We recently found that orexin/ataxin-3 mice, in which <strong>the</strong> hypocretin/orexin (Hcrt)<br />
neurons degenerate, did not exhibit stress-induced <strong>an</strong>algesia (SIA). N/OFQ blocked SIA in wildtype<br />
mice, while co-administration of Hcrt-1 overcame N/OFQ inhibition of SIA. These studies<br />
indicate that generation <strong>an</strong>d modulation of SIA involve interaction between Hcrt <strong>an</strong>d N/OFQ<br />
systems, but <strong>the</strong>y do not show at which level this interaction occurs. We hypo<strong>the</strong>size that N/OFQ<br />
effect on SIA is produced through direct inhibition of Hcrt neurons in <strong>the</strong> lateral hypothalamic<br />
(LH) area. To test this hypo<strong>the</strong>sis, we evaluated SIA in control <strong>an</strong>d injected rats with N/OFQ<br />
locally into <strong>the</strong> LH area in which Hcrt neurons are located.<br />
We used <strong>the</strong> pl<strong>an</strong>tar test (Hargreaves' Method) to evaluate <strong>the</strong>rmal pain responses as indicated by<br />
<strong>the</strong> palm withdrawal latency in adult Sprague-Dawley rats (n=14). The activity of Hcrt neurons<br />
was assessed using a marker of neuronal activity Fos. Direct microinjection of N/OFQ (0.45 <strong>an</strong>d<br />
9 µg each side) or saline in 0.5-1 µl injection volume was made into <strong>the</strong> LH area. Fifteen min<br />
after measuring baseline nociception, <strong>the</strong> rats were injected bilaterally with <strong>the</strong> test solutions via<br />
impl<strong>an</strong>ted guide c<strong>an</strong>nulae <strong>an</strong>d subjected to 30 min of immobilization stress using a corn-shape<br />
plastic bag DecapiCore ® (Braintree Scientific, Inc, Braintree, MA). The pl<strong>an</strong>tar test was repeated<br />
at 0, 30 <strong>an</strong>d 60 min after release of <strong>the</strong> stress. Successively, rats were killed by <strong>an</strong> overdose of<br />
pentobarbital <strong>an</strong>d perfused with PBS followed by 4% para<strong>for</strong>maldehyde in PBS. Brains were<br />
removed <strong>an</strong>d processed <strong>for</strong> Fos/Hcrt immunohistochemical staining. Fos was counted in Hcrt<br />
cells located medially or laterally to <strong>the</strong> <strong>for</strong>nix.<br />
Rats injected with ei<strong>the</strong>r <strong>the</strong> low dose of N/OFQ (n=3) or saline (n=5) had similar extents of SIA<br />
in <strong>the</strong> pl<strong>an</strong>tar test (about 20%) <strong>an</strong>d similar percent of Fos expression in total Hcrt cells counted<br />
(43-46%), though percent of Fos expression after stress was much higher in <strong>the</strong> Hcrt neurons<br />
located medially th<strong>an</strong> laterally to <strong>the</strong> <strong>for</strong>nix. There<strong>for</strong>e, <strong>the</strong>se rats were combined into one group<br />
<strong>an</strong>d compared with rats treated with <strong>the</strong> higher dose of N/OFQ. There were no signific<strong>an</strong>t<br />
differences between baseline <strong>an</strong>d 0, 30 or 60 min after stress in rats injected bilaterally with 9 µg<br />
of N/OFQ, indicating that N/OFQ at <strong>the</strong> high dose blocked SIA. The high dose of N/OFQ
signific<strong>an</strong>tly reduced Fos in Hcrt neurons to 27.7%, particularly in <strong>the</strong> Hcrt neurons located<br />
medially to <strong>the</strong> <strong>for</strong>nix. These results suggest that local injection of N/OFQ is capable of<br />
inhibiting SIA, <strong>an</strong>d that this modulation possibly occurs via direct inhibition of activity of Hcrt<br />
neurons in <strong>the</strong> LH area, particularly those located medially to <strong>the</strong> <strong>for</strong>nix.<br />
Disclosures: D. Gerashchenko, None; X.S. Xie, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.13/FF37<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: Norlien Foundation<br />
C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research<br />
C<strong>an</strong>adi<strong>an</strong> Stroke Network<br />
National Sciences <strong>an</strong>d Engineering Research Council of C<strong>an</strong>ada<br />
Alberta Heritage Foundation <strong>for</strong> Medical Research<br />
Title: Stress modulates motor function <strong>an</strong>d glucocorticoid receptor expression in <strong>an</strong><br />
experimental model of stroke in rodents<br />
Authors: *F. C. ZUCCHI, N. F. MATTHIES, N. BADR, G. A. METZ;<br />
Univ. Lethbridge, Lethbridge, AB, C<strong>an</strong>ada<br />
Abstract: One of <strong>the</strong> major consequences of stroke is perm<strong>an</strong>ent motor disturb<strong>an</strong>ce, such as<br />
postural imbal<strong>an</strong>ce <strong>an</strong>d loss of skilled movement. The degree of neuronal <strong>an</strong>d functional loss <strong>an</strong>d<br />
subsequent recovery after stroke were shown to be influenced by high levels of stress or<br />
glucocorticoids. The purpose of <strong>the</strong> present study was to investigate <strong>the</strong> influence of chronic<br />
restraint stress on recovery <strong>an</strong>d compensation of motor function in a rat model of stroke.<br />
Fur<strong>the</strong>rmore, <strong>the</strong> aim was to determine if stress after stroke modulates negative feedback<br />
mech<strong>an</strong>isms by modulation of glucocorticoid receptor (GR) expression in <strong>the</strong> brain. Adult male<br />
rats were pre-trained <strong>an</strong>d tested in skilled reaching <strong>an</strong>d skilled walking tasks. A focal lesion was<br />
induced by devascularization of <strong>the</strong> motor cortex. Animals were assigned to daily treatment of
estraint stress starting one week prior to lesion up to three weeks post-lesion. One group served<br />
as lesion only control. GR expression was accessed by immunohistochemistry. The results<br />
revealed that <strong>an</strong>imals exposed to restraint stress had signific<strong>an</strong>tly lower reaching success at both<br />
pre- <strong>an</strong>d post-lesion time points, <strong>an</strong>d higher error rates in skilled walking when compared to<br />
lesion controls. Total GR expression was decreased in <strong>the</strong> stress group, when compared to<br />
controls. In <strong>the</strong> lesion hemisphere GR expression increased when compared to non-lesion<br />
hemisphere, <strong>an</strong>d <strong>the</strong> increase was statistically signific<strong>an</strong>t in <strong>the</strong> stress group. Such modulation of<br />
GR density c<strong>an</strong> potentially alter <strong>the</strong> responsiveness to stroke treatment, <strong>an</strong>d compromise<br />
recovery. The present data support <strong>the</strong> notion that chronic stress represents a major modulator of<br />
motor recovery after injury.<br />
Supported by: Norlien Foundation, C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research, C<strong>an</strong>adi<strong>an</strong> Stroke<br />
Network, National Sciences <strong>an</strong>d Engineering Research Council of C<strong>an</strong>ada, <strong>an</strong>d <strong>the</strong> Alberta<br />
Heritage Foundation <strong>for</strong> Medical Research.<br />
Disclosures: F.C. Zucchi, None; N.F. Matthies, None; N. Badr, None; G.A. Metz, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.14/FF38<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: Ministero della Salute (Progetto Finalizzato PS05.15S).<br />
Title: Opiate receptors in ventral tegmental area control stress-induced inhibition of accumbal<br />
dopamine through mesocortical dopamine<br />
Authors: *S. PUGLISI-ALLEGRA 1,2 , T. PASCUCCI 1,2 , C. E. LATAGLIATA 1,2 , S. CABIB 1,2 ;<br />
1 2<br />
Univ. of Rome, 00185 Roma, Italy; Fondazione S<strong>an</strong>ta Lucia, IRCCS, Europe<strong>an</strong> Ctr. <strong>for</strong> Brain<br />
Res. (CERC), Rome, Italy<br />
Abstract: We have recently shown that <strong>the</strong> meso-accumbens Dopamine (DA) response during a<br />
novel stressful experience is determined by <strong>the</strong> opposing influences of norepinephrine (NE) <strong>an</strong>d<br />
DA tr<strong>an</strong>smission within <strong>the</strong> medial pre-Frontal Cortex (mPFC). Thus, rats experiencing restraint<br />
stress <strong>for</strong> <strong>the</strong> first time show <strong>an</strong> initial, short lasting increase of DA release in <strong>the</strong> Nucleus<br />
Accumbens (NAc) followed by a decrease below resting levels that lasts as long as <strong>the</strong> stressful<br />
experience. Moreover, stressed rats show <strong>an</strong> initial, short-lasting increase of NE release that is<br />
followed by a large <strong>an</strong>d sustained increase of DA release in <strong>the</strong> mPFC. By selective NE or DA
depletion we demonstrated that prefrontal cortical NE determines <strong>the</strong> enh<strong>an</strong>ced DA release in <strong>the</strong><br />
NAc, while <strong>the</strong> late increase of prefrontal DA release determines <strong>the</strong> inhibition of<br />
mesoaccumbens DA release (Pascucci et al., Cerebral Cortex 7(12): 2796-2804, 2007).<br />
We report now that intra Ventral Tegmental Area (VTA) infusion of Naltrexone, by reverse<br />
microdialysis, prevents, dose-dependently, <strong>the</strong> late increase of DA in <strong>the</strong> mpFC <strong>an</strong>d <strong>the</strong> decrease<br />
of DA in <strong>the</strong> NAc as shown in triple-probe experiments.<br />
Thus, <strong>the</strong> opioid receptor <strong>an</strong>tagonist produces effects that parallel those produced by selective<br />
mesocortical DA depletion on stress-induced reduction of accumbal DA release.<br />
These results demonstrate that inhibition of accumbal DA release promoted by incontrollable<br />
stressful experience is dependent on stimulation of opioid receptors in <strong>the</strong> VTA through <strong>the</strong><br />
enh<strong>an</strong>cement of prefrontal cortical DA.<br />
Disclosures: S. Puglisi-Allegra, None; T. Pascucci, None; C.E. Latagliata, None; S. Cabib,<br />
None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.15/FF39<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: NIH Gr<strong>an</strong>t DA022830<br />
NIH Gr<strong>an</strong>t DA024817<br />
NIH Gr<strong>an</strong>t MH073930<br />
Title: Depletion of brain-derived neurotrophic factor in <strong>the</strong> ventral tegmental area attenuates<br />
mainten<strong>an</strong>ce of cross-sensitization to amphetamine <strong>an</strong>d modulates social behavior<br />
Authors: *S. FANOUS 1,2 , E. TERWILLIGER 3 , R. P. HAMMER, Jr. 1,2 , E. M. NIKULINA 1,2 ;<br />
1 Basic Med. Sci., Univ. of Arizona Col. of Med. - Phoenix, Phoenix, AZ; 2 Pharmacol., Tufts<br />
Univ. Sch. of Med., Boston, MA; 3 Div. of Exptl. Med., Harvard Inst. of Med., Boston, MA<br />
Abstract: Brain-derived neurotrophic factor (BDNF) in <strong>the</strong> ventral tegmental area (VTA) is<br />
implicated in <strong>the</strong> consequences of chronic social stress on social behavior <strong>an</strong>d modulates drugdirected<br />
behavior. Intermittent social defeat stress produces long-lasting psychomotor crosssensitization<br />
to psychostimul<strong>an</strong>ts. Our laboratory showed that intermittent social defeat stress
increases VTA BDNF, which may influence neuroadaptive processes leading to its sequelae. In<br />
this study, we determined whe<strong>the</strong>r depletion of VTA BDNF would attenuate stress-induced<br />
cross-sensitization to amphetamine or affect social behavior in <strong>the</strong> presence or absence of<br />
intermittent defeat stress. Persistent depletion of VTA BDNF was accomplished by local<br />
infusion of AAV vectors containing shRNA against BDNF. Male Sprague-Dawley rats were<br />
bilaterally infused intra-VTA with AAV-shRNA or a control virus, <strong>an</strong>d 2 weeks later, underwent<br />
4 episodes of control h<strong>an</strong>dling or social defeat stress involving exposure to <strong>an</strong> aggressive Long-<br />
Ev<strong>an</strong>s resident rat. Rats were challenged with amphetamine (1.0 mg/kg, i.p) 4 weeks after stress<br />
or h<strong>an</strong>dling. Depletion of VTA BDNF decreased locomotor response to amphetamine in h<strong>an</strong>dled<br />
control <strong>an</strong>imals, but did not alter cross-sensitization in stressed <strong>an</strong>imals. Animals were<br />
challenged again with amphetamine (1.0 mg/kg, i.p) 6 weeks after stress, whereupon depletion of<br />
VTA BDNF prevented mainten<strong>an</strong>ce of cross-sensitized drug response. Depletion of VTA BDNF<br />
also increased social interaction regardless of stress history, assessed 11-12 weeks after viral<br />
infusion. These results indicate that VTA BDNF modulates amphetamine response <strong>an</strong>d supports<br />
mainten<strong>an</strong>ce of sensitization following stress, although it might not directly mediate crosssensitization,<br />
<strong>an</strong>d alters social behavior associated with social stress. These findings implicate<br />
VTA BDNF in <strong>the</strong> consequences of social stress on behavior.<br />
Disclosures: S. F<strong>an</strong>ous, None; E. Terwilliger, None; R.P. Hammer, None; E.M. Nikulina,<br />
None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.16/FF40<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: Howard Hughes Medical Institute gr<strong>an</strong>t through <strong>the</strong> Undergraduate Biological<br />
Sciences Education Program to Texas Tech University<br />
Title: CRF <strong>an</strong>d CRF R1 are expressed in <strong>the</strong> optic tectum<br />
Authors: *J. A. CARR, N. BERGFELD, S. BULIN, J. LUSTGARTEN;<br />
Texas Tech. Univ., Lubbock, TX<br />
Abstract: The 41 amino acid peptide corticotropin-releasing factor (CRF) inhibits visually<br />
guided feeding in <strong>an</strong>ur<strong>an</strong> amphibi<strong>an</strong>s, but <strong>the</strong> targets <strong>for</strong> CRF action within sub-cortical visual<br />
pathways are unknown. We examined <strong>the</strong> distribution <strong>an</strong>d origin of CRF immunoreactivity in <strong>the</strong>
<strong>an</strong>ur<strong>an</strong> optic tectum, a brain area at <strong>the</strong> interface of sensorimotor integration. CRF<br />
immunoreactive perikarya <strong>an</strong>d fibers were observed in <strong>the</strong> optic tectum of three representative<br />
<strong>an</strong>ur<strong>an</strong> amphibi<strong>an</strong> species, <strong>the</strong> Texas toad Bufo speciosus, <strong>the</strong> South Afric<strong>an</strong> clawed frog<br />
Xenopus laevis, <strong>an</strong>d <strong>the</strong> Kore<strong>an</strong> fire bellied toad Bombina orientalis. Intrinsic CRFimmunoreactive<br />
neurons were observed in tectal layer 8 in all species. These neurons were<br />
pyri<strong>for</strong>m in shape, <strong>an</strong>d had clearly stained projections directed laterally within layer 8 or directed<br />
vertically toward tectal layer 9. The optic tectum expressed mRNA <strong>for</strong> CRF, CRF R1 receptor,<br />
<strong>an</strong>d urocortin-1. CRF was detected in toad optic tectum by radioimmunoassay, although tectal<br />
CRF content was considerably less th<strong>an</strong> that of <strong>the</strong> hypothalamus <strong>an</strong>d <strong>for</strong>ebrain. The r<strong>an</strong>k order<br />
<strong>for</strong> CRF content in toad brain as determined by radioimmunoassay was <strong>for</strong>ebrain,<br />
hypothalamus>brainstem>optic tectum>>olfactory bulb. Unilateral eye ablation did not affect<br />
CRF content of <strong>the</strong> contra lateral optic tectum. We conclude that neurons in <strong>the</strong> <strong>an</strong>ur<strong>an</strong> optic<br />
tectum express mRNA <strong>for</strong> CRF <strong>an</strong>d CRF R1 <strong>an</strong>d that CRF immunoreactivity in <strong>the</strong> optic tectum<br />
arises <strong>from</strong> intrinsic CRF neurons.<br />
Disclosures: J.A. Carr, None; N. Bergfeld, None; S. Bulin, None; J. Lustgarten, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.17/FF41<br />
Topic: E.05.d. Stress-modulated pathways<br />
Title: Chronic variable stress as a rodent model of PTSD: A potential role <strong>for</strong> Neuropeptide Y<br />
Authors: *J. L. MCGUIRE 1 , J. P. HERMAN 2 , F. R. SALLEE 1 , R. SAH 1 ;<br />
1 Psychiatry, 2 Neurosci., Univ. Cincinnati, Cincinnati, OH<br />
Abstract: Post-traumatic stress disorder (PTSD) is a stress-related <strong>an</strong>xiety disorder.<br />
Neuropeptide Y, a peptide neurotr<strong>an</strong>smitter regulating stress <strong>an</strong>d <strong>an</strong>xiety has been proposed as a<br />
potential “stress resilience factor”. Currently, pathophysiological relev<strong>an</strong>ce of NPY in PTSD is<br />
not known. This study intends to a) develop a chronic variable stress (CVS)-recovery model that<br />
elicits delayed expression of “PTSD-like” behavior <strong>an</strong>d b) investigate whe<strong>the</strong>r NPY expression<br />
is dysregulated in <strong>the</strong> CVS-recovery model. Adult male Long Ev<strong>an</strong>s rats were subjected to<br />
variable stressors <strong>for</strong> 7 days followed by 7 d recovery period. Animals were tested <strong>for</strong> PTSDpertinent<br />
behavior (contextual fear conditioning, extinction <strong>an</strong>d re-activation) <strong>an</strong>d NPY peptide<br />
levels. Rats exposed to CVS showed signific<strong>an</strong>t potentiation of freezing response following fearreactivation<br />
(p=.0247, t=2.540, df=13), as well as delayed impairment of fear extinction (p=.008,
t=3.177, df=12). Early <strong>an</strong>d delayed ch<strong>an</strong>ges in NPY expression were observed dependent on<br />
brain region. Signific<strong>an</strong>t reduction in amygdalar NPY was evident after 7 days (38.4% of<br />
controls, p=.045, t=2.258, df=10) <strong>an</strong>d was maintained at 14 days post CVS. Hippocampal NPY<br />
expression was attenuated 1 day post CVS (35.29% of controls, p=.0251, t=2.632, df=10). In <strong>the</strong><br />
mPFC, NPY was signific<strong>an</strong>tly elevated (228% of control p=.02, t=2.761, df=10) after 7 days, but<br />
by 14 days post-CVS was identical to controls. These are regions relev<strong>an</strong>t to contextual fearconditioning<br />
<strong>an</strong>d extinction learning. Separate cohorts of CVS <strong>an</strong>d control rats were tested on <strong>the</strong><br />
EPM. Under non-aversive low light conditions, <strong>an</strong>imals exposed to CVS showed <strong>an</strong> initial<br />
increase in exploratory behavior that normalized after 7 day recovery. However, under aversive<br />
bright light conditions, CVS exposed <strong>an</strong>imals showed a delayed emergence of unusual<br />
hyperlocomotive behavior with a concurrent increase in freezing behavior suggestive of fearful<br />
arousal. Current studies are investigating NPY in conjunction with monoaminergic systems<br />
relev<strong>an</strong>t to PTSD pathophysiology . Our studies indicate that persistent NPY dysregulation in<br />
limbic regions of CVS <strong>an</strong>imals may contribute to enh<strong>an</strong>ced fear responses upon re-exposure,<br />
impaired extinction learning <strong>an</strong>d lower <strong>the</strong> threshold <strong>for</strong> hyperarousal.<br />
Disclosures: J.L. McGuire, None; J.P. Herm<strong>an</strong>, None; F.R. Sallee, None; R. Sah, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.18/FF42<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: MH62621<br />
Title: Assessment of extrinsic sources of neuropeptide Y to <strong>the</strong> basolateral amygdaloid complex<br />
in <strong>the</strong> rat<br />
Authors: *R. J. LEITERMANN, M. R. DE JOSEPH, J. H. URBAN;<br />
Rosalind Fr<strong>an</strong>klin Univ. Med. & Sci., North Chicago, IL<br />
Abstract: The long-st<strong>an</strong>ding belief that all or nearly all <strong>the</strong> Neuropeptide Y (NPY) found within<br />
<strong>the</strong> Basolateral Amygdaloid Complex (BLA) arises <strong>from</strong> BLA interneurons was recently<br />
challenged when a considerable amount of NPY-immunoreactive (ir) fibers were reported in <strong>the</strong><br />
BLA following <strong>the</strong> selective lesioning of NPY-containing BLA interneurons. We have since<br />
observed a signific<strong>an</strong>t number of NPY-ir fibers in <strong>the</strong> BLA that do not colocalize with<br />
somatostatin, a marker <strong>for</strong> interneuronal sources of NPY. Our current studies use multi-label
immunohistochemistry (IHC) on naive rat brain sections to qu<strong>an</strong>titatively assess NPY fibers<br />
within <strong>the</strong> BLA arising <strong>from</strong> interneurons as well as o<strong>the</strong>r sources, <strong>an</strong>d retrograde tract tracing<br />
coupled with in situ hybridization autoradiography to identify <strong>the</strong> regions that send NPY<br />
projections to <strong>the</strong> BLA. IHC <strong>an</strong>alysis revealed ~ 12 <strong>an</strong>d 20 % of NPY-ir fibers within <strong>the</strong> lateral<br />
amygdaloid <strong>an</strong>d basolateral amygdaloid nucleus, respectively, did not arise <strong>from</strong> local<br />
interneurons. While <strong>the</strong>se fibers likely arise <strong>from</strong> sources outside <strong>the</strong> BLA, collaterals <strong>from</strong> BLA<br />
pyramidal projection neurons c<strong>an</strong>not be ruled out. In support of work suggesting brainstem<br />
adrenergic or noradrenergic projections to <strong>the</strong> BLA provide little or no NPY input, we observed<br />
few (< 1 %) BLA NPY-ir fibers that also displayed dopamine beta-hydroxylase-ir in both naive<br />
<strong>an</strong>d stressed rats. While extrinsic NPY projections to <strong>the</strong> BLA remain unknown, our ongoing<br />
tracing studies will provide novel insight into <strong>the</strong>se sources. Ultimately this work will offer new<br />
pathways to consider when assessing <strong>the</strong> endogenous <strong>an</strong>xiolytic properties of NPY within <strong>the</strong><br />
BLA.<br />
Disclosures: R.J. Leiterm<strong>an</strong>n, None; M.R. De Joseph, None; J.H. Urb<strong>an</strong>, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.19/FF43<br />
Topic: E.05.d. Stress-modulated pathways<br />
Support: Korea Research Fund E00230<br />
Title: Evidences <strong>for</strong> glucocorticoid modulation of GABAergic neurons in <strong>the</strong> <strong>an</strong>terior<br />
hypothalamic area <strong>an</strong>d <strong>the</strong> zona incerta<br />
Authors: S. SHIN, T. HAN, S. LEE, S. KIM, *S. LEE, P. RYU;<br />
Col. Vet. Med., Seoul Nat Univ., Seoul, Republic of Korea<br />
Abstract: Ch<strong>an</strong>ges in corticosterone level by adrenalectomy or stress are known to modulate<br />
GABAergic tr<strong>an</strong>smission in <strong>the</strong> paraventricular nucleus (PVN) of hypothalamus. However, it is<br />
not yet known whe<strong>the</strong>r corticosterone (CORT) c<strong>an</strong> directly act on GABAergic neurons in <strong>the</strong><br />
peri-PVN area of <strong>the</strong> hypothalamus. Using GAD65-eGFP tr<strong>an</strong>sgenic mice, we examined <strong>the</strong><br />
expression of glucocorticoid receptor (GR), mineralocorticoid receptor (MR) <strong>an</strong>d <strong>the</strong> effects of<br />
CORT on GABAergic neurons in <strong>the</strong> <strong>an</strong>terior hypothalamic area (AHA) <strong>an</strong>d <strong>the</strong> zona incerta<br />
(ZI), <strong>the</strong> regions rich in GABAergic neurons. For <strong>the</strong> electrophysiological studies, <strong>the</strong> mice were<br />
adrenalectomized <strong>an</strong>d <strong>the</strong> brain slices were incubated ei<strong>the</strong>r in vehicle, 100 nM CORT, 100 nM
CORT with <strong>the</strong> <strong>an</strong>tagonist of GR or MR in vitro.The GR <strong>an</strong>d/or MR mRNA tr<strong>an</strong>scripts in<br />
GAD65 or 67 positive neurons in AHA <strong>an</strong>d ZI were identified by single cell RT-PCR. The<br />
presence of receptor proteins on GABAergic neurons was fur<strong>the</strong>r confirmed by<br />
immunohisochemistry. The firing pattern of GABAergic neurons in response to depolarizing<br />
current pluses was observed in whole cell current clamp mode. A burst firing was showed in<br />
small portion (5-13%) of vehicle, 100 nM CORT <strong>an</strong>d 100 nM CORT+MR <strong>an</strong>tagonist (100 nM of<br />
spironolactone) treated groups. The burst firing was signific<strong>an</strong>tly increased (64%) in 100 nM<br />
CORT with GR <strong>an</strong>tagonist (500 nM of RU486) treated group (p
nucleus (npEW). The intracellular signaling pathways that are activated upon this pain stressor<br />
are not known. We have explored <strong>the</strong> possibility that <strong>the</strong> doublecortin-like kinase (DCLK)<br />
family of proteins is involved in pain stress-induced signaling in <strong>the</strong> PVN <strong>an</strong>d npEW, studying<br />
adult male Wistar rats. Our results show that both DCLK-short <strong>an</strong>d DCLK-long splice vari<strong>an</strong>ts<br />
are present in <strong>the</strong> cytoplasm <strong>an</strong>d proximal dendrites of neurons in both <strong>the</strong> PVN <strong>an</strong>d npEW. Of<br />
<strong>the</strong>se iso<strong>for</strong>ms, DCLK-long but not DCLK-short is phosphorylated in its serine/proline-rich<br />
domain. Fur<strong>the</strong>rmore, we demonstrate that phosphorylation of DCLK-long in <strong>the</strong> npEW is<br />
increased upon acute pain stress, whereas DCLK-long phosphorylation in <strong>the</strong> PVN remains<br />
unaffected. This is <strong>the</strong> first study revealing that DCLK iso<strong>for</strong>ms in <strong>the</strong> PVN <strong>an</strong>d npEW occur in<br />
<strong>the</strong> adult mammali<strong>an</strong> brain <strong>an</strong>d that <strong>the</strong> regulation of DCLK-long through phosphorylation upon<br />
acute pain stress differs between <strong>the</strong>se two brain centers.<br />
Disclosures: T. Rouwette, None; T. Kozicz, None; N. Olde Loohuis, None; B. Gaszner,<br />
None; E. Vreugdenhil, None; G. Scheffer, None; E. Roubos, None; K. Vissers, None; W.<br />
Scheenen, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.21/FF45<br />
Topic: E.01.b. Neuroendocrine Regulation: HPA axis regulation<br />
Support: R01MH077152<br />
Title: Similar regulation of multiple autonomic <strong>an</strong>d endocrine responses to repeated homotypic<br />
stress in rats<br />
Authors: *R. J. NEWSOM, C. V. MASINI, H. E. DAY, S. CAMPEAU;<br />
Psychology, Univ. of Colorado Boulder, Boulder, CO<br />
Abstract: Stress situations elicit a wide r<strong>an</strong>ge of autonomic, endocrine, <strong>an</strong>d behavioral responses<br />
aimed at restoring physiological or psychological homeostasis. Upon repeated exposures to <strong>the</strong><br />
same (homotypic) stress situation, m<strong>an</strong>y autonomic <strong>an</strong>d endocrine responses tend to decrease or<br />
habituate, while available coping behavioral responses c<strong>an</strong> increase or decrease depending on <strong>the</strong><br />
org<strong>an</strong>ism’s repertoire. Whe<strong>the</strong>r all <strong>the</strong>se responses are simult<strong>an</strong>eously <strong>an</strong>d similarly controlled is<br />
still mostly unknown, as most laboratories measure a single response at <strong>an</strong>y one time. The main<br />
goal of <strong>the</strong> current study was to determine <strong>the</strong> extent to which autonomic (heart rate <strong>an</strong>d core<br />
body temperature) <strong>an</strong>d endocrine (corticosterone) responses co-vary in a repeated stressor
paradigm designed to induce variable habituation rates between different groups of rats. Adult<br />
male Sprague-Dawley rats (n = 16) were impl<strong>an</strong>ted with Mini Mitter/Respironics telemetric<br />
sensors (E-mitter monitors) <strong>for</strong> <strong>the</strong> detection of heart rate <strong>an</strong>d core body temperature, in addition<br />
to a jugular ca<strong>the</strong>ter <strong>for</strong> <strong>the</strong> remote sampling of blood during <strong>the</strong> study. Following at least one<br />
week of recovery <strong>from</strong> surgery, rats were exposed to ei<strong>the</strong>r <strong>the</strong> isolation chamber alone (n = 5),<br />
or <strong>the</strong> isolation chamber with 90 dBA (n = 6) or 105 dBA loud white noise (n = 5). Rats were<br />
placed in <strong>the</strong> chambers <strong>for</strong> at least 3.5 hours, <strong>an</strong>d <strong>the</strong> two noise groups received 30 min of <strong>the</strong>ir<br />
respective noise 2 hours after initial placement <strong>an</strong>d connection of <strong>the</strong> rats to <strong>the</strong> blood sampling<br />
apparatus, <strong>for</strong> 5 consecutive days. Heart rate <strong>an</strong>d core body temperature were sampled once a<br />
minute during <strong>the</strong> entire duration of chamber placement. Blood samples (0.2 ml) were taken<br />
immediately prior to, <strong>an</strong>d 15, 30, 45, 60, <strong>an</strong>d 90 min after, noise initiation or at a similar time in<br />
<strong>the</strong> no noise control group. Heart rate <strong>an</strong>d body temperature were reliably <strong>an</strong>d similartly<br />
increased by both intensities of noise, compared to <strong>the</strong> control group, <strong>an</strong>d no reliable habituation<br />
across days were obtained due to <strong>the</strong> large individual differences observed. Additional rats are<br />
being evaluated to reduce variability. The corticosterone determination awaits <strong>the</strong> completion of<br />
all subjects in <strong>the</strong> study. So far, <strong>the</strong> similarity in <strong>the</strong> heart rate <strong>an</strong>d core body temperature<br />
responses support <strong>the</strong> hypo<strong>the</strong>sis that multiple stress responses are regulated in parallel, perhaps<br />
through a unitary central state induced by stress.<br />
Disclosures: R.J. Newsom, None; C.V. Masini, None; H.E. Day, None; S. Campeau, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.22/FF46<br />
Topic: E.05.e. Stress <strong>an</strong>d cognition<br />
Support: NSERC Discovery Gr<strong>an</strong>t<br />
Title: Corticosterone treatment increases <strong>the</strong> age difference in <strong>the</strong> <strong>for</strong>ced swim test in male rats:<br />
Decreased depressive behaviour in adolescence<br />
Authors: *P. WATERS 1 , C. M. THOMAS 2 , I. Z. MATTHEWS 1 , C. M. MCCORMICK 1 ;<br />
1 Psychology, 2 Biol., Brock Univ., St Catharines, ON, C<strong>an</strong>ada<br />
Abstract: Adolescence may be a period of development that is more susceptible to <strong>the</strong> effects of<br />
stress / elevated corticosterone th<strong>an</strong> adulthood. Here, we investigated whe<strong>the</strong>r chronic treatment<br />
with corticosterone in males would lead to increased <strong>an</strong>xiety-like <strong>an</strong>d depressive behaviour when
treated in adolescence compared to in adulthood. O<strong>the</strong>rs have reported that repeated<br />
corticosterone injections in adult males increased depressive behaviour in <strong>the</strong> <strong>for</strong>ced swim test<br />
(FST), but had no effect on <strong>an</strong>xiety in <strong>the</strong> open field test, <strong>an</strong>d its effect in adolescence is<br />
unknown (Gregus et al., 2005). Fur<strong>the</strong>r <strong>an</strong>xiety in elevated plus maze (EPM) has only been<br />
investigated after acute corticosterone injection in adulthood (Shepard et al., 2000). Males<br />
received 16 daily injections of 40 mg/kg corticosterone or vehicle over days 30 - 45, <strong>for</strong><br />
comparison with our adolescent social stress model; (McCormick et al., <strong>2009</strong>) or over days 70 -<br />
85 <strong>an</strong>d were tested on <strong>the</strong> EPM 24 h <strong>an</strong>d on <strong>the</strong> FST 48 h after last injection. Blood samples were<br />
obtained be<strong>for</strong>e <strong>an</strong>d at time intervals after <strong>the</strong> FST to examine differences among <strong>the</strong> groups in<br />
HPA function, <strong>an</strong>d brains were collected 90 min to examine Fos expression. Corticosterone<br />
injections caused weight loss in adults <strong>an</strong>d reduced weight gain in adolescence. There was no<br />
effect of treatment on behaviour in <strong>the</strong> EPM. However, adults were signific<strong>an</strong>tly less <strong>an</strong>xious <strong>an</strong>d<br />
more active th<strong>an</strong> adolescents: Adults spent more time in <strong>the</strong> open arm, more time in <strong>the</strong> center,<br />
reared more often, <strong>an</strong>d made more arm entries th<strong>an</strong> adolescents. Corticosterone treatment<br />
decreased depressive behaviour (decreased immobility <strong>an</strong>d increased swimming in <strong>the</strong> FST) in<br />
adolescents when tested 48 hours after <strong>the</strong> last injection, but <strong>the</strong>re was no effect of corticosterone<br />
treatment in adults. Adults showed more depressive behaviour th<strong>an</strong> adolescents. The extent to<br />
which <strong>the</strong> behavioural differences are paralleled by differences in HPA function <strong>an</strong>d Fos<br />
expression during <strong>the</strong> FST are currently under investigation,<br />
Disclosures: P. Waters, None; C.M. Thomas, None; I.Z. Mat<strong>the</strong>ws, None; C.M.<br />
McCormick, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.23/FF47<br />
Topic: E.05.c. Early life experience<br />
Title: Influence of chronic restraint stress on hippocampal neurogenesis <strong>an</strong>d hippocampusdependent<br />
fear memory in mice<br />
Authors: *K. YAMADA 1 , D. IBI 1 , H. KOIKE 1 , Y. JAESUK 1 , H. MIZOGUCHI 2 , A. NITTA 1 ,<br />
T. NABESHIMA 3 , T. NAGAI 1 ;<br />
1 Neuropsychopharm., Nagoya Univ. Grad Sch. Med., Nagoya, Jap<strong>an</strong>; 2 Futuristic Envrn.<br />
Simulation Ctr., Res. Inst. of Envrn. Medicine, Nagoya Univ., Nagoya city, Jap<strong>an</strong>; 3 Dept. of<br />
Chem. Pharmacol., Grad. Sch. of Pharmaceut. Sciences, Meijo Univ., Nagoya city, Jap<strong>an</strong>
Abstract: Neurogenesis in <strong>the</strong> hippocampus occurs throughout life in a wild r<strong>an</strong>ge of species,<br />
<strong>an</strong>d could be associated with hippocampus-dependent learning <strong>an</strong>d memory. Stress is well<br />
established to seriously perturb <strong>the</strong> physiological/psychological homeostasis, <strong>an</strong>d affect <strong>the</strong><br />
hippocampal function. In <strong>the</strong> present study, to investigate <strong>the</strong> effect of chronic restraint stress in<br />
early life on hippocampal neurogenesis <strong>an</strong>d hippocampus-dependent memory, mice at 3-weekold<br />
were subjected to restraint stress 6 days a week <strong>for</strong> 4 weeks. The chronic restraint stress<br />
signific<strong>an</strong>tly decreased <strong>the</strong> hippocampal volume by 6.3%, <strong>an</strong>d impaired hippocampal<br />
neurogeneis as indicated by <strong>the</strong> reduced number of Ki67-, BrdU- <strong>an</strong>d doublecortin-positive cells<br />
in <strong>the</strong> dentate gyrus (DG). The chronic restraint stress severely impaired hippocampus-dependent<br />
contextual fear memory without affecting hippocampus-independent fear memory. The<br />
expression level of brain-specific tr<strong>an</strong>scription factor Npas4 mRNA in DG of <strong>the</strong> hippocampus<br />
was downregulated by <strong>the</strong> restraint stress. The Npas4 immunoreactivity was detected in<br />
progenitors, immmatue <strong>an</strong>d mature neurons in <strong>the</strong> DG. Our findings suggest that <strong>the</strong> chronic<br />
restraint stress decrease <strong>the</strong> hippocampal neurogenesis, leading <strong>an</strong> impairment of hippocampusdependent<br />
fear memory in mice. Downregulation of Npas4 expression may play a role in stressinduced<br />
impairment of hippocampal function.<br />
Disclosures: K. Yamada, None; D. Ibi, None; H. Koike, None; Y. Jaesuk, None; H.<br />
Mizoguchi, None; A. Nitta, None; T. Nabeshima, None; T. Nagai, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.24/FF48<br />
Topic: E.05.c. Early life experience<br />
Support: NARSAD Young Investigator Award<br />
NIH Gr<strong>an</strong>t DA013159<br />
Title: Isolation rearing <strong>an</strong>d restraint stress produce sex-dependent ch<strong>an</strong>ges in medial prefrontal<br />
cortex immediate early gene expression<br />
Authors: *S. T. BLAND 1 , J. T. BECKLEY 2 , E. K. FISCHER 1 , O. I. IMONEGA 3 , N. C.<br />
ORTIZ 1 , L. R. WATKINS 1 , S. F. MAIER 1 ;<br />
1 2 3<br />
Univ. Colorado, Boulder, CO; Med. Univ. of South Carolina, Charleston, SC; Georgia Inst. of<br />
Technol., Atl<strong>an</strong>ta, GA
Abstract: Isolation rearing (housing rats in isolation after we<strong>an</strong>ing) is a model of early life<br />
adversity that produces ch<strong>an</strong>ges in emotional behaviors, including increased <strong>an</strong>xiety <strong>an</strong>d altered<br />
responses to stress. The purpose of <strong>the</strong> present study was to assess <strong>the</strong> effects of isolation rearing<br />
of male <strong>an</strong>d female rats on stress-induced ch<strong>an</strong>ges in immediate early gene (IEG) expression in<br />
<strong>the</strong> medial prefrontal cortex (mPFC), a brain region essential <strong>for</strong> emotional regulation. Male <strong>an</strong>d<br />
female Sprague-Dawley rats were we<strong>an</strong>ed on postnatal day 21 <strong>an</strong>d housed <strong>for</strong> 4 weeks ei<strong>the</strong>r in<br />
same-sex groups of 4 or individually. Rats were <strong>the</strong>n exposed to 15 min of restraint stress or left<br />
in <strong>the</strong>ir home cage (no stress controls [NS]) <strong>an</strong>d were sacrificed 90 min later.<br />
Immunohistochemistry <strong>an</strong>d stereology were used to assess <strong>the</strong> protein products of <strong>the</strong> IEGs c-fos<br />
<strong>an</strong>d Arc, markers of neuronal activation <strong>an</strong>d plasticity. Restraint stress increased c-fos expression<br />
in <strong>the</strong> mPFC in a sex <strong>an</strong>d housing dependent m<strong>an</strong>ner. To account <strong>for</strong> differences in c-fos<br />
expression between NS groups, data were normalized to a percent of NS. Stress produced<br />
signific<strong>an</strong>t increases in mPFC c-fos expression only in isolation-reared, but not group-reared<br />
females. Conversely, stress produced signific<strong>an</strong>t increases in mPFC c-fos expression only in<br />
group-reared, but not isolation-reared males. NS isolation-reared males had signific<strong>an</strong>tly greater<br />
Arc counts th<strong>an</strong> NS group-reared males. Restraint stress similarly increased Arc expression in<br />
<strong>the</strong> mPFC in a sex <strong>an</strong>d housing dependent m<strong>an</strong>ner. The pattern of stress-induced Arc expression<br />
when counts were normalized was similar to that of c-fos, but <strong>the</strong> increase in Arc expression in<br />
group-reared males was much more pronounced. Most stress-induced IEG expression was<br />
observed in <strong>the</strong> <strong>an</strong>terior cingulate <strong>an</strong>d prelimbic regions, with little in <strong>the</strong> infralimbic region.<br />
IEGs in <strong>the</strong> mPFC appear to be less reactive to stress in females th<strong>an</strong> in males, <strong>an</strong>d isolation<br />
rearing has opposite effects on stress-induced IEG expression in this region, potentiating <strong>the</strong><br />
response in females <strong>an</strong>d attenuating it in males.<br />
Disclosures: S.T. Bl<strong>an</strong>d, None; J.T. Beckley, None; E.K. Fischer, None; O.I. Imonega,<br />
None; N.C. Ortiz, None; L.R. Watkins, None; S.F. Maier, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 573.25/FF49<br />
Topic: E.05.c. Early life experience<br />
Support: IWRH postdoctoral fellowship to SB<br />
NSERC PGS-M funding to TC<br />
CIHR gr<strong>an</strong>t to LAMG
Title: Chronic corticosterone exposure during adolescence: Effects on brain weight <strong>an</strong>d adult<br />
hippocampal neurogenesis in male <strong>an</strong>d female rats<br />
Authors: *S. BRUMMELTE 1 , T. M. CROZIER 1 , L. A. M. GALEA 2 ;<br />
1 2<br />
Dept. of Psychology, Dept. of Psychology <strong>an</strong>d Brain Res. Ctr., Univ. of British Columbia,<br />
V<strong>an</strong>couver, BC, C<strong>an</strong>ada<br />
Abstract: Chronic stress or elevated levels of glucocorticoids are known to cause ch<strong>an</strong>ges in<br />
behaviour <strong>an</strong>d brain morphology. Interestingly, <strong>the</strong> effect of chronic stress on hippocampal<br />
neurogenesis is equivocal in both males <strong>an</strong>d females, with some studies finding no ch<strong>an</strong>ge or <strong>an</strong><br />
increase in neurogenesis in females <strong>an</strong>d usually a decrease in neurogenesis in males. Stress<br />
during adolescence also alters adult neurogenesis levels but in a different pattern in males <strong>an</strong>d<br />
females compared to stress during adulthood with females, but not males, showing a suppression<br />
in adult hippocampal neurogenesis. Prolonged exposure to high levels of <strong>the</strong> stress hormone<br />
corticosterone (CORT: <strong>the</strong> major glucocorticoid in rodents) on <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d reduces<br />
hippocampal cell proliferation <strong>an</strong>d survival in both adult male <strong>an</strong>d female rodents. Thus, <strong>the</strong><br />
current study was conducted to investigate <strong>the</strong> effects of chronic corticosterone exposure during<br />
adolescence on hippocampal neurogenesis to better underst<strong>an</strong>d <strong>the</strong> contribution of high<br />
prolonged CORT during development. For this, 30 day old male <strong>an</strong>d female rats received a daily<br />
s.c. injection of ei<strong>the</strong>r a high dose of CORT (40mg/kg), a low dose of CORT (10mg/kg), or<br />
vehicle (sesame oil) <strong>for</strong> 14 consecutive days. Animals received <strong>an</strong> i.p. injection of BrdU<br />
(50mg/kg) prior to <strong>the</strong> start of treatment <strong>an</strong>d were sacrificed ei<strong>the</strong>r 24 hours after <strong>the</strong> last<br />
injection or in adulthood (day 90). Body weight was measured every 4 days until adulthood <strong>an</strong>d<br />
baseline CORT levels were taken on <strong>the</strong> last day of treatment <strong>an</strong>d in adulthood. Results reveal<br />
that <strong>the</strong> low <strong>an</strong>d high doses of CORT cause a highly signific<strong>an</strong>t interruption of weight gain in<br />
males <strong>an</strong>d females which is not fully recovered in young adulthood. Interestingly, brain weights<br />
at <strong>the</strong> end of <strong>the</strong> CORT treatment were more drastically decreased in males th<strong>an</strong> in females, with<br />
<strong>the</strong> low dose having no effect in females. Subsequently, male CORT rats showed higher brain-tobody<br />
ratios compared to controls, while <strong>the</strong>re was no signific<strong>an</strong>t increase in females. Based on<br />
previous results we hypo<strong>the</strong>size that we will see a suppression in hippocampal cell proliferation<br />
<strong>an</strong>d survival in young <strong>an</strong>d adult rats, but to different degrees in males <strong>an</strong>d females. These results<br />
underline <strong>the</strong> different sensitivities <strong>for</strong> glucocorticoid exposure in males <strong>an</strong>d females, particularly<br />
during a vulnerable time of development.<br />
Disclosures: S. Brummelte, None; T.M. Crozier, None; L.A.M. Galea, None.<br />
Poster<br />
573. Stress-Modulated Brain Pathways III<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 573.26/FF50<br />
Topic: E.05.c. Early life experience<br />
Support: NIH P20 RR15567<br />
NIDA RO1 DA019921<br />
Title: Long-lasting consequences of cortical D2 dopamine receptor activation during adolescence<br />
Authors: *M. J. WATT, J. L. SCHOLL, E. J. HAALAND, G. L. FORSTER;<br />
S<strong>an</strong><strong>for</strong>d Sch. Med., Univ. South Dakota, Vermillion, SD<br />
Abstract: Adolescence is a critical period of behavioral <strong>an</strong>d neural development, making<br />
adolescents vulnerable to detrimental effects of negative social experiences such as bullying. We<br />
have shown that male rats exposed to social defeat in adolescence exhibit decreased medial<br />
prefrontal cortex (mPFC) dopamine (DA) content as adults, which is associated with heightened<br />
sensation-seeking. Accordingly, adult rats defeated in adolescence show enh<strong>an</strong>ced novelty<br />
responses compared to non-defeated controls. We hypo<strong>the</strong>size that <strong>the</strong> reduction in mPFC DA<br />
content <strong>an</strong>d subsequently enh<strong>an</strong>ced novelty responses result <strong>from</strong> heightened defeat stressinduced<br />
activation of DA syn<strong>the</strong>sis-controlling DA D2 autoreceptors in <strong>the</strong> mPFC during<br />
adolescence. Using rats not exposed to social defeat, we investigated whe<strong>the</strong>r repeated<br />
pharmacological activation of mPFC DA D2 autoreceptors during mid-adolescence would result<br />
in increased novelty responses as associated with altered mPFC DA activity. Male rats received<br />
daily bilateral infusions of <strong>the</strong> DA D2 receptor agonist quinpirole (100 ng in 0.3 µl per side) or<br />
vehicle (aCSF) into <strong>the</strong> mPFC, <strong>from</strong> postnatal day (P) 35 to 39. Subjects were <strong>the</strong>n tested <strong>for</strong><br />
responses to a novel object ei<strong>the</strong>r <strong>the</strong> day following last infusion (P40) or upon reaching early<br />
adulthood (P56). Quinpirole-treated rats displayed both greater time in close proximity <strong>an</strong>d more<br />
approaches to <strong>the</strong> novel object compared to vehicle-treated controls, <strong>an</strong>d this effect was apparent<br />
in both adolescence <strong>an</strong>d adulthood. Novelty behavior is currently being related to both mPFC<br />
DA content <strong>an</strong>d DA D2 receptor levels in <strong>the</strong>se rats. Findings thus far suggest that disruption of<br />
adolescent mPFC DA activity via DA D2 autoreceptor activation results in both immediate <strong>an</strong>d<br />
enduring alterations to novelty seeking.<br />
Disclosures: M.J. Watt, None; J.L. Scholl, None; E.J. Haal<strong>an</strong>d, None; G.L. Forster, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 574.1/FF51<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Title: Circadi<strong>an</strong> entrainment <strong>an</strong>d phase-shifting by gravitoinertial stimulation is abolished in<br />
mice lacking functional vestibular inputs<br />
Authors: K. M. RINGGOLD, *C. A. FULLER;<br />
Dept of Neurobiology, Physiol. & Behavior, Univ. of Cali<strong>for</strong>nia, Davis, CA<br />
Abstract: The circadi<strong>an</strong> pacemaker c<strong>an</strong> be phase-shifted <strong>an</strong>d entrained by appropriately timed<br />
nonphotic stimuli, especially locomotor activity. However, <strong>the</strong> mech<strong>an</strong>ism(s) involved remain<br />
poorly understood. Recent work in our lab has suggested <strong>the</strong> involvement of <strong>the</strong> vestibular<br />
otolith org<strong>an</strong>s in activity-induced ch<strong>an</strong>ges within <strong>the</strong> circadi<strong>an</strong> timing system (CTS). For<br />
example, we have shown that ch<strong>an</strong>ges in circadi<strong>an</strong> period <strong>an</strong>d phase in response to wheel<br />
running require input <strong>from</strong> functional macular gravity receptors in <strong>the</strong> vestibular org<strong>an</strong>. On <strong>the</strong><br />
basis of <strong>the</strong>se findings, we hypo<strong>the</strong>size that <strong>the</strong> neurovestibular system is responsible <strong>for</strong> <strong>the</strong><br />
tr<strong>an</strong>sduction of gravitoinertial accelerations associated with locomotor activity <strong>an</strong>d that this<br />
tr<strong>an</strong>sduction process is necessary to evoke phase <strong>an</strong>d period ch<strong>an</strong>ges of <strong>the</strong> CTS. The present<br />
study tested this hypo<strong>the</strong>sis by providing daily, timed gravitoinertial stimuli (i.e., +G via<br />
centrifugation) to <strong>an</strong>imals with <strong>an</strong>d without functional vestibular inputs. +G exposure provides<br />
<strong>an</strong>o<strong>the</strong>r mech<strong>an</strong>ism to stimulate <strong>the</strong> vestibular otoconial receptors, in a m<strong>an</strong>ner similar to <strong>the</strong><br />
tr<strong>an</strong>slation of <strong>the</strong> otoconia that occurs during wheel running. The subjects in this study included<br />
chemically labyrin<strong>the</strong>ctomized (Labx) mice, mice lacking macular otoconia (head tilt or het) <strong>an</strong>d<br />
intact wildtype (WT) littermates. All mice were impl<strong>an</strong>ted i.p. with biotelemetry to continuously<br />
measure body temperature <strong>an</strong>d activity rhythms. The subjects were individually housed in a 4meter<br />
diameter centrifuge in const<strong>an</strong>t darkness (DD). After 2 weeks in DD, <strong>the</strong> mice were<br />
exposed daily to 2G (i.e., 2 x Earth’s gravity) <strong>from</strong> 1000-1200 <strong>for</strong> 9 weeks. Here WT, but not<br />
Labx or het mice, showed entrainment to <strong>the</strong> daily 2G pulses. We <strong>the</strong>n re-set <strong>the</strong> 2G pulse to<br />
occur at 1200-1400 <strong>for</strong> 4 weeks. Again, only WT mice demonstrated a phase shift in response to<br />
<strong>the</strong> re-setting of <strong>the</strong> 2G pulse <strong>an</strong>d subsequent re-entrainment to <strong>the</strong> new centrifugation schedule.<br />
These results provide additional evidence that gravitoinertial stimuli, including +G exposure <strong>an</strong>d<br />
wheel running, require a functional vestibular system to both entrain <strong>an</strong>d phase shift <strong>the</strong> CTS.<br />
Disclosures: K.M. Ringgold, None; C.A. Fuller, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 574.2/FF52<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t GM076430<br />
David <strong>an</strong>d Lucille Packard Foundation<br />
Alfred P. Slo<strong>an</strong> Foundation<br />
Title: An unexpected contribution of rods to circadi<strong>an</strong> photoentrainment at all light intensities<br />
Authors: *C. M. ALTIMUS 1 , A. D. GÜLER 1 , G. LALL 2 , C. ARMAN 3 , N. ALAM 4 , A.<br />
SAMPATH 3 , G. PRUSKEY 4 , R. LUCAS 2 , S. HATTAR 1 ;<br />
1 Johns Hopkins Univ., Baltimore, MD; 2 Univ. of M<strong>an</strong>chester, M<strong>an</strong>chester, United Kingdom;<br />
3 Keck Sch. of Med., Los Angeles, CA; 4 Weill Cornell Med. Col., White Plains, NY<br />
Abstract: Three retinal photoreceptors, rods, cones <strong>an</strong>d mel<strong>an</strong>opsin-containing intrinsically<br />
photosensitive retinal g<strong>an</strong>glion cells (ipRGCs), mediate image <strong>for</strong>ming <strong>an</strong>d image-independent<br />
physiological functions in mammals. In mice lacking mel<strong>an</strong>opsin, light signals originating in <strong>the</strong><br />
outer retinal photoreceptors compensate <strong>for</strong> <strong>the</strong> absence of <strong>the</strong> intrinsic photosensitivity of<br />
ipRGCs <strong>for</strong> circadi<strong>an</strong> photoentrainment. However, <strong>the</strong> high threshold <strong>for</strong> light induced circadi<strong>an</strong><br />
photoreception <strong>an</strong>d <strong>the</strong> need of <strong>the</strong> circadi<strong>an</strong> system to incorporate irradi<strong>an</strong>ce signals <strong>for</strong><br />
prolonged time are at odds with <strong>the</strong> high sensitivity of <strong>the</strong> rods <strong>an</strong>d <strong>the</strong> rapid adaptation of cones.<br />
To address if rods or cones alone could signal circadi<strong>an</strong> photoresponses, we used <strong>an</strong>imals that<br />
were modified to contain only functional rods or functional cones with minimum retinal<br />
degeneration. We show that mice lacking functional rods <strong>an</strong>d mel<strong>an</strong>opsin protein (cone only<br />
<strong>an</strong>imals), weakly photoentrain to high light intensity <strong>an</strong>d are unable to photoentrain at low light<br />
intensities. This result suggests that cones are not <strong>the</strong> domin<strong>an</strong>t input to ipRGCs <strong>for</strong> circadi<strong>an</strong><br />
photoentrainment. To isolate rod contribution, we utilized knockout mouse lines that lack <strong>the</strong><br />
mel<strong>an</strong>opsin protein in combination with two mutations that disable cone photoreception <strong>an</strong>d a<br />
third mutation that eliminates cones. Here we show that rod photoreceptors contribute to<br />
circadi<strong>an</strong> photoentrainment through two distinct retinal pathways that are light intensity<br />
dependent. At low light intensity, rods are <strong>the</strong> only photoreceptors capable of detecting light <strong>for</strong><br />
photoentrainment, but <strong>the</strong>y are also capable of light signaling <strong>for</strong> non-image functions even at<br />
intensities when <strong>the</strong>y are incapable of image discrimination. These findings establish <strong>an</strong><br />
unappreciated role <strong>for</strong> rods in photoentrainment <strong>an</strong>d highlight how <strong>the</strong> use of multiple knockout<br />
lines could reveal properties of neural circuits underlying behavior.<br />
Disclosures: C.M. Altimus, None; A.D. Güler, None; G. Lall, None; C. Arm<strong>an</strong>, None; N.<br />
Alam, None; A. Sampath, None; G. Pruskey, None; R. Lucas, None; S. Hattar, None.<br />
Poster
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.3/FF53<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH gr<strong>an</strong>t NS37919 (to RS)<br />
NSF DBI 320988 to Barnard College<br />
Title: Light exposure induces long-term ch<strong>an</strong>ges in <strong>the</strong> excitability of retinorecipient GRP<br />
neurons in <strong>the</strong> suprachiasmatic nucleus<br />
Authors: *J. J. LE SAUTER 1 , P. WITKOVSKY 2 , R. CLOUES 3 , R. SILVER 4,5 ;<br />
1 Dept Psychol, Barnard Coll, New York, NY; 2 Ophthalmology, New York Univ., New York,<br />
NY; 3 Psychology, Regis Col., Denver, CO; 4 Psychology, Barnard Col., New York, NY;<br />
5 Psychology, Columbia Univ., New York, NY<br />
Abstract: The mammali<strong>an</strong> suprachiasmatic nucleus (SCN) serves as <strong>the</strong> circadi<strong>an</strong> pacemaker<br />
sustaining rhythmicity in m<strong>an</strong>y behavioral <strong>an</strong>d physiological functions <strong>an</strong>d synchronizing <strong>the</strong>se<br />
rhythms to <strong>the</strong> light-dark cycle of <strong>the</strong> local environment. A subset of SCN neurons, among <strong>the</strong>m<br />
gastrin releasing peptide (GRP+) containing cells, receives direct retinal input via <strong>the</strong><br />
retinohypothalamic tract, <strong>an</strong>d is import<strong>an</strong>t in mediating <strong>the</strong> light-induced phase shifts in SCN<br />
rhythmicity. The results point to a surprising long term effect of light exposure on <strong>the</strong>se cells. In<br />
brain slices <strong>from</strong> tr<strong>an</strong>sgenic mice expressing GFP in GRP cells (Karatsoreos et al., J. Neurosci.<br />
24:68, 2004), we examined responses to light of GRP+ <strong>an</strong>d adjacent non-GRP cells.<br />
Experimental <strong>an</strong>imals were exposed to a 1 hr light pulse (LP+) beginning at zeitgeber time 14<br />
(ZT14) <strong>an</strong>d were sacrificed at ZT15 or ZT21, whereas control <strong>an</strong>imals (LP-) were treated<br />
identically but not exposed to light prior to sacrifice. Membr<strong>an</strong>e properties were recorded by<br />
whole cell patch clamp <strong>an</strong>d cells were identified by filling with biocytin <strong>an</strong>d immunostaining <strong>for</strong><br />
biocytin <strong>an</strong>d GFP. There was no effect of light on non-GRP neurons of <strong>the</strong> SCN core. Resting<br />
potentials of GRP+ cells <strong>from</strong> LP+ <strong>an</strong>d LP- groups were statistically indistinguishable between<br />
ZT15.5-17. In contrast, <strong>the</strong> firing rate of LP+ GRP+ cells was elevated at ZT 15.5 -17, <strong>the</strong>n<br />
lowered at ZT17-21 compared to LP-GRP+ cells. Their membr<strong>an</strong>e potential signific<strong>an</strong>tly<br />
hyperpolarized between ZT 17-21, returning to baseline levels <strong>the</strong>reafter. Bicuculline-sensitive<br />
spont<strong>an</strong>eous membr<strong>an</strong>e oscillations were lower in LP+ hyperpolarized cells at ZT17-21 th<strong>an</strong> in<br />
LP- cells, suggesting ei<strong>the</strong>r a reduction in GABA-dependent synaptic input or a loss of GABAA<br />
receptors. Application of GABA to LP+ cells during ZT17-21 reinstated synaptic noise <strong>an</strong>d<br />
depolarized membr<strong>an</strong>e potential, showing that LP+ cells had reduced GABA input during this<br />
time period. The reversal potential of <strong>the</strong> synaptic oscillations was -43 mV, indicating <strong>the</strong><br />
activity of a chloride pump. Given that GRP cells project to <strong>the</strong> SCN shell (Drouyer et al, 2005),<br />
we hypo<strong>the</strong>size that during light-induced phase resetting, activation of oscillator cells results
<strong>from</strong> disinhibition <strong>from</strong> <strong>the</strong> light responsive GRP neurons. The 3h delay between light exposure<br />
<strong>an</strong>d its effect on GRP+ neurons suggests that <strong>the</strong> mech<strong>an</strong>ism involves <strong>the</strong> loss of GABAdependent<br />
inhibition at ZT 17-21 results <strong>from</strong> receptor internalization.<br />
Disclosures: J.J. Le Sauter, None; P. Witkovsky, None; R. Cloues, None; R. Silver, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.4/FF54<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH gr<strong>an</strong>t NS37919<br />
Title: The SCN response to light in <strong>the</strong> subjective day is augmented by activity<br />
Authors: *E. RODRIGUEZ 1 , J. LESAUTER 2 , R. SILVER 1 ;<br />
1 Psychology, Columbia Univ., New York, NY; 2 Psychology, Barnard Col., New York, NY<br />
Abstract: It is well established that <strong>the</strong> SCN responds to photic input during <strong>the</strong> night but not<br />
during <strong>the</strong> day. Here, we investigated whe<strong>the</strong>r <strong>the</strong> SCN’s response to light is determined by <strong>the</strong><br />
prior light-dark (LD) cycle or by <strong>the</strong> <strong>an</strong>imal’s rest/activity cycle. We tested whe<strong>the</strong>r activity<br />
induction by temporal food restriction (FR) or awakening by movement of <strong>the</strong> home cage<br />
influence daytime responses to a light pulse. Mice expressing GFP in gastrin-releasing peptide<br />
(GRP) cells were housed individually with a running wheel, entrained to a 12:12 LD cycle, <strong>an</strong>d<br />
placed in one of 6 groups (n=6/group). Animals in a food <strong>an</strong>ticipatory activity (FAA) paradigm<br />
had food access restricted to ZT 6-14 (FAA+ group), while controls were fed at libitum<br />
(Undisturbed groups). After 5 days FR, <strong>the</strong>y were maintained in const<strong>an</strong>t darkness <strong>from</strong> lights<br />
off (ZT12) until a 30 min light pulse <strong>the</strong> next day starting at CT3.5 (FAA+ LP+ <strong>an</strong>d Undisturbed<br />
LP+); controls received no light pulse (FAA+LP−, Undisturbed LP−). Two o<strong>the</strong>r groups<br />
(Awaken) were fed ad libitum <strong>an</strong>d kept awake (at CT3.5-4) by gentle shaking of <strong>the</strong>ir home cage<br />
by <strong>an</strong> experimenter wearing infrared goggles to see in <strong>the</strong> dark. Of <strong>the</strong>se Awaken <strong>an</strong>imals, one<br />
group was exposed to a light pulse as above (Awaken LP+) while <strong>the</strong> controls received no light<br />
(Awaken LP-). All mice were sacrificed at CT5. For fluorescent immunohistochemistry, every<br />
third 40µm SCN slice was processed <strong>for</strong> c-FOS, arginine-vasopressin (AVP) <strong>an</strong>d GFP. AVP was<br />
used to delineate <strong>the</strong> core <strong>an</strong>d shell SCN. AVP-outlined images were qu<strong>an</strong>tified by two<br />
independent experimenters. Contrary to expectation, a light pulse during <strong>the</strong> subjective day led to<br />
a small but signific<strong>an</strong>t increase in <strong>the</strong> number of c-FOS expressing cells. The number of light
induced c-FOS cells was signific<strong>an</strong>tly increased if <strong>an</strong>imals were active due to FAA or awakened<br />
by <strong>the</strong> experimenter. The effect of activity is supported by a positive correlation between <strong>the</strong><br />
number of wheel revolutions <strong>an</strong>d <strong>the</strong> number of c-FOS cells in <strong>the</strong> SCN. Surprisingly, c-FOS<br />
was not observed in GRP cells, a subpopulation of SCN cells that respond to light during <strong>the</strong><br />
subjective night. In <strong>the</strong> absence of a light-pulse, <strong>the</strong> <strong>an</strong>imals in <strong>the</strong> Awakened condition had<br />
more shell (not core) c-FOS compared to <strong>the</strong> Undisturbed condition. There was no difference<br />
between FAA <strong>an</strong>d Awakened groups in c-FOS in ei<strong>the</strong>r <strong>the</strong> presence or absence of light. These<br />
results suggest that <strong>the</strong> SCN responds to light during <strong>the</strong> subjective day <strong>an</strong>d that activity, whe<strong>the</strong>r<br />
endogenously org<strong>an</strong>ized or exogenously driven, enh<strong>an</strong>ces this response.<br />
Disclosures: E. Rodriguez, None; J. LeSauter, None; R. Silver, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.5/FF55<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: UMDNJ foundation<br />
Title: Membr<strong>an</strong>e gu<strong>an</strong>ylate cyclase in circadi<strong>an</strong> rhythms<br />
Authors: *V. VENKATARAMAN 1 , R. SWANSON 1 , A. KRISHNAN 1 , T. DUDA 2 ;<br />
1 Cell Biol., UMDNJ, Strat<strong>for</strong>d, NJ; 2 Salus Univ., Elkins Park, PA<br />
Abstract: Cyclic GMP plays a critical role in setting <strong>the</strong> phase in circadi<strong>an</strong> rhythms. However,<br />
<strong>the</strong> mech<strong>an</strong>ism by which it is regulated is unclear. The results <strong>from</strong> this study suggest that<br />
membr<strong>an</strong>e gu<strong>an</strong>ylate cyclase is <strong>the</strong> source <strong>an</strong>d that its activity is regulated by <strong>the</strong> neuronal<br />
calcium sensor protein neurocalcin δ. This would also enable a tight coupling of <strong>the</strong> system to<br />
calcium, which is already <strong>an</strong> established regulator of generation <strong>an</strong>d mainten<strong>an</strong>ce of circadi<strong>an</strong><br />
rhythms.<br />
Disclosures: V. Venkataram<strong>an</strong>, None; R. Sw<strong>an</strong>son, None; A. Krishn<strong>an</strong>, None; T. Duda,<br />
None.<br />
Poster
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.6/FF56<br />
Topic: E.07.e. Monoamine regulators<br />
Title: Free-running behavior, melatonin levels, metabolism, <strong>an</strong>d behavior in rats exposed to<br />
continuous light<br />
Authors: *H. M. MURPHY 1 , C. H. WIDEMAN 2 ;<br />
1 Dept Psychol, 2 Dept Biol., John Carroll Univ., Clevel<strong>an</strong>d, OH<br />
Abstract: Melatonin is a hormone that is syn<strong>the</strong>sized <strong>an</strong>d released <strong>from</strong> <strong>the</strong> pineal gl<strong>an</strong>d. Serum<br />
melatonin levels show a clear-cut circadi<strong>an</strong> rhythm pattern in vertebrates, including hum<strong>an</strong>s <strong>an</strong>d<br />
rodents, <strong>an</strong>d <strong>the</strong> rhythmicity exists regardless of whe<strong>the</strong>r <strong>the</strong> <strong>an</strong>imals have a diurnal or nocturnal<br />
activity pattern. In rats, melatonin secretion is at its peak in <strong>the</strong> dark when <strong>the</strong> <strong>an</strong>imals are<br />
normally active. In contrast to studies that examine <strong>the</strong> effects of melatonin on various<br />
physiological parameters by administering <strong>the</strong> hormone, <strong>the</strong> present study utilized a<br />
methodology that suppresses <strong>the</strong> level of <strong>the</strong> hormone. Three group of rats were studied: 1)<br />
<strong>an</strong>imals subjected to a 12h/12h light/dark cycle (LD), 2) <strong>an</strong>imals exposed to continuous dark<br />
(DD), <strong>an</strong>d 3) <strong>an</strong>imals exposed to continuous light (LL). The presentation of continuous light was<br />
shown to suppress melatonin levels in both <strong>the</strong> subjective light <strong>an</strong>d subjective dark periods of LL<br />
<strong>an</strong>imals; thus, providing a me<strong>an</strong>s of studying <strong>the</strong> effects of lowered melatonin levels on<br />
metabolic, circadi<strong>an</strong> rhythm, <strong>an</strong>d behavioral patterns in rats. In addition to being similar in<br />
melatonin levels, LD <strong>an</strong>d DD <strong>an</strong>imals were similar in food intake, relative food intake, feed<br />
efficiency, water intake, circadi<strong>an</strong> activity levels, <strong>an</strong>d behavior. Compared with LD <strong>an</strong>d DD<br />
<strong>an</strong>imals, LL rats had less food intake, less relative food intake <strong>an</strong>d less water intake. LL <strong>an</strong>imals<br />
also exhibited a more positive feed efficiency <strong>an</strong>d greater visceral adiposity th<strong>an</strong> <strong>the</strong> o<strong>the</strong>r two<br />
groups. The lower level of relative food intake <strong>an</strong>d more positive feed efficiency in LL <strong>an</strong>imals<br />
indicate altered metabolism in LL <strong>an</strong>imals that could have led to increased visceral adiposity.<br />
Circadi<strong>an</strong> rhythmicity of activity became free-running in LL <strong>an</strong>imals <strong>an</strong>d <strong>the</strong>re was a decrease in<br />
overall activity in <strong>the</strong>se <strong>an</strong>imals. The ch<strong>an</strong>ge in activity levels should also be considered as a<br />
factor in <strong>the</strong> development of visceral adiposity. Ano<strong>the</strong>r laboratory has reported that melatonin<br />
acts on <strong>the</strong> nucleus accumbens in <strong>the</strong> brain of rats to increase acetylcholine release <strong>an</strong>d thus<br />
increase motor activity. Consequently, if melatonin levels are low, as in <strong>the</strong> present study,<br />
melatonin would not be present in sufficient qu<strong>an</strong>tity to act on <strong>the</strong> nucleus accumbens to increase<br />
acetylcholine release <strong>an</strong>d activity would decrease. An increase in irritability <strong>an</strong>d excitability were<br />
notable behavioral ch<strong>an</strong>ges observed in LL <strong>an</strong>imals. Results of <strong>the</strong> present study indicate that, in<br />
rats, a decrease in melatonin levels <strong>an</strong>d concomit<strong>an</strong>t ch<strong>an</strong>ges in metabolism, circadi<strong>an</strong> rhythms,<br />
<strong>an</strong>d behavior are consequences of exposure to continuous light.<br />
Disclosures: H.M. Murphy, None; C.H. Widem<strong>an</strong>, None.
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.7/FF57<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t MH079084<br />
Title: Prior daily practice of a sustained attention task during <strong>the</strong> light phase evokes a diurnal<br />
behavioral activity pattern <strong>an</strong>d a task time-synchronized increase in prefrontal cholinergic<br />
neurotr<strong>an</strong>smission<br />
Authors: *T. M. LEE, G. PAOLONE, M. SARTER;<br />
Univ. Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: We recently observed that daily practice of a sustained attention task (SAT) during <strong>the</strong><br />
light phase of <strong>the</strong> light/dark cycle causes a stable, entrained, diurnal behavioral activity pattern<br />
(Gritton et al. <strong>2009</strong>). As SAT per<strong>for</strong>m<strong>an</strong>ce increases cortical acetylcholine (ACh) release, this<br />
experiment assessed <strong>the</strong> influence of <strong>the</strong> prefrontal ACh on SAT practice-induced diurnality.<br />
Circadi<strong>an</strong> behavioral activity was recorded to verify <strong>the</strong> SAT effect on circadi<strong>an</strong> activity, <strong>an</strong>d<br />
prefrontal ACh release was measured, using microdialysis, 3 days following <strong>the</strong> last practice<br />
session. SAT practice occurred ei<strong>the</strong>r during <strong>the</strong> light phase [ZT4] or during <strong>the</strong> dark phase,<br />
[ZT16]. A control group practiced a daily fixed interval 9 s [FI-9] schedule of rein<strong>for</strong>cement at<br />
ZT4. A second control group was h<strong>an</strong>dled at r<strong>an</strong>domly selected times but was nei<strong>the</strong>r waterdeprived<br />
nor per<strong>for</strong>med a task [NP]. Dialysates were collected every 15 min <strong>for</strong> 180 min total,<br />
beginning 90 min be<strong>for</strong>e <strong>the</strong> prior onset of task practice <strong>an</strong>d again during <strong>the</strong> equivalent time<br />
period twelve hours later. For all <strong>an</strong>imals, ACh release was higher during <strong>the</strong> dark phase when<br />
compared with <strong>the</strong> light period. Fur<strong>the</strong>rmore, in previously SAT-per<strong>for</strong>ming <strong>an</strong>imals, ACh levels<br />
increased <strong>for</strong> 45 min at ZT4 <strong>an</strong>d ZT16. Collectively <strong>the</strong>se results indicate that <strong>the</strong> diurnal activity<br />
pattern that results <strong>from</strong> SAT practice during <strong>the</strong> light phase is not mediated via global<br />
alterations in <strong>the</strong> circadi<strong>an</strong> regulation of ACh release. However, prior practice of <strong>the</strong> SAT<br />
established a stable increase in ACh release that lasted as long as <strong>the</strong> prior SAT sessions, <strong>an</strong>d this<br />
task time-synchronized increase in prefrontal cholinergic activity may contribute to <strong>the</strong> induction<br />
or mainten<strong>an</strong>ce of diurnality in ZT4 <strong>an</strong>imals. Future research is testing whe<strong>the</strong>r removal of<br />
cholinergic neurons interferes with ZT4 SAT practice-induced diurnality. Fur<strong>the</strong>rmore, it will be<br />
import<strong>an</strong>t to determine <strong>the</strong> time point after cessation of SAT practice at which ZT4 <strong>an</strong>imals<br />
reverse to a nocturnal pattern <strong>an</strong>d whe<strong>the</strong>r this coincides with <strong>the</strong> loss of task time-synchronized<br />
cholinergic activity. This research provides new insights in underst<strong>an</strong>ding cognitive work-
induced shifts in circadi<strong>an</strong> rhythms, <strong>the</strong> cause <strong>an</strong>d role of circadi<strong>an</strong> abnormalities in<br />
neuropsychiatric disorders, <strong>an</strong>d it eventually will in<strong>for</strong>m <strong>the</strong> development of treatments of such<br />
disorders.<br />
Disclosures: T.M. Lee, None; G. Paolone, None; M. Sarter, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.8/FF58<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NEI (CLC)<br />
NHLBI (MUG)<br />
Title: Ca2+ signaling induced by glutamate in rat suprachiasmatic nucleus neurons<br />
Authors: *T. WANG 1,2 , G. GOVINDAIAH 2,3 , C. L. COX 2,3 , M. U. GILLETTE 2,4,3 ;<br />
2 3 4 1<br />
Mol. <strong>an</strong>d Integrative Physiol., Neurosci. Program, Cell <strong>an</strong>d Developmental Biol., Univ. of<br />
Illinois at Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a, IL<br />
Abstract: Mammals’ daily rhythms of physiology <strong>an</strong>d behavior are synchronized to day <strong>an</strong>d<br />
night by <strong>the</strong> action of <strong>the</strong> signal of environmental light on suprachiasmatic nucleus (SCN).<br />
Glutamate (Glu) is <strong>the</strong> primary neurotr<strong>an</strong>smitter that conveys photic in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> retina<br />
to SCN. The effect on SCN state is specific to <strong>the</strong> time of day: in early night (CT 14), this signal<br />
induces phase-delay of <strong>the</strong> circadi<strong>an</strong> clock, but phase-adv<strong>an</strong>ces it in late night (CT 19). Ca2+<br />
plays a pivotal role in Glu signaling <strong>an</strong>d is a key second messenger, mediating signal specificity<br />
by its complex spatial-temporal distribution. Using two-photon laser microscopy <strong>an</strong>d whole-cell<br />
patch-clamp recording, we studied neuronal Ca2+ signaling in <strong>the</strong> rat SCN brain slice. We find<br />
that at CT 14 Glu induces a tr<strong>an</strong>sient increase in Ca2+i (amplitude: 20%-30%, duration 40-50 s,<br />
n=20, p
Ca2+i leak rate through RyR in vivo. The RyR-mediated Ca2+i release is much more sensitive to<br />
excitatory stimulation at CT 14 th<strong>an</strong> CT 19 (3 fold, n=21, p
MTEP = 1.7 ± 0.1 hr; veh = 1.3 ± 0.2 h vs 10 mg/kg fenobam = 1.6 ± 0.1 h, both p < 0.05,<br />
ANOVA. In contrast, <strong>the</strong> group II mGluR <strong>an</strong>tagonist LY341,495 (0.6 - 10 mg/kg) did not<br />
signific<strong>an</strong>tly affect light-induced phase adv<strong>an</strong>ces. Since mGluR5 <strong>an</strong>tagonists also have efficacy<br />
as <strong>an</strong>xiolytics in <strong>an</strong>imal models, we tested <strong>the</strong> ability of MTEP <strong>an</strong>d fenobam to reduce <strong>an</strong>xiety in<br />
<strong>the</strong> Thatcher-Britton Conflict test. As a validation of this test in hamsters, diazepam (2 mg/kg)<br />
reduced <strong>the</strong> latency to eat in <strong>an</strong> open field <strong>from</strong> 101 ± 17 s following vehicle injections to 22 ± 5<br />
s, <strong>an</strong>d this dose of diazepam did not signific<strong>an</strong>tly affect locomotion levels. In contrast, MTEP (10<br />
mg/kg) non-signific<strong>an</strong>tly leng<strong>the</strong>ned <strong>the</strong> time to eat <strong>from</strong> 28 ± 8 s following vehicle injections to<br />
48 ± 13 s, <strong>an</strong>d fenobam likewise leng<strong>the</strong>ned <strong>the</strong> time to eat <strong>from</strong> 104 ± 22 s following vehicle<br />
injections to 130 ± 19 s. There<strong>for</strong>e, nei<strong>the</strong>r MTEP nor fenobam displayed <strong>an</strong>y <strong>an</strong>xiolytic activity<br />
in <strong>the</strong> Thatcher-Britton Conflict test. These results do however; support a role <strong>for</strong> mGluR5<br />
receptors in modulating <strong>the</strong> photic entrainment of circadi<strong>an</strong> activity rhythms in <strong>the</strong> hamster.<br />
Supported by NSF IOB 05499880 (RLG).<br />
Disclosures: R.L. G<strong>an</strong>non, NSF, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Servier, C. O<strong>the</strong>r Research<br />
Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); E.A. Lungwitz,<br />
None; N. Batista, None; M.J. Mill<strong>an</strong>, Servier, A. Employment (full or part-time).<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.10/FF60<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: SIU School of Medicine Clinici<strong>an</strong> Scientist Award<br />
Title: Diurnal variation in NMDA receptor subunits <strong>an</strong>d pro-survival factors in <strong>the</strong> hippocampus<br />
Authors: S. W. KARMARKAR 1 , *K. M. BOTTUM 2 , S. A. TISCHKAU 1 ;<br />
1 Pharmacol., 2 SIU Sch. Med., Springfield, IL<br />
Abstract: Ischemic stroke is a major cause of death <strong>an</strong>d disability. Hum<strong>an</strong>s exhibit diurnal<br />
susceptibility to stroke, with increased incidence during <strong>the</strong> early morning. This laboratory has<br />
demonstrated diurnal variation in neuronal damage incurred by ischemic insult. Neuronal death<br />
after stroke involves N-methyl-D-aspartate receptor (NMDAR) overactivation, with subunit<br />
specific effects. This study was designed to test <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong>re is diurnal variation in 1)<br />
NMDAR subunit expression <strong>an</strong>d 2) downstream signaling events associated with
neuroprotection or neuronal death. 1 month old Sprague-Dawley rats were entrained to a 12:12<br />
light/dark cycle <strong>an</strong>d sacrificed at 4h intervals between ZT0 <strong>an</strong>d ZT20 (ZT=Zeitgeber time, i.e.<br />
time after lights come on). Hippocampi were dissected <strong>an</strong>d stored in RNA later at -80°C. RNA<br />
was isolated by Trizol extraction. NMDAR subunits (NR1, NR2A, NR2B, NR2C <strong>an</strong>d NR2D),<br />
neuritin <strong>an</strong>d Bcl2 tr<strong>an</strong>scripts were qu<strong>an</strong>tified using qu<strong>an</strong>titative PCR. NR1 <strong>an</strong>d NR2D tr<strong>an</strong>script<br />
levels did not ch<strong>an</strong>ge over <strong>the</strong> 24 h period. NR2A was elevated at ZT4 <strong>an</strong>d ZT16. NR2B <strong>an</strong>d<br />
NR2C peaked at ZT8. Bcl2 <strong>an</strong>d neuritin peaked at ZT4. Bcl2 is a well studied pro-survival<br />
factor. Neuritin is required <strong>for</strong> axon arbor elaboration <strong>an</strong>d is implicated in synaptic plasticity.<br />
Circadi<strong>an</strong> variation in stroke susceptibility may involve NMDAR subunit variation with time-ofday<br />
<strong>an</strong>d <strong>the</strong> downstream pathways activated. More data is required to firmly establish time-ofday<br />
variation in NMDAR subunits <strong>an</strong>d downstream pathways be<strong>for</strong>e ultimately developing a<br />
suitable NMDAR <strong>an</strong>tagonist <strong>for</strong> <strong>the</strong>rapeutic use in stroke.<br />
Disclosures: S.W. Karmarkar, None; K.M. Bottum, None; S.A. Tischkau, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.11/FF61<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: UT small gr<strong>an</strong>t<br />
Title: tPA modulates glutamate-induced phase shifts of <strong>the</strong> mouse suprachiasmatic circadi<strong>an</strong><br />
clock in vitro<br />
Authors: *X. MOU, J. LAIRD, C. PETERSON, R. PROSSER;<br />
Univ. Tennes-Knoxville, Knoxville, TN<br />
Abstract: Mammali<strong>an</strong> circadi<strong>an</strong> rhythms are controlled by a clock located in <strong>the</strong><br />
suprachiasmatic nucleus (SCN). The mech<strong>an</strong>isms through which light phase-shifts <strong>the</strong> SCN<br />
circadi<strong>an</strong> clock are similar to those underlying memory <strong>for</strong>mation <strong>an</strong>d long-term potentiation<br />
(LTP). Several secreted proteins, including tissue plasminogen activator (tPA), plasminogen, <strong>an</strong>d<br />
brain-derived neurotrophic factor (BDNF), have been implicated in this process. tPA<br />
enzymatically converts plasminogen into plasmin, which in turn converts pro-BDNF into<br />
m(ature) BDNF. mBDNF activation of its TrkB receptor is critical <strong>for</strong> LTP expression. BDNF is<br />
also necessary <strong>for</strong> photic phase-shifts of <strong>the</strong> SCN circadi<strong>an</strong> clock. We have shown tPA, plasmin<br />
<strong>an</strong>d BDNF modulate glutamate-induced phase delays during early night in mouse SCN in vitro.
Here we investigated <strong>the</strong>ir roles in glutamate-induced phase adv<strong>an</strong>ces during late night. For <strong>the</strong>se<br />
experiments we prepared acute mouse brain slices containing <strong>the</strong> SCN. We monitored clock<br />
phase through extracellular recordings of SCN neuronal activity. SCN neurons exhibit a 24 h<br />
rhythm in neuronal activity that continues in vitro. Neurochemical stimuli applied in vitro reset<br />
<strong>the</strong> SCN clock in a m<strong>an</strong>ner that mimics <strong>the</strong>ir effects in vivo. Here we show that glutamate<br />
applied to SCN slices during <strong>the</strong> late night adv<strong>an</strong>ces <strong>the</strong> neuronal activity rhythm by 3 h. These<br />
phase adv<strong>an</strong>ces are blocked by <strong>the</strong> tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1).<br />
Future experiments will assess whe<strong>the</strong>r plasmin activation of BDNF is necessary <strong>for</strong> glutamateinduced<br />
phase adv<strong>an</strong>ces.<br />
We also investigated endogenous glutamate-induced modulation of tPA levels in SCN tissue. We<br />
have demonstrated <strong>the</strong> presence of vitronectin, PAI-1, plasminogen, tPA <strong>an</strong>d BDNF in protein<br />
extracts <strong>from</strong> mouse SCN tissue collected at Zeitgeber time (ZT) 16. Using western-blot <strong>an</strong>alyses<br />
to assay <strong>the</strong> circadi<strong>an</strong> expression pattern of tPA in <strong>the</strong> SCN, we find higher tPA levels in <strong>the</strong><br />
night vs. <strong>the</strong> day. Also, 10 min glutamate treatment at ZT16 increases tPA levels to ~2.5 times<br />
control levels (n=5), while treatment at ZT23 <strong>an</strong>d ZT6 did not alter tPA expression. These results<br />
indicate that glutamate modulates tPA expression in <strong>the</strong> SCN during early night. These ch<strong>an</strong>ges<br />
may be part of <strong>the</strong> mech<strong>an</strong>ism through which glutamate resets SCN clock phase.<br />
Disclosures: X. Mou, None; J. Laird, None; C. Peterson, None; R. Prosser, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.12/FF62<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t K99 GM086683 (KLG)<br />
NIH Gr<strong>an</strong>t P50 MH078028 (DGM)<br />
Title: Fast delayed rectifier potassium current underlies photic signaling via gastrin releasing<br />
peptide within <strong>the</strong> mammali<strong>an</strong> circadi<strong>an</strong> clock<br />
Authors: *K. L. GAMBLE, D. G. MCMAHON;<br />
Dept Biol Sci., V<strong>an</strong>derbilt Univ., Nashville, TN<br />
Abstract: The mammali<strong>an</strong> circadi<strong>an</strong> clock in <strong>the</strong> suprachiasmatic nucleus (SCN) is not only<br />
responsible <strong>for</strong> driving <strong>an</strong>d maintaining 24-h rhythms in physiology, but also <strong>for</strong> resetting <strong>the</strong>
phase to one that is consistent with <strong>the</strong> local environmental light-dark cycle. We have previously<br />
shown that <strong>an</strong> intra-SCN neuropeptide, gastrin releasing peptide (GRP), communicates latenight,<br />
photic phase resetting signals within <strong>the</strong> SCN network by inducing a persistent increase in<br />
neurophysiological activity of SCN neurons. Here, we investigated <strong>the</strong> ionic basis <strong>for</strong> GRPinduced<br />
ch<strong>an</strong>ges in neuronal activity. SCN slice cultures <strong>from</strong> <strong>the</strong> Per1::GFP reporter mouse<br />
model were prepared during <strong>the</strong> late day <strong>an</strong>d treated <strong>for</strong> 1 hour with GRP or vehicle during <strong>the</strong><br />
projected early night. Loose patch extracellular recordings of Per1-fluorescent neurons <strong>from</strong><br />
GRP-treated slices three hours after GRP treatment revealed a signific<strong>an</strong>tly greater action<br />
potential frequency th<strong>an</strong> those <strong>from</strong> vehicle-treated slices. Voltage clamp recordings <strong>from</strong> GRPtreated<br />
cells showed a signific<strong>an</strong>t increase in voltage-activated outward current at depolarized<br />
potentials compared to vehicle-treated cells. Itri, et al (2005) showed a clock-regulated increase<br />
in <strong>the</strong> fast delayed rectifier (FDR) current within SCN neurons that accomp<strong>an</strong>ies elevated spike<br />
rates during <strong>the</strong> day. Isolation of FDR currents in GRP-treated, Per1::GFP expressing neurons<br />
revealed a signific<strong>an</strong>t increase in FDR current that persisted hours after GRP washout when<br />
compared to vehicle-treated, Per1::GFP expressing neurons. These results support <strong>the</strong> hypo<strong>the</strong>sis<br />
that GRP delays <strong>the</strong> phase of <strong>the</strong> clock during <strong>the</strong> early night by prolonging day-like membr<strong>an</strong>e<br />
properties of SCN cells. Fur<strong>the</strong>rmore, <strong>the</strong>se findings implicate FDR currents in <strong>the</strong> ionic basis <strong>for</strong><br />
photic entrainment of <strong>the</strong> primary mammali<strong>an</strong> pacemaker.<br />
Disclosures: K.L. Gamble, None; D.G. McMahon, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.13/FF63<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t T32 MH/AG-19957<br />
NIH Gr<strong>an</strong>t 1P30DA018310<br />
NIH Gr<strong>an</strong>t HL67007<br />
Title: Functional neuropeptidomics: Exploring peptide communication of phase-resetting cues in<br />
<strong>the</strong> rat circadi<strong>an</strong> clock through mass spectrometry <strong>an</strong>d physiology<br />
Authors: *N. ATKINS, Jr. 1 , S. REN 2 , N. G. HATCHER 2 , J. LEE 3 , D. MORGAN 4 , T.<br />
COMINSKI 4 , J. E. PINTAR 4 , N. L. KELLEHER 3 , J. V. SWEEDLER 1 , M. U. GILLETTE 1 ;<br />
1 Neurosci Prgm, 2 Beckm<strong>an</strong> Inst. <strong>for</strong> Adv<strong>an</strong>ced Sci. <strong>an</strong>d Technol., 3 Dept. of Chem., Univ. Illinois<br />
Urb<strong>an</strong>a Champaign, Urb<strong>an</strong>a, IL; 4 Dept. of Cell Biol. <strong>an</strong>d Neurosci., Univ. of Med. <strong>an</strong>d Dent. of<br />
New Jersey, Newark, NJ<br />
Abstract: Peptides have well known physiological roles in neuromodulation <strong>an</strong>d<br />
neurotr<strong>an</strong>smission. Recent adv<strong>an</strong>ces in mass spectrometry-based peptidomic strategies have<br />
enabled researchers to investigate more exhaustive profiles of neuropeptide expression <strong>an</strong>d<br />
release under physiological conditions. In <strong>the</strong> suprachiasmatic nucleus (SCN), <strong>the</strong> central<br />
mammali<strong>an</strong> circadi<strong>an</strong> clock, peptides have signific<strong>an</strong>t influences on communication of<br />
timekeeping signals. Using a combination of strategies, we evaluated <strong>the</strong> dynamic properties of<br />
<strong>the</strong> suites of neuropeptides within <strong>the</strong> rat SCN clock. Fourier tr<strong>an</strong>s<strong>for</strong>m mass spectrometry<br />
(FTMS) <strong>an</strong>alysis of whole-tissue peptide extracts confirmed <strong>the</strong> identity of over 100 peptide <strong>an</strong>d<br />
prohormone fragments in <strong>the</strong> SCN. A second strategy, employing matrix-assisted laser<br />
desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with<br />
electrical stimulation of SCN afferent nerve fibers, demonstrated that time-of-day- <strong>an</strong>d stimulusspecific<br />
peptide release <strong>from</strong> in vitro SCN corresponds with clock phase-resetting. These peptide<br />
signatures included both reported <strong>an</strong>d unreported SCN neuropeptides. One peptide, little SAAS,<br />
was selected <strong>for</strong> fur<strong>the</strong>r characterization. Little SAAS immunohisochemistry reveals localization<br />
to <strong>the</strong> SCN region implicated in relay <strong>an</strong>d processing of light in<strong>for</strong>mation. Electrophysiological<br />
studies of SCN tissue in vitro suggest a functional role <strong>for</strong> <strong>the</strong> little SAAS peptide in circadi<strong>an</strong><br />
clock regulation. Exogenous little SAAS is capable of shifting <strong>the</strong> phase of rhythmic firing rates<br />
of SCN brain slices in a dose- <strong>an</strong>d time-of-day-dependent m<strong>an</strong>ner. Glutamate-induced phase<br />
shifting is blocked by exogenous treatment of <strong>the</strong> SCN slice with little SAAS <strong>an</strong>tiserum. Little<br />
SAAS peptide-induced phase shifting is not blocked by NMDA receptor <strong>an</strong>tagonist APV. These<br />
approaches to exploring peptidomics of <strong>the</strong> SCN present a promising template <strong>for</strong> exp<strong>an</strong>ding<br />
underst<strong>an</strong>ding of peptidergic function in physiological function <strong>an</strong>d dysfunction.
Disclosures: N. Atkins, None; S. Ren, None; N.G. Hatcher, None; J. Lee, None; D. Morg<strong>an</strong>,<br />
None; T. Cominski, None; J.E. Pintar, None; N.L. Kelleher, None; J.V. Sweedler,<br />
None; M.U. Gillette, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.14/FF64<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: FRSQ Fellowship<br />
Title: Ghrelin receptor knock-out mice display alterations in circadi<strong>an</strong> rhythms<br />
Authors: *E. WADDINGTON LAMONT 1 , I. D. BLUM 2 , A. ABIZAID 2 ;<br />
1 Dept. of Psychology, Inst. of Neuroscience, Carleton Univ., Ottawa, ON, C<strong>an</strong>ada; 2 Inst. of<br />
Neuroscience, Dept. of Psychology, Carleton Univ., Ottawa, ON, C<strong>an</strong>ada<br />
Abstract: The 24-hour rhythms of hormones, enzymes, stomach motility, <strong>an</strong>d food seeking<br />
behaviors are physiological processes governed by <strong>the</strong> master circadi<strong>an</strong> clock, located in <strong>the</strong><br />
suprachiasmatic nucleus (SCN) of <strong>the</strong> hypothalamus. However, temporal restriction of food<br />
availability to <strong>the</strong> day, or “inactive period” of nocturnal rodents, has been shown to have a robust<br />
effect on circadi<strong>an</strong> rhythms, inducing <strong>an</strong> increase in activity in <strong>an</strong>ticipation of <strong>the</strong> daily meal <strong>an</strong>d<br />
synchronizing molecular clock markers in peripheral org<strong>an</strong>s <strong>an</strong>d brain regions outside of <strong>the</strong><br />
master circadi<strong>an</strong> clock. These effects occur independently of <strong>the</strong> SCN, but may have <strong>an</strong> influence<br />
on <strong>the</strong> master clock. A role <strong>for</strong> <strong>the</strong> orexigenic stomach peptide ghrelin has been postulated in <strong>the</strong><br />
synchronization of <strong>the</strong> circadi<strong>an</strong> system by meal timing. For this reason, we investigated <strong>the</strong><br />
circadi<strong>an</strong> phenotype of ghrelin receptor knock-out mice (GHSR KO) <strong>an</strong>d wild-type controls<br />
(WT). Both groups showed normal synchronization of running-wheel activity to a 24-hour day<br />
when on a 12-hour light-dark cycle <strong>an</strong>d no obvious differences were observed in <strong>the</strong> time<br />
required to adapt to <strong>an</strong> acute phase adv<strong>an</strong>ce or delay of <strong>the</strong> light-dark cycle. The circadi<strong>an</strong> free<br />
running periods under conditions of const<strong>an</strong>t darkness were similar <strong>for</strong> both groups. However,<br />
under conditions of const<strong>an</strong>t light, both groups showed a leng<strong>the</strong>ning of <strong>the</strong> period, consistent<br />
with previous observations in mice, but GHSR KOs showed <strong>an</strong> average period that was about 30<br />
minutes longer th<strong>an</strong> that of <strong>the</strong>ir wild-type counterparts. This provides fur<strong>the</strong>r evidence that<br />
ghrelin c<strong>an</strong> modulate <strong>the</strong> effect of light on <strong>the</strong> master circadi<strong>an</strong> clock.<br />
Disclosures: E. Waddington Lamont, None; I.D. Blum, None; A. Abizaid, None.
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.15/FF65<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t NS057583<br />
AFOSR 07NL172<br />
Title: Relationship between <strong>the</strong> phase <strong>an</strong>gle of entrainment <strong>an</strong>d <strong>the</strong> free running period in rats<br />
Authors: *L. S. ZHANG, C. HART, T. LIU, S. AHMED, J. BORJIGIN;<br />
Univ. Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Animals have intrinsic circadi<strong>an</strong> periods of close to 24 hours. In order to synchronize<br />
to <strong>the</strong> 24-hour day, circadi<strong>an</strong> rhythms adapt or entrain to external time cues <strong>an</strong>d establish a<br />
consistent daily rhythm. The mainten<strong>an</strong>ce of a stable phase relationship between circadi<strong>an</strong><br />
rhythms <strong>an</strong>d external time cues, such as <strong>the</strong> light-dark cycle, is necessary <strong>for</strong> entrainment to<br />
occur. It is commonly thought that <strong>the</strong>re is a fixed relationship between timing of entrained<br />
rhythms (or <strong>the</strong> phase <strong>an</strong>gle of entrainment) <strong>an</strong>d <strong>the</strong> intrinsic free-running period, where <strong>an</strong><br />
earlier phase <strong>an</strong>gle reflects a shorter period. We reevaluated this relationship in rats. In our<br />
studies, <strong>the</strong> phase <strong>an</strong>gle of entrainment was determined by monitoring melatonin rhythms<br />
entrained in light-dark cycle of 12:12h, <strong>an</strong>d defined as <strong>the</strong> precise time difference between dark<br />
onset, <strong>an</strong>d <strong>the</strong> melatonin onset or offset, whereas <strong>the</strong> period was determined in <strong>the</strong> same <strong>an</strong>imals<br />
<strong>from</strong> both temperature <strong>an</strong>d locomotor activity rhythms after <strong>an</strong>imals were released into complete<br />
darkness. Using in vivo pineal microdialysis, we found that melatonin onset <strong>an</strong>d offset timing did<br />
not correlate with each o<strong>the</strong>r. Fur<strong>the</strong>rmore, <strong>the</strong> entrained melatonin duration was associated with<br />
melatonin onset, but not offset. Most import<strong>an</strong>tly, <strong>the</strong> phase <strong>an</strong>gle <strong>from</strong> ei<strong>the</strong>r melatonin onset or<br />
offset did not correlate signific<strong>an</strong>tly with <strong>the</strong> free-running period <strong>from</strong> temperature or activity<br />
rhythms. There was also no apparent relationship found between <strong>the</strong> entrained melatonin<br />
duration <strong>an</strong>d <strong>the</strong> free-running period <strong>from</strong> temperature or activity. These results demonstrate that<br />
<strong>the</strong> phase <strong>an</strong>gle obtained <strong>from</strong> melatonin rhythms in entrained rats do not exhibit a fixed<br />
relationship to <strong>the</strong> free-running period. As <strong>the</strong> light-dark cycle is <strong>the</strong> domin<strong>an</strong>t time cue used in<br />
nature to entrain to <strong>the</strong> 24-h day, this finding has import<strong>an</strong>t implications <strong>for</strong> underst<strong>an</strong>ding <strong>the</strong><br />
physiological org<strong>an</strong>ization of <strong>the</strong> circadi<strong>an</strong> timing system.
Disclosures: L.S. Zh<strong>an</strong>g , None; C. Hart, None; T. Liu, None; S. Ahmed, None; J. Borjigin,<br />
None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.16/FF66<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: PHS gr<strong>an</strong>t P30 DA 018310<br />
Title: Clock-to-clock coupling of SCN <strong>an</strong>d non-SCN cells by diffusible factors<br />
Authors: *J. W. MITCHELL 1 , K. E. WEIS 1 , H. ROSENBERG 2 , N. G. HATCHER 3 , N.<br />
ATKINS, Jr. 4 , J. V. SWEEDLER 3 , M. U. GILLETTE 1 ;<br />
1 Dept. of Cell <strong>an</strong>d Developmental Biol., 2 Dept. of Mol. <strong>an</strong>d Intergrative Physiol., 3 Dept. of<br />
Chem., 4 Neurosci. Program, Univ. of Illinois At Urb<strong>an</strong>a-Champaign, Urb<strong>an</strong>a, IL<br />
Abstract: The suprachiasmatic nucleus (SCN) is <strong>the</strong> body’s central circadi<strong>an</strong> clock <strong>an</strong>d is<br />
involved in synchronizing a myriad of peripheral clocks in unknown ways. Loss of coordination<br />
between <strong>the</strong> central circadi<strong>an</strong> clock in <strong>the</strong> hypothalamic SCN <strong>an</strong>d non-SCN clocks has been<br />
implicated in increased incidences of metabolic disorders, cardiovascular disease, c<strong>an</strong>cer, <strong>an</strong>d<br />
sleep disorders. Studies of central nervous system <strong>an</strong>d peripheral cells <strong>an</strong>d tissues cultured in<br />
isolation have revealed that in <strong>the</strong> SCN’s absence, cellular rhythms continue, but <strong>the</strong>y become<br />
desynchronized within <strong>the</strong> population as phase <strong>an</strong>d period properties ch<strong>an</strong>ge. Such SCNentrainable<br />
targets include <strong>the</strong> paraventricular nucleus (PVN) <strong>an</strong>d cortical glia <strong>from</strong> brain, as<br />
well as liver, lung, muscle, kidney <strong>an</strong>d tail fibroblasts <strong>from</strong> <strong>the</strong> periphery. With decoupling, <strong>the</strong><br />
various tissues <strong>an</strong>d cells lose temporal coherence, as well as appropriate alignment to <strong>the</strong> daily<br />
cycle of sleep <strong>an</strong>d wakefulness. Little is known about what couples <strong>an</strong> org<strong>an</strong>ism’s circadi<strong>an</strong><br />
clocks, except that m<strong>an</strong>y c<strong>an</strong> be entrained by diffusible factor(s). Here we show that org<strong>an</strong>otypic<br />
cultures of SCN brain slices are capable of synchronizing <strong>an</strong>d entraining rhythms in heart<br />
fibroblasts <strong>an</strong>d cortical glia, when separated in co-culture by a MilliCell-CM membr<strong>an</strong>e.<br />
Induction of circadi<strong>an</strong> rhythms is assessed via expression of clock gene tr<strong>an</strong>scripts. Phasing of<br />
<strong>the</strong> peaks of Per2 <strong>an</strong>d Bmal1 expression in non-SCN brain <strong>an</strong>d peripheral cells is dependent on<br />
<strong>the</strong> phase of <strong>the</strong> SCN, as determined <strong>from</strong> spont<strong>an</strong>eous peak electrical activity of SCN brain<br />
slices prepared <strong>from</strong> alternate lighting schedules. Based on <strong>the</strong> results of <strong>the</strong>se<br />
SCN:fibroblast/glia co-cultures, we have undertaken peptidomic <strong>an</strong>alysis of <strong>the</strong> releasate <strong>from</strong>
SCN cultures. We have identified a number of known <strong>an</strong>d unknown peptides released <strong>from</strong> <strong>the</strong><br />
SCN that provide a basis <strong>for</strong> discovering c<strong>an</strong>didate coupling factors.<br />
Disclosures: J.W. Mitchell, None; K.E. Weis, None; H. Rosenberg, None; N.G. Hatcher,<br />
None; N. Atkins, None; J.V. Sweedler, None; M.U. Gillette, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.17/FF67<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: CIHR<br />
NSERC<br />
CURC<br />
Title: Recovery of PER2 expression in limbic <strong>for</strong>ebrain <strong>an</strong>d striatum following morphine<br />
withdrawal: Effects of withdrawal time <strong>an</strong>d quinpirole injection<br />
Authors: *S. J. HOOD, P. CASSIDY, J. STEWART, S. AMIR;<br />
Concordia Univ., Montreal, QC, C<strong>an</strong>ada<br />
Abstract: Disturb<strong>an</strong>ces in circadi<strong>an</strong> rhythms are implicated in pathologies of motivation <strong>an</strong>d<br />
emotion, including drug abuse. For example, m<strong>an</strong>ipulations of clock gene expression c<strong>an</strong><br />
dramatically alter behavioral responses to drugs of abuse in drosophila <strong>an</strong>d rodents (Abarca et<br />
al., 2002; Roybal et al., 2007). Fur<strong>the</strong>rmore, chronic exposure to <strong>an</strong>d withdrawal <strong>from</strong> drugs of<br />
abuse disrupt circadi<strong>an</strong> regulation of activity <strong>an</strong>d normal rhythmic expression of clock genes in<br />
rodents <strong>an</strong>d hum<strong>an</strong>s (Kosobud et al., 1998; Nikado et al., 2001; Li et al., 2008; Li et al., <strong>2009</strong>).<br />
We reported previously that <strong>the</strong> normal rhythm of expression of <strong>the</strong> clock protein PER2 in <strong>the</strong> rat<br />
<strong>for</strong>ebrain is altered during daily injections of morphine (5-40 mg/kg, i.p.) <strong>an</strong>d during <strong>the</strong> early<br />
phase of withdrawal (46-58 h after last injection). In withdrawal, <strong>the</strong> peak of PER2 expression<br />
normally seen in <strong>the</strong> dorsal striatum <strong>an</strong>d basolateral amygdala (BLA) at zeitgeber time (ZT) 1 is<br />
strongly reduced <strong>an</strong>d <strong>the</strong> peak normally seen in <strong>the</strong> bed nucleus of <strong>the</strong> stria terminalis (BNSTov)<br />
<strong>an</strong>d <strong>the</strong> central nucleus of <strong>the</strong> amygdala (CEA) at ZT 13 is also blunted. In <strong>the</strong> suprachiasmatic<br />
nucleus (SCN), no ch<strong>an</strong>ges are seen during withdrawal <strong>from</strong> morphine. Here, we show that<br />
although <strong>the</strong> normal rhythm of PER2 expression returns in dorsal striatum, <strong>an</strong>d BLA after 7 days
of withdrawal <strong>from</strong> morphine, <strong>the</strong> blunted PER2 rhythm persists in BNSTov <strong>an</strong>d CEA after 7<br />
days of withdrawal. Because opiate withdrawal is associated with a profound reduction in<br />
mesolimbic dopamine activity (Rosetti et al., 1992), we attempted to restore normal PER2<br />
expression in early withdrawal by stimulating dopamine D2 receptors with injections of <strong>the</strong> D2<br />
dopamine receptor agonist, quinpirole (0.5 mg/kg, i.p.), given at ZT 13 <strong>for</strong> two days beginning<br />
after <strong>the</strong> last morphine injection. Quinpirole maintained PER2 expression in BNSTov <strong>an</strong>d CEA<br />
at ZT 13 at levels seen in morphine-naïve control rats. These injections of quinpirole did not,<br />
however, affect <strong>the</strong> reductions of PER2 seen in dorsal striatum <strong>an</strong>d BLA. Surprisingly,<br />
quinpirole reduced PER2 expression at ZT 1 in <strong>the</strong>se latter regions in morphine-naïve rats. Taken<br />
toge<strong>the</strong>r, <strong>the</strong>se results suggest that <strong>the</strong> recovery of PER2 expression over <strong>an</strong> extended period of<br />
morphine withdrawal differs as a function of <strong>for</strong>ebrain region <strong>an</strong>d that different regions are<br />
differentially sensitive to D2 dopamine receptor stimulation.<br />
Disclosures: S.J. Hood, None; P. Cassidy, None; J. Stewart, None; S. Amir, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.18/FF68<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t MH067752<br />
DoD FA9550-05-1-0464<br />
Title: A QTL <strong>for</strong> early activity in a mouse model of adv<strong>an</strong>ced sleep phase syndrome<br />
Authors: P. JIANG 1 , M. STRIZ 1 , K. SHIMOMURA 2 , J. S. TAKAHASHI 3 , J. P. WISOR 4 , *B.<br />
F. O'HARA 1 ;<br />
1 Dept. of Biol., Univ. of Kentucky, Lexington, KY; 2 Ctr. <strong>for</strong> Functional Genomics <strong>an</strong>d Dept. of<br />
Neurobio. <strong>an</strong>d Physiol., Northwestern Univ., Ev<strong>an</strong>ston, IL; 3 Howard Hughes Med. Institute,<br />
Dept. of Neurosci., Univ. of Texas Southwestern Med. Ctr. at Dallas, Dallas, TX; 4 Dept. of Vet.<br />
Comparative Anatomy, Pharmacol. <strong>an</strong>d Physiol. <strong>an</strong>d Med. Educ. Program, Washington State<br />
Univ., Spok<strong>an</strong>e, WA<br />
Abstract: The adaptive values of circadi<strong>an</strong> rhythms require <strong>the</strong> endogenously generated rhythms<br />
to <strong>for</strong>m stable phase relations to cycles of <strong>the</strong> environmental timing cues. In extreme cases such<br />
as adv<strong>an</strong>ced or delayed sleep phase syndrome (ASPS or DSPS), shift work, or jet lag, <strong>the</strong> phase
<strong>an</strong>gle of entrainment may be incompatible with work requirements or o<strong>the</strong>r social dem<strong>an</strong>ds.<br />
Recently, we reported “early-runner” behavior, similar to hum<strong>an</strong> ASPS, in a subset of mice<br />
derived <strong>from</strong> a backcross of two genetically diverse strains - CAST/EiJ (CE) x C57BL/6J (B6).<br />
These “early-runners” typically initiate wheel-running 4-6 hours prior to dark onset, whereas<br />
most mice begin at dark. We identified a QTL on chromosome 18 that accounts <strong>for</strong> a subst<strong>an</strong>tial<br />
portion of <strong>the</strong> vari<strong>an</strong>ce in this trait. Here, we tested similar mice under a wider r<strong>an</strong>ge of<br />
conditions. <strong>When</strong> entrained under 100-lux green light, all B6 mice displayed normal activity<br />
onsets at dark, while CE mice displayed activity onsets about 3 hours earlier. This was observed<br />
in both wheel-running activity <strong>an</strong>d by percentage of sleep-wake using a validated piezoelectric<br />
technology. The adv<strong>an</strong>ced activity phase has been consistently observed in three independent<br />
laboratories. Upon release to const<strong>an</strong>t dark (DD), all mice maintained <strong>the</strong> phase determined by<br />
<strong>the</strong> prior LD cycle. Under 12:12LD conditions, unlike B6 mice, wheel-running activities in CE<br />
mice were not inhibited by a 1-hour light pulse at ZT12.5 when 100-lux green light was used, but<br />
were moderately suppressed when white florescent light was used. This result indicates <strong>an</strong><br />
impaired light masking response in CE mice. Moreover, <strong>the</strong> PRC was phase-lagged by about 3<br />
hrs relative to <strong>the</strong> daily activity onset in CE mice, which suggests that <strong>the</strong> endogenous pacemaker<br />
may also be phase-lagged relative to <strong>the</strong> behavioral rhythm in <strong>the</strong>se mice. To confirm <strong>an</strong>d finemap<br />
<strong>the</strong> chromosome 18 QTL, we obtained 3 strains of B6.CE.18 GTM mice <strong>from</strong> UCLA,<br />
which contain CE segments introgressed on proximal, middle <strong>an</strong>d distal portions of chromosome<br />
18, respectively. The earlier phase <strong>an</strong>gle of entrainment in B6.CE.18P <strong>an</strong>d B6.CE.18M mice<br />
confirmed <strong>the</strong> QTL <strong>an</strong>d narrowed it down to a 10Mb genomic region where <strong>the</strong> CE alleles in<br />
<strong>the</strong>se two strains overlap. Surprisingly, this QTL region also confers longer ra<strong>the</strong>r th<strong>an</strong> shorter<br />
free running periods. In addition, <strong>the</strong>se mice also exhibit excessive early morning activities that<br />
last even after lights-on. By identifying <strong>the</strong> gene(s) that contribute to this earlier phase <strong>an</strong>gle,<br />
light masking, <strong>an</strong>d o<strong>the</strong>r traits, our work may provide insights into aspects of <strong>the</strong> circadi<strong>an</strong><br />
system that are poorly understood, <strong>an</strong>d also may suggest novel <strong>the</strong>rapeutics <strong>for</strong> ASPS <strong>an</strong>d<br />
problems related to shift work <strong>an</strong>d jet lag.<br />
Disclosures: P. Ji<strong>an</strong>g, None; M. Striz, None; K. Shimomura, None; J.S. Takahashi,<br />
None; J.P. Wisor, None; B.F. O'Hara, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.19/FF69<br />
Topic: F.02.c. Timing <strong>an</strong>d temporal processing<br />
Support: PAPIIT IN207008
Title: Preliminary study of <strong>the</strong> expression of two clock genes in <strong>the</strong> brain of <strong>the</strong> crayfish<br />
procambarus clarkii<br />
Authors: *J. PRIETO-SAGREDO 1 , V. TOWNS 2 , M. FANJUL-PEÑA 2 ;<br />
1 Facultad De Ciencias, UNAM, Mexico, Mexico; 2 Facultad de Ciencias, Univ. Nacional<br />
Autónoma de México, México DF, Mexico<br />
Abstract: Circadi<strong>an</strong> clocks in eukaryotes rely on tr<strong>an</strong>scriptional feedback loops, in which clock<br />
genes repress <strong>the</strong>ir own tr<strong>an</strong>scription resulting in molecular oscillations with a period of<br />
approximately 24 h. In <strong>the</strong> invertebrate model Drosophila, <strong>the</strong> proteins Clock (CLK) <strong>an</strong>d Cycle<br />
(CYC), activate <strong>the</strong> tr<strong>an</strong>scription of period (per) <strong>an</strong>d timeless (tim). PER <strong>an</strong>d TIM, dimerize in<br />
<strong>the</strong> cytoplasm of <strong>the</strong> clock cells <strong>an</strong>d interact with CLK-CYC inside <strong>the</strong> nucleus, inhibiting it´s<br />
own tr<strong>an</strong>scription thus per <strong>an</strong>d tim are tr<strong>an</strong>scribed rhythmically. In addition clock is inhibited by<br />
VRI in <strong>an</strong>tiphase with per <strong>an</strong>d tim expression. As compared to <strong>the</strong> molecular clock of insect’s<br />
knowledge <strong>the</strong> molecular clock of Procambarus clarkii is poor, but previous works gives us<br />
insight of some clock proteins behavior. Hence here we tried to characterize per <strong>an</strong>d clk’s mRNA<br />
in a putative pacemaker of crayfish, <strong>the</strong> brain, determining whe<strong>the</strong>r <strong>the</strong>re is <strong>an</strong> effect of time on<br />
<strong>the</strong> expression of both genes. Primers designed were based on conserved sequences of similar<br />
genes of different groups of <strong>an</strong>imals. Two groups of 15 crayfish each, were kept in light dark<br />
photoperiod 12:12, brain RNA was extracted at two hours (1100h <strong>an</strong>d 2200h). Different<br />
amplicons obtained <strong>from</strong> <strong>the</strong> PCR reactions were excised <strong>from</strong> agarose gels <strong>an</strong>d sequenced. The<br />
sequences showing similarity with per or clk of o<strong>the</strong>r species were cloned <strong>an</strong>d reamplified. To<br />
determine possible daily variations of both genes mRNA abund<strong>an</strong>ce during <strong>the</strong> subjective day<br />
<strong>an</strong>d night, we qu<strong>an</strong>tified <strong>the</strong> gels b<strong>an</strong>d density at <strong>the</strong> above mentioned hours. Results showed that<br />
<strong>the</strong> different products sequence share diverse degrees of identity with <strong>the</strong> base <strong>an</strong>d amino acid<br />
sequence of clock genes <strong>an</strong>d proteins reported <strong>for</strong> o<strong>the</strong>r invertebrates. Procambarus clk is similar<br />
to <strong>the</strong> conserved region in clock gene of decapod Machrobrachium rosenbergii (20%). Per<br />
shows identity with <strong>the</strong> same gene in Apis mellifera <strong>an</strong>d D. mel<strong>an</strong>ogaster (24% <strong>an</strong>d 31%). We<br />
found a maximum level of per mRNA at <strong>the</strong> beginning of <strong>the</strong> subjective day, at <strong>the</strong> same time as<br />
clk mRNA. Owing to in this work we only qu<strong>an</strong>tified two hours, so we c<strong>an</strong>’t make conclusions<br />
about gene oscillations, especially clk. However o<strong>the</strong>r results of our laboratory show PER<br />
maximum expression at 1900h indicating that <strong>the</strong> difference between <strong>the</strong> expression of <strong>the</strong><br />
messenger <strong>an</strong>d <strong>the</strong> protein is about 6 hours suggesting that molecular circadi<strong>an</strong> clock in P. clarkii<br />
could be based in a negative feedback loop similar to those proposed <strong>for</strong> o<strong>the</strong>r species, where<br />
PER protein inhibits <strong>the</strong> expression of its own messenger.<br />
Disclosures: J. Prieto-Sagredo, None; V. Towns, None; M. F<strong>an</strong>jul-Peña, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.20/FF70<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: CONACYT 43950-M<br />
PAPIIT UNAM IN-203907<br />
Title: Phase adv<strong>an</strong>ce of nocturnal activity by feeding schedules is independent of SCN clock<br />
genes<br />
Authors: K. RODRIGUEZ, *M. ANGELES-CASTELLANOS, A. BLANCAS, R.<br />
SALGADO, C. ESCOBAR;<br />
UNAM, DF, Mexico<br />
Abstract: The suprachiasmatic nucleus (SCN) is entrained by <strong>the</strong> light/dark cycle (LD), while<br />
feeding schedules selectively entrain peripherals oscillators. Under special conditions, feeding<br />
schedules c<strong>an</strong> modify <strong>the</strong> SCN suggesting that <strong>the</strong> SCN is in<strong>for</strong>med of food related signals.<br />
An intense locomotor activity, arousal <strong>an</strong>d correlative phase relation with <strong>the</strong> endogenous<br />
rhythm facilitate non-photic entrainment of <strong>the</strong> SCN.. Projections of <strong>the</strong> arousal system inhibit<br />
<strong>the</strong> SCN promoting a phase shift.<br />
The aim of this study was to explore <strong>the</strong> effects of normocaloric feeding <strong>an</strong>d arousal on <strong>the</strong> SCN<br />
activity on locomotor <strong>an</strong>d physiological circadi<strong>an</strong> rhythms.<br />
Adult male rats were maintained in a light /dark cycle, 12:12 (ZT0:lights on). Rats were<br />
r<strong>an</strong>domly assigned to one of three groups <strong>for</strong> 3 weeks. Control: ad libitum food. Const<strong>an</strong>t<br />
Arousal (RF3-9): food access twice a day <strong>for</strong> 1 hour at ZT3 <strong>an</strong>d ZT9. Phase relation (RF9): food<br />
access <strong>for</strong> 2 hours at ZT9.<br />
The last day of <strong>the</strong> feeding protocol, lights were switched off in order to observe <strong>the</strong> free running<br />
activity <strong>an</strong>d rats were left ad libitum <strong>for</strong> 3 days. Rats were sacrificed every 3 hours (n=3-4) <strong>from</strong><br />
ZT3 to ZT15. The activity onset <strong>for</strong> locomotion, c-Fos <strong>an</strong>d Pe1 were compared among <strong>the</strong> 3<br />
groups.<br />
By eliminating <strong>the</strong> L/D cycle RF3-9 <strong>an</strong>d RF 9 produced phase adv<strong>an</strong>ce of <strong>the</strong> nocturnal activity<br />
to food time. However Per1 in <strong>the</strong> SCN was not shifted. The dorsal SCN exhibited inhibition of<br />
c-Fos at <strong>the</strong> moment that food was delivered. While <strong>the</strong> ventral SCN showed similar activation in<br />
<strong>the</strong> three groups, Dorsal SCN seems to be sensitive to food or arousal signaling <strong>an</strong>d responsible<br />
of <strong>the</strong> phase adv<strong>an</strong>ce showed on locomotor activity.<br />
A schedule of RF 3-9 in const<strong>an</strong>t dark conditions also entrained <strong>the</strong> nocturnal activity which<br />
showed a phase adv<strong>an</strong>ce towards food access time. We conclude that const<strong>an</strong>t arousal produced<br />
by double scheduled feeding c<strong>an</strong> modify <strong>the</strong> SCN c-Fos activity, but not Per1, <strong>an</strong>d allows phase<br />
shift of locomotor activity.<br />
Disclosures: K. Rodriguez, None; M. Angeles-Castell<strong>an</strong>os, None; A. Bl<strong>an</strong>cas, None; R.<br />
Salgado, None; C. Escobar, None.
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.21/FF71<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research on Priority Areas Molecular Brain Science <strong>from</strong><br />
MEXT<br />
Title: Approach <strong>for</strong> photic entrainment mech<strong>an</strong>ism of circadi<strong>an</strong> clock via SCOP related<br />
signaling<br />
Authors: *K. SHIMIZU, T. KOBAYASHI, Y. KOBAYASHI, Y. FUKADA;<br />
Dept. Biophysics <strong>an</strong>d Biochemistry, The Univ. of Tokyo, Tokyo, Jap<strong>an</strong><br />
Abstract: SCOP (SCN Circadi<strong>an</strong> Oscillatory Protein) was originally identified as a protein<br />
whose expression is regulated in a circadi<strong>an</strong> m<strong>an</strong>ner within <strong>the</strong> rat SCN (suprachiasmatic<br />
nucleus). SCOP interacts directly through its LRR domain with K-Ras in <strong>the</strong> gu<strong>an</strong>ine nucleotidefree<br />
<strong>for</strong>m in <strong>the</strong> membr<strong>an</strong>e rafts, <strong>an</strong>d negatively regulates K-Ras function <strong>an</strong>d its down stream<br />
signaling of ERK/MAPK. SCOP is proteolyzed by calpain when hippocampal neurons are<br />
stimulated by BDNF, KCl or NMDA. Noticeably, training of mice <strong>for</strong> novel object memory<br />
downregulates SCOP protein level in <strong>the</strong> hippocampus. Overexpression of SCOP inhibited<br />
ERK/MAPK activation in <strong>the</strong> hippocampus <strong>an</strong>d completely blocked long-term memory <strong>for</strong> novel<br />
objects.<br />
The master circadi<strong>an</strong> clock in <strong>the</strong> SCN is entrained by daily light/dark cycles. In <strong>the</strong> SCN,<br />
calcium concentration <strong>an</strong>d ERK/MAPK activity are oscillating in a circadi<strong>an</strong> m<strong>an</strong>ner. It is known<br />
that ERK/MAPK activity contributes to photic entrainment. Since SCOP controls ERK/MAPK<br />
activity, SCOP would contribute to photic entrainment in <strong>the</strong> SCN. We applied light pulse to<br />
mice during <strong>the</strong> dark period to test if light affects SCOP level in <strong>the</strong> SCN. We found that photic<br />
stimulation during <strong>the</strong> dark period stimulates SCOP protein degradation <strong>an</strong>d ERK activation in<br />
<strong>the</strong> SCN. Photic stimulation appear to increase in calcium concentration, followed by <strong>an</strong> increase<br />
calpain activity that downregulates SCOP protein level in <strong>the</strong> SCN. To assess <strong>the</strong> role of SCOP<br />
in clock entrainment by light, we are currently pursuing a virus infusion approach to deliver <strong>the</strong><br />
SCOP shRNA to <strong>the</strong> SCN.<br />
Disclosures: K. Shimizu, None; T. Kobayashi, None; Y. Kobayashi, None; Y. Fukada, None.
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.22/FF72<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NSF Gr<strong>an</strong>t IBN-0090974<br />
NIMH Gr<strong>an</strong>t MH62335<br />
Title: mTOR signaling couples light to entrainment of <strong>the</strong> suprachiasmatic circadi<strong>an</strong> clock<br />
Authors: *R. CAO 1 , A. LI 2 , H.-Y. CHO 2 , B. LEE 2 , K. OBRIETAN 2 ;<br />
2 Neurosci., 1 Ohio State Univ., Columbus, OH<br />
Abstract: Inducible gene expression appears to be a critical event in light entrainment of <strong>the</strong><br />
mammali<strong>an</strong> suprachiasmatic nucleus (SCN) clock. Recently, we found that photic stimulation<br />
leads to robust activation of <strong>the</strong> mammali<strong>an</strong> target of rapamycin (mTOR) signaling pathway, in<br />
<strong>the</strong> SCN (Mol Cell Neurosci. 2008, 38:312). Given that mTOR is a key regulator of inducible<br />
mRNA tr<strong>an</strong>slation, <strong>the</strong>se data raised <strong>the</strong> prospect that inducible mTOR-evoked protein<br />
expression contributes to light-actuated clock entrainment. Here we continued to investigate <strong>the</strong><br />
role of this pathway in <strong>the</strong> clock resetting process. To this end, we report that <strong>the</strong> in vivo<br />
abrogation mTOR triggered a signific<strong>an</strong>t attenuation of <strong>the</strong> phase-delaying effect of <strong>an</strong> early<br />
night light pulse, whereas mTOR inhibition during <strong>the</strong> late night facilitated <strong>the</strong> phase-adv<strong>an</strong>cing<br />
effect of light. To assess <strong>the</strong> role of mTOR signaling within <strong>the</strong> context of molecular clock, <strong>the</strong><br />
effects of mTOR inhibition on light-induced expression of PERIOD1 <strong>an</strong>d PERIOD2 proteins<br />
were examined. At both <strong>the</strong> early <strong>an</strong>d late night time points, abrogation of mTOR signaling led<br />
to a signific<strong>an</strong>t attenuation of light-evoked PERIOD protein expression. We also provide data<br />
showing that light-induced mTOR activation leads to expression of mRNAs with a 5'-TOP tracts.<br />
A subset of <strong>the</strong>se mRNAs encode <strong>for</strong> tr<strong>an</strong>slational machinery include ribosomal proteins <strong>an</strong>d<br />
elongation factors, which have been shown to enh<strong>an</strong>ce mRNA tr<strong>an</strong>slation efficiency.<br />
Collectively, <strong>the</strong>se finding indicate that mTOR functions as potent regulator of light-evoked<br />
protein tr<strong>an</strong>slation <strong>an</strong>d SCN clock entrainment.<br />
Key words: circadi<strong>an</strong> clock; entrainment; light; mTOR; mRNA tr<strong>an</strong>slation; PERIOD protein;<br />
phase shift<br />
This work was supported by <strong>an</strong> NSF gr<strong>an</strong>t (IBN-0090974) <strong>an</strong>d a gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> NIMH<br />
(MH62335).<br />
Disclosures: R. Cao, None; A. Li, None; H. Cho, None; B. Lee, None; K. Obriet<strong>an</strong>, None.
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.23/FF73<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NSF Gr<strong>an</strong>t IOS 08-18555<br />
NIH Gr<strong>an</strong>t P30 DA018310<br />
Title: Proteomic characterization of <strong>the</strong> SCN<br />
Authors: K. E. WEIS 1 , J. W. MITCHELL 1 , B. S. IMAI 2 , *J. V. SWEEDLER 3 , P. M. YAU 1 , M.<br />
U. GILLETTE 1 ;<br />
1 Dept. of Cell. <strong>an</strong>d Developmental Biol., 2 Biotech. Ctr., Univ. Illinois, Urb<strong>an</strong>a-Champaign,<br />
Urb<strong>an</strong>a, IL; 3 Dept Chem., Univ. Illinois, Urb<strong>an</strong>a, IL<br />
Abstract: The timing of m<strong>an</strong>y biological processes in mammals is orchestrated by <strong>the</strong> core<br />
circadi<strong>an</strong> clock situated in <strong>the</strong> suprachiasmatic nucleus (SCN) of <strong>the</strong> brain hypothalamus. A<br />
small group of essential genes constitute a molecular feedback loop which drives biochemical,<br />
metabolic <strong>an</strong>d behavioral rhythms throughout <strong>the</strong> org<strong>an</strong>ism. These clock genes typically create<br />
<strong>an</strong> oscillating pattern of protein expression over a 24 hour day. Beyond this h<strong>an</strong>dful of clock<br />
elements, it is estimated that between 2-10 % of all mammali<strong>an</strong> genes display circadi<strong>an</strong><br />
rhythmicity in tr<strong>an</strong>scriptional levels (P<strong>an</strong>da et. al. [2002] Cell, 109,3: 307-320). Clearly a vast<br />
number of unknown players may contribute to biological timekeeping.<br />
We have undertaken a proteomic survey of <strong>the</strong> entire rat SCN to identify novel proteins which<br />
show daily variation in expression. We compared SCN gene products at 4 different times of day<br />
by subjecting tissue lysates to two-dimensional difference in gel electrophoresis (2D-DIGE)<br />
followed by mass spectrometry. We identified numerous proteins that show differential<br />
expression between day <strong>an</strong>d night. These gene products mediate a variety of cellular functions,<br />
including metabolism, cytoskeletal structure <strong>an</strong>d stress response. Fur<strong>the</strong>rmore, we are applying<br />
<strong>the</strong>se techniques to specific, functional subsets of <strong>the</strong> SCN proteome. Dynamic reorg<strong>an</strong>ization of<br />
<strong>the</strong> actin cytoskeleton has proven to be <strong>an</strong> unexpected me<strong>an</strong>s to regulate <strong>the</strong> circadi<strong>an</strong> clock. We<br />
are using <strong>the</strong>se proteomic methods to identify SCN gene products which show differential<br />
expression upon actin remodeling. These technologies offer us a new tool-set <strong>for</strong> circadi<strong>an</strong> gene<br />
discovery <strong>an</strong>d characterization.
Disclosures: K.E. Weis, None; J.W. Mitchell, None; B.S. Imai, None; J.V. Sweedler,<br />
None; P.M. Yau, None; M.U. Gillette, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.24/FF74<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Title: Filamentous (F)-actin reorg<strong>an</strong>ization is necessary <strong>for</strong> melatonin to induce a phase adv<strong>an</strong>ce<br />
in <strong>the</strong> rat SCN at dusk<br />
Authors: *P. C. KANDALEPAS 1 , M. U. GILLETTE 2 ;<br />
1 2<br />
Neurosci. Program, Univ. Illinois, Urb<strong>an</strong>a-Champaign, IL; Cell <strong>an</strong>d Developmental Biol.,<br />
Univ. of Illinois, Urb<strong>an</strong>a-Champaign, IL<br />
Abstract: Extracelluar signals encoding time-of-day are integrated by <strong>the</strong> mammali<strong>an</strong><br />
suprachiasmatic nucleus (SCN) to synchronize <strong>an</strong> org<strong>an</strong>ism with day <strong>an</strong>d night. Previous work<br />
has established that <strong>the</strong> pineal hormone melatonin c<strong>an</strong> directly modulate <strong>the</strong> intrinsic<br />
timekeeping mech<strong>an</strong>ism of <strong>the</strong> SCN within two windows of sensitivity corresponding to dusk<br />
<strong>an</strong>d dawn. At <strong>the</strong>se times, melatonin adv<strong>an</strong>ces <strong>the</strong> peak of neuronal firing rhythms in <strong>the</strong> SCN<br />
via <strong>the</strong> melatonin type 2 (MT2) receptor <strong>an</strong>d downstream activation of protein kinase C (PKC).<br />
Beyond this, <strong>the</strong> intracellular mech<strong>an</strong>isms by which melatonin alters circadi<strong>an</strong> timekeeping are<br />
largely undefined. Melatonin has been shown to alter <strong>the</strong> org<strong>an</strong>ization of cytoskeletal proteins<br />
across various cell types. Intracellular scaffoding networks c<strong>an</strong> rapidly ch<strong>an</strong>ge in response to<br />
extracellular stimuli, <strong>an</strong>d actively participate in cell signaling. This led to <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong><br />
cytoskeletal network may act as a plat<strong>for</strong>m <strong>for</strong> melatonin signal tr<strong>an</strong>sduction in <strong>the</strong> SCN.<br />
Melatonin applied to <strong>the</strong> SCN at CT 10 in vitro results in a rapid, tr<strong>an</strong>sient increase in<br />
filamentous (F)-actin assembly that is inhibited when a chemical stabilizer of F-actin,<br />
Jasplakinolide (Jasp), is added prior to melatonin stimulation. Experiments on rat SCN slices<br />
reveal that Per1 <strong>an</strong>d Per2 tr<strong>an</strong>scripts increase when melatonin is applied to <strong>the</strong> SCN at dusk, a<br />
time when melatonin treatment also adv<strong>an</strong>ces <strong>the</strong> peak of neuronal firing rate that is intrinsic to<br />
<strong>the</strong> SCN. This increase in Per1 <strong>an</strong>d Per2 mRNA is essential <strong>for</strong> <strong>the</strong> phase-resetting effect of<br />
melatonin on <strong>the</strong> clock. <strong>When</strong> F-actin assembly is inhibited in <strong>the</strong> SCN using Jasp, <strong>the</strong> increases<br />
in Per1 <strong>an</strong>d Per2 by melatonin at CT 10 are abolished. These data suggest that reorg<strong>an</strong>ization of<br />
<strong>the</strong> actin cytoskeleton is upstream of <strong>the</strong> ch<strong>an</strong>ges in clock gene expression, <strong>an</strong>d that actin<br />
remodeling is necessary <strong>for</strong> melatonin to induce Per1 <strong>an</strong>d Per2 tr<strong>an</strong>scripts in <strong>the</strong> SCN at CT 10.<br />
Fur<strong>the</strong>rmore, we investigated <strong>the</strong> role of actin state in phase resetting by melatonin in <strong>the</strong> SCN.
Similar to <strong>the</strong> results with clock gene expression, pre-treatment of SCN slices with Jasp<br />
effectively blocks melatonin-induced adv<strong>an</strong>ces in neuronal activity rhythms. Thus, activation of<br />
<strong>the</strong> melatonin signaling pathway in SCN cells leads to F-actin assembly, <strong>an</strong>d this cytoskeletal<br />
reorg<strong>an</strong>ization is necessary <strong>for</strong> melatonin to induce ch<strong>an</strong>ges in clock gene expression <strong>an</strong>d to elicit<br />
a phase adv<strong>an</strong>ce in rat SCN neuronal activity rhythms at dusk.<br />
Disclosures: P.C. K<strong>an</strong>dalepas, None; M.U. Gillette, None.<br />
Poster<br />
574. Entrainment <strong>an</strong>d Phase Shifts: Neurons, Tr<strong>an</strong>smitters, Genes, <strong>an</strong>d Proteins<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 574.25/FF75<br />
Topic: E.08.a. Entrainment <strong>an</strong>d phase shifts<br />
Support: NIH Gr<strong>an</strong>t HL086870<br />
Title: Light signaling via actin in early-night phase shifts of <strong>the</strong> rodent SCN<br />
Authors: *J. ARNOLD, S.-H. TYAN, J. W. MITCHELL, M. U. GILLETTE;<br />
Univ. of Illinois, Urb<strong>an</strong>a, IL<br />
Abstract: The cells of <strong>the</strong> circadi<strong>an</strong> clock in <strong>the</strong> mammali<strong>an</strong> suprachiasmatic nucleus (SCN)<br />
undergo a nearly 24-hour cycle of tightly controlled cellular events. Light signals <strong>from</strong> <strong>the</strong> eye<br />
are capable of ch<strong>an</strong>ging <strong>the</strong> state of <strong>the</strong>se cells in a time-of-day dependent m<strong>an</strong>ner. In <strong>the</strong> early<br />
night, light signals are tr<strong>an</strong>smitted <strong>from</strong> <strong>the</strong> eye to <strong>the</strong> SCN by <strong>the</strong> neurotr<strong>an</strong>smitter glutamate<br />
(Glu), which acts via <strong>an</strong> NMDA-R/nitric oxide (NO) pathway to cause a delay in clock phase.<br />
This pathway also requires <strong>the</strong> release of stored calcium (Ca2+) through neuronal ry<strong>an</strong>odine<br />
receptors (RyR), which in turn activates <strong>the</strong> Ca2+-sensitive protease, calpain. Calpain c<strong>an</strong> act on<br />
cytoskeletal actin filaments (F-actin), dynamic structures that <strong>for</strong>m intracellular networks<br />
involved in a variety of processes, including cell migration, neuronal plasticity, <strong>an</strong>d even cell<br />
signaling. Our data support <strong>the</strong> hypo<strong>the</strong>sis that activating <strong>the</strong> light signaling pathway in early<br />
night leads to a reorg<strong>an</strong>ization of <strong>the</strong> actin cytoskeleton, <strong>an</strong>d that this cytoskeletal rearr<strong>an</strong>gement<br />
is a necessary component of <strong>the</strong> pathway leading to delay of clock phasing. We have shown that<br />
applying Glu in vitro to brain slices containing <strong>the</strong> SCN causes <strong>an</strong> immediate, tr<strong>an</strong>sient decrease<br />
in F-actin. This phenomenon c<strong>an</strong> be replicated in vivo with light exposure. Fur<strong>the</strong>rmore,<br />
applying <strong>the</strong> actin stabilizer, Jasplakinolide, blocks <strong>the</strong> Glu-induced phase delay in vitro, while<br />
applying actin disrupters, Latrunculin A <strong>an</strong>d Cytochalasin D, alone leads to a phase delay. We<br />
are evaluating <strong>the</strong>se reagents in vivo to assess how directly altering actin state affects phasing of
circadi<strong>an</strong> rhythms of wheel-running locomotor activity. These findings suggest that ch<strong>an</strong>ges in<br />
<strong>the</strong> actin cytoskeleton mediate SCN phase shifts in <strong>the</strong> early night in response to light exposure.<br />
Disclosures: J. Arnold, None; S. Ty<strong>an</strong>, None; J.W. Mitchell, None; M.U. Gillette, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.1/FF76<br />
Topic: F.01.f. Working memory<br />
Support: NIH P01 NS40813<br />
Title: Hemispheric differences in working memory capacity <strong>an</strong>d filtering<br />
Authors: *P. A. BUTCHER, R. B. IVRY;<br />
Psychology Dept., Cognition <strong>an</strong>d Action Lab., Berkeley, CA<br />
Abstract: A network sp<strong>an</strong>ning prefrontal cortex <strong>an</strong>d <strong>the</strong> basal g<strong>an</strong>glia (BG) have been<br />
implicated in <strong>an</strong> fMRI study in filtering in<strong>for</strong>mation that gains access to visual working memory<br />
(WM). Specifically, bilateral middle frontal gyrus <strong>an</strong>d <strong>the</strong> left BG were more active on trials in<br />
which distractors were present with trials compared to trials in which no distractors were<br />
presented. The lateralized activity in <strong>the</strong> BG suggests that filtering may be better when<br />
distractors are presented in <strong>the</strong> right visual field (RVF). We conducted a behavioral study to test<br />
this prediction. Particip<strong>an</strong>ts were shown a display composed of 14 squares arr<strong>an</strong>ged in a circle<br />
centered at <strong>the</strong> fixation point. On each trial, red <strong>an</strong>d yellow circles appeared in a subset of <strong>the</strong><br />
squares. Prior to <strong>the</strong> memory display, one of two instruction cues had been presented. In <strong>the</strong> “red<br />
only” condition, particip<strong>an</strong>ts had to remember only <strong>the</strong> locations that had red circles inside of<br />
<strong>the</strong>m; <strong>the</strong>y were to ignore <strong>the</strong> yellow circles (if present). In <strong>the</strong> “all” condition, particip<strong>an</strong>ts were<br />
instructed to remember <strong>the</strong> locations of both <strong>the</strong> red <strong>an</strong>d yellow circles. After a two-second<br />
delay, a question mark appeared in one location <strong>an</strong>d particip<strong>an</strong>ts indicated with a 2-AFC<br />
judgment whe<strong>the</strong>r <strong>the</strong> probed location had contained one of <strong>the</strong> circles in <strong>the</strong> working memory<br />
set. The memory display was presented <strong>for</strong> only 200 ms to minimize <strong>the</strong> utility of eye<br />
movements, <strong>an</strong>d thus, keep <strong>the</strong> stimuli lateralized. Particip<strong>an</strong>ts were consistently better on <strong>the</strong><br />
memory test when locations in <strong>the</strong> left visual field were probed (80% vs. 78% overall accuracy).<br />
However, <strong>the</strong> visual field adv<strong>an</strong>tage was similar on filtering (4%) <strong>an</strong>d non-filtering (4%) trials.<br />
While <strong>the</strong> results indicate <strong>the</strong>re may be hemispheric (or visual field) differences in WM<br />
per<strong>for</strong>m<strong>an</strong>ce, <strong>the</strong> results failed to demonstrate <strong>an</strong>y asymmetries in filtering effects. Fur<strong>the</strong>r
investigation is necessary to identify components of working memory that may account <strong>for</strong> <strong>the</strong><br />
LVF adv<strong>an</strong>tage.<br />
Disclosures: P.A. Butcher, None; R.B. Ivry, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.2/FF77<br />
Topic: F.01.f. Working memory<br />
Support: DFG/KFO 163, TP3<br />
Title: Neural generators of sustained activity differ <strong>for</strong> stimulus-encoding <strong>an</strong>d delay mainten<strong>an</strong>ce<br />
Authors: *U. MALECKI 1 , S. STALLFORTH 2 , D. HEIPERTZ 1 , N. LAVIE 3,4 , E. DÜZEL 1,3 ;<br />
1 Institue of Cognitive Neurol. <strong>an</strong>d Dementia Research, Univ. Magdeburg, Magdeburg, Germ<strong>an</strong>y;<br />
2 Dept. of Neurol., Otto-von-Guericke Univ., Magdeburg, Germ<strong>an</strong>y; 3 Inst. of Cognitive<br />
Neurosci., 4 Dept. of Psychology, Univ. Col. London, London, United Kingdom<br />
Abstract: The ability to maintain in<strong>for</strong>mation online beyond sensory stimulation is regarded as a<br />
key contribution of working memory to goal-directed behavior. It is widely accepted that<br />
sustained neural activity is a key mech<strong>an</strong>ism of stimulus mainten<strong>an</strong>ce, but it is unclear to what<br />
extent <strong>the</strong> neural generators of sustained activity ch<strong>an</strong>ge <strong>from</strong> stimulus-encoding to mainten<strong>an</strong>ce.<br />
Using event-related potentials (ERP) in hum<strong>an</strong>s, we show that, in a delayed match-to-sample<br />
task, slow-shifts over parieto-occipital electrode sites had a different topography <strong>an</strong>d polarity<br />
during encoding <strong>an</strong>d delay-mainten<strong>an</strong>ce of images depicting scenes. This clearly demonstrates<br />
that neural generators of sustained activity associated with stimulus-encoding <strong>an</strong>d delay<br />
mainten<strong>an</strong>ce differed <strong>an</strong>d that <strong>the</strong> ch<strong>an</strong>ge between <strong>the</strong>se generators occurred time-locked to <strong>the</strong><br />
onset of <strong>the</strong> delay period. We also investigated how monetary reward incentives modulated <strong>the</strong><br />
amplitude <strong>an</strong>d topography of sustained delay activity <strong>an</strong>d <strong>the</strong> ability to suppress irrelev<strong>an</strong>t<br />
distracting in<strong>for</strong>mation during <strong>the</strong> delay. Reward incentives improved mainten<strong>an</strong>ce per<strong>for</strong>m<strong>an</strong>ce<br />
<strong>an</strong>d this was correlated with <strong>an</strong> exp<strong>an</strong>sion of <strong>the</strong> parieto-occipital electrode sites that were<br />
entrained into sustained delay activity (ra<strong>the</strong>r th<strong>an</strong> improved distractor suppression), suggesting<br />
that under <strong>the</strong> influence of reward, <strong>the</strong> parieto-occipital regions that contributed to delay<br />
mainten<strong>an</strong>ce exp<strong>an</strong>ded in size.
Disclosures: U. Malecki, None; S. Stall<strong>for</strong>th, None; D. Heipertz, None; N. Lavie, None; E.<br />
Düzel, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.3/FF78<br />
Topic: F.01.f. Working memory<br />
Title: The left temporoparietal junction in working memory: Storage or attention?<br />
Authors: S. RUIZ 1 , *S. M. RAVIZZA 2 ;<br />
2 Psychology, 1 Michig<strong>an</strong> State Univ., East L<strong>an</strong>sing, MI<br />
Abstract: Working memory allows in<strong>for</strong>mation to be actively maintained <strong>for</strong> short periods of<br />
time, <strong>an</strong>d lesion studies have suggested that <strong>the</strong> left temporoparietal junction (TPJ) may play a<br />
crucial role in maintaining verbal in<strong>for</strong>mation. In a previous neuroimaging study, we have shown<br />
that activity in <strong>the</strong> left TPJ was sensitive to <strong>the</strong> type of in<strong>for</strong>mation to be recalled, but insensitive<br />
to <strong>the</strong> amount of in<strong>for</strong>mation to be maintained (Ravizza, et al., 2004). These results are<br />
inconsistent with attributions of <strong>the</strong> left TPJ as a verbal store, but may instead imply that this<br />
region is import<strong>an</strong>t <strong>for</strong> bottom-up attention as is its right hemisphere homologue. In <strong>the</strong> present<br />
experiment, we use fMRI to assess activity in <strong>the</strong> left TPJ in working memory at different stages<br />
(i.e., encoding, mainten<strong>an</strong>ce <strong>an</strong>d retrieval). We also m<strong>an</strong>ipulated two types of in<strong>for</strong>mation<br />
(verbal <strong>an</strong>d non verbal) <strong>an</strong>d two memory loads (high <strong>an</strong>d low). If this area is import<strong>an</strong>t <strong>for</strong><br />
bottom-up attention, we predict that <strong>the</strong> left TPJ will be active during <strong>the</strong> encoding process,<br />
particularly <strong>for</strong> verbal items, but become deactivated during mainten<strong>an</strong>ce in order to prohibit<br />
distracting in<strong>for</strong>mation <strong>from</strong> entering short term memory. In contrast, <strong>the</strong> storage hypo<strong>the</strong>sis<br />
would predict that activity would be sustained across <strong>the</strong> delay as <strong>the</strong> store is refreshed, <strong>an</strong>d that<br />
<strong>the</strong> region should be more active <strong>for</strong> high th<strong>an</strong> low amounts of in<strong>for</strong>mation. As in our previous<br />
study, we find that <strong>the</strong> left TPJ is insensitive to working memory load. Additionally, this region<br />
was active during encoding of verbal in<strong>for</strong>mation, but this activity fell during mainten<strong>an</strong>ce. This<br />
pattern of activity is more consistent with <strong>an</strong> attentional th<strong>an</strong> a storage account of left TPJ<br />
function <strong>an</strong>d implies that this region may be import<strong>an</strong>t <strong>for</strong> bottom-up attention to speech-like<br />
stimuli.<br />
Disclosures: S. Ruiz, None; S.M. Ravizza, None.
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.4/FF79<br />
Topic: F.01.f. Working memory<br />
Support: NIMH Gr<strong>an</strong>t MH065395-01<br />
NIH Training Gr<strong>an</strong>t 3T32NS041231-03S1<br />
C<strong>an</strong>adi<strong>an</strong> Institutes <strong>for</strong> Health Research (CIHR) doctoral research award<br />
Georgetown University institutional funds<br />
Title: Differential load-dependent suppression of activity within default-mode regions during<br />
working memory<br />
Authors: *E. M. GORDON 1 , M. STOLLSTORFF 2 , C. J. VAIDYA 2 ;<br />
2 Psychology, 1 Georgetown, Washington, DC<br />
Abstract: Cognitive processes such as working memory require both engagement of “taskpositive”<br />
brain areas as well as suppression of “default-mode” areas. During per<strong>for</strong>m<strong>an</strong>ce of<br />
working memory tasks, increases in “load” (in<strong>for</strong>mation mainten<strong>an</strong>ce <strong>an</strong>d updating<br />
requirements) increase task difficulty <strong>an</strong>d increase recruitment of task-positive areas, with<br />
activity in <strong>the</strong>se areas scaling in proportion to load. However, it is unknown whe<strong>the</strong>r <strong>the</strong><br />
suppression of default-mode areas during working memory is also modulated by load. To test<br />
this possibility, we used fMRI in 42 subjects (age 19-22) while <strong>the</strong>y per<strong>for</strong>med a blocked-design<br />
n-back working memory task in which n varied <strong>from</strong> 1 to 2 to 3 in different task blocks. The nback<br />
task requires subjects to maintain <strong>an</strong>d update <strong>the</strong> identity of n stimuli on each trial. This<br />
task thus allows parametric m<strong>an</strong>ipulation of working memory load via m<strong>an</strong>ipulation of n.<br />
To determine whe<strong>the</strong>r <strong>the</strong> working memory task engaged activity in task-positive regions <strong>an</strong>d<br />
suppressed activity in default-mode regions, we conducted a whole-brain <strong>an</strong>alysis across subjects<br />
to find voxels in which activity positively (task-positive) <strong>an</strong>d negatively (default-mode)<br />
correlated with <strong>the</strong> task wave<strong>for</strong>m. We found a robust set of classic task-positive regions<br />
activated by <strong>the</strong> task, including dorsal middle cingulate cortex, bilateral dorsolateral prefrontal<br />
cortex, bilateral superior parietal lobule, <strong>an</strong>d bilateral striatum. We also found robust suppression<br />
of activity in classic default-mode areas, including dorsal <strong>an</strong>d ventral medial prefrontal cortex,<br />
posterior cingulate cortex, bilateral inferior parietal lobule, <strong>an</strong>d bilateral medial temporal lobe.<br />
To determine which task-positive <strong>an</strong>d default-mode areas are modulated by working memory<br />
load, we conducted <strong>an</strong>o<strong>the</strong>r whole-brain <strong>an</strong>alysis across subjects to find voxels which positively
<strong>an</strong>d negatively correlated with a modified task wave<strong>for</strong>m that scaled with load. We found that<br />
<strong>the</strong> set of regions which demonstrated increased activity as load increased had high spatial<br />
concord<strong>an</strong>ce with <strong>the</strong> previously-identified task-positive regions, suggesting that increased task<br />
dem<strong>an</strong>ds require activity increases in all task-related areas. However, within default-mode areas,<br />
we found that only <strong>the</strong> subgenual ventromedial prefrontal cortex (vmPFC) exhibited increased<br />
suppression as load increased. These results suggest a functional dissociation between <strong>the</strong><br />
vmPFC <strong>an</strong>d <strong>the</strong> o<strong>the</strong>r default-mode areas, such that while <strong>the</strong> majority of <strong>the</strong>se regions are<br />
suppressed equally at all loads, only <strong>the</strong> vmPFC must be more suppressed to per<strong>for</strong>m a more<br />
difficult working memory task.<br />
Disclosures: E.M. Gordon, None; M. Stollstorff, None; C.J. Vaidya, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.5/FF80<br />
Topic: F.01.f. Working memory<br />
Support: BMBF Gr<strong>an</strong>t 01GQ0411<br />
DFG Gr<strong>an</strong>t GSC86/1-<strong>2009</strong><br />
Title: Decoding delayed intentions during secondary task execution<br />
Authors: *I. MOMENNEJAD 1,2 , J. HAYNES 1,2,3 ;<br />
1 BCCN Berlin, Berlin, Germ<strong>an</strong>y; 2 Grad. Sch. of Mind <strong>an</strong>d Brain, Humboldt-Universität zu<br />
Berlin, Berlin, Germ<strong>an</strong>y; 3 Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Hum<strong>an</strong> Cognitive <strong>an</strong>d Brain Sci., Leipzig,<br />
Germ<strong>an</strong>y<br />
Abstract: Our ‘volitional’ capacities largely depend upon our ability to pursue ‘delayed<br />
intentions’ to per<strong>for</strong>m a task at a later point in time. Successful execution of prospective task-sets<br />
requires a capacity to maintain <strong>the</strong> prior intention while our attentional resources are dedicated to<br />
per<strong>for</strong>ming conflicting ongoing tasks; <strong>an</strong>d later retrieve <strong>an</strong>d execute our delayed intention when<br />
appropriate conditions are detected. Here we investigated a) whe<strong>the</strong>r it is possible to decode<br />
delayed intentions during a conflicting ongoing task (current results), b) whe<strong>the</strong>r it is possible to<br />
dissociate <strong>the</strong> spatiotemporal patterns of brain activity while encoding, maintaining, <strong>an</strong>d<br />
retrieving delayed intentions (ongoing research). We used a modified task-switching paradigm in<br />
combination with functional magnetic reson<strong>an</strong>ce imaging. Subjects had to per<strong>for</strong>m one task
(color judgment) while at <strong>the</strong> same time memorizing one of two o<strong>the</strong>r possible prospective tasks<br />
(magnitude or parity judgment). The subjects were required to switch to <strong>the</strong> delayed task in a<br />
self-paced fashion after <strong>an</strong> instructed time of 15s, 20s or 25s (time-based prospective memory).<br />
A finite impulse response model was applied to <strong>the</strong> imaging data with no assumption of <strong>the</strong><br />
shape of <strong>the</strong> hemodynamic function. A support vector machine was trained to ‘classify’ <strong>the</strong> brain<br />
patterns associated with <strong>the</strong> delayed intention during <strong>the</strong> mainten<strong>an</strong>ce interval prior to switching<br />
to <strong>the</strong> delayed task-set. <strong>When</strong> tested on novel imaging data, our method could successfully<br />
predict <strong>the</strong> delayed intention up to 20 seconds prior to <strong>the</strong> switch, <strong>an</strong>d with up to 85% accuracy<br />
in single subjects. The in<strong>for</strong>mation concerning <strong>the</strong> prospective task-set could best be decoded<br />
<strong>from</strong> <strong>the</strong> spatiotemporal patterns of activity in <strong>the</strong> premotor <strong>an</strong>d prefrontal cortices. Notably,<br />
high decoding accuracies were obtained in <strong>the</strong> premotor cortex <strong>an</strong>d DLPFC during <strong>the</strong><br />
mainten<strong>an</strong>ce phase <strong>an</strong>d orbitofrontal areas at <strong>the</strong> time of switch to <strong>the</strong> prospective task-set. The<br />
current results suggest that in<strong>for</strong>mation about a delayed intention c<strong>an</strong> be decoded <strong>from</strong> ongoing<br />
brain activity while per<strong>for</strong>ming a highly dem<strong>an</strong>ding conflicting task-set tens of seconds prior to<br />
<strong>the</strong> execution of <strong>the</strong> delayed intention.<br />
Disclosures: I. Momennejad, None; J. Haynes, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.6/FF81<br />
Topic: F.01.f. Working memory<br />
Support: National Science Foundation of China Gr<strong>an</strong>t 30630022<br />
Title: Brain networks in working memory mainten<strong>an</strong>ce <strong>an</strong>d action org<strong>an</strong>ization: An<br />
electrocorticography study<br />
Authors: *Y. KU 1 , W. ZHOU 2 , B. HONG 1 , X. GAO 1 , S. GAO 1 ;<br />
1 Dept. of Biomed. Engin., Tsinghua Univ., Beijing, China; 2 Dept. of Neurosurg., The Second<br />
Affiliated Hospital, Tsinghua Univ., Beijing, China<br />
Abstract: It has long been suggested that prefrontal <strong>an</strong>d parietal network played a central role in<br />
working memory process. However, most studies used delayed response paradigm in which <strong>the</strong><br />
memory storage <strong>an</strong>d <strong>the</strong> action org<strong>an</strong>ization could not be separated. Here we used “double<br />
delay/double response” paradigm on epileptic patients with intracr<strong>an</strong>ial electrodes, to determine<br />
<strong>the</strong> brain networks <strong>an</strong>d <strong>the</strong> dynamic process during <strong>the</strong> two delay periods. The mainten<strong>an</strong>ce of
memorized items involves bilateral frontal <strong>an</strong>d parietal cortices while <strong>the</strong> action org<strong>an</strong>ization<br />
mainly contributes left frontal cortices. Event related synchronization/desynchronization<br />
(ERD/ERS) were observed during both delays. Gamma (60~100Hz) ERS <strong>an</strong>d alpha (8~13Hz)<br />
ERD were prominent in both frontal <strong>an</strong>d parietal cortices during mainten<strong>an</strong>ce process. The action<br />
org<strong>an</strong>ization induced mainly alpha <strong>an</strong>d beta (14~30Hz) ERD/ERS in frontal cortices. These<br />
results suggest that working memory mainten<strong>an</strong>ce <strong>an</strong>d action org<strong>an</strong>ization involve different brain<br />
networks <strong>an</strong>d show different oscillatory patterns.<br />
Disclosures: Y. Ku, None; W. Zhou, None; B. Hong, None; X. Gao, None; S. Gao, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.7/FF82<br />
Topic: F.01.f. Working memory<br />
Support: Ataxia UK<br />
Title: Identification of critical cerebellar subregions <strong>for</strong> distinct cognitive functions<br />
Authors: *F. E. COOPER 1 , M. GRUBE 1 , K. KRIEGSTEIN 2 , P. ENGLISH 3 , P. F.<br />
CHINNERY 1 , T. D. GRIFFITHS 1 ;<br />
1 Inst. of Neurosci., Newcastle Univ., Newcastle Upon Tyne, United Kingdom; 2 Max Pl<strong>an</strong>ck Inst.<br />
<strong>for</strong> Hum<strong>an</strong> Cognitive <strong>an</strong>d Brain Sci., Leipzig, Germ<strong>an</strong>y; 3 Neuroradiology, Newcastle Gen.<br />
Hosp., Newcastle Upon Tyne, United Kingdom<br />
Abstract: This study sought evidence <strong>for</strong> a cerebellar contribution to cognitive processes by<br />
investigating correlations between grey matter degeneration <strong>an</strong>d cognitive per<strong>for</strong>m<strong>an</strong>ce in a<br />
group of patients with Spinocerebellar Ataxia type 6 (SCA6); <strong>an</strong> autosomal domin<strong>an</strong>t genetic<br />
disease that causes a highly specific, late onset degeneration of <strong>the</strong> cerebellum. High resolution<br />
<strong>an</strong>atomical sc<strong>an</strong>s were collected <strong>from</strong> 15 subjects with genetically confirmed SCA6, who also<br />
underwent a comprehensive, internationally st<strong>an</strong>dardised, neuropsychological assessment<br />
(Wechsler Adult Intelligence Scale-III, Wechsler Memory Scale-III, Delis <strong>an</strong>d Kapl<strong>an</strong> Executive<br />
Function System, Brixton Spatial Anticipation test). Optimised Voxel Based Morphometry<br />
(VBM) was used to investigate a relationship between localised grey matter loss <strong>an</strong>d decreased<br />
cognitive per<strong>for</strong>m<strong>an</strong>ce.<br />
Signific<strong>an</strong>t positive correlations (p = 0.01 uncorrected <strong>for</strong> multiple comparisons) were found<br />
between cognitive indices <strong>an</strong>d cerebellar grey matter volume in specific areas. Verbal
Intelligence <strong>an</strong>d Processing Speed correlated with bilateral grey matter increases in lobes III-VI.<br />
Immediate Auditory Memory correlated with increases in left lobule IX. Non-verbal Concept<br />
Formation correlated with bilateral increases in lobules VI <strong>an</strong>d VIIB. The most striking<br />
correlations were found between verbal working memory subtests <strong>an</strong>d cerebellar grey matter<br />
volume as follows; digit sp<strong>an</strong>, letter-number sequencing (bilateral superior <strong>an</strong>d inferior<br />
cerebellum) <strong>an</strong>d arithmetic (left lobule IV <strong>an</strong>d VIIB), but not visuospatial working memory (as<br />
measured by <strong>the</strong> spatial sp<strong>an</strong>).<br />
The results support a role <strong>for</strong> both <strong>the</strong> superior <strong>an</strong>d inferior cerebellum in cognitive processes,<br />
particularly in working memory as predicted by <strong>the</strong> Cerebellar Cognitive Affective Syndrome<br />
(CCAS) (Schmahm<strong>an</strong>n & Sherm<strong>an</strong>, 1998) <strong>an</strong>d more specifically as part of <strong>the</strong> phonological<br />
rehearsal loop of <strong>the</strong> working memory model (Baddely, 1992).<br />
References:<br />
Baddely, A. (1992). Science, 255. Schmahm<strong>an</strong>n, J. D., & Sherm<strong>an</strong>, J. C. (1998). Brain, 121.<br />
Disclosures: F.E. Cooper, None; M. Grube, None; K. Kriegstein, None; P. English,<br />
None; P.F. Chinnery, None; T.D. Griffiths, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.8/FF83<br />
Topic: F.01.f. Working memory<br />
Support: NTNU<br />
Title: Neural correlates of spatial working memory encoding<br />
Authors: *I. K. ANTONSEN 1 , J. XU 1 , A. HABERG 1,2 ;<br />
1 NTNU, Trondheim, Norway; 2 St. Olav's Hosp., Trondheim, Norway<br />
Abstract: Working memory (WM) refers to <strong>the</strong> retention of in<strong>for</strong>mation relev<strong>an</strong>t or needed to<br />
guide ongoing behavior or tasks being per<strong>for</strong>med. The neural correlates underlying WM consist<br />
of several brain areas, including <strong>the</strong> prefrontal, parietal, <strong>an</strong>d cingulated cortices. Recent studies<br />
indicate that also <strong>the</strong> hippocampus is essential <strong>for</strong> WM. However, <strong>the</strong> specific role of<br />
hippocampus in WM is still undetermined. The aim of <strong>the</strong> present study was to use fMRI to<br />
identify ch<strong>an</strong>ges in activity in <strong>the</strong> hippocampus <strong>an</strong>d o<strong>the</strong>r brain areas related to different phases<br />
of WM, e.g. encoding, delay, <strong>an</strong>d response selection, in a spatial WM task.<br />
Eighteen healthy teenagers (ages 13-16 years of age, 10 males) were included in <strong>the</strong> study. The
spatial WM paradigm was event designed <strong>an</strong>d consisted of 60 tasks of varying difficulty. Each<br />
task consisted of <strong>an</strong> encoding period where <strong>the</strong> task was presented, a delay period, a responseselection<br />
period, <strong>an</strong>d a null event consisting of a fixation cross. During <strong>the</strong> encoding period, <strong>the</strong><br />
particip<strong>an</strong>ts were presented with three to six spatial locations to be remembered on a three by<br />
three grid of white circles. The circles would light up in red one at <strong>the</strong> time in various places,<br />
orders, <strong>an</strong>d numbers. After a delay period, <strong>the</strong> subject was to point <strong>an</strong>d click on <strong>the</strong> circles in <strong>the</strong><br />
same places <strong>an</strong>d order as presented.<br />
A r<strong>an</strong>dom-effects group <strong>an</strong>alysis of <strong>the</strong> overall brain activation related to <strong>the</strong> task conditions<br />
taken as a whole (encoding, delay, <strong>an</strong>d response selection) in contrast with <strong>the</strong> null event showed<br />
bilateral signific<strong>an</strong>t BOLD increases in <strong>the</strong> inferior frontal lobe, parietal <strong>an</strong>d occipital lobes,<br />
<strong>an</strong>terior cingulate gyrus, supplementary motor area, primary motor cortex, <strong>an</strong>d thalamus. Overall<br />
brain activation related specifically to <strong>the</strong> encoding period of <strong>the</strong> spatial WM task showed<br />
increased activity in <strong>the</strong> left <strong>an</strong>terior hippocampus, <strong>the</strong> dorsolateral prefrontal cortex, <strong>the</strong> <strong>an</strong>terior<br />
prefrontal cortex, <strong>an</strong>d <strong>the</strong> <strong>an</strong>terior cingulated cortex. These findings demonstrate that <strong>the</strong><br />
hippocampus is involved in spatial WM, specifically during encoding of spatial in<strong>for</strong>mation.<br />
Disclosures: I.K. Antonsen, None; J. Xu, None; A. Haberg, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.9/FF84<br />
Topic: F.01.f. Working memory<br />
Support: University of Illinois Graduate Student Fellowship<br />
NIH/NICHD 1P50HD055751-01<br />
NIH/NIMH MH077862<br />
K23 MH083126<br />
Title: Differential mainten<strong>an</strong>ce of activity across widely distributed neural circuitry during delay<br />
periods in a spatial working memory task<br />
Authors: *A.-M. D'CRUZ 1 , J. L. REILLY 2 , K. KAPUR 2 , S. K. KEEDY 2 , J. A. SWEENEY 2 ;<br />
1 Dept. of Psychiatry, Chicago, IL; 2 Dept. of Psychiatry, Univ. of Illinois at Chicago, Chicago, IL
Abstract: How <strong>the</strong> brain maintains spatial in<strong>for</strong>mation in working memory <strong>for</strong> intended action<br />
over time remains to be fully clarified. In <strong>the</strong> oculomotor delayed response task (ODR) subjects<br />
make saccades to remembered locations after a delay period. Sustained neuronal activity in<br />
dorsolateral prefrontal cortex (DLPFC) during relatively brief delays is accepted as a neural<br />
representation of working memory mainten<strong>an</strong>ce. Using a long delay in <strong>an</strong> event-related fMRI<br />
study, we examined <strong>the</strong> localization of sustained BOLD activation over ODR delay periods.<br />
Twenty-four healthy individuals per<strong>for</strong>med <strong>an</strong> ODR task with a 10s delay <strong>an</strong>d a visually-guided<br />
saccade (VGS) control task during <strong>an</strong> fMRI sc<strong>an</strong>ning session. The latency <strong>an</strong>d accuracy of eye<br />
movements were measured. We used r<strong>an</strong>dom-effects regression models <strong>an</strong>d Bayes estimation to<br />
identify voxels with a signific<strong>an</strong>t hemodynamic response. Within <strong>the</strong>se voxels <strong>the</strong> percent signal<br />
increase <strong>an</strong>d rate of decay of <strong>the</strong> hemodynamic response was measured to characterize patterns<br />
of activation over <strong>the</strong> delay period. Regions of interest included DLPFC, frontal <strong>an</strong>d<br />
supplementary eye fields, <strong>an</strong>terior cingulate, intraparietal sulcus, caudate <strong>an</strong>d thalamus. Me<strong>an</strong><br />
activation parameters of active voxels during <strong>the</strong> ODR delay period were contrasted with those<br />
<strong>from</strong> <strong>the</strong> visually-guided control task in which immediate saccadic responses were made to<br />
similar sensory stimuli.<br />
Percent signal ch<strong>an</strong>ge was signific<strong>an</strong>tly greater in <strong>the</strong> DLPFC, pre-supplementary motor area,<br />
dorsal <strong>an</strong>terior cingulate cortex <strong>an</strong>d thalamus during <strong>the</strong> ODR delay th<strong>an</strong> during <strong>the</strong> VGS task.<br />
However, <strong>the</strong> persistence of activation was signific<strong>an</strong>tly greater during <strong>the</strong> ODR task only in <strong>the</strong><br />
frontal <strong>an</strong>d supplementary eye fields compared to <strong>the</strong> decay in <strong>the</strong> VGS task. These findings<br />
suggest that DLPFC, toge<strong>the</strong>r with attentional <strong>an</strong>d motor-coding systems in <strong>an</strong>terior cingulate<br />
<strong>an</strong>d pre-supplementary eye fields, may encode spatial location <strong>an</strong>d pl<strong>an</strong> responses. However with<br />
sufficient time, ra<strong>the</strong>r th<strong>an</strong> maintaining spatial in<strong>for</strong>mation <strong>an</strong>d response pl<strong>an</strong>s in prefrontal<br />
systems over long delay periods, premotor cortex may maintain response pl<strong>an</strong>s which would free<br />
prefrontal systems to evaluate ongoing environmental dem<strong>an</strong>ds <strong>an</strong>d pl<strong>an</strong> fur<strong>the</strong>r responses. This<br />
suggests a temporal-dynamic aspect to <strong>the</strong> functional <strong>an</strong>atomy of mainten<strong>an</strong>ce operations in<br />
hum<strong>an</strong> spatial working memory.<br />
Disclosures: A. D'Cruz, None; J.L. Reilly, None; K. Kapur, None; S.K. Keedy, None; J.A.<br />
Sweeney, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.10/FF85<br />
Topic: F.01.f. Working memory<br />
Support: CONACYT
DGAPA-PAPIIT<br />
Title: Remembering <strong>the</strong> exact position of a target in space: An fMRI study<br />
Authors: J. GUTIERREZ-GARRALDA, *J. FERNANDEZ-RUIZ;<br />
Dept Fisiologia. Facultad Medicina, Univ. Nacional Autonoma Mexico, Mexico D.F., Mexico<br />
Abstract: Spatial working memory is a phenomenon that has been tackled in different ways in<br />
<strong>an</strong> attempt to underst<strong>an</strong>d <strong>the</strong> underlying mech<strong>an</strong>ism behind it. Different experiments have been<br />
created trying to undertake this issue. However, <strong>the</strong>se protocols usually rely on fixed targets <strong>an</strong>d<br />
n-back procedures which may not necessarily measure spatial working memory, but a different<br />
kind of memory in which <strong>the</strong> exact positioning of <strong>the</strong> stimuli is not relev<strong>an</strong>t to <strong>the</strong> task. We<br />
designed a task which deals with this variable <strong>an</strong>d evaluates spatial working memory without <strong>the</strong><br />
possibility of using o<strong>the</strong>r cues. After obtaining psychophysical data, we were able to establish <strong>the</strong><br />
spatial working memory absolute threshold <strong>for</strong> this task. Afterwards, a protocol was designed in<br />
order to test subjects while <strong>the</strong>y underwent functional magnetic reson<strong>an</strong>ce imaging (fMRI). Our<br />
data <strong>from</strong> <strong>the</strong> fMRI study reveals new in<strong>for</strong>mation on how <strong>the</strong> brain processes spatial working<br />
memory.<br />
Disclosures: J. Gutierrez-garralda, None; J. Fern<strong>an</strong>dez-Ruiz, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.11/FF86<br />
Topic: F.01.f. Working memory<br />
Support: CELEST, <strong>an</strong> NSF Science of Learning Center (SBE-0354378)<br />
SyNAPSE program of DARPA (HR001109-03-0001, HR001-09-C-0011<br />
Title: Interactions between r<strong>an</strong>k-selective spatial working memory <strong>an</strong>d <strong>the</strong> supplementary eye<br />
fields during <strong>the</strong> dynamic control of sequential eye movements<br />
Authors: *M. R. SILVER 1 , D. BULLOCK 1 , S. GROSSBERG 1 , M. H. HISTED 2 , E. K.<br />
MILLER 2 ;<br />
1 Dept. of Cognitive <strong>an</strong>d Neural Systems <strong>an</strong>d Ctr. <strong>for</strong> Adaptive Systems, Boston Univ., Boston,
MA; 2 The Picower Inst. <strong>for</strong> Learning <strong>an</strong>d Memory <strong>an</strong>d Dept. of Brain <strong>an</strong>d Cognitive Sci., MIT,<br />
Boston, MA<br />
Abstract: How do working memory circuits store multiple spatial locations <strong>for</strong> <strong>the</strong> control of<br />
pl<strong>an</strong>ned sequences of eye movements? How does using r<strong>an</strong>k-sensitive coding contribute to<br />
sequence storage <strong>an</strong>d recall when <strong>the</strong> same movement repeats at multiple list positions, or r<strong>an</strong>ks,<br />
during <strong>the</strong> sequence? We develop a competitive queuing model of working memory to explain<br />
<strong>an</strong>d simulate microstimulation, behavioral, electrophysiological, <strong>an</strong>d <strong>an</strong>atomical data that clarify<br />
<strong>the</strong>se processes. The model utilizes r<strong>an</strong>k-sensitive prefrontal working memory representations<br />
(Averbeck et al, 2003), which depend upon r<strong>an</strong>k-related activity in parietal cortex (Sawamura et<br />
al., 2002, see also Grossberg & Pearson, 2008), to produce spatial sequences in which <strong>the</strong> same<br />
action is repeated several times. The model shows how <strong>the</strong> supplementary eye fields (SEF;<br />
Schlag & Schlag-Rey, 1987) could mediate <strong>the</strong> selection of saccade pl<strong>an</strong>s <strong>from</strong> sequential<br />
working memory by simulating both behavioral results <strong>an</strong>d a number of SEF cell types (Isoda &<br />
T<strong>an</strong>ji, 2002, Lu et al., 2002). In addition, model simulations illustrate how microstimulation<br />
(Histed & Miller, 2006; Y<strong>an</strong>g et al., 2008) may alter <strong>the</strong> order of per<strong>for</strong>m<strong>an</strong>ce of sequences of<br />
eye movements stored in spatial working memory, but not which movements (items) are<br />
generated. Finally, <strong>the</strong> model proposes how SEF interacts with downstream regions such as <strong>the</strong><br />
frontal eye fields (FEF), during memory-guided sequential saccade tasks. The model explicates a<br />
functional role <strong>for</strong> <strong>the</strong> SEF <strong>an</strong>d its interactions with interconnected cortical areas, <strong>an</strong>d makes<br />
predictions <strong>for</strong> novel experimental conditions. The explicit circuit model clarifies how r<strong>an</strong>k-order<br />
sensitive working memories may solve <strong>the</strong> problem of representing sequential pl<strong>an</strong>s in which<br />
component actions repeat at later points in <strong>the</strong> sequence.<br />
Disclosures: M.R. Silver, None; D. Bullock, None; S. Grossberg, None; M.H. Histed,<br />
None; E.K. Miller, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.12/FF87<br />
Topic: F.01.f. Working memory<br />
Support: MEXT KAKENHI 20220003<br />
MEXT KAKENHI 21680024
Title: Dynamic cortical networks with <strong>the</strong>ta oscillation <strong>for</strong> <strong>the</strong> scene memory mainten<strong>an</strong>ce: A<br />
simult<strong>an</strong>eous fMRI <strong>an</strong>d EEG study<br />
Authors: *H. MIZUHARA 1,2 , N. SATO 2,3 , Y. YAMAGUCHI 2 ;<br />
1 2 3<br />
Kyoto Univ., Kyoto-shi, Jap<strong>an</strong>; RIKEN Brain Sci. Inst., Saitama, Jap<strong>an</strong>; Future Univ.<br />
Hakodate, Hakodate, Hokkaido, Jap<strong>an</strong><br />
Abstract: The medial temporal cortex including <strong>the</strong> hippocampus is known to be crucial <strong>for</strong> <strong>the</strong><br />
episodic memory. For <strong>the</strong> inst<strong>an</strong>tiation of <strong>the</strong> memory, <strong>the</strong> medial temporal cortex should work<br />
toge<strong>the</strong>r with distributed cortices to give a global network. However, little is known on <strong>the</strong><br />
mech<strong>an</strong>ism of global network <strong>for</strong>mation. A previous <strong>the</strong>oretical study predicted that <strong>the</strong> objectplace<br />
association memory is <strong>for</strong>med by <strong>the</strong> integration of <strong>the</strong> objects <strong>an</strong>d <strong>the</strong> place in<strong>for</strong>mation in<br />
<strong>the</strong> distributed cortices into <strong>the</strong> medial temporal cortex, <strong>an</strong>d that this integration is realized by <strong>the</strong><br />
<strong>the</strong>ta phase coding of <strong>the</strong> rat’s or primate’s local field potential (Sato & Yamaguchi,<br />
Hippocampus 2005). The <strong>the</strong>ta EEG enh<strong>an</strong>cement (4~8Hz) was also found during <strong>the</strong> objectplace<br />
association memory task in hum<strong>an</strong> subjects (Sato & Yamaguchi, Neuroreport 2007). To<br />
examine whe<strong>the</strong>r <strong>the</strong> in<strong>for</strong>mation represented in <strong>the</strong> distributed cortices is dynamically linked to<br />
<strong>the</strong> medial temporal cortex in <strong>the</strong> time window of <strong>the</strong> <strong>the</strong>ta EEG appear<strong>an</strong>ce during <strong>the</strong> memory<br />
mainten<strong>an</strong>ce, here we measured <strong>the</strong> simult<strong>an</strong>eous fMRI <strong>an</strong>d EEG during <strong>the</strong> natural scene<br />
memory task. In <strong>the</strong> current study, to m<strong>an</strong>ipulate <strong>the</strong> conscious access to <strong>the</strong> sensory inputs of <strong>the</strong><br />
natural scene, we used <strong>the</strong> attentional blink procedure. First, by comparing <strong>the</strong> EEG power<br />
among <strong>the</strong> short <strong>an</strong>d long SOA conditions, <strong>the</strong> results showed that <strong>the</strong> EEG <strong>the</strong>ta at <strong>the</strong> scalp<br />
frontal sites enh<strong>an</strong>ced when <strong>the</strong> subjects successfully retained <strong>the</strong> natural scene. Secondly, <strong>the</strong><br />
BOLD signals in <strong>the</strong> right parahippocampal place area, <strong>the</strong> left frontal eye field <strong>an</strong>d <strong>the</strong> left<br />
visual cortex showed <strong>the</strong> signific<strong>an</strong>t positive correlation to <strong>the</strong> <strong>the</strong>ta EEG power at <strong>the</strong> frontal<br />
sites. In addition, <strong>the</strong> signals in <strong>the</strong> medial prefrontal cortices showed <strong>the</strong> signific<strong>an</strong>t negative<br />
correlation. Finally, based on <strong>the</strong> correlation <strong>an</strong>alysis of <strong>the</strong> BOLD signal <strong>an</strong>d <strong>the</strong> EEG power,<br />
we estimated <strong>the</strong> hemodynamic response function to <strong>the</strong> <strong>the</strong>ta EEG enh<strong>an</strong>cement. The signals in<br />
<strong>the</strong> parahippocampal place area <strong>an</strong>d frontal eye field showed <strong>the</strong> sustained BOLD responses,<br />
while <strong>the</strong> c<strong>an</strong>onical positive or negative hemodynamic responses were also found in <strong>the</strong> <strong>the</strong>ta<br />
EEG power correlated regions. Our previous study reported that <strong>the</strong> negative BOLD response in<br />
<strong>the</strong> medial prefrontal cortices is tightly coupled with <strong>the</strong> central executive function to integrate<br />
<strong>the</strong> in<strong>for</strong>mation represented in <strong>the</strong> distributed cortices (Mizuhara & Yamaguchi, NeuroImage<br />
2007). Thus, toge<strong>the</strong>r with <strong>the</strong> previous studies, our results indicate that memory inst<strong>an</strong>tiation is<br />
a process of dynamical integration of <strong>the</strong> parahippocampal place area <strong>an</strong>d <strong>the</strong> frontal eye field<br />
under <strong>the</strong> control of <strong>the</strong> central executive function via frontal <strong>the</strong>ta EEG oscillation.<br />
Disclosures: H. Mizuhara, None; N. Sato, None; Y. Yamaguchi, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.13/FF88<br />
Topic: F.01.f. Working memory<br />
Support: NIH Gr<strong>an</strong>t R01 MH060234<br />
Title: Functional dissociations in neocortical <strong>an</strong>d cerebellar activations in verbal working<br />
memory<br />
Authors: C. L. MARVEL, D. M. ECHAVARRIA, M. L. FAULKNER, *J. E. DESMOND;<br />
Dept Neurol, Johns Hopkins, Baltimore, MD<br />
Abstract: It has been suggested that <strong>the</strong> inferior parietal lobe (IPL) is import<strong>an</strong>t <strong>for</strong> maintaining<br />
in<strong>for</strong>mation in <strong>the</strong> phonological store during working memory. In particular, <strong>the</strong> IPL is activated<br />
as a function of phonological load, such as when <strong>the</strong> verbal content contains unfamiliar phoneme<br />
sequences (i.e., pseudowords). Previous research has demonstrated concord<strong>an</strong>t activity between<br />
<strong>the</strong> left IPL <strong>an</strong>d <strong>the</strong> right inferior cerebellum (lobule VIII/VIIb) during <strong>the</strong> short-term rehearsal<br />
of verbal content. However, it is unclear whe<strong>the</strong>r inferior cerebellar activity is specific to<br />
phonological load or to a more general aspect of mainten<strong>an</strong>ce load, such as <strong>an</strong> increasing number<br />
of items to hold in memory. There<strong>for</strong>e, it is unclear whe<strong>the</strong>r <strong>the</strong> IPL <strong>an</strong>d inferior cerebellum<br />
work toge<strong>the</strong>r as part of a shared function or if <strong>the</strong>se two regions make separate contributions to<br />
maintaining in<strong>for</strong>mation in <strong>the</strong> phonological store.<br />
In this study, we examined verbal working memory <strong>for</strong> words <strong>an</strong>d pseudowords. We increased<br />
working memory load by varying <strong>the</strong> qu<strong>an</strong>tity of in<strong>for</strong>mation (i.e., number of items) <strong>an</strong>d <strong>the</strong><br />
phonological dem<strong>an</strong>d (i.e., words vs. pseudowords) in order to elicit differential activity in <strong>the</strong>se<br />
two brain regions.<br />
We used fMRI to examine verbal working memory in sixteen healthy young adults. Particip<strong>an</strong>ts<br />
were visually presented with 2 or 4 targets <strong>for</strong> 2 seconds. Targets consisted of ei<strong>the</strong>r words or<br />
pseudowords within a trial, 1-3 syllables, 4-7 letters, <strong>an</strong>d (<strong>for</strong> words) did not belong to <strong>the</strong> same<br />
sem<strong>an</strong>tic category. Two-target trials contained one phoneme in common; four-target trials<br />
contained two phonemes in common. Following presentation, <strong>the</strong>re was a 4-6 second delay<br />
period in which particip<strong>an</strong>ts covertly rehearsed <strong>the</strong> targets. Then a probe was presented <strong>for</strong> 1<br />
second. Particip<strong>an</strong>ts were instructed to decide whe<strong>the</strong>r <strong>the</strong> probe matched <strong>an</strong>y of <strong>the</strong> targets.<br />
Behavioral results yielded signific<strong>an</strong>t main effects of target load (4 > 2) <strong>an</strong>d trial type<br />
(pseudowords > words) reflected as lower accuracy <strong>an</strong>d slower response times in <strong>the</strong> higher load<br />
conditions. Event-related fMRI data <strong>an</strong>alysis focused on target load <strong>an</strong>d trial type contrasts<br />
during <strong>the</strong> rehearsal phase.<br />
Greater BOLD signal in response to phonological load (pseudowords) was observed in <strong>the</strong> right<br />
inferior cerebellum <strong>an</strong>d <strong>the</strong> left IPL. However, greater BOLD signal related to target load was<br />
specific to <strong>the</strong> inferior cerebellum. These results indicate that <strong>the</strong> functions of <strong>the</strong>se two regions<br />
are dissociable, <strong>an</strong>d that each makes a distinct contribution to <strong>the</strong> short-term rehearsal of verbal<br />
content. The cerebellum may respond to more general aspects of phonological complexity,<br />
involving both <strong>the</strong> number <strong>an</strong>d familiarity of <strong>the</strong> phonemes held in memory.
Disclosures: C.L. Marvel, None; D.M. Echavarria, None; M.L. Faulkner, None; J.E.<br />
Desmond, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.14/FF89<br />
Topic: F.01.f. Working memory<br />
Support: Ministerio de Ciencia e Innovacion, programa Ramon y Cajal.<br />
Title: Causal connectivity <strong>an</strong>alysis of tracking faces in presence of repeated distracters during<br />
working memory tasks<br />
Authors: *J. M. CORTES 1 , D. MARINAZZO 2 , E. MADRID 3 , P. TUDELA 3 ;<br />
1 Dept. Ciencias Computacion E Inteligencia Artificial. Univ. of, Gr<strong>an</strong>ada, Spain; 2 CNRS, UMR<br />
8119, Paris, Fr<strong>an</strong>ce; 3 Dept. de Psicologia Exptl. y Fisiologia del Comportamiento., Univ. of<br />
Gr<strong>an</strong>ada, Gr<strong>an</strong>ada, Spain<br />
Abstract: Recognition of a specific visual target among equally familiar distracters requires<br />
neural mech<strong>an</strong>isms <strong>for</strong> tracking items in working memory. High Density EEG recordings were<br />
obtained <strong>from</strong> sixteen right-h<strong>an</strong>ded normal particip<strong>an</strong>ts while <strong>the</strong>y per<strong>for</strong>med a visual working<br />
memory task in which familiarity <strong>for</strong> targets <strong>an</strong>d distracters was similar. Each trial consisted of a<br />
target face to be remembered, presented <strong>for</strong> 3.5 s, followed by 13 faces presented at a rate of 1.5<br />
s per face. The target <strong>an</strong>d one of <strong>the</strong> distracters were repeated up to five times in a given trial,<br />
separated by 3 to 15 s.<br />
Gr<strong>an</strong>ger causality states that a given signal A has causal influence on B if <strong>the</strong> predictability on B<br />
improves adding to B <strong>the</strong> past of A. We have applied Gr<strong>an</strong>ger connectivity <strong>an</strong>alysis (Seth 2005)<br />
to study <strong>the</strong> dynamics of <strong>the</strong> neural networks involved on <strong>the</strong> recognition of a visual target<br />
stimulus among equally familiar distracter, obtaining <strong>the</strong> causal interactions among <strong>the</strong> evenly<br />
spaced array of 128 EEG electrodes on <strong>the</strong> scalp. By using Kernel methods (Marinazzo et al<br />
2008), we extended <strong>the</strong> results in (Seth 2005) to achieve detection of non-linear causal<br />
relationships. Causal connectivity <strong>an</strong>alysis is a new tool to look at EEG data <strong>an</strong>d it allows <strong>for</strong><br />
establishing new correlates among electrical brain activity <strong>an</strong>d cognitive processes.<br />
Disclosures: J.M. Cortes, None; D. Marinazzo, None; E. Madrid, None; P. Tudela, None.
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.15/FF90<br />
Topic: F.01.f. Working memory<br />
Title: Bimodal occipital EEG gamma spectrum during visual choice RT: Artifact or sc<strong>an</strong>ning<br />
cognitive code?<br />
Authors: *R. B. GLASSMAN, C. P. HARTLEY, C. VELASQUEZ;<br />
Dept Psychol, Lake Forest Col., Lake Forest, IL<br />
Abstract: Brain waves are larger scale phenomena th<strong>an</strong> single-neuron events. Is <strong>the</strong>re sufficient<br />
resolution in <strong>an</strong> EEG sample a second long, to code hints of <strong>an</strong> "engram" of knowledge in<br />
individual ch<strong>an</strong>nels, or must coherence among ch<strong>an</strong>nels always be measured? We observed<br />
bimodal power spectral density (PSD) of EEG at occipital points O1 <strong>an</strong>d O2 in subjects who<br />
quickly click <strong>the</strong> mouse when a “correct” color picture appears (Click: rowboat or eagle. Do not<br />
click: balloon or cardinal.) One set of peaks r<strong>an</strong>ges <strong>from</strong> 20 to 70 Hz in different runs of 10 trials<br />
<strong>an</strong>d <strong>the</strong> o<strong>the</strong>r between 70 <strong>an</strong>d 150 Hz. In some runs <strong>the</strong> spectra remain steady <strong>from</strong> trial to trial;<br />
o<strong>the</strong>rs drift. Our <strong>an</strong>alysis program (SfN 2008) took one-second segments of EEG <strong>from</strong> each trial,<br />
based on behavioral event time stamps. Most choice reaction times are 300-700 ms. On<br />
preliminary examination of <strong>the</strong> PSD graphs, it does not seem to matter much which portion of<br />
<strong>the</strong> trial is sampled. Each of <strong>the</strong> figures below is <strong>from</strong> ten successive trials, top to bottom (left O1<br />
electrode), each 1s sample beginning 0.5s after a correct click (abscissas 0-200 Hz). The subject<br />
on <strong>the</strong> left (RBG) has often done <strong>the</strong> task, while <strong>the</strong> subject on <strong>the</strong> right (MB) was new at it - but<br />
it is premature to infer level of experience as relev<strong>an</strong>t. Our programs <strong>for</strong> data acquisition <strong>an</strong>d<br />
<strong>an</strong>alysis are written in LabVIEW (National Instruments, Austin, TX); two ch<strong>an</strong>nels of brain<br />
waves (monopolar; earlobe reference) are acquired to a laptop computer via instrumentation<br />
amplifier chips (INA128, Texas Instruments) <strong>an</strong>d PCMCIA card (NI 6024E). A notch filter is not<br />
needed because we use DC battery power. The subject elects when to begin each trial; <strong>the</strong><br />
computer <strong>the</strong>n r<strong>an</strong>domly selects one of <strong>the</strong> four pictures, to appear between 2 <strong>an</strong>d 7 s later. EEG<br />
is acquired at 1K samples/s with digital b<strong>an</strong>dpass 1 to 110 Hz. If our results are valid (& not<br />
artifacts of <strong>the</strong> amplifier, software timing, aliasing, EMG, etc.) <strong>the</strong>y suggest a fluid, sc<strong>an</strong>ning<br />
EEG code <strong>for</strong> cognitive items, not highly localized in specific brain regions or EEG frequencies.
What might be <strong>the</strong> secret of how such <strong>an</strong> engram occupies brain geography <strong>an</strong>d time?<br />
Disclosures: R.B. Glassm<strong>an</strong>, None; C.P. Hartley, None; C. Velasquez, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.16/FF91<br />
Topic: F.01.f. Working memory<br />
Support: NIH Gr<strong>an</strong>t MH064498<br />
Title: Investigating <strong>the</strong> effects of rTMS to perisylvi<strong>an</strong> brain regions during verbal working<br />
memory task per<strong>for</strong>m<strong>an</strong>ce with EEG<br />
Authors: *D. ACHESON, B. POSTLE;<br />
Univ. Wisconsin, Madison, WI<br />
Abstract: Regions of <strong>the</strong> posterior superior temporal lobe are critical to representing <strong>an</strong>d<br />
maintaining phonological representations in l<strong>an</strong>guage production, comprehension <strong>an</strong>d verbal<br />
working memory (WM). Previous research has shown that repetitive tr<strong>an</strong>scr<strong>an</strong>ial magnetic<br />
stimulation (rTMS) of <strong>the</strong> posterior superior temporal gyrus (pSTG) increases error rates on<br />
l<strong>an</strong>guage production <strong>an</strong>d WM tasks, yet <strong>the</strong> neural bases of this rTMS effect remain unclear.<br />
Fur<strong>the</strong>rmore, it is unclear why rTMS to left hemisphere perisylvi<strong>an</strong> regions has reliably produced<br />
impairment across several verbal WM tasks, whereas <strong>an</strong>alogous rTMS protocols targeting<br />
dorsal-stream regions during spatial WM have not. In <strong>the</strong> present study <strong>the</strong> temporal <strong>an</strong>d spatial<br />
dynamics of this rTMS effect were explored by using functional magnetic reson<strong>an</strong>ce imaging<br />
(fMRI)-guided rTMS while simult<strong>an</strong>eously recording electroencephalographic (EEG) activity as<br />
subjects per<strong>for</strong>med delayed recognition of nonword tongue twister stimuli. Tongue twisters (e.g.<br />
kif geev gif keev) are defined by <strong>the</strong> pattern of onsets (ABBA; /k/ /g/ /g/ /k/) <strong>an</strong>d rhymes<br />
(CDCD; /If/ /iv/ /If/ /iv/) in syllables, <strong>an</strong>d induce serial ordering errors during phonological<br />
encoding <strong>an</strong>d articulation in speech production.
The study proceeded in two stages. First, event-related fMRI was used to elicit activation in <strong>the</strong><br />
pSTG as individuals per<strong>for</strong>med delayed serial recognition of nonword tongue twister stimuli.<br />
Second, 10 Hz rTMS (110% of motor threshold) was applied to <strong>the</strong> functionally-defined area of<br />
<strong>the</strong> pSTG <strong>an</strong>d to <strong>the</strong> leg area of somatosensory cortex (<strong>the</strong> control region) during <strong>the</strong> delay<br />
period of <strong>the</strong> same task. EEG recording (60 ch<strong>an</strong>nels) was taken during task per<strong>for</strong>m<strong>an</strong>ce, <strong>an</strong>d<br />
rTMS artifacts were removed prior to <strong>an</strong>alysis. Previous research has shown<br />
that10 Hz rTMS of <strong>the</strong> superior parietal lobule during <strong>the</strong> delay period of a spatial WM task<br />
affects alpha-b<strong>an</strong>d power, <strong>an</strong>d that this rTMS effect is negatively correlated with accuracy. We<br />
predicted that stimulation of <strong>the</strong> pSTG but not <strong>the</strong> control region would lead to entrainment of<br />
task-related neuronal oscillations, <strong>an</strong>d that <strong>the</strong> magnitude of this rTMS effect would be correlated<br />
with rTMS-induced disruption on task-per<strong>for</strong>m<strong>an</strong>ce. Thus, simult<strong>an</strong>eously recording EEG with<br />
rTMS during verbal WM tasks allows one to observer <strong>the</strong> mech<strong>an</strong>isms by which rTMS disrupts<br />
task per<strong>for</strong>m<strong>an</strong>ce, as well as to examine how neuronal oscillations influence <strong>the</strong> mainten<strong>an</strong>ce of<br />
phonological representations over short periods of time.<br />
Disclosures: D. Acheson, None; B. Postle, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.17/FF92<br />
Topic: F.01.f. Working memory<br />
Support: IDO 05/010 EEG-fMRI<br />
Title: Alpha <strong>an</strong>d executive functioning: A simult<strong>an</strong>eous EEG-fMRI study<br />
Authors: *J. RAMAUTAR 1 , N. NOVITSKIY 2 , K. VANDERPERREN 3 , B. VANRUMSTE 3,6 ,<br />
L. LAGAE 4 , J. WAGEMANS 2 , S. VAN HUFFEL 3 , S. SUNAERT 5 , P. STIERS 7 ;<br />
2 Dept. of Psychology, 3 Dept. of Electrical Engin., 4 Dept. of Paediatric Neurol., 5 Dept. of<br />
Radiology, 1 Catholic Univ. of Leuven|910001106|0, Leuven, Belgium; 6 Biosci. <strong>an</strong>d Technol.<br />
Dept., Katholieke Hogeschool Kempen, Geel, Belgium; 7 Fac. of Psychology, Univ. of<br />
Maastricht, Maastricht, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Electroencephalography related functional magnetic reson<strong>an</strong>ce imaging (EEG-fMRI)<br />
c<strong>an</strong> be applied to identify blood oxygen level dependent (BOLD) signal ch<strong>an</strong>ges associated with<br />
physiological EEG events. EEG studies of spont<strong>an</strong>eous brain activity have indicated alpha<br />
activity (8-12 Hz) as <strong>an</strong> idling rhythm because it marks <strong>the</strong> state of relaxed wakefulness. Studies
investigating <strong>the</strong> BOLD signals associated with alpha amplitude during passive tasks such as<br />
resting state (eyes open/closed) paradigms have shown that <strong>the</strong> posterior visual regions as well as<br />
o<strong>the</strong>r regions across <strong>the</strong> cortex correlate with posterior alpha power fluctuations. Extending <strong>the</strong>se<br />
studies, we w<strong>an</strong>ted to investigate <strong>the</strong> relation between alpha power fluctuations with areas<br />
involved in more specific cognitive functioning such as executive functioning. The expectation is<br />
that areas related to executive functioning will increase in activity in periods of high alpha<br />
compared to low alpha.<br />
In this study, sixty-four ch<strong>an</strong>nels of EEG were recorded in 9 healthy subjects during a Go-NoGo<br />
task (20% Nogo) inside a 3T sc<strong>an</strong>ner. After artifact removal, individual alpha power fluctuations<br />
at posterior sites were extracted <strong>from</strong> <strong>the</strong> EEG by applying a continuous wavelet tr<strong>an</strong>s<strong>for</strong>m<br />
(CWT). To assess <strong>the</strong> distribution of BOLD signal modulations related to alpha fluctuations, <strong>the</strong><br />
resulting alpha amplitude time course was convolved with <strong>the</strong> c<strong>an</strong>onical hemodynamic response<br />
function (HRF) <strong>an</strong>d used as a regressor in <strong>the</strong> general linear model. In order to investigate <strong>the</strong><br />
influence of alpha power fluctuations on <strong>the</strong> activation of task-relev<strong>an</strong>t brain regions, alpha<br />
power in a time window around trial start was directly related to <strong>the</strong> strength of <strong>the</strong> BOLDresponse<br />
invoked during each trial in selected Regions of Interests. ROI <strong>an</strong>alyses based on a<br />
separate prefrontal mapping task in combination with alpha power magnitude were per<strong>for</strong>med<br />
<strong>an</strong>d <strong>the</strong> extracted BOLD responses were sorted across stimulus type.<br />
GLM <strong>an</strong>alysis of Go <strong>an</strong>d Nogo events replicates Go-Nogo findings as <strong>the</strong> inferior frontal<br />
junction <strong>an</strong>d (pre)supplementary motor areas. In addition, areas in visual cortex <strong>an</strong>d sub-cortical<br />
regions associated with alpha fluctuations were in line with previous alpha findings during<br />
resting state (eyes open/closed) studies. ROI extracted time courses <strong>from</strong> areas associated with<br />
executive functioning showed a complex relationship with alpha power: high amplitude BOLD<br />
responses were related to periods of low <strong>an</strong>d high alpha power <strong>an</strong>d were specific <strong>for</strong> stimulus<br />
type. These findings provide insights into <strong>the</strong> fluctuations induced by alpha during task<br />
per<strong>for</strong>m<strong>an</strong>ce.<br />
Disclosures: J. Ramautar, None; N. Novitskiy, None; K. V<strong>an</strong>derperren, None; B.<br />
V<strong>an</strong>rumste, None; L. Lagae, None; J. Wagem<strong>an</strong>s, None; S. V<strong>an</strong> Huffel, None; S. Sunaert,<br />
None; P. Stiers, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.18/FF93<br />
Topic: F.01.f. Working memory<br />
Support: NIH Gr<strong>an</strong>t AG15793
NIH Gr<strong>an</strong>t AG0925<br />
NSF Graduate Research Fellowship<br />
Title: Electrophysiological correlates of refreshing: Event-related potentials associated with<br />
directing reflective attention to face, scene, or word representations<br />
Authors: *M. R. JOHNSON, G. MCCARTHY, K. A. MULLER, M. K. JOHNSON;<br />
Yale Univ., New Haven, CT<br />
Abstract: Refreshing is a brief act of reflective attention that serves to <strong>for</strong>eground one active<br />
mental representation relative to o<strong>the</strong>rs. Refreshing is a simple executive process thought to be a<br />
component of more complex mental processes such as those engaged by working memory.<br />
FMRI studies have revealed that refreshing is associated with dorsolateral prefrontal cortex<br />
activity, <strong>an</strong>d refreshing c<strong>an</strong> also modulate activity in posterior category-selective extrastriate<br />
areas. However, little is known about <strong>the</strong> time scale over which refreshing operates <strong>an</strong>d whe<strong>the</strong>r<br />
this already simple cognitive operation comprises subcomponents. Here we sought to determine<br />
whe<strong>the</strong>r event-related potentials (ERPs) could be used to differentiate between different<br />
categories of items being refreshed, as previous fMRI work suggests. Thus, we conducted <strong>an</strong><br />
ERP study in which particip<strong>an</strong>ts were shown pairs of faces, scenes, or words, followed by a cue<br />
to ei<strong>the</strong>r refresh (briefly think back to, direct reflective attention towards) one of <strong>the</strong> stimuli<br />
(Refresh condition), to press a button (Act condition), or to do nothing (NoAct condition).<br />
Across stimulus categories, at multiple electrode sites, refreshing was associated with a positive<br />
peak with a shorter latency (~ 400ms) th<strong>an</strong> <strong>the</strong> corresponding peak in <strong>the</strong> baseline NoAct<br />
condition. In addition, a later sustained potential (~ 800-1400ms) was observed which remained<br />
positive in <strong>the</strong> Refresh condition while returning to baseline in NoAct. At several sites,<br />
refreshing words was associated with greater positivity at <strong>the</strong> earlier peak th<strong>an</strong> refreshing faces<br />
or scenes, whereas refreshing scenes was associated with <strong>the</strong> least positivity during <strong>the</strong> later<br />
sustained component. Potentials associated with refreshing faces tended to resemble potentials<br />
<strong>for</strong> refreshing scenes at <strong>the</strong> earlier peak but potentials <strong>for</strong> refreshing words during <strong>the</strong> later<br />
component. These results are consistent with prior fMRI studies that suggest refreshing may<br />
entail at least two components, one of initiating <strong>the</strong> shift of reflective attention (associated with<br />
earlier activity in <strong>an</strong>terior prefrontal cortex) <strong>an</strong>d one of actually modulating activity relev<strong>an</strong>t to<br />
maintaining <strong>the</strong> indicated representation (associated with later activity in lateral<br />
prefrontal/parietal cortices <strong>an</strong>d enh<strong>an</strong>cement of activity in stimulus-specific extrastriate cortex).<br />
Disclosures: M.R. Johnson, None; G. McCarthy, None; K.A. Muller, None; M.K. Johnson,<br />
None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.19/FF94<br />
Topic: F.01.f. Working memory<br />
Support: BMBF/BCCN gr<strong>an</strong>t 01GQ0411<br />
Title: Compositionality of rule representations in hum<strong>an</strong> fronto-parietal cortex<br />
Authors: *C. REVERBERI 1,2 , J.-D. HAYNES 1,3 ;<br />
1<br />
Bernstein Ctr. For Computat. Neuroscience, Charite - Universitaetsmedizin, Berlin, Germ<strong>an</strong>y;<br />
2 3<br />
Dept. of Psychology, Univ. Mil<strong>an</strong>o - Bicocca, Mil<strong>an</strong>o, Italy; Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Cognitive<br />
<strong>an</strong>d Brain Sci., Leipzig, Germ<strong>an</strong>y<br />
Abstract: Conditional rules (“if x <strong>the</strong>n y”) are widely used in daily life to org<strong>an</strong>ize <strong>an</strong>d control<br />
actions <strong>an</strong>d thoughts. At <strong>the</strong> simplest level, conditional rules may be used to link percepts (a<br />
green traffic light) to actions (go). The neural bases of rule-guided behavior have been<br />
extensively studied recently. A question that has received less attention is that simple rules are<br />
often combined to compound rule sets. Are <strong>the</strong> same brain structures involved in representation<br />
of compound rules as in simple rules? And is <strong>the</strong> neural coding of compound rules<br />
compositional, me<strong>an</strong>ing that it is represented by combining <strong>the</strong> codes <strong>for</strong> its constituent parts?<br />
A modified cued task switching paradigm was administered during fMRI sc<strong>an</strong>ning. Subjects<br />
were required to retrieve, maintain during a delay, <strong>an</strong>d apply <strong>the</strong> appropriate set of conditional<br />
rules (e.g. “if <strong>the</strong>re is a house press left”) to a series of target stimuli. Two visual cues coded <strong>for</strong><br />
each rule set, allowing us to disent<strong>an</strong>gle between rule representation <strong>an</strong>d <strong>the</strong> visual features of<br />
<strong>the</strong> cue. Six rule sets belonging to two different classes were used. Simple rules (n = 4) consisted<br />
of a unique conditional rule linking a visual stimulus (houses or faces) to <strong>an</strong> action (press left or<br />
press right). Compound rules (n = 2) consisted of a pair of single rules en<strong>for</strong>ced at <strong>the</strong> same time.<br />
Each compound rule involved <strong>the</strong> same motor acts <strong>an</strong>d visual stimuli but in different<br />
combinations.<br />
We applied multivariate classifiers to <strong>the</strong> local pattern of activation during <strong>the</strong> delay between<br />
rule cue <strong>an</strong>d task execution. We were able to decode which compound rule was active <strong>from</strong> <strong>the</strong><br />
left superior parietal lobe, <strong>the</strong> precuneus, <strong>an</strong>d <strong>the</strong> inferior frontal lobe bilaterally, with <strong>an</strong> average<br />
accuracy r<strong>an</strong>ging between 60% <strong>an</strong>d 64%. The same areas, except <strong>for</strong> <strong>the</strong> right inferior frontal<br />
lobe were also involved in single rule representation. Given this overlap, we assessed whe<strong>the</strong>r<br />
<strong>the</strong> activity patterns of <strong>the</strong> simple rules could be used to determine which compound rule was<br />
active. This was possible in <strong>the</strong> left superior parietal lobe <strong>an</strong>d precuneus.<br />
Our findings show that left parietal, precuneus <strong>an</strong>d left inferior frontal cortex maintain<br />
in<strong>for</strong>mation on which simple <strong>an</strong>d complex rules are controlling behavior. Fur<strong>the</strong>rmore, <strong>the</strong><br />
parietal lobe <strong>an</strong>d precuneus use a “compositional” code, so that a conditional rule is coded in <strong>the</strong><br />
same way both when is represented alone (A) <strong>an</strong>d along with <strong>an</strong>o<strong>the</strong>r rule (A+B). By contrast,<br />
frontal cortex does not use a compositional activity code. This dissociation may help clarifying<br />
<strong>the</strong> respective role of <strong>the</strong> two brain areas in rule guided behavior.<br />
Disclosures: C. Reverberi, None; J. Haynes, None.
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.20/FF95<br />
Topic: F.01.f. Working memory<br />
Support: ZonMW 016.036.401<br />
Title: Context representation <strong>an</strong>d <strong>the</strong> Working Memory system<br />
Authors: *N. F. RAMSEY 1 , T. R. VAN RAALTEN 2 , J. M. JANSMA 3 ;<br />
1 Dept Neurol. & Neurosurg., 2 Dept Child Psychiatry, Rudolf Magnus Institute, Univ. of Utrecht,<br />
Utrecht, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 Mood <strong>an</strong>d Anxiety Disorders Br., Natl. Inst. of Mental Hlth., Be<strong>the</strong>sda,<br />
MD<br />
Abstract: The Working Memory (WM) system of <strong>the</strong> hum<strong>an</strong> brain is typically associated with<br />
short-term storage of in<strong>for</strong>mation. Ano<strong>the</strong>r critical function of this system, notably <strong>the</strong><br />
dorsolateral prefrontal cortex, is to m<strong>an</strong>ipulation of in<strong>for</strong>mation in order to make a decision<br />
about its nature, <strong>an</strong>d regulate sensory <strong>an</strong>d motor systems in order to generate appropriate actions.<br />
Because features of a stimulus are highly dependent on <strong>the</strong> context, <strong>an</strong> import<strong>an</strong>t question is<br />
whe<strong>the</strong>r <strong>the</strong> WM system also represents context (as has been postulated) <strong>an</strong>d if so, how.<br />
In a series of studies in hum<strong>an</strong>s, using fMRI, electrocorticography <strong>an</strong>d TMS, we show that <strong>the</strong><br />
WM system appears to be reactive ra<strong>the</strong>r th<strong>an</strong> proactive in responding to stimuli. We show that<br />
evaluation of novel stimuli engages <strong>the</strong> WM system, but engagement declines with practise <strong>an</strong>d<br />
eventually completely disappears. This practise effect is rapid <strong>for</strong> mainten<strong>an</strong>ce of in<strong>for</strong>mation,<br />
<strong>an</strong>d slower <strong>for</strong> processing of <strong>the</strong> stimulus. Moreover, r<strong>an</strong>domly intermixed overlearned stimuli<br />
are accurately processed with virtually no detectable brainresponse. In addition, stimulation of<br />
WM regions using TMS impairs per<strong>for</strong>m<strong>an</strong>ce with novel stimuli, but impairment declines with<br />
practise <strong>an</strong>d eventually disappears completely.<br />
The findings indicate that <strong>the</strong> WM system responds to <strong>the</strong> presence or absence of context<br />
relev<strong>an</strong>t features of stimuli. We argue that <strong>the</strong> WM system is <strong>the</strong>re<strong>for</strong>e not in control of<br />
in<strong>for</strong>mation processing, but instead functions secondary to requests posed by <strong>an</strong> as of yet<br />
unknown system. We postulate that <strong>the</strong> WM system does not represent context, but ra<strong>the</strong>r<br />
responds to situations where stimuli c<strong>an</strong>not be evaluated by lower-level processes in sensory <strong>an</strong>d<br />
motor systems.<br />
The WM systems' function may be to endow stimuli with task-relev<strong>an</strong>t features by me<strong>an</strong>s of<br />
regulating sensory <strong>an</strong>d motor systems to couple stimulus features with context-relev<strong>an</strong>t<br />
properties. Once stimuli have been endowed with <strong>the</strong> appropriate feature(s), <strong>the</strong> WM system is
no longer called upon, <strong>an</strong>d processing of incoming stimuli is h<strong>an</strong>dled without involvement of <strong>the</strong><br />
WM system.<br />
Taken toge<strong>the</strong>r, we argue that <strong>the</strong>re is <strong>an</strong> as-of-yet unknown system in <strong>the</strong> brain that supercedes<br />
<strong>the</strong> WM system hierarchically <strong>an</strong>d decides if WM involvement is needed, given <strong>the</strong> specific<br />
context <strong>an</strong>d stimuli features.<br />
Disclosures: N.F. Ramsey, None; T.R. V<strong>an</strong> Raalten, None; J.M. J<strong>an</strong>sma, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.21/FF96<br />
Topic: F.01.f. Working memory<br />
Support: JSPS Gr<strong>an</strong>t#19203032<br />
JSPS Gr<strong>an</strong>t#18330156<br />
JSPS Gr<strong>an</strong>t#21653077<br />
JSPS 20・5480<br />
Title: Incidental encoding of goal-irrelev<strong>an</strong>t in<strong>for</strong>mation c<strong>an</strong> be predicted by activation of <strong>the</strong><br />
left middle frontal gyrus: A preliminary fMRI study<br />
Authors: *T. MINAMOTO 1 , M. OSAKA 2 , N. OSAKA 1 ;<br />
1 Kyoto Univ., Kyoto, Jap<strong>an</strong>; 2 Osaka Univ., Suita, Jap<strong>an</strong><br />
Abstract: Efficient filtering of goal-irrelev<strong>an</strong>t in<strong>for</strong>mation is essential to lead goal-oriented<br />
behavior; however, behavioral studies have shown that our attentional system sometimes fail to<br />
filter out goal-irrelev<strong>an</strong>t in<strong>for</strong>mation, <strong>an</strong>d encode <strong>the</strong>m into long-term memory. In <strong>the</strong> present<br />
fMRI study, we aimed to reveal neural structures that determine <strong>the</strong> incidental encoding of goalirrelev<strong>an</strong>t<br />
in<strong>for</strong>mation. Eight healthy adults were participated in <strong>the</strong> study. In <strong>an</strong> MRI sc<strong>an</strong>ner,<br />
two memory tasks were administered: a delayed matching-to-sample (DMS) task with distraction<br />
<strong>an</strong>d a surprising recognition task. In <strong>the</strong> DMS task, particip<strong>an</strong>ts were required to memorize 3<br />
face stimuli, while ignoring 3 face distractors presented during retention phase. Two types of<br />
arrow stimuli (right or left) were presented with <strong>the</strong> distractors, <strong>an</strong>d particip<strong>an</strong>ts were instructed<br />
to judge <strong>the</strong> direction of <strong>the</strong> arrow presented after <strong>the</strong> third distractor stimulus. Following 10 min
eak, a surprising recognition task was given. In <strong>the</strong> task, 4 types of stimuli were presented: face<br />
stimuli to-be-remembered in <strong>the</strong> DMS task (FR), face stimuli to-be-ignored in <strong>the</strong> DMS task<br />
(FI), novel face stimuli (FN), <strong>an</strong>d numbers. In <strong>the</strong> face conditions, particip<strong>an</strong>ts were instructed to<br />
judge if <strong>the</strong>y saw <strong>the</strong> face picture in <strong>the</strong> DMS task. In <strong>the</strong> number condition, <strong>the</strong>y needed to judge<br />
if <strong>the</strong> number is odd or even. As <strong>for</strong> experimental designs, a trial-based design was employed in<br />
<strong>the</strong> DMS task while a rapid event-related design was used in <strong>the</strong> recognition task. Behavioral<br />
result showed that particip<strong>an</strong>ts memorized both FR <strong>an</strong>d FI stimuli above ch<strong>an</strong>ce level while <strong>the</strong><br />
per<strong>for</strong>m<strong>an</strong>ce of FN was around ch<strong>an</strong>ce level. In <strong>an</strong> fMRI <strong>an</strong>alysis, we mainly focused on <strong>the</strong> two<br />
conditions: FI subsequently remembered (incidental encoding) <strong>an</strong>d FI not-remembered<br />
(successful filtering) in <strong>the</strong> recognition task. As a result, during <strong>the</strong> distraction phase of <strong>the</strong> DMS<br />
task, <strong>the</strong> left middle frontal gyrus showed a signific<strong>an</strong>tly greater activation in FI subsequently<br />
not-remembered th<strong>an</strong> those remembered. We also made <strong>the</strong> same contrast in <strong>the</strong> recognition task,<br />
<strong>an</strong>d found that <strong>the</strong> right <strong>an</strong>terior <strong>an</strong>d posterior hippocampus showed a greater activation in FI<br />
subsequently remembered th<strong>an</strong> ones not-remembered. Those results suggest that <strong>the</strong> left middle<br />
frontal gyrus plays <strong>an</strong> import<strong>an</strong>t role in suppressing <strong>the</strong> encoding of goal-irrelev<strong>an</strong>t in<strong>for</strong>mation<br />
into long-term memory, possibly modulating <strong>the</strong> activity of <strong>the</strong> medial temporal lobe.<br />
Disclosures: T. Minamoto, None; M. Osaka, None; N. Osaka, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.22/FF97<br />
Topic: F.01.f. Working memory<br />
Title: Rapid task learning as a window into <strong>the</strong> neural basis of executive control<br />
Authors: *M. W. COLE 1 , W. SCHNEIDER 2 ;<br />
1 Neurosci., 2 Psychology, Univ. Pittsburgh, Pittsburgh, PA<br />
Abstract: Rapidly learning a task <strong>from</strong> instructions is a key aspect of nearly every study of<br />
hum<strong>an</strong> cognition, yet very little is known about <strong>the</strong> executive control processes underlying this<br />
behavior. Research in this area could provide import<strong>an</strong>t new details <strong>for</strong> neurocognitive modeling<br />
of a wide r<strong>an</strong>ge of behaviors. More broadly, rapid instructed task learning (RITL) is extremely<br />
import<strong>an</strong>t <strong>for</strong> everyday life. Indeed, <strong>the</strong> inability to adapt to novel situations is exceptionally<br />
debilitating, affecting everything <strong>from</strong> using new technologies, playing new games, or learning<br />
new tasks at work, <strong>an</strong>d is perhaps <strong>the</strong> defining characteristic of disorders of both executive<br />
function <strong>an</strong>d prefrontal cortex . The present research uses novel situations as a window into <strong>the</strong>
inner workings of executive control in hum<strong>an</strong>s <strong>an</strong>d has implications <strong>for</strong> studies of cognition<br />
generally. We developed a cognitive paradigm to systematically investigate <strong>the</strong> neural basis of<br />
RITL <strong>for</strong> <strong>the</strong> first time. We discovered a double dissociation between a network including<br />
dorsolateral prefrontal cortex (DLPFC) <strong>an</strong>d posterior parietal cortex (PPC), involved in loading<br />
individual task sem<strong>an</strong>tics into working memory, <strong>an</strong>d a network including <strong>an</strong>terior prefrontal<br />
cortex (aPFC) <strong>an</strong>d <strong>an</strong>terior inferior temporal cortex (aIT), involved in integrating task sem<strong>an</strong>tics<br />
in preparation <strong>for</strong> task per<strong>for</strong>m<strong>an</strong>ce. We found that both networks are involved in novel task set<br />
<strong>for</strong>mation <strong>an</strong>d practiced task set retrieval, but that task set <strong>for</strong>mation occurs in a bottom-up<br />
m<strong>an</strong>ner (sem<strong>an</strong>tic loading, <strong>the</strong>n integration) while task set retrieval occurs in a top-down m<strong>an</strong>ner<br />
(integration retrieval, <strong>the</strong>n sem<strong>an</strong>tic loading). These findings suggest that RITL relies upon<br />
sem<strong>an</strong>tic loading by DLPFC <strong>an</strong>d PPC, but that aPFC <strong>an</strong>d aIT are necessary to support sem<strong>an</strong>tic<br />
integration both dynamically during RITL <strong>an</strong>d <strong>from</strong> long-term memory after extensive practice.<br />
These findings have implications <strong>for</strong> cognitive studies using instructions in preparation <strong>for</strong> task<br />
per<strong>for</strong>m<strong>an</strong>ce, as well as <strong>for</strong> underst<strong>an</strong>ding <strong>the</strong> m<strong>an</strong>y everyday behaviors utilizing rapid task<br />
learning.<br />
Disclosures: M.W. Cole, None; W. Schneider, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.23/FF98<br />
Topic: F.01.f. Working memory<br />
Support: NSERC PDF
Title: Behavioral <strong>an</strong>d neural evidence <strong>for</strong> item-specific per<strong>for</strong>m<strong>an</strong>ce monitoring<br />
Authors: *C. BLAIS, M. HARRIS, S. A. BUNGE;<br />
Univ. Calif, Berkeley, Berkeley, CA<br />
Abstract: How cognitive control is recruited <strong>an</strong>d implemented has become a major focus of<br />
researchers in cognitive psychology <strong>an</strong>d neuroscience. Current <strong>the</strong>ories posit that cognitive<br />
control operates at <strong>the</strong> level of general rules - <strong>for</strong> example, in a Stroop task, “attend to <strong>the</strong> color<br />
of <strong>the</strong> stimulus”. Here we present a series of experiments designed to tap global <strong>an</strong>d local<br />
components of cognitive control independently.<br />
In <strong>the</strong>se studies, <strong>the</strong> overall proportion of congruent trials is m<strong>an</strong>ipuated across a series of trial<br />
blocks to index <strong>the</strong> global component of control. Critically, embedded within each block is <strong>an</strong><br />
item-level proportion congruent m<strong>an</strong>ipulation that serves to index <strong>the</strong> local component of<br />
control. Behavioral results suggest that cognitive control is implemented locally, operating at <strong>the</strong><br />
level of specific stimuli appearing in a task block. Additionally, fMRI data provide evidence that<br />
m<strong>an</strong>y of <strong>the</strong> regions implicated in cognitive control on <strong>the</strong> Stroop task, including <strong>an</strong>terior<br />
cingulate cortex <strong>an</strong>d dorsolateral prefrontal cortex, also operate at a local level. These results fit<br />
well with current computational modeling ef<strong>for</strong>ts by Blais et al. (2007, Psychological Review)<br />
<strong>an</strong>d Notebaert & Verguts (2008, Psychological Review). This presentation will review <strong>the</strong><br />
implications of <strong>the</strong>se results <strong>for</strong> ext<strong>an</strong>t <strong>the</strong>ories, which assume that cognitive control is adjusted<br />
globally.<br />
Disclosures: C. Blais, None; M. Harris, None; S.A. Bunge, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.24/FF99<br />
Topic: F.01.f. Working memory<br />
Support: NIH CTSA TL1RR024147<br />
NIH R01 MH065492<br />
PEO National Scholar Award<br />
Title: Saccadic measures of inhibitory control <strong>an</strong>d working memory in Tourette syndrome<br />
subtypes
Authors: *C. B. JETER 1 , S. S. PATEL 1 , I. J. BUTLER 2 , A. B. SERENO 1 ;<br />
1 Neurobio. & Anat., 2 Neurol., Univ. Texas Med. Sch., Houston, TX<br />
Abstract: INTRODUCTION: Tourette Syndrome (TS) has a childhood onset <strong>an</strong>d results in<br />
uncontrollable motor (e.g., blinking, mouth opening) <strong>an</strong>d verbal (e.g., sniffing, grunting)<br />
behaviors that often lead to social stigma. An additional complication is <strong>the</strong> prevalence of<br />
concurrent conditions like attention deficit hyperactive disorder (ADHD) or obsessivecompulsive<br />
disorder (OCD) in over 50% of patients. These comorbid conditions are known to be<br />
associated with cognitive deficits. Because eye movements share common brain areas with<br />
higher cognitive abilities, eye movements are a good tool to measure certain cognitive functions.<br />
OBJECTIVE: We sought to evaluate inhibitory control <strong>an</strong>d working memory in TS using eye<br />
movements. METHOD: Using <strong>an</strong> infrared eye tracker, we measured eye movement per<strong>for</strong>m<strong>an</strong>ce<br />
of unmedicated TS patients <strong>an</strong>d healthy controls aged 10-16 years on tasks of movement<br />
generation, inhibition, <strong>an</strong>d working memory. These tasks included prosaccade (automatically<br />
look to a peripheral target when it appears), <strong>an</strong>tisaccade (voluntarily look opposite to <strong>the</strong><br />
suddenly illuminated target), 0-back memory (look to <strong>the</strong> last stimulus in a variable series of<br />
flashes), <strong>an</strong>d 1-back memory (look to <strong>the</strong> next-to-last stimulus in a variable series of flashes).<br />
Whereas <strong>the</strong> prosaccade is a control task, <strong>the</strong> <strong>an</strong>tisaccade, 0-back memory <strong>an</strong>d 1-back memory<br />
tasks are powerful measures of inhibition <strong>an</strong>d working memory. In addition to suppression of<br />
automatic <strong>an</strong>d early responses, <strong>the</strong>se tasks require remembering <strong>an</strong>d generating voluntary eye<br />
movements at <strong>the</strong> proper time to a specific non-illuminated location. The Yale Global Tic<br />
Severity Scale, Obsessive Compulsive Inventory - Child Version, <strong>an</strong>d ADHD-IV rating scales<br />
evaluated TS, OCD, <strong>an</strong>d ADHD severity, respectively. Error rate <strong>an</strong>d response time of each task<br />
were included in a multivariate linear model with TS status (healthy control, TS-only, TS-OCD,<br />
or TS-ADHD) as a main factor covaried by age. RESULTS: TS-ADHD patients had<br />
signific<strong>an</strong>tly more errors th<strong>an</strong> controls on three tasks (<strong>an</strong>tisaccade, 0-back memory, <strong>an</strong>d 1-back<br />
memory), <strong>an</strong>d greater difference between 1-back <strong>an</strong>d 0-back response times th<strong>an</strong> controls. Errors<br />
on <strong>the</strong> 0-back memory task differentiated TS-only patients <strong>from</strong> those with comorbidities.<br />
CONCLUSIONS: Errors more sensitively differentiated among controls <strong>an</strong>d patient subtypes<br />
th<strong>an</strong> did response times. TS patients with comorbidities showed impaired inhibition <strong>an</strong>d working<br />
memory compared to TS-only patients, <strong>an</strong>d TS-ADHD children per<strong>for</strong>med <strong>the</strong> worst when<br />
compared to controls.<br />
Disclosures: C.B. Jeter, None; S.S. Patel, None; I.J. Butler, None; A.B. Sereno, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.25/FF100
Topic: F.01.f. Working memory<br />
Support: JSPS Gr<strong>an</strong>t#19203032 to NO<br />
Title: Training of inhibitory control of attention introduce effective control of executive function<br />
in elderly working memory: An fMRI study<br />
Authors: M. OSAKA 1 , Y. OTSUKA 2 , Y. KINOSHITA 1 , K. YAOI 2 , *N. OSAKA 2 ;<br />
1 Psychology, Osaka Univ., Mino city, Jap<strong>an</strong>; 2 Psychology, Kyoto Univ., Kyoto, Jap<strong>an</strong><br />
Abstract: The effect of training on inhibitory control of attention was investigated in <strong>the</strong> elderly,<br />
with special reference to executive function of working memory. During training period, 13<br />
healthy elderly particip<strong>an</strong>ts practiced to inhibit preprogrammed responses using four kinds of <strong>the</strong><br />
Stroop tasks. To explore <strong>the</strong> neural substrates underlying inhibitory training effects, two kinds of<br />
working memory task, i.e., focused <strong>an</strong>d non-focused reading sp<strong>an</strong> tests (RSTs), were per<strong>for</strong>med.<br />
In both reading sp<strong>an</strong> tests, <strong>the</strong> elderly were requested to memorize <strong>the</strong> target words while <strong>the</strong>y<br />
read a few sentences (dual task). In <strong>the</strong> focused RST (F-RST), <strong>the</strong> target word to be memorized<br />
was a focus word (most critical word to comprehend <strong>the</strong> sentence) of <strong>the</strong> sentence. In <strong>the</strong> nonfocused<br />
RST (NF-RST), <strong>the</strong> target word was not a focus word of <strong>the</strong> sentence, in which<br />
inhibitory control of attention is strongly required to per<strong>for</strong>m <strong>the</strong> task th<strong>an</strong> in F-RST (Osaka, et<br />
al., 2002; 2007). fMRI activations were serially measured while elderly particip<strong>an</strong>ts per<strong>for</strong>med<br />
F- <strong>an</strong>d NF- RST, be<strong>for</strong>e <strong>an</strong>d after a training period. After training, behavioral data showed that<br />
recognition accuracy increased <strong>an</strong>d reaction time decreased only in NF-RST but not in F-RST. In<br />
NF-RST condition, fMRI data showed that <strong>the</strong>re were signific<strong>an</strong>t activations in <strong>the</strong> left<br />
dorsolateral prefrontal cortex (DLPFC), bilateral superior parietal lobule (SPL) <strong>an</strong>d <strong>an</strong>terior<br />
cingulate cortex (ACC) in post-training session compared with that of pretraining. However,<br />
<strong>the</strong>re was no such signific<strong>an</strong>t activation increase in <strong>the</strong> elderly groups without training. These<br />
findings suggest that inhibitory training in <strong>the</strong> elderly leads to improve attentional control <strong>an</strong>d<br />
increase <strong>the</strong> activations which underlie executive functions of working memory.<br />
Disclosures: M. Osaka, None; Y. Otsuka, None; Y. Kinoshita, None; K. Yaoi, None; N.<br />
Osaka, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.26/FF101<br />
Topic: F.01.f. Working memory
Title: An event-related potential (ERP) study of proactive interference resolution in spatial<br />
working memory<br />
Authors: *C. HAU, H.-C. LEUNG;<br />
SUNY At Stony Brook, Stony Brook, NY<br />
Abstract: Successful working memory per<strong>for</strong>m<strong>an</strong>ce requires reducing interference <strong>from</strong> nolonger-relev<strong>an</strong>t<br />
or irrelev<strong>an</strong>t in<strong>for</strong>mation. Proactive interference (PI) in working memory arises<br />
when memory of <strong>an</strong> earlier event intrudes memory of a more recent event. While m<strong>an</strong>y studies<br />
have examined <strong>the</strong> neural correlates of PI, <strong>the</strong> temporal aspect of brain activity, especially on<br />
spatial working memory, remains unclear. The present study attempts to identify <strong>the</strong> ERP<br />
components in correspondence to PI in spatial working memory.<br />
ERP data were collected <strong>from</strong> 7 college students while <strong>the</strong>y were per<strong>for</strong>ming a delayed spatial<br />
recognition task with <strong>an</strong> updating cue. Each trial beg<strong>an</strong> with a fixation period (1s) followed by a<br />
study phase (1s), during which four dots were presented in four different locations on a 4x4 grid.<br />
After a 2-sec delay, two of <strong>the</strong> studied dots reappeared, cueing <strong>the</strong> to-be-ignored locations.<br />
Following a second delay period (2s), a probe (a circle) was presented <strong>an</strong>d subjects made a<br />
judgment whe<strong>the</strong>r <strong>the</strong> probe matches one of <strong>the</strong> locations in <strong>the</strong> final memory set. There were<br />
three probe conditions: (1) “Yes” (50%), probe location matches one of <strong>the</strong> remembered<br />
locations; (2) “No-HF” (25%), probe location matches one of <strong>the</strong> cued locations but no-longerrelev<strong>an</strong>t<br />
<strong>for</strong> <strong>the</strong> recognition test (<strong>the</strong> familiarity level of this No probe is high); (3) “No-LF”<br />
condition (25%), probe location does not match <strong>an</strong>y of <strong>the</strong> dot locations in initial memory set<br />
(<strong>the</strong> familiarity level of this probe is low). The probe conditions were presented pseudor<strong>an</strong>domly<br />
without repetition.<br />
Response time (RT) data revealed a PI effect, with a longer RT to <strong>the</strong> No-HF probe compared to<br />
<strong>the</strong> No-LF <strong>an</strong>d Yes probes. Averaged stimulus-locked ERP wave<strong>for</strong>ms revealed a positive peak<br />
between 200-700ms across all recording sites. The Yes condition peaked earlier (around 250 ms)<br />
<strong>an</strong>d showed a larger positivity th<strong>an</strong> <strong>the</strong> No conditions. The two No conditions peaked later, with<br />
<strong>the</strong> peak in No-LF shows a larger positivity th<strong>an</strong> in No-HF. It is possible that this positive peak<br />
reflects <strong>the</strong> recollection process, with <strong>an</strong> earlier recollection <strong>for</strong> <strong>the</strong> Yes probe. In contrast,<br />
averaged response-locked ERP wave<strong>for</strong>ms revealed a larger positivity <strong>for</strong> <strong>the</strong> No-LF condition<br />
compared to <strong>the</strong> No-HF <strong>an</strong>d Yes conditions. In addition, our data revealed a slow negative wave<br />
component starting around 350ms after response <strong>an</strong>d last till <strong>the</strong> end of <strong>the</strong> epoch over <strong>the</strong><br />
fronto-central regions, with <strong>the</strong> No-HF condition showing a larger negativity th<strong>an</strong> <strong>the</strong> Yes <strong>an</strong>d<br />
No-LF conditions. This late component may be associated with post-response evaluation or<br />
monitoring. In sum, our data reveal <strong>an</strong> early process in response to <strong>the</strong> Yes probe <strong>an</strong>d separate<br />
processes <strong>for</strong> <strong>the</strong> No-LF <strong>an</strong>d No-HF probes.<br />
Disclosures: C. Hau, None; H. Leung, None.<br />
Poster<br />
575. Mainten<strong>an</strong>ce <strong>an</strong>d Executive Control in Working Memory
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 575.27/FF102<br />
Topic: F.01.f. Working memory<br />
Support: Americ<strong>an</strong> Psychological Association Gr<strong>an</strong>t #5 T32 MH18882<br />
NINDS Gr<strong>an</strong>t NS21135 Supplement<br />
NINDS Gr<strong>an</strong>t NS21135<br />
Title: Working memory deficits <strong>an</strong>d functional recovery after unilateral prefrontal or basal<br />
g<strong>an</strong>glia lesions in hum<strong>an</strong>s<br />
Authors: *B. VOYTEK 1 , E. K. VOGEL 2 , R. T. KNIGHT 3,4 ;<br />
1 Univ. Cali<strong>for</strong>nia, Berkeley, Berkeley, CA; 2 Dept. of Psychology, Univ. of Oregon, Eugene, OR;<br />
3 Helen Wills Neurosci. Inst., 4 Dept. of Psychology, Univ. of Cali<strong>for</strong>nia, Berkeley, Berkeley, CA<br />
Abstract: Hum<strong>an</strong> visual working memory (VWM) is proposed to rely on active mainten<strong>an</strong>ce of<br />
goal-relev<strong>an</strong>t in<strong>for</strong>mation by <strong>the</strong> prefrontal cortex (PFC) with input <strong>from</strong> extrastriate cortex (EC)<br />
updating goal-relev<strong>an</strong>t PFC-dependent representations. In computational models (Ashby et al.,<br />
2005 <strong>an</strong>d Hazy et al., 2006) updating <strong>an</strong>d mainten<strong>an</strong>ce functions though recurrent PFC/basal<br />
g<strong>an</strong>glia (BG) loops.<br />
PFC lesions in non-hum<strong>an</strong> primates lead to per<strong>for</strong>m<strong>an</strong>ce deficits in delayed match to sample<br />
tasks while research on hum<strong>an</strong>s with PFC lesions is less conclusive (Hillary et al., 2006). Here<br />
we recorded scalp EEG <strong>from</strong> patients with unilateral PFC (N=7) or BG (N=6) lesions <strong>an</strong>d agematched<br />
controls (N=13) per<strong>for</strong>ming a lateralized VWM paradigm with three levels of visual<br />
load. All groups had a main effect with load resulting in decreased behavioral per<strong>for</strong>m<strong>an</strong>ce<br />
(p
correct trials--may be accounted <strong>for</strong> by compensatory load-dependent frontal <strong>the</strong>ta activity in <strong>the</strong><br />
intact PFC.<br />
Disclosures: B. Voytek, None; E.K. Vogel, None; R.T. Knight, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.1/FF103<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: NIH Gr<strong>an</strong>t 5K08DC007653<br />
NIH Gr<strong>an</strong>t 1R01DC010014<br />
Title: Olfactory perceptual decision-making in <strong>the</strong> hum<strong>an</strong> brain<br />
Authors: *N. E. BOWMAN 1 , J. D. HOWARD 1 , K. P. KORDING 2 , J. A. GOTTFRIED 1 ;<br />
1 CNADC, 2 Northwestern Univ., Chicago, IL<br />
Abstract: Little is known about how percepts of odor quality develop in <strong>the</strong> context of olfactory<br />
decision-making. Using functional magnetic reson<strong>an</strong>ce imaging (fMRI) <strong>an</strong>d psychophysical<br />
testing we investigated <strong>the</strong> role of perceptual decision-making in <strong>the</strong> disambiguation of odor<br />
mixtures. An intensity-matched binary odor-mixture set of citral (lemon) <strong>an</strong>d eugenol (clove)<br />
was assembled, systematically varying between 100% citral <strong>an</strong>d 100% eugenol (9 mixtures).<br />
Particip<strong>an</strong>ts were instructed to make as m<strong>an</strong>y sniffs as needed to confidently identify which of<br />
<strong>the</strong> two odors was more prevalent on a given odor mixture trial. Particip<strong>an</strong>ts <strong>the</strong>n made <strong>an</strong> <strong>an</strong>alog<br />
rating on a continuum between pure lemon <strong>an</strong>d pure clove, indicating <strong>the</strong> extent to which <strong>the</strong>y
perceived each odor contributed to <strong>the</strong> mixture. Psychometric data <strong>from</strong> 12 particip<strong>an</strong>ts showed<br />
that <strong>an</strong>alog perceptual decisions reflected <strong>the</strong> ratio of <strong>the</strong> pure odor<strong>an</strong>ts in a sigmoidal fashion,<br />
<strong>an</strong>d reaction times increased with <strong>the</strong> ambiguity of <strong>the</strong> odor. Particip<strong>an</strong>ts varied in <strong>the</strong>ir ability to<br />
identify <strong>the</strong> domin<strong>an</strong>t odor<strong>an</strong>t with a me<strong>an</strong> accuracy of 90.3% (s.d. 5.6%) <strong>for</strong> binary perceptual<br />
decisions (Figure). Preliminary fMRI <strong>an</strong>alysis shows that odor-evoked activation in <strong>the</strong> piri<strong>for</strong>m<br />
cortex <strong>an</strong>d olfactory OFC con<strong>for</strong>med to a "U-shaped" curve, such that <strong>the</strong> two pure odor<strong>an</strong>ts at<br />
<strong>the</strong> extremes of <strong>the</strong> scale (least ambiguous) had <strong>the</strong> largest activation, while those at <strong>the</strong> middle<br />
of <strong>the</strong> scale (most ambiguous) had reduced activation. Conversely, activation in <strong>the</strong> <strong>an</strong>terior<br />
cingulate cortex <strong>an</strong>d areas in prefrontal cortex con<strong>for</strong>med to <strong>an</strong> "inverted U-shaped" curve, with<br />
increasing neural responses <strong>for</strong> <strong>the</strong> more ambiguous odor mixtures. These results suggest that<br />
<strong>the</strong>se regions may play different roles in evaluating <strong>the</strong> content of odor mixtures. Analyses will<br />
explore whe<strong>the</strong>r ensemble patterns in piri<strong>for</strong>m <strong>an</strong>d orbitofrontal cortices c<strong>an</strong> predict particip<strong>an</strong>ts’<br />
perceptual decisions on a trial-by-trial basis, <strong>an</strong>d neural correlates of confidence <strong>an</strong>d ambiguity<br />
will be <strong>an</strong>alyzed to elucidate what role <strong>the</strong>y play in perceptual decision-making, <strong>an</strong>d how <strong>the</strong>y<br />
contribute to trial accuracy.<br />
Disclosures: N.E. Bowm<strong>an</strong>, None; J.D. Howard, None; K.P. Kording, None; J.A. Gottfried,<br />
None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.2/FF104<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: Modeling brain behavior using concurrent autonomous computer agents<br />
Authors: *J. A. PARRA;<br />
Computer Sci., Pontificia Univ. Javeri<strong>an</strong>a Cali, Cali, Colombia<br />
Abstract: Current computer systems have become increasingly complex structures made up of<br />
hundreds, even thous<strong>an</strong>ds of small programs known as agents. In this way, <strong>the</strong>ir similarity with<br />
biological brains have allowed <strong>the</strong> conception of more natural computational architectures both<br />
<strong>for</strong> doing computation as well as <strong>for</strong> modeling biological high level processes such as behavior<br />
<strong>an</strong>d cognition.<br />
An agent-based computational architecture is developed under <strong>the</strong> assumption that a more<br />
flexible, complex functionality c<strong>an</strong> be obtained by <strong>the</strong> use of a multitude of small programs<br />
working concurrently ra<strong>the</strong>r th<strong>an</strong> <strong>from</strong> a monolithic centralized program.<br />
These agents work in a coordinated fashion in order to satisfy <strong>the</strong>ir objectives. Dynamically <strong>the</strong>y<br />
switch between a tendency to cooperate of compete. The way <strong>the</strong>y communicate is through<br />
diffusion of messages into a common medium, resembling diffusion of neurotr<strong>an</strong>smitters.<br />
Moreover, each agent follows similar strategies to those of real neurons, <strong>for</strong> it has a set of genes<br />
interacting as a regulatory genetic network, which are in charge of <strong>the</strong> current behavior of that<br />
agent. Artificial proteins may act as tr<strong>an</strong>scription factors inside this networks, or travel outside<br />
<strong>the</strong> neuron to interact with o<strong>the</strong>r neurons as second or third messengers.<br />
Considering <strong>the</strong>se concepts, a computational architecture, named NeuroAgent, has been built <strong>an</strong>d<br />
used to train artificial robots, which evolve (phylogenetically) <strong>an</strong>d develop (ontogenetically)<br />
following strategies built upon sets of neurons (agents) which develop connections<br />
(epigenetically) to store learnt patterns <strong>an</strong>d behaviors.<br />
Given its unique dual aspect, NeuroAgent c<strong>an</strong> be used to test neurobiological <strong>an</strong>d<br />
neuropsychological models of cognition as well as to develop computational systems which<br />
work in a similar way as <strong>the</strong>y biological counterparts.<br />
Disclosures: J.A. Parra, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 576.3/FF105<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: EJLB Foundation<br />
FYSSEN Foundation<br />
CIHR<br />
Title: Hum<strong>an</strong> perceptual decisions in noisy, ch<strong>an</strong>ging conditions<br />
Authors: *D. THURA, P. CISEK;<br />
Dept Physiol., Univ. Montreal, Montreal, QC, C<strong>an</strong>ada<br />
Abstract: In recent years, signific<strong>an</strong>t progress has been made toward underst<strong>an</strong>ding <strong>the</strong> neural<br />
basis of primate decision-making. Most decision-making studies <strong>an</strong>d models have suggested that<br />
simple decisions are made through a process of ‘bounded integration’, in which neurons integrate<br />
sensory evidence until a threshold is reached. However, nearly all of <strong>the</strong> results supporting this<br />
<strong>the</strong>ory have been obtained in tasks where sensory evidence was const<strong>an</strong>t during <strong>the</strong> course of<br />
each trial. In this particular situation, behavioral <strong>an</strong>d neural data are also compatible with a<br />
model in which <strong>the</strong>re is no integration of sensory evidence, but instead a multiplication of current<br />
evidence by a growing ‘urgency’ signal. In a recent study, Cisek et al. (SfN <strong>abstract</strong> 2008)<br />
presented hum<strong>an</strong> subjects with a task (‘tokens’ task) in which evidence ch<strong>an</strong>ged over time.<br />
Results were more consistent with <strong>the</strong> ‘urgency-gating’ model th<strong>an</strong> with “integrator” models, but<br />
it was not clear whe<strong>the</strong>r this was task-dependent.<br />
Here, 11 hum<strong>an</strong> subjects per<strong>for</strong>med a new task, conceptually similar to <strong>the</strong> ‘tokens’ task but<br />
perceptually close to <strong>the</strong> well-known ‘direction-discrimination’ task, in which subjects per<strong>for</strong>m<br />
two-alternative perceptual decisions about <strong>the</strong> direction of motion in a dynamic r<strong>an</strong>dom-dot<br />
display. In our task, each trial beg<strong>an</strong> with a centrally-located visual stimulus consisting of 200<br />
dots moving in r<strong>an</strong>dom directions. After 200ms, 6 of <strong>the</strong> dots beg<strong>an</strong> to move coherently in one of<br />
two opposite directions (right or left). After <strong>an</strong>o<strong>the</strong>r 200ms, <strong>an</strong>o<strong>the</strong>r 6 of <strong>the</strong> r<strong>an</strong>dom dots beg<strong>an</strong><br />
to move coherently in one of <strong>the</strong> two directions, <strong>an</strong>d so on <strong>for</strong> a total of 15 steps. The subject’s<br />
task was to select, as soon as <strong>the</strong>y felt confident enough, <strong>the</strong> target corresponding to <strong>the</strong> net<br />
direction of motion <strong>the</strong>y predicted to see at <strong>the</strong> end of <strong>the</strong> trial. Once <strong>the</strong> choice was made, <strong>the</strong><br />
remaining steps of coherence were reduced to a 20ms duration. To distinguish <strong>the</strong> models, we<br />
embedded in a full pseudor<strong>an</strong>dom sequence some trials whose first 6 motion steps provided<br />
ei<strong>the</strong>r a perceptual bias <strong>for</strong> or against <strong>the</strong> correct target.<br />
Consistent with <strong>the</strong> ‘urgency-gating’ model, decision times as well as success probabilities of<br />
8/11 subjects were not signific<strong>an</strong>tly influenced by <strong>the</strong>se early biases, suggesting that noisy<br />
sensory evidence in favor of a given choice was not integrated with a long time-const<strong>an</strong>t.<br />
Additional <strong>an</strong>alyses suggested that <strong>the</strong> level of certainty at which most subjects made <strong>the</strong>ir<br />
decisions decreased signific<strong>an</strong>tly during <strong>the</strong> course of a trial.<br />
Our results suggest that hum<strong>an</strong>s <strong>for</strong>m decisions by comparing to a threshold <strong>the</strong> product of <strong>the</strong><br />
momentary in<strong>for</strong>mation provided by <strong>the</strong> environment with a growing signal related to elapsed<br />
time (‘urgency’).
Disclosures: D. Thura, None; P. Cisek, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.4/FF106<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: Two separate decision systems affected by <strong>the</strong> value of subliminal <strong>an</strong>d supraliminal<br />
stimulus in hum<strong>an</strong> brain<br />
Authors: *M. MATSUMOTO 1 , K. MATSUMOTO 1 , N. HAYAMIZU 2 , M. SAKAGAMI 1 ;<br />
1 Tamagawa univ, Tokyo, Jap<strong>an</strong>; 2 Toyota Central R&D Labs., Inc., Aichi, Jap<strong>an</strong><br />
Abstract: Behavioral economic studies suggest <strong>the</strong> existence of two separate systems of<br />
decision-making, intuition (unconscious) <strong>an</strong>d deliberate reasoning (conscious). To discriminate<br />
<strong>the</strong> brain areas that are involved in <strong>the</strong> two systems, we investigated brain activity by functional<br />
magnetic reson<strong>an</strong>ce imaging while hum<strong>an</strong> subjects were per<strong>for</strong>ming a subliminal/supraliminal<br />
monetary choice task. In <strong>the</strong> task, two different monetary coins among 1-, 10-, <strong>an</strong>d 500-yen were<br />
presented left <strong>an</strong>d right simult<strong>an</strong>eously <strong>for</strong> 33 ms (subliminal condition) or 100 ms (supraliminal<br />
condition) with <strong>for</strong>ward <strong>an</strong>d backward masks. Actually, <strong>the</strong> particip<strong>an</strong>ts reported later in most<br />
cases that <strong>the</strong>y did not recognize what coins were presented under <strong>the</strong> subliminal condition<br />
whereas <strong>the</strong>y did under <strong>the</strong> supraliminal condition. They had been explicitly instructed to choose<br />
<strong>the</strong> coin with higher value within 3 s following <strong>the</strong> monetary cue onset by pressing <strong>the</strong><br />
corresponding left or right button. After <strong>the</strong> choice between <strong>the</strong> two coins, <strong>the</strong> chosen one was<br />
presented as feedback. The duration of <strong>the</strong> monetary stimuli (33 ms or 100 ms) as well as <strong>the</strong><br />
pairing of <strong>the</strong> coins were determined at pseudo-r<strong>an</strong>dom, so that <strong>the</strong> particip<strong>an</strong>ts could not predict<br />
which condition or what pair of <strong>the</strong> coins would come next. Subjects were in<strong>for</strong>med that <strong>the</strong>ir<br />
rewards were calculated <strong>from</strong> <strong>the</strong> sum of <strong>the</strong> chosen money during one session that was<br />
r<strong>an</strong>domly selected among three by a lottery <strong>an</strong>d <strong>the</strong>y will obtain <strong>the</strong> half of <strong>the</strong> monetary sum.<br />
The particip<strong>an</strong>ts won more th<strong>an</strong> 10,000 yen on average. In <strong>the</strong> behavioral per<strong>for</strong>m<strong>an</strong>ce, even<br />
under <strong>the</strong> subliminal condition, <strong>the</strong> action selection was biased to <strong>the</strong> coin with higher value. The<br />
activity of <strong>the</strong> orbitofrontal cortex (OFC) signific<strong>an</strong>tly correlated with <strong>the</strong> value of coins<br />
presented subliminally, whereas <strong>the</strong> activity of <strong>the</strong> medial prefrontal cortex (MPFC) signific<strong>an</strong>tly<br />
did with <strong>the</strong> value of coins presented supraliminally. Moreover, <strong>the</strong> difference in <strong>the</strong> effect sizes<br />
of <strong>the</strong> OFC activity between 500-yen vs. 10-yen <strong>an</strong>d 10-yen vs. 1-yen was signific<strong>an</strong>tly<br />
correlated with <strong>the</strong> difference in correct rates between 500-yen vs. 10-yen <strong>an</strong>d 10-yen vs. 1-yen
in <strong>the</strong> subliminal condition. These results suggest that <strong>the</strong> OFC is involved in unconscious<br />
behavioral bias whereas <strong>the</strong> MPFC is involved in conscious behavioral decision.<br />
Disclosures: M. Matsumoto, None; K. Matsumoto, None; N. Hayamizu, None; M.<br />
Sakagami, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.5/FF107<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: NIH Gr<strong>an</strong>t P50-MH62196 (NY)<br />
Deutsche Forschungsgemeinschaft gr<strong>an</strong>t STE 1708/1-1 (MS)<br />
Title: Neural correlates of error processing <strong>an</strong>d error awareness<br />
Authors: *N. YEUNG 1 , G. HUGHES 1 , M. STEINHAUSER 2 ;<br />
1 Univ. of Ox<strong>for</strong>d, Ox<strong>for</strong>d, United Kingdom; 2 Univ. of Konst<strong>an</strong>z, Konst<strong>an</strong>z, Germ<strong>an</strong>y<br />
Abstract: People demonstrate <strong>an</strong> ability to detect <strong>the</strong>ir own errors <strong>an</strong>d enact appropriate<br />
behavioral adjustments, <strong>an</strong> ability of obvious adaptive signific<strong>an</strong>ce. In <strong>the</strong> scalp-recorded eventrelated<br />
potential (ERP), error processing is reflected in two components, <strong>the</strong> error-related<br />
negativity (Ne/ERN) <strong>an</strong>d <strong>the</strong> later error positivity (Pe). A consensus view holds that <strong>the</strong> Ne/ERN<br />
<strong>an</strong>d Pe reflect successive stages of error processing. However, debate remains about <strong>the</strong> precise<br />
nature of <strong>the</strong> stages reflected in <strong>the</strong>se components: ei<strong>the</strong>r or both could reflect precursers to<br />
explicit error detection such as conflict monitoring, <strong>the</strong> error detection process itself, evaluation<br />
of <strong>the</strong> signific<strong>an</strong>ce of a detected error, or initiation of subsequent behavioral adjustments. In <strong>the</strong><br />
present research we adopted a novel approach to this issue in which we treated error processing<br />
as a noisy decision process, <strong>an</strong>d investigated <strong>the</strong> in<strong>for</strong>mational content of <strong>the</strong> Ne/ERN <strong>an</strong>d Pe in<br />
relation to objective measures <strong>an</strong>d subjective ratings of per<strong>for</strong>m<strong>an</strong>ce accuracy. In our<br />
experiments, subjects per<strong>for</strong>med speeded perceptual discriminations <strong>an</strong>d indicated with a<br />
keypress when <strong>the</strong>y judged <strong>the</strong>y had made <strong>an</strong> error. We varied <strong>the</strong> difficulty of <strong>the</strong> perceptual<br />
decision <strong>an</strong>d <strong>the</strong> monetary reward associated with accurate error signaling, <strong>an</strong>d <strong>an</strong>alyzed <strong>the</strong><br />
resulting ERP data using conventional averaging toge<strong>the</strong>r with pattern-classification <strong>an</strong>alysis of<br />
neural activity on individual trials. Analysis of <strong>the</strong> averaged ERP indicated that <strong>the</strong> Ne/ERN <strong>an</strong>d<br />
Pe were differentially affected by m<strong>an</strong>ipulations of task difficulty <strong>an</strong>d error signaling incentive.
Converging results <strong>from</strong> our single-trial data <strong>an</strong>alysis suggested <strong>an</strong> in<strong>for</strong>mation tr<strong>an</strong>s<strong>for</strong>mation<br />
<strong>from</strong> <strong>the</strong> Ne/ERN to <strong>the</strong> Pe, with <strong>the</strong> <strong>for</strong>mer reflecting <strong>the</strong> dynamics of task processing <strong>an</strong>d <strong>the</strong><br />
latter correlating with subjective judgments of response accuracy. The results suggested fur<strong>the</strong>r<br />
that <strong>the</strong> later Pe component reflects <strong>the</strong> strength of evidence that <strong>an</strong> error has occurred, ra<strong>the</strong>r<br />
th<strong>an</strong> <strong>the</strong> output of <strong>an</strong> error signaling process or a subsequent evaluation of <strong>the</strong> signific<strong>an</strong>ce of <strong>the</strong><br />
error. Taken toge<strong>the</strong>r, <strong>the</strong>se findings provide new insight into <strong>the</strong> neural correlates of error<br />
processing, <strong>an</strong>d demonstrate <strong>the</strong> relev<strong>an</strong>ce of emerging concepts <strong>an</strong>d methods in decision making<br />
research to <strong>the</strong> study of hum<strong>an</strong> per<strong>for</strong>m<strong>an</strong>ce monitoring.<br />
Disclosures: N. Yeung, None; G. Hughes, None; M. Steinhauser, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.6/FF108<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: Complexity-based functional connectivity <strong>an</strong>alysis in visuo-spatial reasoning<br />
Authors: E. SHOKRI KOJORI, M. MOTES, M. MCCLELLAND, B. RYPMA, *D. C.<br />
KRAWCZYK;<br />
Univ. of Texas at Dallas, Dallas, TX<br />
Abstract: We investigated <strong>the</strong> functional connectivity of cortical regions related to <strong>the</strong><br />
complexity of a visuo-spatial reasoning task. Particip<strong>an</strong>ts per<strong>for</strong>med a visuo-spatial reasoning<br />
task that included three complexity levels during fMRI sc<strong>an</strong>ning. Within each trial, multiple<br />
shapes with different ch<strong>an</strong>ging patterns were shown simult<strong>an</strong>eously to subjects. Then <strong>the</strong>y were<br />
asked to judge whe<strong>the</strong>r or not <strong>the</strong> ch<strong>an</strong>ges occurred in accord<strong>an</strong>ce with predefined rules. The<br />
complexity level was m<strong>an</strong>ipulated by increasing or decreasing <strong>the</strong> number of shapes that<br />
ch<strong>an</strong>ged toge<strong>the</strong>r. Six different blocks corresponding to three complexity levels were distributed<br />
pseudo-r<strong>an</strong>domly throughout each of two runs of <strong>the</strong> sc<strong>an</strong>ning time. The group results showed<br />
signific<strong>an</strong>t activation ch<strong>an</strong>ges to increased complexity in right dorsolateral PFC (rDLPFC), right<br />
parietal cortex (rPC), right inferior frontal gyrus (rIFG), right premotor cortex (rPMC), <strong>an</strong>d<br />
dorsal <strong>an</strong>terior cingulate (dACC). Average time series of top twenty activated voxels<br />
corresponding to each condition block <strong>for</strong> <strong>the</strong>se regions were correlated toge<strong>the</strong>r. Statistical<br />
<strong>an</strong>alysis was per<strong>for</strong>med to explore whe<strong>the</strong>r <strong>the</strong> correlations between each ROI-pair would<br />
increase with task complexity. Signific<strong>an</strong>t increases (p < 0.05) in correlations were seen among<br />
several regions. These correlations included rDLPFC with rPC, rDLPFC with dACC, rDLPFC
with rPMC, <strong>an</strong>d rPMC with rPC. There was also a signific<strong>an</strong>t positive linear trend in rDLPFC<br />
with rPC correlations, having <strong>the</strong> strongest evidence of increased connectivity with complexity<br />
level (p < 0.05). These results indicate regions of <strong>the</strong> cortex which have increased correlation in<br />
<strong>the</strong>ir activation in response to increases in complexity during visuo-spatial reasoning. However<br />
in m<strong>an</strong>y ROI-pairs, <strong>the</strong> strength of connectivity was not ch<strong>an</strong>ged by increase in complexity.<br />
These findings along with Gr<strong>an</strong>ger Causality <strong>an</strong>alyses revealed a cortical network between<br />
<strong>an</strong>terior regions <strong>an</strong>d right premotor, prefrontal <strong>an</strong>d parietal cortices in visuo-spatial reasoning<br />
that is modulated by task complexity.<br />
Disclosures: E. Shokri Kojori, None; M. Motes, None; M. McClell<strong>an</strong>d, None; B. Rypma,<br />
None; D.C. Krawczyk, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.7/FF109<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: An optimal decision-making model of visual search per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: K. ISHIBASHI 1,2 , S. KITA 1 , Y. OHTSUBO 1 ;<br />
1 2<br />
Dept. of Psychology, Kobe Univ., Kobe/Hyogo, Jap<strong>an</strong>; Jap<strong>an</strong> <strong>Society</strong> <strong>for</strong> <strong>the</strong> Promotion of Sci.,<br />
Tokyo, Jap<strong>an</strong><br />
Abstract: In <strong>the</strong> present study, we propose a simple ma<strong>the</strong>matical model to describe hum<strong>an</strong><br />
visual search per<strong>for</strong>m<strong>an</strong>ce. The model is developed to explain a phenomenon that target<br />
probabilities modulate <strong>the</strong> termination time in visual search tasks. In reference to <strong>the</strong> optimal<br />
<strong>for</strong>aging <strong>the</strong>ory in ethology (org<strong>an</strong>isms <strong>for</strong>age in such a way as to maximize <strong>the</strong>ir energy intake<br />
per unit time), <strong>the</strong> model assumes that hum<strong>an</strong> visual search strategies are tuned to maximize <strong>the</strong><br />
number of targets searched in a fixed period of time. In <strong>the</strong> model, we assume that <strong>an</strong> evaluation<br />
function is jointly determined by 1) positive reaction time in target-present trials, 2) negative<br />
reaction time in target-absent trials <strong>an</strong>d 3) target probability. The maximization of <strong>the</strong> evaluation<br />
function provides <strong>the</strong> optimal reaction time <strong>for</strong> search termination in target-absent trials. To test<br />
<strong>the</strong> model, we conducted behavioral experiments of visual search tasks m<strong>an</strong>ipulating <strong>the</strong><br />
difficulty level <strong>an</strong>d target probability. The target probability r<strong>an</strong>ged <strong>from</strong> 10% to 90% with<br />
increment of 20%. We compared <strong>the</strong> observed search termination times with <strong>the</strong> model’s<br />
predictions, which were calculated using <strong>the</strong> observed positive reaction times in target-present<br />
trials <strong>for</strong> each level of <strong>the</strong> target probability. As a result, <strong>for</strong> <strong>the</strong> relatively difficult search tasks
(i.e., <strong>the</strong> conjunctive search task <strong>an</strong>d <strong>the</strong> large set size task), <strong>the</strong> model fitted <strong>the</strong> observed data<br />
well: negative reaction times in target-absent trials were almost equivalent to <strong>the</strong> predicted times.<br />
On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, <strong>for</strong> relatively easy search tasks (i.e., <strong>the</strong> feature search task <strong>an</strong>d <strong>the</strong> small set<br />
size task), <strong>the</strong> model yielded erroneously longer negative reaction times th<strong>an</strong> <strong>the</strong> observed times.<br />
Those results indicate that <strong>the</strong> difficulty levels of <strong>the</strong> tasks have <strong>an</strong> effect on <strong>the</strong> decision-making<br />
strategy in visual search. The results showed that <strong>the</strong> model fitted <strong>the</strong> data <strong>for</strong> relatively difficult<br />
tasks (i.e., people use <strong>the</strong> decision-making strategy relev<strong>an</strong>t to survival only when tasks are<br />
difficult). It may reflect <strong>an</strong> evolutionary competence, which hum<strong>an</strong>s have acquired through<br />
hunting <strong>an</strong>d ga<strong>the</strong>ring.<br />
Disclosures: K. Ishibashi, None; S. Kita, None; Y. Ohtsubo, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.8/FF110<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: NIH Gr<strong>an</strong>t MH63901<br />
Title: Evidence accumulation <strong>an</strong>d <strong>the</strong> speed-accuracy tradeoff in a decision-making task<br />
Authors: V. VAN VEEN 1 , D. T. ERICKSON 2 , B. R. BUCHSBAUM 3 , *A. S. KAYSER 2 , M. T.<br />
D'ESPOSITO 1 ;<br />
1 Univ. Cali<strong>for</strong>nia, Berkeley, Berkeley, CA; 2 Neurol., Univ. Cali<strong>for</strong>nia, S<strong>an</strong> Fr<strong>an</strong>cisco,<br />
Emeryville, CA; 3 Rotm<strong>an</strong> Inst., Toronto, ON, C<strong>an</strong>ada<br />
Abstract: Computational models of decision-making have suggested that decisions are made by<br />
a baseline-to-threshold accumulation process. Two relev<strong>an</strong>t parameters are <strong>the</strong> drift rate, which is<br />
determined by <strong>the</strong> strength of <strong>the</strong> sensory evidence, <strong>an</strong>d <strong>the</strong> baseline-to-threshold dist<strong>an</strong>ce, which<br />
determines <strong>the</strong> speed-accuracy tradeoff (SAT). Previous studies have found support <strong>for</strong> <strong>the</strong>se two<br />
processes. In <strong>the</strong> present study, we m<strong>an</strong>ipulated both <strong>the</strong> strength of <strong>the</strong> evidence <strong>an</strong>d <strong>the</strong> SAT, to<br />
investigate potential differences <strong>an</strong>d overlap in <strong>the</strong> brain regions engaged. Particip<strong>an</strong>ts per<strong>for</strong>med<br />
a version of <strong>the</strong> dot motion coherence task; cues instructed <strong>the</strong> particip<strong>an</strong>t to emphasize ei<strong>the</strong>r<br />
speed or accuracy. Per<strong>for</strong>m<strong>an</strong>ce data were <strong>an</strong>alyzed by fitting <strong>the</strong> data to <strong>the</strong> diffusion-drift<br />
model. Functional MRI data were <strong>an</strong>alyzed by using <strong>the</strong> obtained parameters as constraints.<br />
Consistent with earlier data, <strong>the</strong> effect of coherence engaged superior parietal <strong>an</strong>d premotor<br />
regions, supporting <strong>the</strong> notion that <strong>the</strong>se regions act as accumulators. The SAT also engaged
premotor regions, but modulated more laterally located superior parietal areas. These results<br />
provide fur<strong>the</strong>r support <strong>for</strong> decision making by accumulation of evidence, <strong>an</strong>d additionally<br />
suggest a dissociation in parietal cortex between top-down <strong>an</strong>d bottom-up modulation of <strong>the</strong><br />
accumulation process.<br />
Disclosures: V. V<strong>an</strong> Veen, None; D.T. Erickson, None; B.R. Buchsbaum, None; A.S.<br />
Kayser, None; M.T. D'Esposito, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.9/FF111<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: AFOSR gr<strong>an</strong>t FA 9550-07-1-0537<br />
Title: Event-related fMRI during slow decision making c<strong>an</strong> reveal temporal structure in neural<br />
activity<br />
Authors: *P. A. SIMEN, L. NYSTROM, M. VAN VUGT, P. KRUEGER, J. D. COHEN;<br />
Princeton Neurosci. Inst., Princeton Univ., Princeton, NJ<br />
Abstract: The neural basis of perceptual decision making has traditionally been investigated<br />
using single-cell recording in non-hum<strong>an</strong> <strong>an</strong>imals. In contrast, non-invasive methods suitable <strong>for</strong><br />
use with hum<strong>an</strong> subjects, such as functional magnetic reson<strong>an</strong>ce imaging (fMRI), have strong<br />
limitations when compared to electrophysiology. While m<strong>an</strong>y aspects of decision making c<strong>an</strong><br />
<strong>an</strong>d have been investigated with fMRI, low temporal resolution makes it difficult to use fMRI to<br />
infer <strong>the</strong> specific temporal profile of neural activity underlying rapid perceptual decision making<br />
(e.g, with response times on <strong>the</strong> order of 1 second). To address this problem, we have developed<br />
a behavioral method that successfully slows average hum<strong>an</strong> response times into <strong>the</strong> 4-5 second<br />
r<strong>an</strong>ge. In this r<strong>an</strong>ge, different temporal profiles of neural activity over <strong>the</strong> course of individual<br />
decisions (such as boxcars <strong>an</strong>d linear ramps) c<strong>an</strong> be reliably distinguished even when <strong>the</strong>y are<br />
convolved with a hemodynamic response function. We accomplished this slowing by imposing<br />
stiff penalties <strong>for</strong> errors in a two-alternative <strong>for</strong>ced choice, dot-motion discrimination task.<br />
Theoretical work examining optimal per<strong>for</strong>m<strong>an</strong>ce in this task predicted that <strong>the</strong> imposition of<br />
such penalties should induce subjects to increase response thresholds (to favor accuracy) <strong>an</strong>d<br />
<strong>the</strong>reby slow per<strong>for</strong>m<strong>an</strong>ce. This was confirmed behaviorally. Using this behavioral method with<br />
fMRI, we have obtained evidence <strong>for</strong> ramping activity in parietal <strong>an</strong>d prefrontal cortex, as
opposed to const<strong>an</strong>t, sustained activity in motion-sensitive area V5/MT+, in accord with results<br />
<strong>from</strong> similar experiments using nonhum<strong>an</strong> primates.<br />
Disclosures: P.A. Simen, None; L. Nystrom, None; M. v<strong>an</strong> Vugt, None; P. Krueger,<br />
None; J.D. Cohen, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.10/FF112<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: SFB TRR 62<br />
Title: Similiar fMRI activation by delayed <strong>an</strong>d omitted visual feedback<br />
Authors: C. KOHRS, N. BEHNE, H. SCHEICH, *A. BRECHMANN;<br />
Leibniz-Institute <strong>for</strong> Neurobio., Magdeburg, Germ<strong>an</strong>y<br />
Abstract: Feedback provides <strong>the</strong> basis <strong>for</strong> communication <strong>an</strong>d is used to adjust one´s own<br />
behavior during a dialog situation. Not much is known about <strong>the</strong> neuronal processing of neutral<br />
feedback, i.e. feedback which only provides in<strong>for</strong>mation about <strong>the</strong> registration of <strong>an</strong> action by<br />
<strong>an</strong>o<strong>the</strong>r agent. In <strong>the</strong> current study we investigated <strong>the</strong> effects of delayed <strong>an</strong>d missing neutral<br />
feedback on fMRI activation in comparison to immediate feedback.<br />
Particip<strong>an</strong>ts (n=13) per<strong>for</strong>med a well-characterized auditory categorization paradigm, in which<br />
<strong>the</strong>y had to decide whe<strong>the</strong>r <strong>the</strong> pitch of frequency-modulated (FM) tones was rising or falling.<br />
These FM tones were presented in <strong>an</strong> event related design with <strong>an</strong> intertrial interval of 6, 8 or 10<br />
s. Independent of <strong>the</strong> correctness of <strong>the</strong> subjects response <strong>the</strong>y received <strong>an</strong> immediate neutral<br />
visual feedback in <strong>the</strong> <strong>for</strong>m of a green checkmark in 76% of <strong>the</strong> cases. In 12% of all trials this<br />
feedback was delayed by 500 ms after <strong>the</strong> button press <strong>an</strong>d in <strong>an</strong>o<strong>the</strong>r 12% <strong>the</strong> feedback was<br />
omitted. The particip<strong>an</strong>ts did not know that this was part of <strong>the</strong> experiment <strong>an</strong>d expected <strong>an</strong><br />
immediate feedback.<br />
During omitted compared to immediate feedback we found stronger activations bilateral in <strong>the</strong><br />
<strong>an</strong>terior insula/inferior frontal gyrus, inferior parietal lobe, precuneus, in <strong>the</strong> posterior medial<br />
frontal cortex, <strong>an</strong>d in <strong>the</strong> right dorsolateral prefontal cortex. We also found stronger deactivations<br />
in <strong>the</strong> <strong>an</strong>terior <strong>an</strong>d posterior cingulate cortex during omitted feedback. These regions are known<br />
to be part of <strong>an</strong> attentional network activated during different task sets. Import<strong>an</strong>tly, <strong>the</strong> effects<br />
of delayed feedback were very similar to omitted feedback <strong>an</strong>d even in those two subjects, who
did not notice <strong>the</strong> delay. From this result we c<strong>an</strong> assume that a delay of 500ms has very similar<br />
neuronal effects as <strong>an</strong> unexpected omission of a neutral feedback.<br />
A fur<strong>the</strong>r result of our study revealed <strong>an</strong> involvement of <strong>the</strong> putamen during delayed <strong>an</strong>d<br />
immediate feedback compared to missing feedback, indicating that <strong>the</strong> midbrain dopamine<br />
system is not only activated by reward but may already be activated by a neutral visual feedback.<br />
Disclosures: C. Kohrs, None; N. Behne, None; H. Scheich, None; A. Brechm<strong>an</strong>n , None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.11/FF113<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: The effects of multiple stimulus features on <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of a hum<strong>an</strong> perceptual<br />
decision-making circuit<br />
Authors: *D. T. ERICKSON 1 , A. KAYSER 1 , B. BUCHSBAUM 2 , M. D'ESPOSITO 3 ;<br />
1 Ernest Gallo Clin. <strong>an</strong>d Res. Ctr., Emeryville, CA; 2 The Rotm<strong>an</strong> Res. Inst., Toronto, ON,<br />
C<strong>an</strong>ada; 3 Univ. of Cali<strong>for</strong>nia, Berkeley, Berkeley, CA<br />
Abstract: The mech<strong>an</strong>isms by which we accumulate perceptual in<strong>for</strong>mation in <strong>the</strong> service of a<br />
decision accord with simple models in which sensory evidence builds until a decision threshold<br />
is reached. Exp<strong>an</strong>ding upon results <strong>from</strong> macaque <strong>an</strong>d o<strong>the</strong>r hum<strong>an</strong> studies, our previous fMRI<br />
work demonstrated that a network of brain regions varies parametrically with <strong>the</strong> coherence of a<br />
dot motion stimulus, including areas whose activity c<strong>an</strong> be grouped under putative sensory (e.g.<br />
MT+), integrative (e.g. intraparietal sulcus), <strong>an</strong>d response-related (e.g. middle frontal gyrus)<br />
functions. However, we have yet to explicitly evaluate <strong>the</strong> role of attentional processes, or to<br />
fur<strong>the</strong>r characterize <strong>the</strong> relationship of IPS to its sensory inputs. To differentiate such functions<br />
within this circuit, we created stimuli in which two visual features -- dot motion <strong>an</strong>d dot color --<br />
were parametrically <strong>an</strong>d independently modulated. Highly-trained subjects were required to<br />
attend to <strong>an</strong>d select ei<strong>the</strong>r <strong>the</strong> direction of coherent dot motion or <strong>the</strong> predomin<strong>an</strong>t dot color,<br />
while ignoring <strong>the</strong> o<strong>the</strong>r feature. Per<strong>for</strong>m<strong>an</strong>ce (accuracy) was matched across features. In<br />
keeping with <strong>the</strong>ir training, our subjects' reaction times showed strongly signific<strong>an</strong>t trends with<br />
respect to <strong>the</strong> attended feature (all ps 0.10). Statistically signific<strong>an</strong>t behavioral effects of ei<strong>the</strong>r<br />
sensory or response conflict were not noted. As expected, BOLD activity revealed signific<strong>an</strong>t<br />
main effects of task (p < 0.001 uncorrected) in a network of areas including independentlylocalized<br />
V4/color regions <strong>an</strong>d MT+, as well as IPS, MFG, ACC, <strong>an</strong>d <strong>an</strong>terior insula.
Disclosures: D.T. Erickson, None; A. Kayser, None; B. Buchsbaum, None; M. D'Esposito,<br />
None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.12/FF114<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Title: How does <strong>the</strong> hum<strong>an</strong> brain deal with uncertainty?<br />
Authors: *I. VILARES, H. L. FERNANDES, K. KORDING;<br />
Rehabil. Inst. of Chicago/ Northwestern Univ., Chicago, IL<br />
Abstract: The world is uncertain. Both <strong>the</strong> prior knowledge that we have of <strong>the</strong> world <strong>an</strong>d <strong>the</strong><br />
sensory in<strong>for</strong>mation we receive have some associated variability. The hum<strong>an</strong> brain thus has to<br />
rely on both <strong>the</strong>se sources <strong>for</strong> successful motor control. Several studies have shown that hum<strong>an</strong>s<br />
c<strong>an</strong> often integrate in<strong>for</strong>mation <strong>from</strong> both previous knowledge (<strong>the</strong> prior) <strong>an</strong>d current feedback<br />
(<strong>the</strong> likelihood) in a way that is close to <strong>the</strong> statistically optimal way. Here we present a novel<br />
task in which both <strong>the</strong> uncertainty about <strong>the</strong> prior <strong>an</strong>d about <strong>the</strong> feedback are varied. Subjects<br />
have to find <strong>the</strong> hidden position of a coin, about which <strong>the</strong>y receive noisy feedback in <strong>the</strong> <strong>for</strong>m<br />
of scattered dots. We compare behavior between small vari<strong>an</strong>ce <strong>an</strong>d large vari<strong>an</strong>ce conditions of<br />
both <strong>the</strong> feedback dots <strong>an</strong>d <strong>the</strong> prior. We measure subjects’ behavior ch<strong>an</strong>ges in each of <strong>the</strong> 4<br />
combinations of prior <strong>an</strong>d feedback conditions. We find that people readily combine in<strong>for</strong>mation<br />
<strong>from</strong> both <strong>the</strong> position of feedback dots as well as previous knowledge of coin distribution in<br />
order to predict where <strong>the</strong> next coin will appear. Moreover, <strong>the</strong>y rely more on <strong>the</strong> feedback when<br />
<strong>the</strong> prior vari<strong>an</strong>ce is larger, <strong>an</strong>d more on <strong>the</strong> prior when it is smaller. Measured behavior was<br />
similar to <strong>the</strong> predictions <strong>from</strong> Bayesi<strong>an</strong> decision <strong>the</strong>ory. Our results thus indicate that <strong>the</strong><br />
nervous system is able to weigh prior <strong>an</strong>d likelihood in a m<strong>an</strong>ner that is close to optimal in a<br />
probabilistic sense.<br />
Disclosures: I. Vilares, None; H.L. Fern<strong>an</strong>des, None; K. Kording, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.13/FF115<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: CIHR 144079<br />
Title: Reward expectation modulates motion discrimination per<strong>for</strong>m<strong>an</strong>ce via <strong>the</strong> decisionmaking<br />
threshold <strong>an</strong>d evidence-accumulation-speed. A dopamine depletion fMRI study<br />
Authors: *A. NAGANO 1,2 , P. CISEK 4 , A. S. PERNA 2 , F. Z. SHIRDEL 1 , M. LEYTON 2 , C.<br />
BENKELFAT 3 , A. DAGHER 1 ;<br />
1 Montreal Neurol Ins, Montreal, QC, C<strong>an</strong>ada; 2 Dept. of Psychiatry, 3 Dept. of Psychiatry, Neurol.<br />
<strong>an</strong>d Neurosurg., McGill Univ., Montreal, QC, C<strong>an</strong>ada; 4 Dept. de physiologie, Univ. de Montréal,<br />
Montreal, QC, C<strong>an</strong>ada<br />
Abstract: We aimed to investigate how dopamine <strong>an</strong>d prior in<strong>for</strong>mation modulates task-related<br />
brain activity during a motion discrimination task. 17 subjects underwent fMRI sc<strong>an</strong>s on two<br />
different days: once after drinking <strong>an</strong> amino acid mixture deficient in <strong>the</strong> dopamine precursors<br />
tyrosine <strong>an</strong>d phenylal<strong>an</strong>ine (TPD), <strong>an</strong>d once after drinking a bal<strong>an</strong>ced amino acid mixture<br />
(BAL).<br />
The task involved detecting <strong>the</strong> motion direction of a r<strong>an</strong>dom-dot motion stimulus. Prior to each<br />
trial, auditory <strong>an</strong>d visual (AV) cues in<strong>for</strong>med subjects of (1) <strong>the</strong> probability of <strong>the</strong> coherent<br />
direction (left:right; 75:25, 50:50, 25:75) <strong>an</strong>d (2) whe<strong>the</strong>r <strong>the</strong>re would be a monetary reward <strong>for</strong> a<br />
correct decision. (3) In addition, <strong>the</strong>re were two types of 50:50 cues: those where both <strong>the</strong> AV<br />
cues signaled 50:50 (congruent) <strong>an</strong>d those where <strong>the</strong> AV cues signaled opposite directions at<br />
75% likelihood (incongruent 50:50 cues).<br />
We hypo<strong>the</strong>sized that <strong>the</strong> decision to choose ei<strong>the</strong>r left or right direction is based on a linear<br />
accumulation of evidence <strong>from</strong> <strong>an</strong> initial level related to prior probability. A decision is made<br />
when evidence reaches a certain threshold. We used <strong>the</strong> method of Carpenter <strong>an</strong>d Williams<br />
(Nature 1995) to estimate a decision-making-threshold index (T-index), <strong>an</strong>d <strong>an</strong> evidenceaccumulation-speed<br />
index (A-index). We also measured <strong>the</strong> reaction time (RT) on each trial.<br />
During <strong>the</strong> fMRI, we investigated <strong>the</strong> effects of (1) expected probability of <strong>the</strong> direction, (2)<br />
congruency of <strong>the</strong> cues, <strong>an</strong>d (3) reward expectation. The statistical threshold was set at p =<br />
0.0001 <strong>for</strong> fMRI <strong>an</strong>alyses.<br />
In both BAL <strong>an</strong>d TPD conditions, incongruent cues <strong>an</strong>d reward expectation both increased RT.<br />
In addition, RT was shorter when subjects responded to <strong>the</strong> congruent-cued direction. In <strong>the</strong><br />
BAL session only, <strong>the</strong>re was <strong>an</strong> inverse correlation between <strong>the</strong> RT difference <strong>an</strong>d activation in<br />
MT area while observing <strong>the</strong> motion stimulus.<br />
<strong>When</strong> comparing rewarded to non-rewarded trials, reward expectation was associated with<br />
greater activation in extensive cortico-striatal areas (dorsolateral prefrontal, <strong>an</strong>terior insula,<br />
bilateral caudate) <strong>an</strong>d visual areas (fusi<strong>for</strong>m gyrus <strong>an</strong>d visual cortex) while observing <strong>the</strong> motion<br />
stimulus. The <strong>for</strong>mer was observed only in <strong>the</strong> BAL session. T-index difference was positively
correlated with activity in <strong>the</strong> cortico-striatal areas. This suggests that <strong>the</strong> cortico-striatal system<br />
may be involved in setting <strong>the</strong> decision-making threshold or <strong>the</strong> initial activity level using cue<br />
in<strong>for</strong>mation, <strong>an</strong>d that this phenomenon is dopamine-dependent. In <strong>the</strong> BAL session, <strong>the</strong> A-index<br />
difference (rewarded versus non-rewarded trials) was negatively correlated with activation in <strong>the</strong><br />
inferior parietal sulcus during <strong>the</strong> response period.<br />
Disclosures: A. Nag<strong>an</strong>o, None; P. Cisek, None; A.S. Perna, None; F.Z. Shirdel, None; M.<br />
Leyton, None; C. Benkelfat, None; A. Dagher, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.14/FF116<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: NIMH IRP<br />
Title: The nature of <strong>the</strong> evidence ch<strong>an</strong>ges how <strong>the</strong> brain uses prior probability in decisions<br />
Authors: *K. A. HANSEN, S. F. HILLENBRAND, L. G. UNGERLEIDER;<br />
NIMH, NIH, Be<strong>the</strong>sda, MD<br />
Abstract: It is well known that people use prior probability (PP) to bias decisions. We<br />
characterized <strong>the</strong> brain mech<strong>an</strong>isms mediating this robust behavioral effect by identifying fMRI<br />
interactions between PP <strong>an</strong>d target discriminability during perceptual decisions about two target<br />
types: lower-level (signal-to-noise-modulated) <strong>an</strong>d higher-level (<strong>for</strong>m-modulated) <strong>abstract</strong><br />
shapes. We observed two double dissociations. (1) Anatomical location: Given <strong>the</strong> lower-level<br />
perceptual evidence <strong>an</strong> interaction occurred in middle occipital gyrus (MOG) only, while given<br />
<strong>the</strong> higher-level perceptual evidence interactions occurred in a network of prefrontal <strong>an</strong>d parietal<br />
regions but not in MOG. (2) Activity pattern: The lower-level interaction was driven by<br />
increased activity when <strong>the</strong> target discriminability was low <strong>an</strong>d <strong>the</strong> PP of one alternative was<br />
high, while <strong>the</strong> higher-level interactions were driven by increased activity when both <strong>the</strong> target<br />
discriminability <strong>an</strong>d <strong>the</strong> PP of one alternative were high <strong>an</strong>d when <strong>the</strong> target was of <strong>the</strong> type<br />
contraindicated by PP. Additional <strong>an</strong>alyses ruled out task difficulty, stimulus unexpectedness <strong>an</strong>d<br />
time to task as expl<strong>an</strong>ations <strong>for</strong> <strong>the</strong> frontoparietal results. A control dataset ruled out cognitive<br />
load as <strong>an</strong> expl<strong>an</strong>ation <strong>for</strong> <strong>the</strong> frontoparietal results <strong>an</strong>d confirmed that <strong>the</strong> activity increases<br />
driving <strong>the</strong> observed interactions depended on PP, not on evidence type itself. The interactions<br />
suggest a novel hypo<strong>the</strong>sis: that PP affects decisions based on lower-level vs. higher-level
evidence by influencing <strong>the</strong> representation of <strong>the</strong> evidence vs. <strong>the</strong> behavioral criterion on <strong>the</strong><br />
evidence. Acknowledgement: This research was supported by <strong>the</strong> NIMH IRP.<br />
Disclosures: K.A. H<strong>an</strong>sen, None; S.F. Hillenbr<strong>an</strong>d, None; L.G. Ungerleider, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.15/FF117<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: The Burroughs-Wellcome Fund<br />
The McKnight Endowment Fund <strong>for</strong> Neuroscience<br />
R01 EY015260<br />
T32 EY007035<br />
T32 GM12453-03<br />
Russell Ackoff Doctoral Student Fellowship<br />
Title: Updating beliefs in a ch<strong>an</strong>ging world<br />
Authors: *M. R. NASSAR, R. WILSON, B. HEASLY, J. GOLD;<br />
Neurosci., Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: The brain faces a major computational challenge in determining how much to update a<br />
belief about <strong>the</strong> world after obtaining new, relev<strong>an</strong>t in<strong>for</strong>mation. The challenge arises <strong>from</strong> <strong>the</strong><br />
various <strong>for</strong>ms of uncertainty inherent in natural environments. Some in<strong>for</strong>mation is noisy, <strong>an</strong>d<br />
thus new samples c<strong>an</strong>not be trusted completely. Natural environments are also prone to ch<strong>an</strong>ge,<br />
<strong>an</strong>d thus beliefs based on old in<strong>for</strong>mation c<strong>an</strong> quickly become obsolete <strong>an</strong>d new in<strong>for</strong>mation<br />
should be favored. To better underst<strong>an</strong>d how <strong>the</strong> brain assigns influence to incoming in<strong>for</strong>mation<br />
in <strong>the</strong> presence of <strong>the</strong>se <strong>for</strong>ms of uncertainty, we have developed a task in which subjects must<br />
estimate <strong>the</strong> value of a noisy <strong>an</strong>d sometimes ch<strong>an</strong>ging variable. The influence of each<br />
sequentially presented piece of in<strong>for</strong>mation on subject estimates c<strong>an</strong> be qu<strong>an</strong>tified directly. This<br />
task, along with a Bayesi<strong>an</strong> ideal observer model, has allowed us to develop heuristic
descriptions of hum<strong>an</strong> belief updating in noisy <strong>an</strong>d dynamic environments.<br />
The heuristic models update beliefs using a delta-rule framework in which <strong>an</strong> updated belief<br />
depends on <strong>the</strong> previous belief, <strong>the</strong> newly observed datum, <strong>an</strong>d <strong>the</strong> learning rate. Here we focus<br />
on selection of <strong>the</strong> learning rate, which describes <strong>the</strong> relative influence of <strong>the</strong> newest datum on<br />
<strong>the</strong> updated belief. Like <strong>the</strong> ideal observer model, <strong>the</strong> most effective heuristic models choose a<br />
learning rate according to <strong>the</strong> probability that <strong>an</strong> environmental ch<strong>an</strong>ge has occurred <strong>an</strong>d <strong>the</strong><br />
number of data points observed since <strong>the</strong> last such ch<strong>an</strong>ge. However, unlike <strong>the</strong> ideal-observer<br />
model, <strong>the</strong> heuristic models approximate <strong>the</strong>se values by estimating only a single predictive<br />
distribution.<br />
The most effective heuristic models describe several salient features of subject behavior<br />
including increased influence of unexpected in<strong>for</strong>mation, scale invari<strong>an</strong>t ch<strong>an</strong>gepoint detection,<br />
decay in <strong>the</strong> influence of in<strong>for</strong>mation after ch<strong>an</strong>gepoints, <strong>an</strong>d decay in estimate uncertainty after<br />
ch<strong>an</strong>gepoints. In addition, <strong>the</strong> models describe sub-optimal subject behaviors <strong>an</strong>d offer unique<br />
insights into variability in learning rates used by different subjects: some subjects see errors as<br />
noise <strong>an</strong>d are hardly influenced by new in<strong>for</strong>mation, whereas o<strong>the</strong>r subjects see errors as ch<strong>an</strong>ge<br />
<strong>an</strong>d are heavily influenced by new in<strong>for</strong>mation.<br />
Disclosures: M.R. Nassar, None; R. Wilson, None; B. Heasly, None; J. Gold, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.16/FF118<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning<br />
Support: The Burroughs-Wellcome Fund<br />
The McKnight Endowment Fund <strong>for</strong> Neuroscience<br />
R01 EY015260<br />
T32 EY007035<br />
P50 MH062196<br />
Title: Correlates of decision commitment in measurements of pupil diameter using <strong>the</strong><br />
counterm<strong>an</strong>ding task
Authors: *R. M. KALWANI, J. I. GOLD;<br />
Univ. Pennsylv<strong>an</strong>ia, Philadelphia, PA<br />
Abstract: Previous studies have suggested that event-related ch<strong>an</strong>ges in pupil diameter are <strong>an</strong><br />
indirect measure of activity of noradrenergic neurons in <strong>the</strong> brainstem nucleus locus coeruleus<br />
(LC), particularly under low-lumin<strong>an</strong>ce conditions. Single-unit recordings <strong>from</strong> <strong>the</strong> LC, in alert<br />
subjects suggest that LC phasic activity is correlated with a goal-directed motor commitment. In<br />
particular, LC neurons burst prior to <strong>an</strong> overt motor response to obtain a reward but fail to fire in<br />
no-go trials in which <strong>an</strong>imals are rewarded <strong>for</strong> inhibiting <strong>the</strong>ir motor response. Based on <strong>the</strong><br />
proposed relationship between ch<strong>an</strong>ges in pupil diameter <strong>an</strong>d phasic LC activity, we hypo<strong>the</strong>size<br />
that pupil diameter should ch<strong>an</strong>ge on trials in which subjects overtly respond to achieve <strong>the</strong>ir<br />
reward. However, pupil diameter should not ch<strong>an</strong>ge on trials in which subjects inhibit <strong>the</strong>ir<br />
motor response.<br />
We measured pupil diameter of hum<strong>an</strong> subjects per<strong>for</strong>ming <strong>an</strong> isolumin<strong>an</strong>t version of <strong>the</strong><br />
counterm<strong>an</strong>ding task. For this task, <strong>the</strong> subject fixates <strong>an</strong>d <strong>the</strong>n is shown a peripheral target that<br />
indicates which of two levers to pull. However, on a small fraction of <strong>the</strong> trials, sometime after<br />
<strong>the</strong> target is shown but be<strong>for</strong>e <strong>the</strong> lever response is initiated, a “stop-signal” is given. The exact<br />
time of occurrence of this signal is <strong>the</strong> critical variable that determines whe<strong>the</strong>r or not it is<br />
effective <strong>an</strong>d stops <strong>the</strong> subject <strong>from</strong> pulling <strong>the</strong> lever. Per<strong>for</strong>m<strong>an</strong>ce is typically modeled as a race<br />
between dynamic “go” <strong>an</strong>d “stop” processes, <strong>the</strong> winner of which commits <strong>the</strong> subject to initiate<br />
or withhold <strong>the</strong> response, respectively, on a given trial.<br />
We have collected pupil-diameter data <strong>from</strong> three subjects per<strong>for</strong>ming <strong>the</strong> task. Phasic pupil<br />
diameter consistently increased around <strong>the</strong> time of <strong>the</strong> subject’s response <strong>for</strong> both visually<br />
guided lever release trials <strong>an</strong>d incorrect stop-signal trials. Pupil diameter also increased around<br />
<strong>the</strong> time of stopping on correct stop-signal trials. More work is needed to determine whe<strong>the</strong>r<br />
<strong>the</strong>re are qu<strong>an</strong>titative differences in <strong>the</strong> phasic pupil response <strong>for</strong> different trial types. However,<br />
preliminary <strong>an</strong>alyses suggest that <strong>the</strong>re is little difference in pupil responses <strong>for</strong> different trial<br />
types, suggesting a reflection of commitments to ei<strong>the</strong>r stop or go on <strong>the</strong> counterm<strong>an</strong>ding task.<br />
These results are inconsistent with data collected <strong>from</strong> LC neurons, which are thought to fire<br />
prior to a motor response but not during no-response trials.<br />
Disclosures: R.M. Kalw<strong>an</strong>i, None; J.I. Gold, None.<br />
Poster<br />
576. Perceptual Decisions <strong>an</strong>d Models<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 576.17/FF119<br />
Topic: F.01.g. Decision making <strong>an</strong>d reasoning
Support: Office of Naval Research<br />
Title: Neural correlates of visual per<strong>for</strong>m<strong>an</strong>ce improvement in action video-game players<br />
Authors: W. KHOE 1 , *D. BAVELIER 2 , S. HILLYARD 1 ;<br />
1 2<br />
Dept. of Neurosciences, UCSD, S<strong>an</strong> Diego, La Jolla, CA; Brain & Cog Sci., Univ. Rochester,<br />
Rochester, NY<br />
Abstract: na<br />
Disclosures: W. Khoe, None; D. Bavelier, None; S. Hillyard, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.1/FF120<br />
Topic: F.01.l. Social cognition<br />
Title: Involvement of amygdala in gaze-triggered reflexive shift of attention<br />
Authors: *M. TOICHI 1 , T. OKADA 2 , W. SATO 3 ;<br />
1 Fac. of Hum<strong>an</strong> Hlth. Sci., 2 Dept. of Neuropsychiatry, Kyoto Univ. Grad. Sch. of Med., Kyoto,<br />
Jap<strong>an</strong>; 3 Kyoto Univ. Primate Res. Inst., Kyoto, Jap<strong>an</strong><br />
Abstract: Introduction: Recent studies have consistently reported that eye gaze of <strong>an</strong>o<strong>the</strong>r<br />
individual triggers shift of <strong>the</strong> observer’s visuospatial attention to its direction, which is called<br />
joint attention. The aims of <strong>the</strong> current study were to confirm <strong>the</strong> robustness of <strong>the</strong> joint attention<br />
phenomenon in healthy adults (Study 1), <strong>an</strong>d to examine whe<strong>the</strong>r or not <strong>the</strong> amygdala is involved<br />
in joint attention.<br />
Study 1<br />
Methods: Subjects were twenty healthy young adults. Facial stimuli consisted of line drawings<br />
<strong>an</strong>d photographs. Gaze triggered attention shift was examined using a Posner’s paradigm. The<br />
gaze direction was expressed by <strong>the</strong> eye gaze direction (line drawings), or by <strong>the</strong> eye gaze <strong>an</strong>d<br />
head direction (photographs). After a warning signal, <strong>the</strong> gaze cue was presented (200 msec) in<br />
<strong>the</strong> center of <strong>the</strong> visual field, be<strong>for</strong>e target presentation in <strong>the</strong> peripheral field. Subjects were<br />
instructed that gaze directions were not predictive of <strong>the</strong> location of <strong>the</strong> targets. The task <strong>for</strong><br />
subjects was to localize <strong>the</strong> target as soon as possible, <strong>an</strong>d <strong>the</strong> reaction time (RT) needed to<br />
localize <strong>the</strong> target was measured.
Results: RT <strong>for</strong> correct responses was signific<strong>an</strong>tly shorter <strong>for</strong> valid th<strong>an</strong> invalid gaze cues <strong>for</strong><br />
both types of gaze cues.<br />
Discussion: The result confirmed that gaze triggered reflexive shift of attention occurs in adults<br />
under photograph conditions as well as line drawing conditions.<br />
Study 2<br />
Methods: Subjects were seven patients with unilateral amygdala incision (including <strong>an</strong>terior<br />
lobectomy) <strong>for</strong> <strong>the</strong> treatment of medication-resist<strong>an</strong>t epilepsy <strong>an</strong>d nine age- <strong>an</strong>d IQ-matched<br />
controls with epilepsy. Gaze triggered attention shift was examined using <strong>the</strong> same Posner’s<br />
paradigm as Study 1, with a few modifications. Gaze cues were presented <strong>for</strong> 200 ms to <strong>the</strong><br />
unilateral visual field, after which subjects were required to localize targets as quickly as<br />
possible.<br />
Results: RT <strong>for</strong> correct responses was signific<strong>an</strong>tly shorter when gaze directions were toward <strong>the</strong><br />
targets th<strong>an</strong> away <strong>from</strong> <strong>the</strong> targets. This cuing effect was not m<strong>an</strong>ifested following stimulation of<br />
<strong>the</strong> lesioned hemisphere in patients with unilateral amygdale incision.<br />
Discussion: The result strongly suggest that <strong>the</strong> amygdala plays <strong>an</strong> import<strong>an</strong>t role in spatial<br />
attention shift triggered by <strong>an</strong>o<strong>the</strong>r person’s gaze direction.<br />
General Discussion: Recent studies have reported <strong>an</strong> import<strong>an</strong>t role of <strong>the</strong> amygdala in a variety<br />
of social functions, which may include joint attention.<br />
Disclosures: M. Toichi, None; T. Okada, None; W. Sato, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.2/FF121<br />
Topic: F.01.l. Social cognition<br />
Support: EU-Project ‘‘Joint Action Science <strong>an</strong>d Technology’’ (IST-FP6- 003747)<br />
Title: Pl<strong>an</strong>ning joint actions <strong>an</strong>d achieving shared goals<br />
Authors: *I. TONI 1,2 , J. MAJDANDŽIć 2 , H. BEKKERING 2 ;<br />
2 Radboud Univ. Nijmegen, 1 Donders Inst., Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: We have investigated <strong>the</strong> cerebral bases of <strong>the</strong> hum<strong>an</strong> ability to act with o<strong>the</strong>rs<br />
according to shared goals. Sharing goals refer to <strong>the</strong> ability to coordinate movements, attentional<br />
states, <strong>an</strong>d inferred knowledge with o<strong>the</strong>r agents in order to achieve a given outcome, <strong>an</strong>d to<br />
achieve it toge<strong>the</strong>r (Tomasello et al, 2005). We used BOLD-sensitive fMRI to compare <strong>the</strong>
cerebral consequences of pl<strong>an</strong>ning actions according to a goal shared with a co-actor (Joint<br />
condition) or alone (Solo condition). In each condition, subjects (N=16) per<strong>for</strong>med grasping<br />
actions as specified on <strong>the</strong> basis of ei<strong>the</strong>r <strong>the</strong> object to be grasped (Immediate Goal) or <strong>the</strong> endposition<br />
of <strong>the</strong> object (Final Goal). The rationale is that pl<strong>an</strong>ning joint actions on <strong>the</strong> basis of a<br />
given final goal requires <strong>the</strong> integration of <strong>the</strong> co-actor’s predicted action into <strong>the</strong> overall action<br />
pl<strong>an</strong>. In contrast, pl<strong>an</strong>ning joint actions on <strong>the</strong> basis of a given immediate goal, or pl<strong>an</strong>ning solo<br />
actions, does not require <strong>the</strong> actor to adapt his own movement pl<strong>an</strong> to <strong>the</strong> predicted response of<br />
<strong>the</strong> co-actor.<br />
We found that pl<strong>an</strong>ning a joint action according to a shared goal (Joint Final Goal condition)<br />
increases BOLD signals in left ventrolateral prefrontal cortex, over <strong>an</strong>d above <strong>the</strong> effects of<br />
pl<strong>an</strong>ning <strong>the</strong> same action in a joint setting that does not require shared goals (Joint Immediate<br />
Goal condition) or during <strong>the</strong> pl<strong>an</strong>ning of Solo actions (Fig. 1).<br />
These results illustrate how integrating action pl<strong>an</strong>s <strong>an</strong>d goals across multiple agents might be <strong>an</strong><br />
inst<strong>an</strong>ce of <strong>the</strong> general ability of this region to unify spatio-temporally independent events by<br />
<strong>abstract</strong>ing over individual sensorimotor tokens (Koechlin & Jubault, 2006; Hagoort, 2005).<br />
Figure 1. fMRI data showing differential activity <strong>for</strong> Final-Goal versus Immediate-Goal action<br />
preparation, <strong>an</strong>d specific <strong>for</strong> actions per<strong>for</strong>med with a co-actor. A, C) Parameter estimates [in<br />
st<strong>an</strong>dard error (SE) units] of preparatory activity within <strong>the</strong> left ventrolateral prefrontal cortex. B)<br />
Statistical parametric map (P
Title: Dissociable components of observational learning during virtual navigation in <strong>the</strong> Morris<br />
water maze<br />
Authors: M. A. HENDRICKS, L. BERGERON, A. POE, B. NACE, *B. D. DEVAN;<br />
Psychology, Towson Univ., Towson, MD<br />
Abstract: Recent interest in observational learning has resulted <strong>from</strong> <strong>the</strong> discovery of mirror<br />
neurons in ventral premotor <strong>an</strong>d <strong>the</strong> posterior parietal cortices. The present study was conducted<br />
to determine <strong>the</strong> relative import<strong>an</strong>ce of observing <strong>the</strong> h<strong>an</strong>d movements of a joystick in producing<br />
virtual movement during place navigation in <strong>the</strong> water maze. Following a pretraining phase,<br />
subjects were r<strong>an</strong>domly assigned to one of four conditions: 1) no observation, 2) joystick only, 3)<br />
virtual screen only <strong>an</strong>d 4) joystick <strong>an</strong>d virtual screen. Contrary to expectations, <strong>the</strong> group<br />
receiving simult<strong>an</strong>eous viewing of joystick <strong>an</strong>d virtual movement did not outper<strong>for</strong>m <strong>the</strong> virtual<br />
screen only group. This result may be attributable to bottlenecking where <strong>the</strong> two types of<br />
observation compete <strong>for</strong> attention resources thus causing interference in allocentric spatial<br />
learning. Alternatively, optimum per<strong>for</strong>m<strong>an</strong>ce occurred in <strong>the</strong> group observing only <strong>the</strong> virtual<br />
screen whereas inferior per<strong>for</strong>m<strong>an</strong>ce resulted <strong>from</strong> observing joystick movements only. The<br />
results suggest that ra<strong>the</strong>r th<strong>an</strong> a simple representation of overt movement, observational learning<br />
of <strong>the</strong> water maze place response may more import<strong>an</strong>tly involve a representation of virtual<br />
movement through allocentric space.<br />
Disclosures: M.A. Hendricks, None; L. Bergeron, None; A. Poe, None; B. Nace, None; B.D.<br />
Dev<strong>an</strong>, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.4/FF123<br />
Topic: F.01.l. Social cognition<br />
Support: NSF #0811450: Exploring <strong>the</strong> Unc<strong>an</strong>ny Valley<br />
Title: The neural correlates of goal-directed action underst<strong>an</strong>ding<br />
Authors: *E. J. CARTER;<br />
Psychology, Carnegie Mellon Univ., Pittsburgh, PA
Abstract: Prior research has used functional magnetic reson<strong>an</strong>ce imaging (fMRI) to examine<br />
viewer’s brain responses when watching <strong>an</strong>o<strong>the</strong>r hum<strong>an</strong> miss or achieve a goal <strong>an</strong>d found<br />
increased posterior superior temporal sulcus (pSTS) <strong>an</strong>d mirror neuron activity <strong>for</strong> failures (e.g.,<br />
Buccino et al., 2007; Pelphrey et al., 2004). In this fMRI study, adult particip<strong>an</strong>ts viewed<br />
reaching behaviors in which one of 4 <strong>an</strong>imated characters moved a h<strong>an</strong>d towards one of two<br />
objects <strong>an</strong>d ei<strong>the</strong>r (a) picked up <strong>the</strong> object, (b) missed <strong>the</strong> object, or (c) ch<strong>an</strong>ged his path halfway<br />
to move towards <strong>an</strong>d lift <strong>the</strong> o<strong>the</strong>r object. The <strong>an</strong>imated characters included a hum<strong>an</strong>, a<br />
hum<strong>an</strong>oid robot, a set of stacked boxes with a moving arm, <strong>an</strong>d a machine with a grasping claw.<br />
Using motion capture technology (Park & Hodgins, 2006), movement patterns recorded <strong>from</strong> a<br />
live actor’s reaching behaviors were overlaid onto <strong>the</strong> hum<strong>an</strong>, hum<strong>an</strong>oid robot, <strong>an</strong>d boxes in<br />
order <strong>for</strong> <strong>the</strong>m to all move biologically <strong>an</strong>d identically despite <strong>the</strong>ir differing appear<strong>an</strong>ces. For<br />
<strong>the</strong> hum<strong>an</strong> character, pSTS activity increased when he ei<strong>the</strong>r shifted goals or missed <strong>the</strong> original<br />
target. Similar results were found <strong>for</strong> <strong>the</strong> robot. However, <strong>the</strong> boxes character <strong>an</strong>d mech<strong>an</strong>ical<br />
arm did not elicit this pattern of response, suggesting that <strong>the</strong> pSTS is engaged differentially <strong>for</strong><br />
figures that appear more hum<strong>an</strong>like regardless of biological motion. Additionally, medial<br />
prefrontal areas previously reported to be part of a protagonist-monitoring network (e.g., Mason<br />
& Just, 2006) were most engaged <strong>for</strong> <strong>the</strong> hum<strong>an</strong> character, followed by <strong>the</strong> robot character, <strong>for</strong><br />
all reaching behaviors. These results suggest that <strong>the</strong> neural correlates of goal-directed action<br />
underst<strong>an</strong>ding respond similarly to different cases in which <strong>the</strong> original goal is not achieved <strong>an</strong>d<br />
that activity c<strong>an</strong> be modulated by type of actor.<br />
Disclosures: E.J. Carter, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.5/FF124<br />
Topic: F.01.l. Social cognition<br />
Support: NSF Gr<strong>an</strong>t BCS0826897<br />
NIMH Gr<strong>an</strong>t MH80038<br />
US Office of Naval Research Contract N000140510117<br />
Title: The (weird?) coordination dynamics of boxing
Authors: *G. C. DE GUZMAN 1 , J. LAGARDE 2 , Y. PALUT 2 , O. OULLIER 3 , J. A. S.<br />
KELSO 1 ;<br />
1 The Hum<strong>an</strong> Brain & Behavior Lab. - Ctr. <strong>for</strong> Complex Systems <strong>an</strong>d Brain Sci., Florida Atl<strong>an</strong>tic<br />
Univ., Boca Raton, FL; 2 Lab. Efficience Defficience Motrices, Univ. Montpellier 1, Montpellier,<br />
Fr<strong>an</strong>ce; 3 The Hum<strong>an</strong> Neurobio. Lab., Aix-Marseille Univ., Marseille, Fr<strong>an</strong>ce<br />
Abstract: The d<strong>an</strong>ce of two boxers per<strong>for</strong>ming <strong>the</strong>ir respective tasks presents a paradoxical<br />
social situation: each opponent tries to be unpredictable yet at <strong>the</strong> same time must coordinate<br />
his/her movements with <strong>the</strong> o<strong>the</strong>r to gain <strong>the</strong> upper h<strong>an</strong>d. Here, we studied time series of head<br />
movements of boxers engaged in a controlled match to explore <strong>the</strong>ir behavioral patterns.<br />
Specifically, we used <strong>the</strong> framework of coupled r<strong>an</strong>dom walks to probe <strong>the</strong> statistical properties<br />
of absolute <strong>an</strong>d relative head movement patterns <strong>an</strong>d <strong>the</strong>ir tr<strong>an</strong>sitions. At each time point of <strong>the</strong><br />
timeseries, a 3-bit word is produced that grossly describes three pieces of movement in<strong>for</strong>mation<br />
relative to a fixed lab frame: (1) relative phase (sense of movement in lab frame - same or<br />
opposite), (2) comparison of speed, (3) direction (sense of movement away/towards each o<strong>the</strong>r)<br />
along <strong>the</strong> line of sight vector. The tr<strong>an</strong>sition matrix of <strong>the</strong> 3-bit words was constructed <strong>an</strong>d<br />
<strong>an</strong>alyzed <strong>for</strong> convergence of patterns <strong>an</strong>d pattern switches. Analysis was also per<strong>for</strong>med using a<br />
map based minimal model in which <strong>the</strong> probability of a directed movement step is based on <strong>the</strong><br />
line of sight dist<strong>an</strong>ce between <strong>the</strong> opponents. The probability distribution used is uni<strong>for</strong>m <strong>an</strong>d is<br />
defined in <strong>an</strong> interval with <strong>an</strong>gular end-points dependent on primarily two dist<strong>an</strong>ce parameters<br />
(one <strong>for</strong> each boxer). Stationary properties of <strong>the</strong> model qualitatively consistent with <strong>the</strong> data<br />
have been found <strong>for</strong> <strong>the</strong> separation dist<strong>an</strong>ce, <strong>an</strong>gular velocity of line of sight, average center of<br />
mass motion including <strong>the</strong> dispersion of orbit points over time about <strong>the</strong> center of mass. A<br />
comparison of <strong>the</strong> behavior of <strong>the</strong> tr<strong>an</strong>sition matrices <strong>for</strong> both data <strong>an</strong>d simulation will be<br />
presented.<br />
Disclosures: G.C. De Guzm<strong>an</strong>, None; J. Lagarde, None; Y. Palut, None; O. Oullier,<br />
None; J.A.S. Kelso, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.6/FF125<br />
Topic: F.01.l. Social cognition<br />
Support: Gr<strong>an</strong>t-in Aid <strong>for</strong> Scientific Research S#17100003 (to NS) <strong>from</strong> <strong>the</strong> Jap<strong>an</strong> <strong>Society</strong> <strong>for</strong><br />
<strong>the</strong> Promotion of Science.
Title: “Like me”: The role of extrastriate body area <strong>for</strong> reciprocal imitation<br />
Authors: *Y. OKAMOTO 1,2 , R. KITADA 1,2 , A. SASAKI 1,2 , T. MORITA 3 , S. ITAKURA 4,5 , T.<br />
KOCHIYAMA 6 , H. C. TANABE 1,2 , N. SADATO 1,2,3,7 ;<br />
1 Dept. of Cerebral Res., Natl. Inst. For Physiological Sci., Aichi, Jap<strong>an</strong>; 2 Dept. of Physiological<br />
Sci., The Grad. Univ. <strong>for</strong> Adv<strong>an</strong>ced Studies (Sokendai), Aichi, Jap<strong>an</strong>; 3 Jap<strong>an</strong> Sci. <strong>an</strong>d Technol.<br />
Agency, Saitama, Jap<strong>an</strong>; 4 Dept. of Psychology, Grad. Sch. of Letters, Kyoto Univ., Kyoto,<br />
Jap<strong>an</strong>; 5 Intelligent Robotics <strong>an</strong>d Communication laboratories, 6 Brain Activity Imaging Ctr.,<br />
Adv<strong>an</strong>ced Telecommunications Res. Inst. Intl., Kyoto, Jap<strong>an</strong>; 7 Biomed. imaging research center,<br />
Univ. of Fukui, Fukui, Jap<strong>an</strong><br />
Abstract: Introduction : It is well known that both imitating o<strong>the</strong>r’s action <strong>an</strong>d being imitated<br />
are import<strong>an</strong>t <strong>for</strong> development of social ability (Meltzoff 2005; Escalona et al. 2002). How do<br />
we know our movement is similar to that of <strong>the</strong> observed action, or how do we know our<br />
movement is being imitated? Iacoboni et al. (2001) reported that <strong>the</strong> lateral occipito-temporal<br />
region was activated by imitation. These authors suggested that <strong>the</strong>se regions receive efference<br />
copy <strong>from</strong> mirror neuron system (MNS), <strong>an</strong>d monitor <strong>the</strong> congruence between self action <strong>an</strong>d<br />
o<strong>the</strong>r’s action. However it is unknown whe<strong>the</strong>r <strong>the</strong>se regions are also activated by being imitated.<br />
Recently, extrastriate body area (EBA) in <strong>the</strong> occipital cortex was shown to be not only involved<br />
in visual perception of moving body parts, but also responds to <strong>the</strong> self-generation of actions or<br />
even to <strong>the</strong> motor imagery with <strong>the</strong> same body parts (Astafiev et al. 2004). These findings lead<br />
us to hypo<strong>the</strong>size that while MNS is critical <strong>for</strong> action underst<strong>an</strong>ding by internally emulating<br />
observed action, <strong>the</strong> EBA may be devoted to evaluating <strong>the</strong> observed action in <strong>the</strong> context of <strong>the</strong><br />
action of self. Our hypo<strong>the</strong>sis is based on <strong>the</strong> assumption that <strong>the</strong> working memory <strong>for</strong> <strong>the</strong> action<br />
is generated both by observation <strong>an</strong>d execution. If this is true, regardless of doing imitation or<br />
being imitated, <strong>the</strong> EBA should show differential activation due to <strong>the</strong> congruence/incongruence.<br />
Method : Fourteen subjects participated in <strong>the</strong> functional MRI. Subjects underwent visuallycued<br />
h<strong>an</strong>d shape <strong>for</strong>mation task with <strong>the</strong>ir right h<strong>an</strong>d while viewing <strong>the</strong> video-clips of <strong>the</strong> h<strong>an</strong>d<br />
shape <strong>for</strong>mation by o<strong>the</strong>r person. We have m<strong>an</strong>ipulated <strong>the</strong> timing of <strong>the</strong> execution <strong>an</strong>d<br />
observation, <strong>an</strong>d <strong>the</strong> congruency of <strong>the</strong> executed <strong>an</strong>d observed action, thus constituting <strong>the</strong> 2<br />
(timing: preceding (P) vs. following (F)) x 2 (congruency: congruent (C) vs. incongruent (I))<br />
factorial design. The main effect <strong>an</strong>d <strong>the</strong> interaction were tested across <strong>the</strong> whole brain, as well<br />
as specifically in <strong>the</strong> EBA that was functionally defined.<br />
Results <strong>an</strong>d Discussion : The bilateral lateral occipito-temporal cortex showed signific<strong>an</strong>t<br />
congruency effect without interaction. The activated areas were markedly overlapped with<br />
functionally defined EBA. Subsequent functional region-of-interest (ROI) <strong>an</strong>alysis revealed that<br />
<strong>the</strong> activity that is related to being imitated (PC) was negatively correlated with autistic-spectrum<br />
quotient (AQ). These findings imply <strong>the</strong> import<strong>an</strong>ce of EBA as <strong>the</strong> visual “social brain” that is<br />
relev<strong>an</strong>t <strong>for</strong> inter-subjective communication.<br />
Disclosures: Y. Okamoto, None; R. Kitada, None; A. Sasaki, None; T. Morita, None; S.<br />
Itakura, None; T. Kochiyama, None; H.C. T<strong>an</strong>abe, None; N. Sadato, None.
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.7/FF126<br />
Topic: F.01.l. Social cognition<br />
Title: Neural correlates of <strong>an</strong>imacy detection<br />
Authors: *C. E. LOOSER, T. WHEATLEY;<br />
Dartmouth Col., H<strong>an</strong>over, NH<br />
Abstract: Deciding what is alive <strong>an</strong>d capable of interacting with us is one of <strong>the</strong> most import<strong>an</strong>t<br />
decisions our brains make. Recent work has indicated that specific cortices are activated when<br />
something is deemed <strong>an</strong>imate versus in<strong>an</strong>imate. This putative ‘social network’ includes <strong>the</strong><br />
medial prefrontal, posterior cingulate <strong>an</strong>d insular cortices as well as <strong>the</strong> lateral fusi<strong>for</strong>m gyrus,<br />
superior temporal sulcus, <strong>an</strong>d amygdalae. Previous research investigating <strong>the</strong>se areas has used<br />
<strong>an</strong>imate <strong>an</strong>d in<strong>an</strong>imate exemplars (e.g.,faces, houses -- Haxby et al., 2001; K<strong>an</strong>wisher, et al.,<br />
1997) that differ on whe<strong>the</strong>r <strong>the</strong>y are alive as well as in <strong>the</strong>ir basic visual features (e.g., curves,<br />
<strong>an</strong>gles). Although visual <strong>for</strong>m may be <strong>an</strong> import<strong>an</strong>t component of <strong>an</strong>imacy, <strong>the</strong>re are m<strong>an</strong>y<br />
in<strong>an</strong>imate objects that have <strong>an</strong>imate <strong>for</strong>m (statues, dolls, stuffed <strong>an</strong>imals, etc.). Such objects<br />
provide a more stringent test <strong>for</strong> which regions within <strong>the</strong> social network are recruited to<br />
discriminate <strong>an</strong>imacy beyond <strong>the</strong> basic recognition of <strong>an</strong>imate <strong>for</strong>m.<br />
We created four stimulus categories wherein <strong>an</strong>imacy <strong>an</strong>d visual similarity were orthogonal<br />
properties. These categories were <strong>an</strong>imate hum<strong>an</strong> faces (hum<strong>an</strong>s), in<strong>an</strong>imate hum<strong>an</strong> faces (dolls),<br />
<strong>an</strong>imate <strong>an</strong>imal faces (dogs), in<strong>an</strong>imate <strong>an</strong>imal faces (stuffed <strong>an</strong>imal dogs). Import<strong>an</strong>tly, <strong>the</strong>se<br />
categories c<strong>an</strong> be compared in terms of <strong>the</strong>ir <strong>an</strong>imacy (alive; not alive) or visual similarity<br />
(hum<strong>an</strong> <strong>for</strong>m; dog <strong>for</strong>m). Subjects passively viewed <strong>the</strong>se images while lying in a 3T fMRI<br />
sc<strong>an</strong>ner <strong>an</strong>d catch trials ensured attention.<br />
Consistent with previous research, a GLM <strong>an</strong>alysis showed that <strong>the</strong> lateral fusi<strong>for</strong>m cortex<br />
preferred faces to clocks but did not appear to differentiate between face categories (Tong et al.,<br />
2000). In contrast, <strong>the</strong> amygdala, portions of <strong>the</strong> medial prefrontal cortex, <strong>an</strong>d <strong>the</strong> posterior<br />
cingulate cortex showed greater activation to <strong>an</strong>imate agents regardless of <strong>the</strong>ir <strong>for</strong>m. That is,<br />
<strong>the</strong>se regions activated more strongly to hum<strong>an</strong>s <strong>an</strong>d dogs th<strong>an</strong> to dolls <strong>an</strong>d stuffed <strong>an</strong>imals.<br />
These results suggest that <strong>the</strong> social network is divided between areas that respect <strong>the</strong> concept of<br />
<strong>an</strong>imacy <strong>an</strong>d areas that recognize <strong>an</strong>imate <strong>for</strong>m regardless of <strong>an</strong>imacy proper.<br />
Disclosures: C.E. Looser, None; T. Wheatley, None.
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.8/FF127<br />
Topic: F.01.l. Social cognition<br />
Title: <strong>When</strong> music becomes l<strong>an</strong>guage. Left-lateralized frontal brain activity during call-response<br />
improvisation<br />
Authors: *M. D. FRIIS-OLIVARIUS 1,2 , M. WALLENTIN 3,4 , A. ROEPSTORFF 3,5 , P.<br />
VUUST 2,6 ;<br />
1 Copenhagen Business Sch. / Dept. of Marketing, Copenhagen, Denmark; 2 Ctr. of Functionally<br />
Integrated neuroscience, Aarhus University Hospital, Denmark; 3 Aarhus Univ. Hosp., Ctr. of<br />
Functionally Integrated neuroscience, Aarhus, Denmark; 4 Univ. of Aarhus, Ctr. <strong>for</strong> Semiotics,<br />
Aarhus, Denmark; 5 Dept. of Social Anthrop., University of Aarhus, Denmark; 6 Royal Acad. of<br />
Music, Aarhus, Denmark<br />
Abstract: Nearly all neuroimaging studies of creative behavior investigate verbal associations<br />
between sem<strong>an</strong>tic knowledge. These studies find <strong>an</strong> involvement of <strong>the</strong> left dorsolateral<br />
prefrontal cortex (DLPFC). In contrast, two recent studies of improvisation in classically trained<br />
pi<strong>an</strong>ists found <strong>the</strong> right DLPFC to be linked with this non-verbal creative behavior. Although<br />
m<strong>an</strong>y studies of basic music processing show a greater right hemispheric involvement, especially<br />
in relation to <strong>the</strong> processing of pitch, brain responses to auditory stimuli are not only determined<br />
by physical properties of <strong>the</strong> stimuli or <strong>the</strong> nature of cognitive operations involved. In m<strong>an</strong>y<br />
cases, <strong>the</strong> listeners’ competence <strong>an</strong>d familiarity with <strong>the</strong> stimuli affect neuronal processing. For<br />
example, Kujala et al. (2003) showed that after 3 months of intensive training in Morse code (<strong>an</strong><br />
acoustic message tr<strong>an</strong>smitted by <strong>the</strong> use of tone patterns), neuronal processing reversed<br />
lateralization <strong>from</strong> <strong>the</strong> right hemisphere to <strong>the</strong> left hemisphere, suggesting that once a rhythmic<br />
pattern conveys me<strong>an</strong>ing, it is processed by <strong>the</strong> left hemisphere. An interesting question is<br />
<strong>the</strong>re<strong>for</strong>e whe<strong>the</strong>r <strong>the</strong> brain regions involved in improvisational music would be more left<br />
lateralized if <strong>the</strong> improvisations were used as communication?<br />
Jazz <strong>an</strong>d rock musici<strong>an</strong>s often communicate on stage through <strong>the</strong>ir playing <strong>an</strong>d this is in<br />
particular facilitated through improvisation. We used this non-verbal ability in a functional<br />
magnetic reson<strong>an</strong>ce imaging (fMRI) experiment <strong>an</strong>d created, what in musical terms is known as<br />
a call-response situation. Twenty-three professional musici<strong>an</strong>s were asked to improvise a<br />
rhythmic <strong>an</strong>swer to a given rhythmic phrase <strong>an</strong>d as control to imitate that given phrase. Our task<br />
was aimed at mimicking <strong>an</strong> ecological communicative situation between musici<strong>an</strong>s, <strong>an</strong>d we<br />
<strong>the</strong>re<strong>for</strong>e hypo<strong>the</strong>sized that although studying a non-verbal creative behavior, brain activations<br />
would be left lateralized in this mimicry task.<br />
We found that only <strong>the</strong> superior frontal gyrus in <strong>the</strong> left DLPFC (BA 8) was activated during
esponse creation compared to control. The left DLPFC is usually tied to l<strong>an</strong>guage processing<br />
tasks, but has not yet been linked to a non-verbal divergent thinking task. In particular, <strong>the</strong> left<br />
BA 8 is known to be involved in speech production, speech improvisation <strong>an</strong>d implemented in<br />
sem<strong>an</strong>tic memory retrieval. Thus, this indicates that musical communication resembles that of<br />
linguistic behavior ra<strong>the</strong>r th<strong>an</strong> strictly non-linguistic behavior.<br />
Disclosures: M.D. Friis-Olivarius, None; M. Wallentin, None; A. Roepstorff, None; P.<br />
Vuust, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.9/FF128<br />
Topic: F.01.l. Social cognition<br />
Support: Europe<strong>an</strong> Union Joint-Action Science <strong>an</strong>d Technology (IST-FP6-003747)<br />
Dutch Org<strong>an</strong>isation <strong>for</strong> Scientific Research (NWO Rubicon 446-08-008)<br />
Niels Stensen Foundation<br />
Title: Dissociating linguistic <strong>an</strong>d communicative abilities in <strong>the</strong> hum<strong>an</strong> brain<br />
Authors: *R. WILLEMS 1,2 , M. DE BOER 2 , J.-P. DE RUITER 3,4 , M. NOORDZIJ 2,5 , P.<br />
HAGOORT 2,3 , I. TONI 2 ;<br />
1 Helen Wills Neurosci. Inst., Berkeley, CA; 2 Donders Inst. <strong>for</strong> Brain, Cognition <strong>an</strong>d Behaviour,<br />
Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 Max Pl<strong>an</strong>ck Inst. <strong>for</strong> Psycholinguistics, Nijmegen, Ne<strong>the</strong>rl<strong>an</strong>ds;<br />
4 Bielefeld Univ., Bielefeld, Germ<strong>an</strong>y; 5 Univ. of Twente, Enschede, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Although l<strong>an</strong>guage is a very effective vehicle <strong>for</strong> communication, it is unclear how<br />
linguistic <strong>an</strong>d communicative abilities relate to each o<strong>the</strong>r. Some have argued that<br />
communicative message generation involves perspective taking (‘mentalizing’), <strong>an</strong>d - crucially -<br />
that mentalizing depends on l<strong>an</strong>guage. We employed a verbal communication paradigm to<br />
directly test A) whe<strong>the</strong>r <strong>the</strong> generation of a communicative action relies on mentalizing <strong>an</strong>d B)<br />
whe<strong>the</strong>r <strong>the</strong> cerebral bases of communicative message generation are distinct <strong>from</strong> those<br />
sensitive to linguistic variables.<br />
We measured cerebral activity (using fMRI) in healthy particip<strong>an</strong>ts while <strong>the</strong>y engaged in a<br />
verbal communication task derived <strong>from</strong> <strong>the</strong> 'Taboo-game'. Particip<strong>an</strong>ts had to describe a target-
word (e.g. 'beard') without using certain pre-specified taboo-words (e.g. 'hair', 'moustache', 'face',<br />
'chin'). Communicative intent was m<strong>an</strong>ipulated by having particip<strong>an</strong>ts make descriptions <strong>for</strong> a<br />
specific o<strong>the</strong>r person ('Targeted') or not ('Non-Targeted'). Linguistic difficulty was m<strong>an</strong>ipulated<br />
by ch<strong>an</strong>ging <strong>the</strong> sem<strong>an</strong>tic dist<strong>an</strong>ce between Target <strong>an</strong>d Taboo-words, making it easier or harder<br />
to come up with a description.<br />
Behaviorally, <strong>the</strong>re was no difference between Targeted <strong>an</strong>d Non-targeted trials. However, a<br />
confined region of dorso-medial prefrontal cortex, a brain area associated with mentalizing<br />
abilities, was sensitive to <strong>the</strong> communicative intent of utter<strong>an</strong>ces, irrespective of linguistic<br />
difficulty (Fig. 1A). On <strong>the</strong> contrary, left inferior frontal cortex, <strong>an</strong> area known to be involved in<br />
l<strong>an</strong>guage, was sensitive to <strong>the</strong> linguistic dem<strong>an</strong>ds of utter<strong>an</strong>ces, but indifferent to communicative<br />
intent (Fig. 1B). Our results argue against a proposal in which mentalizing abilities are ultimately<br />
dependent upon our l<strong>an</strong>guage abilities. Ra<strong>the</strong>r, <strong>the</strong>se findings show that communicative <strong>an</strong>d<br />
linguistic abilities, despite being closely related during day-to-day verbal communication, rely on<br />
cerebrally (<strong>an</strong>d computationally) distinct mech<strong>an</strong>isms.<br />
Disclosures: R. Willems, None; M. de Boer, None; J. de Ruiter, None; M. Noordzij,<br />
None; P. Hagoort, None; I. Toni, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.10/FF129<br />
Topic: F.01.l. Social cognition<br />
Support: A gr<strong>an</strong>t <strong>from</strong> <strong>the</strong> Tamagawa University Center of Excellence <strong>from</strong> <strong>the</strong> MEXT<br />
A gr<strong>an</strong>t <strong>from</strong> CREST (No. 17022035)
A Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research on Priority Areas—System Study on Higherorder<br />
Brain Functions <strong>from</strong> <strong>the</strong> MEXT (No. 20020026)<br />
A Showa University Gr<strong>an</strong>t-in-Aid <strong>for</strong> Innovative Collaborative Research Projects <strong>an</strong>d<br />
a Special Research Gr<strong>an</strong>t-in-Aid <strong>for</strong> Development of Characteristic Education <strong>from</strong> MEXT<br />
Title: Facial emotion recognition <strong>an</strong>d limbic lesions in myotonic dystrophy type 1<br />
Authors: *M. KOBAYAKAWA 1,2 , N. TSURUYA 1,2 , A. TAKEDA 1 , A. SUZUKI 3 , M.<br />
KAWAMURA 1 ;<br />
1 2 3<br />
Showa Univ. Sch. of Med., Tokyo, Jap<strong>an</strong>; Tamagawa Univ., Tokyo, Jap<strong>an</strong>; Nagoya Univ.,<br />
Nagoya, Jap<strong>an</strong><br />
Abstract: In order to investigate <strong>the</strong> cognitive <strong>an</strong>d neurological bases of social cognitive<br />
impairment in myotonic dystrophy type 1 (DM1) patients, we examined <strong>the</strong> facial expression<br />
recognition abilities of <strong>the</strong>se patients <strong>an</strong>d <strong>an</strong>alysed <strong>the</strong> correlation between sensitivity to<br />
emotions <strong>an</strong>d cerebral white matter lesions.<br />
We investigated <strong>the</strong> facial expression recognition ability of 9 DM1 patients (5 men, 4 women).<br />
We determined <strong>the</strong> sensitivity to facial emotions <strong>an</strong>d compared <strong>the</strong> findings with magnetic<br />
reson<strong>an</strong>ce imaging (MRI) findings that were evaluated using a semi-qu<strong>an</strong>titative method.<br />
The DM1 patients showed lower sensitivity to disgusted <strong>an</strong>d <strong>an</strong>gry faces as compared to <strong>the</strong><br />
healthy controls. (1)The assessment of brain lesions revealed that more severe lesions occurred<br />
in subcortices of <strong>the</strong> frontal, temporal <strong>an</strong>d insular areas. (2)The sensitivity to <strong>the</strong> emotion of<br />
disgust was correlated with temporal lesions, <strong>an</strong>d sensitivity to <strong>an</strong>ger was correlated with frontal,<br />
temporal, <strong>an</strong>d insular lesions.<br />
The results of this study indicated <strong>an</strong> association between lesions in <strong>the</strong> frontal, temporal, <strong>an</strong>d<br />
insular subcortices <strong>an</strong>d decreased emotional sensitivity to disgust <strong>an</strong>d <strong>an</strong>ger in DM1 patients.<br />
These areas are considered to play <strong>an</strong> import<strong>an</strong>t role in emotional processing in <strong>the</strong> normal brain.<br />
Our results suggest that social cognitive impairment in DM1 patients is attributed to impaired<br />
emotional processing due to white matter lesions in <strong>the</strong> brain.<br />
Disclosures: M. Kobayakawa, None; N. Tsuruya, None; A. Takeda, None; A. Suzuki,<br />
None; M. Kawamura, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.11/FF130
Topic: F.01.l. Social cognition<br />
Support: R01 DA022549<br />
NSERC-CRSNG PGS D<br />
Title: Ventromedial prefrontal cortex damage <strong>an</strong>d <strong>the</strong> fundamental attribution error<br />
Authors: *T. R. KOSCIK, D. TRANEL;<br />
Neurol., Univ. of Iowa, Iowa City, IA<br />
Abstract: The ventromedial prefrontal cortex (VMPC) is known to be involved in social<br />
processing, <strong>an</strong>d damage to <strong>the</strong> VMPC frequently results in major deficits in social conduct.<br />
Despite <strong>the</strong> large <strong>an</strong>d growing body of literature examining <strong>the</strong> role of <strong>the</strong> VMPC in social<br />
processing, however, it is still not clear what social-cognitive processes require <strong>an</strong> intact VMPC.<br />
In nonhum<strong>an</strong> mammals, brain regions that are grossly homologous to <strong>the</strong> hum<strong>an</strong> VMPC are<br />
necessary <strong>for</strong> processing chemosensory in<strong>for</strong>mation <strong>from</strong> conspecifics, providing in<strong>for</strong>mation<br />
about social <strong>an</strong>d reproductive value of o<strong>the</strong>rs. We speculate that <strong>the</strong> functional purview of <strong>the</strong><br />
hum<strong>an</strong> VMPC reflects this evolutionary heritage of conspecific chemical communication.<br />
Namely, <strong>the</strong> VMPC is necessary to appropriately assign biologically relev<strong>an</strong>t values to o<strong>the</strong>r<br />
hum<strong>an</strong>s, or in social psychology terms, to make social attributions.<br />
Normal, healthy persons tend to overestimate dispositional (or internal) causes of <strong>an</strong>o<strong>the</strong>r’s<br />
behavior, even when situational in<strong>for</strong>mation is <strong>the</strong> obvious cause, known as <strong>the</strong> Fundamental<br />
Attribution Error (FAE, also referred to as <strong>the</strong> “correspondence bias”). We predicted that if <strong>the</strong><br />
VMPC is required <strong>for</strong> making social attributions, damage to <strong>the</strong> VMPC would result in a lack of<br />
this normal bias, i.e., a decreased FAE. To test this prediction, VMPC, brain-damaged<br />
comparisons, <strong>an</strong>d normal healthy comparisons completed a version of <strong>the</strong> classic, Ross, et al.<br />
(1978), Quiz Game experiment. Particip<strong>an</strong>ts observe a Questioner asking a Contest<strong>an</strong>t (both<br />
confederates) 10 challenging questions that <strong>the</strong> Questioner created based on his/her own esoteric<br />
knowledge (thus putting <strong>the</strong> Questioner in a biased position of appearing more knowledgeable).<br />
The particip<strong>an</strong>ts rate <strong>the</strong> general knowledge of both <strong>the</strong> Questioner <strong>an</strong>d Contest<strong>an</strong>t after<br />
observing <strong>the</strong> question <strong>an</strong>d <strong>an</strong>swer session. The comparison particip<strong>an</strong>ts reliably committed <strong>the</strong><br />
FAE; <strong>the</strong>y rated <strong>the</strong> Questioner as more knowledgeable th<strong>an</strong> <strong>the</strong> Contest<strong>an</strong>t. In contrast,<br />
particip<strong>an</strong>ts with VMPC damage did not reliably make <strong>the</strong> FAE, <strong>an</strong>d were no more likely to rate<br />
<strong>the</strong> Questioner as more knowledgeable th<strong>an</strong> <strong>the</strong> Contest<strong>an</strong>t th<strong>an</strong> vice versa.<br />
So far, this evidence suggests that <strong>the</strong> ventromedial prefrontal cortex is necessary <strong>for</strong> making<br />
appropriate social attributions. An inability to do so may cause <strong>the</strong> social dysfunction that is<br />
often observed following VMPC damage.<br />
Disclosures: T.R. Koscik, None; D. Tr<strong>an</strong>el, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.12/FF131<br />
Topic: F.01.l. Social cognition<br />
Support: The Met<strong>an</strong>exus Institute<br />
John Templeton Foundation<br />
Title: Socially gifted? Resting RSA predicts greater engagement of social cognitive areas in<br />
BOLD response to emotionally heterogeneous slides<br />
Authors: *E. CASTLE 1 , E. SIMON-THOMAS 1 , J. GRUBER 2 , A. KOGAN 3 , D. KELTNER 1 ;<br />
1 Psychology, Univ. of Cali<strong>for</strong>nia Berkeley, Berkeley, CA; 2 Psychology, Yale Univ., New Haven,<br />
CT; 3 Psychology, Univ. of Hong Kong, Hong Kong, Hong Kong<br />
Abstract: The myelinated br<strong>an</strong>ch of <strong>the</strong> vagus nerve slows heart rate during expiration in a<br />
process <strong>the</strong>orized to foster calm behavioral states <strong>an</strong>d spur social engagement (Porges, 2007).<br />
One measure of this effect is resting respiratory sinus arrhythmia (rRSA). Studies have shown<br />
associations between rRSA <strong>an</strong>d increased emotion regulation <strong>an</strong>d decreased negative response to<br />
stressors (Fabes & Eisenberg, 1997), heightened experience of pro-social states like compassion<br />
(Oveis et al., <strong>2009</strong>), <strong>an</strong>d neural activation related to reminiscence ra<strong>the</strong>r th<strong>an</strong> personal pain<br />
during grief responses to pictures of deceased loved-ones (O'Connor, Gundel, McRae, & L<strong>an</strong>e,<br />
2007). This study investigated <strong>the</strong> relationship between rRSA <strong>an</strong>d neural correlates of emotional<br />
<strong>an</strong>d social cognitive processes. To achieve this aim, subjects (n=10; 6 female) viewed blocks of<br />
slides designed to elicit several social <strong>an</strong>d nonsocial states (pleasure, compassion, pride, <strong>an</strong>d<br />
neutral) during fMRI sc<strong>an</strong>ning, <strong>an</strong>d provided heart rate <strong>an</strong>d respiration measures during a<br />
separate, non-fMRI resting condition. Regression <strong>an</strong>alyses between individual rRSA values <strong>an</strong>d<br />
neural responses to slides were per<strong>for</strong>med. With respect to <strong>the</strong> pleasure slides, rRSA predicted<br />
bilateral TPJ which has been implicated in <strong>the</strong>ory of mind, mirroring <strong>an</strong>d perspective taking<br />
(Saxe & Powell, 2006), as well as nucleus accumbens, a classic reward response that indicates<br />
hedonic pleasure (Knutson & Cooper, 2005). In <strong>the</strong> compassion condition, rRSA predicted<br />
activation both in <strong>the</strong> mPFC, <strong>an</strong> area involved in inferring one’s own, <strong>an</strong>d o<strong>the</strong>rs’ mental states<br />
(Gusnard, Akbudak, Shulm<strong>an</strong>, & Raichle, 2001; Mitchell, B<strong>an</strong>aji, & Macrae, 2005), <strong>an</strong>d in <strong>the</strong><br />
superior parietal cortex, which has been implicated in evaluating social dist<strong>an</strong>ce (Yamakawa,<br />
K<strong>an</strong>ai, Matsumura, & Naito, <strong>2009</strong>). In response to both <strong>the</strong> pride <strong>an</strong>d neutral slides, rRSA<br />
predicted activation in <strong>the</strong> left temporal parietal junction (TPJ), <strong>an</strong>d <strong>the</strong> medial prefrontal cortex<br />
(mPFC). These patterns of activation suggest that greater tonic influence of <strong>the</strong> myelinated vagus<br />
on heart rate, as indexed by rRSA, relates to more socially attuned responding; individuals with<br />
higher rRSA more readily engage social cognitive brain areas in response to both affective <strong>an</strong>d<br />
neutral stimuli.
Disclosures: E. Castle, None; E. Simon-Thomas, None; J. Gruber, None; A. Kog<strong>an</strong>,<br />
None; D. Keltner, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.13/FF132<br />
Topic: F.01.l. Social cognition<br />
Support: ESRC (to SPT <strong>an</strong>d PED)<br />
Wellcome Trust (to SPT)<br />
Title: Multivoxel pattern <strong>an</strong>alysis reveals mirror-like responses in hum<strong>an</strong> parietal <strong>an</strong>d<br />
occipitotemporal cortex<br />
Authors: *N. OOSTERHOF, A. J. WIGGETT, J. DIEDRICHSEN, S. P. TIPPER, P. E.<br />
DOWNING;<br />
B<strong>an</strong>gor Univ., B<strong>an</strong>gor, United Kingdom<br />
Abstract: There is a strong body of behavioural evidence showing that observing o<strong>the</strong>rs’ actions<br />
c<strong>an</strong> modulate one’s own actions, a finding that is generally consistent with <strong>the</strong> notion of a hum<strong>an</strong><br />
“mirror system”. This suggests a common neural code <strong>for</strong> action representation across <strong>the</strong> visual<br />
<strong>an</strong>d motor domains. Single-cell studies in <strong>the</strong> macaque have revealed <strong>the</strong> existence of “mirror<br />
neurons” - neurons that are selective to <strong>the</strong> observation <strong>an</strong>d execution of specific actions. In<br />
hum<strong>an</strong>s, however, <strong>the</strong>re is as yet little direct evidence <strong>for</strong> a mirror-like system where <strong>the</strong> visual<br />
<strong>an</strong>d motor aspects of specific actions are represented in a common neural code.<br />
In <strong>the</strong> present study, we provide compelling evidence <strong>for</strong> such a code in <strong>the</strong> hum<strong>an</strong> brain.<br />
Particip<strong>an</strong>ts per<strong>for</strong>med <strong>an</strong>d observed three me<strong>an</strong>ingless, intr<strong>an</strong>sitive h<strong>an</strong>d actions while <strong>the</strong>y<br />
were sc<strong>an</strong>ned using fMRI. We combined a whole-brain “searchlight” algorithm with a multivoxel<br />
pattern (MVPA) <strong>an</strong>alysis classifier. This approach allows us to identify brain regions that<br />
meet criteria that are essential to <strong>the</strong> core of <strong>the</strong> “mirror system” idea: 1) <strong>the</strong> pattern of neural<br />
responses to a given action should be similar, whe<strong>the</strong>r it is viewed or per<strong>for</strong>med, but 2) <strong>the</strong><br />
pattern of neural responses evoked by different actions should be different.<br />
We report such mirror-like regions in <strong>an</strong>d around <strong>the</strong> intraparietal sulcus (previously implicated<br />
in visuo-motor functions) <strong>an</strong>d, to a lesser extent, in <strong>the</strong> lateral occipitotemporal cortex (where<br />
visual representations of objects, bodies, <strong>an</strong>d motion overlap); see <strong>the</strong> Figure <strong>for</strong> MVPA<br />
accuracies <strong>an</strong>d corresponding p-values. We did not find mirror-like representations in o<strong>the</strong>r
egions previously implicated as <strong>the</strong> hum<strong>an</strong> “mirror system” (premotor cortex, inferior parietal<br />
lobule), which may be due to <strong>the</strong> me<strong>an</strong>ingless <strong>an</strong>d intr<strong>an</strong>sitive aspects of <strong>the</strong> h<strong>an</strong>d actions used in<br />
<strong>the</strong> current study.<br />
The present findings may help account <strong>for</strong> behavioural action-priming effects, while <strong>from</strong> a<br />
methodological perspective <strong>the</strong>y show <strong>the</strong> feasibility of applying a searchlight-MVPA approach<br />
to studying <strong>the</strong> visual-motor coding of actions in <strong>the</strong> hum<strong>an</strong> brain.<br />
Disclosures: N. Oosterhof, None; A.J. Wiggett, None; J. Diedrichsen, None; S.P. Tipper,<br />
None; P.E. Downing, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.14/FF133<br />
Topic: F.01.l. Social cognition<br />
Support: ESRC Gr<strong>an</strong>t RES-000-22-2262<br />
Title: Implicit learning in a social decision-making task<br />
Authors: *E. A. HEEREY, H. VELANI;<br />
B<strong>an</strong>gor University, Sch. of Psychology, B<strong>an</strong>gor, United Kingdom<br />
Abstract: Hum<strong>an</strong>s are capable of complex, highly coordinated social behavior. One way people<br />
may accomplish such behavior is by making predictions about o<strong>the</strong>rs based on behavioral cues.<br />
Here, we asked whe<strong>the</strong>r people could learn to predict a partner’s behavior using a social cue <strong>an</strong>d<br />
whe<strong>the</strong>r this learning is implicit.<br />
We created a computerized game of rock-paper-scissors in which particip<strong>an</strong>ts viewed <strong>an</strong><br />
“opponent” via a “video link.” The opponent was actually <strong>an</strong> avatar, constructed of pre-recorded<br />
video sequences of <strong>an</strong>o<strong>the</strong>r person that included expressions such as smiles <strong>an</strong>d frowns, as well<br />
as <strong>the</strong> non-me<strong>an</strong>ingful movements that are present in normal behavior (e.g., blinks, postural
adjustments, small head movements). A hidden Markov chain controlled <strong>the</strong> avatar’s behavior,<br />
playing <strong>the</strong> video clips in a continuous m<strong>an</strong>ner such that <strong>the</strong> avatar’s behavior resembled that of<br />
a person. In 3 phases of each trial, particip<strong>an</strong>ts (N=35) decided which move to play (e.g., rock),<br />
made <strong>the</strong> play via a key press, <strong>an</strong>d viewed <strong>the</strong> trial outcome. Particip<strong>an</strong>ts won money when <strong>the</strong>y<br />
beat or tied <strong>the</strong> opponent <strong>an</strong>d lost money when <strong>the</strong>y lost <strong>the</strong> round. Be<strong>for</strong>e beginning <strong>the</strong> task, <strong>the</strong><br />
computer r<strong>an</strong>domly chose 1 of 2 nonverbal cues (brow-raise or lateral mouth movement) to be<br />
predictive of one of <strong>the</strong> avatar’s moves. The cue was reliable <strong>an</strong>d always occurred during <strong>the</strong><br />
decision-making phase of <strong>the</strong> trial (e.g., <strong>the</strong> avatar might make a brow-raise be<strong>for</strong>e playing<br />
‘rock’). After <strong>the</strong> first 10 trials of <strong>the</strong> game (in which <strong>the</strong>re were no predictive trials), particip<strong>an</strong>ts<br />
played three, 75-trial blocks with 25 predictive trials each.<br />
As expected, particip<strong>an</strong>ts won signific<strong>an</strong>tly more often on predictive, relative to non-predictive<br />
trials (F(2,34)=12.35, p=0.001). <strong>When</strong> asked to identify <strong>the</strong> predictive cue <strong>from</strong> a set of 3 cues,<br />
45% of particip<strong>an</strong>ts chose <strong>the</strong> correct cue. However, particip<strong>an</strong>ts who correctly identified <strong>the</strong> cue<br />
<strong>an</strong>d those who did not both showed above ch<strong>an</strong>ce win-rates (p-values
NINDS gr<strong>an</strong>t NS048229-04<br />
ONR Contract N000140510117<br />
Title: Exploring <strong>the</strong> innards of hum<strong>an</strong> social coordination via brain coordination dynamics<br />
Authors: *J. KELSO 1 , E. TOGNOLI 2 , D. BENITES 2 , G. C. DE GUZMAN 2 ;<br />
2 Ctr. <strong>for</strong> Complex Systems <strong>an</strong>d Brain Sci., 1 Florida Atl<strong>an</strong>tic Univ., Boca Raton, FL<br />
Abstract: To underst<strong>an</strong>d <strong>the</strong> brain mech<strong>an</strong>isms unfolding during behavioral coordination<br />
between people, we focused on <strong>the</strong> tr<strong>an</strong>sition between coordinated <strong>an</strong>d uncoordinated social<br />
behavior. Pairs of subjects per<strong>for</strong>med rhythmic right index finger movements in full view of one<br />
<strong>an</strong>o<strong>the</strong>r under instructions to establish one of three collective behaviors: inphase (both fingers<br />
flex/extend in synchrony), <strong>an</strong>tiphase (one subject extends while <strong>the</strong> o<strong>the</strong>r flexes) <strong>an</strong>d intrinsic<br />
(both subject maintain <strong>the</strong>ir own movement <strong>an</strong>d do not intend to synchronize). Behavioral<br />
tr<strong>an</strong>sitions were identified <strong>an</strong>d classified according to collective behavior (tr<strong>an</strong>sition to<br />
synchronized or unsynchronized behavior), agency (i.e., who effects <strong>the</strong> tr<strong>an</strong>sition), task goal <strong>an</strong>d<br />
behavioral strategy. We applied a 4D colorimetric mapping to both subjects’ EEG to identify<br />
brain spatiotemporal patterns associated with behavioral tr<strong>an</strong>sitions. Each tr<strong>an</strong>sient network of<br />
brain activity creates a spatiotemporal pattern of EEG that recurs under renewed functional<br />
dem<strong>an</strong>d. In each family of spatiotemporal patterns, we studied <strong>the</strong> distribution of dependent <strong>an</strong>d<br />
independent behavioral variables <strong>an</strong>d identified combinations of factors that were signific<strong>an</strong>tly<br />
more frequent th<strong>an</strong> ch<strong>an</strong>ce. We also examined <strong>the</strong> interactions between activities in both brains<br />
by <strong>an</strong>alyzing <strong>the</strong> tendency <strong>for</strong> coincident switching between oscillatory patterns. Our results are<br />
likely to shed new light on how <strong>the</strong> temporal org<strong>an</strong>ization within <strong>an</strong>d between brains is<br />
connected to functional processes of social behavior.<br />
Disclosures: J. Kelso, NIH, NSF, ONR, B. Research Gr<strong>an</strong>t (principal investigator, collaborator<br />
or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); E. Tognoli, NIH, NSF,<br />
ONR, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as<br />
well as gr<strong>an</strong>ts already received); D. Benites, None; G.C. de Guzm<strong>an</strong>, NIH, NSF, B. Research<br />
Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts<br />
already received).<br />
Poster
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.16/FF135<br />
Topic: F.01.l. Social cognition<br />
Support: ESRC<br />
Title: Observing touch: fMRI explorations of body perspective <strong>an</strong>d ownership<br />
Authors: *J. WARD, H. HOLLE;<br />
Univ. of Sussex, Brighton, United Kingdom<br />
Abstract: <strong>When</strong> you see a body part, how c<strong>an</strong> you tell whe<strong>the</strong>r it belongs to you or someone<br />
else? There are likely to be multiple sources of relev<strong>an</strong>t in<strong>for</strong>mation including: The perspective<br />
of <strong>the</strong> body part (ego- v. allo-centric), <strong>the</strong> shape <strong>an</strong>d appear<strong>an</strong>ce of <strong>the</strong> body part (varying <strong>from</strong><br />
individual to individual), <strong>an</strong>d how <strong>the</strong> seen body part links with motor <strong>an</strong>d bodily senses. The<br />
present study examines how <strong>the</strong>se different sources of in<strong>for</strong>mation are combined in <strong>the</strong> brain<br />
using fMRI. Healthy particip<strong>an</strong>ts were sc<strong>an</strong>ned while observing movies of h<strong>an</strong>ds being touched<br />
(or not touched as a control condition). The h<strong>an</strong>ds were ei<strong>the</strong>r those of <strong>the</strong> particip<strong>an</strong>t (recorded<br />
in a previous session) or those of <strong>an</strong>o<strong>the</strong>r person. The perspective was varied orthogonally<br />
between self-perspective (facing away) <strong>an</strong>d o<strong>the</strong>r perspective (facing towards). A region in <strong>the</strong><br />
left intra-parietal sulcus may be responsible <strong>for</strong> discriminating between self <strong>an</strong>d o<strong>the</strong>r using<br />
multiple cues. The region responded more to observing touch th<strong>an</strong> no-touch; more to selfperspective<br />
th<strong>an</strong> o<strong>the</strong>r-perspective; <strong>an</strong>d more to observing ones own h<strong>an</strong>d th<strong>an</strong> <strong>an</strong>o<strong>the</strong>r h<strong>an</strong>d.<br />
Previous studies have emphasized <strong>the</strong> import<strong>an</strong>ce of visual regions (e.g. <strong>the</strong> ‘extra-striate body<br />
area’) in distinguishing perspective <strong>an</strong>d self/o<strong>the</strong>r. However, o<strong>the</strong>r higher cortical regions may<br />
have equal import<strong>an</strong>ce. In a separate condition, particip<strong>an</strong>ts’ own h<strong>an</strong>ds were touched (without<br />
seeing <strong>an</strong>y stimuli) to enable localisation of somatosensory regions on <strong>an</strong> individual basis. This<br />
also enabled us to examine <strong>the</strong> mirror system <strong>for</strong> touch (i.e. common neural substrates <strong>for</strong><br />
observing <strong>an</strong>d feeling touch). Areas that were common to observed touch <strong>an</strong>d actual touch<br />
included <strong>the</strong> primary <strong>an</strong>d secondary somatosensory regions, but not <strong>the</strong> IPS region identified in<br />
<strong>the</strong> previous conjunction. This suggests that <strong>the</strong> IPS may be a convergence zone that provides<br />
top-down modulation of <strong>the</strong> somatosensory system in response to observations of touch <strong>an</strong>d<br />
body parts.<br />
Disclosures: J. Ward, None; H. Holle, None.<br />
Poster
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.17/FF136<br />
Topic: F.01.l. Social cognition<br />
Title: Inferior lateral parietal <strong>an</strong>d prefrontal activity is elicited when making judgments about <strong>the</strong><br />
virtual self<br />
Authors: *K. L. CAUDLE, K. E. DEMOS, T. F. HEATHERTON, W. M. KELLEY;<br />
Dartmouth Col., H<strong>an</strong>over, NH<br />
Abstract: Each day, millions of individuals immerse <strong>the</strong>mselves in online digital environments<br />
known as Massively-Multiplayer Online Games (MMOGs) that provide a pseudo-naturalistic<br />
setting <strong>for</strong> social interaction through <strong>the</strong> use of avatars (visual character representations of users<br />
in <strong>the</strong> virtual environment). Participation in <strong>the</strong>se environments requires extensive control of<br />
one's avatar in a variety of ways (including bodily movement, emotion <strong>an</strong>d dialogue) using a<br />
keyboard <strong>an</strong>d mouse. Here we used functional magnetic reson<strong>an</strong>ce imaging (fMRI) to examine<br />
neural activity during trait judgments in individuals with considerable experience in <strong>the</strong> virtual<br />
environment "World of Warcraft," a popular MMOG. During sc<strong>an</strong>ning, subjects viewed trait<br />
adjectives <strong>an</strong>d judged how well <strong>the</strong> adjective described one of <strong>the</strong> following: <strong>the</strong> self, a real-life<br />
friend, <strong>the</strong> virtual self, or <strong>an</strong> online friend. In this way, judgments could be made <strong>for</strong> both real<br />
world <strong>an</strong>d virtual representations of self <strong>an</strong>d o<strong>the</strong>r, resulting in a two-way factorial design that<br />
crossed domain (virtual versus real) <strong>an</strong>d target (self versus o<strong>the</strong>r). Results revealed a main effect<br />
of domain in bilateral regions of <strong>the</strong> inferior lateral parietal lobe (ilPL) that demonstrated greater<br />
activity during judgments of virtual characters th<strong>an</strong> real life individuals. Consistent with prior<br />
work on self-referencing, a main effect of target (self > o<strong>the</strong>r) was observed in <strong>the</strong> medial<br />
prefrontal cortex (MPFC) <strong>an</strong>d posterior cingulate (PC). This effect was present <strong>for</strong> both real <strong>an</strong>d<br />
virtual self versus o<strong>the</strong>r comparisons. Taken toge<strong>the</strong>r, <strong>the</strong>se results suggest that experienced<br />
MMOG particip<strong>an</strong>ts may have <strong>an</strong> extended representation of <strong>the</strong> self that includes mech<strong>an</strong>isms<br />
to both differentiate (e.g. ilPL) <strong>an</strong>d incorporate (e.g., MPFC <strong>an</strong>d PC) real <strong>an</strong>d virtual experiences.<br />
Disclosures: K.L. Caudle, None; K.E. Demos, None; T.F. Hea<strong>the</strong>rton, None; W.M. Kelley,<br />
None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.18/FF137<br />
Topic: F.01.l. Social cognition<br />
Support: Gr<strong>an</strong>t-in-Aid <strong>for</strong> Scientific Research (KAKENHI) on Innovative Areas by <strong>the</strong> MEXT<br />
(no.20119003),Jap<strong>an</strong><br />
Gr<strong>an</strong>d-in-Aid <strong>for</strong> Comprehensive Research on Disability Health <strong>an</strong>d Welfare by<br />
MHLW (no.H20-shougaiipp<strong>an</strong>-009),Jap<strong>an</strong><br />
Title: The event-related oscillation as <strong>an</strong> index of self face recognition<br />
Authors: *K. SAKIHARA, A. GUNJI, W. FURUSHIMA, M. INAGAKI;<br />
Dept. of Developmental Disorders, Natl. Ctr. of Neurol. <strong>an</strong>d Psychiatry, Kodaira, Tokyo, Jap<strong>an</strong><br />
Abstract: Objective<br />
Faces are outst<strong>an</strong>dingly rich sources of in<strong>for</strong>mation <strong>for</strong> social interaction, providing about person<br />
identity, familiarity, mood, gender, age <strong>an</strong>d even self awareness. Facial in<strong>for</strong>mation helps us to<br />
underst<strong>an</strong>d o<strong>the</strong>rs <strong>an</strong>d we are capable to behave suitably in communication. Recently, <strong>the</strong><br />
hemodynamic studies have demonstrated that <strong>the</strong> right hemisphere, especially prefrontal cortex,<br />
plays <strong>an</strong> import<strong>an</strong>t role <strong>for</strong> <strong>the</strong> self face recognition. However, <strong>the</strong> temporal ch<strong>an</strong>ge of neural<br />
network has not been clarified yet. We in <strong>the</strong> present study aimed to investigate <strong>the</strong> cerebral<br />
oscillations related to self face recognition.<br />
Methods<br />
Nine healthy adults participated in <strong>the</strong> experiment <strong>an</strong>d <strong>the</strong>y were instructed to see three facial<br />
images (self, familiar <strong>an</strong>d unfamiliar faces). In order to determine <strong>the</strong> neural correlations of self<br />
awareness, we compared <strong>the</strong> cerebral oscillations between <strong>the</strong> stimuli. EEG was recorded <strong>from</strong><br />
19 locations on <strong>the</strong> scalp with a sampling rate of 250Hz. The oscillatory power ch<strong>an</strong>ge was<br />
calculated by me<strong>an</strong>s of MBFA (multiple b<strong>an</strong>d frequency <strong>an</strong>alysis) software in 1-45Hz with<br />
squared amplitude. The <strong>an</strong>alyzed time was 1000 ms after stimulus onset, with 100 ms baseline<br />
collection. Analyzed time resolution was 100 ms in 1-3.9Hz <strong>an</strong>d 40 ms in 4-45Hz. The positive<br />
<strong>an</strong>d negative oscillation was defined as <strong>an</strong> ERS (event-related synchronization) <strong>an</strong>d <strong>an</strong> ERD<br />
(event-related desynchronization), respectively. The power ch<strong>an</strong>ges were statistically <strong>an</strong>alyzed<br />
between <strong>the</strong> stimuli.<br />
Results<br />
In <strong>the</strong> right prefrontal area (especially at F4), beta ERS was signific<strong>an</strong>tly greater in <strong>the</strong> self face<br />
compared to that in <strong>the</strong> familiar face within 400-800 ms after <strong>the</strong> stimulus presentation (p
compared to that in <strong>the</strong> familiar face. The region was consistent with cortical areas strongly<br />
related to self face recognition reported in <strong>the</strong> previous studies (Keen<strong>an</strong> et al., 2001; Uddin et al.,<br />
2005). Present results imply <strong>the</strong> specificity in <strong>the</strong> right hemisphere related to <strong>the</strong> self-awareness<br />
of adult face.<br />
Disclosures: K. Sakihara, None; A. Gunji, None; W. Furushima, None; M. Inagaki, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.19/FF138<br />
Topic: F.01.l. Social cognition<br />
Support: NSF BCS-0720312 to J.Y.C.<br />
NSF BCS-0722326 to J.Y.C.<br />
Title: Neural responses within medial prefrontal cortex modulated by self-construal style during<br />
first- <strong>an</strong>d third-person perspective takings: An fMRI study<br />
Authors: *T. HARADA 1 , J. Y. CHIAO 2 ;<br />
1 2<br />
Psychiatry/Psychobiology, Nagoya Univ., Nagoya/Aichi, Jap<strong>an</strong>; Psychology, Northwestern<br />
Univ., Ev<strong>an</strong>ston, IL<br />
Abstract: Self-construal style refers to cultural values that influence how people think about<br />
<strong>the</strong>mselves <strong>an</strong>d <strong>the</strong>ir relation to <strong>the</strong> social <strong>an</strong>d physical environment. Cultural psychologists have<br />
identified two main styles of self-construal: individualism <strong>an</strong>d collectivism. Individualists think<br />
of people as relatively independent of each o<strong>the</strong>r, whereas collectivists think of people as highly<br />
interconnected with one <strong>an</strong>o<strong>the</strong>r. Taking <strong>an</strong>o<strong>the</strong>r person's perspective is a fundamental<br />
component of hum<strong>an</strong> social life. First-person perspective taking is related to self-referential<br />
processing, while third-person perspective taking is related to mentalizing which is <strong>an</strong> ability to<br />
infer o<strong>the</strong>rs’ mental states. Numerous neuroimaging studies investigating self-referential<br />
processing, mentalizing <strong>an</strong>d perspective taking have implicated import<strong>an</strong>t role of medial<br />
prefrontal cortex (MPFC) in <strong>the</strong>se functions, <strong>an</strong>d some of <strong>the</strong>m have suggested differential<br />
functionality in ventral (VMPFC) <strong>an</strong>d dorsal (DMPFC) medial subregions within MPFC. To<br />
investigate how self-construal style modulates neural responses within medial prefrontal cortex<br />
during perspective taking, we conducted <strong>an</strong> fMRI experiment in which particip<strong>an</strong>ts took firstperson<br />
perspective targeting <strong>the</strong>mselves (1Ps) or <strong>the</strong>ir close friend (1Po) <strong>an</strong>d took a third-person
perspective targeting <strong>the</strong>ir close friend (3Po). The results indicate that VMPFC activity was<br />
positively correlated with individualistic score <strong>for</strong> <strong>the</strong> 1Ps <strong>an</strong>d collectivistic score <strong>for</strong> <strong>the</strong> 1Po<br />
conditions, while <strong>the</strong> activity within DMPFC was positively correlated with collectivistic score<br />
<strong>for</strong> <strong>the</strong> 3Po condition. The results demonstrate cultural variation of neural activity within<br />
subregions of MPFC (i.e. VMPFC <strong>an</strong>d DMPFC) as a function of first- or third-person<br />
perspective taking.<br />
Disclosures: T. Harada, None; J.Y. Chiao, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.20/FF139<br />
Topic: F.01.l. Social cognition<br />
Title: Difference in neural representation between <strong>the</strong> self <strong>an</strong>d <strong>the</strong> social self through facial<br />
makeup<br />
Authors: *A. ONZO 1 , K. SARUWATARI 2 , Y. TANAKA 2 , S. OKAZAKI 2 , A. SASAKI 3 , K.<br />
MOGI 4 ;<br />
1 none, Had<strong>an</strong>o, Jap<strong>an</strong>; 2 K<strong>an</strong>ebo Cosmetics Inc., K<strong>an</strong>agawa, Jap<strong>an</strong>; 3 Dentsu Inc., Tokyo, Jap<strong>an</strong>;<br />
4 Sony computer science laboratories, Inc, Tokyo, Jap<strong>an</strong><br />
Abstract: Facial perception is import<strong>an</strong>t in <strong>the</strong> social construction of <strong>the</strong> self. The perception of<br />
<strong>the</strong> self <strong>an</strong>d o<strong>the</strong>r persons are influenced largely by <strong>the</strong> appear<strong>an</strong>ces of faces (Cole 1998).<br />
Hum<strong>an</strong>s have applied facial makeup since early times in civilization, although <strong>the</strong> social <strong>an</strong>d<br />
psychological signific<strong>an</strong>ce of facial makeup might have ch<strong>an</strong>ged over time. Anecdotal evidences<br />
suggest that females apply facial makeup considering how <strong>the</strong>y would like to be seen by o<strong>the</strong>r<br />
persons. Facial makeup has <strong>an</strong> essential role in establishing <strong>the</strong> self in relation to o<strong>the</strong>rs,<br />
affecting <strong>the</strong> subject's behavior both consciously <strong>an</strong>d unconsciously. Here we investigate how<br />
female subjects perceive <strong>the</strong> face of <strong>the</strong> self <strong>an</strong>d o<strong>the</strong>rs with or without makeup using imaging by<br />
a 3.0 tesra fMRI. The particip<strong>an</strong>ts were healthy female subjects (ages 20-35) <strong>from</strong> <strong>the</strong> greater<br />
Tokyo area who had <strong>the</strong> custom of applying facial makeup on a daily basis. We previously<br />
reported in <strong>an</strong> fMRI study that a female subject tends to recognize her own face with makeup as<br />
if it was a face of o<strong>the</strong>r person (Onzo et al. 2008). Our result, however, was based on subjects<br />
sc<strong>an</strong>ned without facial makeup, a factor which could have possibly affected <strong>the</strong> result. The<br />
presence of magnetically active materials in commercially available makeup materials have<br />
made it difficult <strong>for</strong> <strong>the</strong> subject to be sc<strong>an</strong>ned in <strong>the</strong> fMRI machine with facial makeup. In order
to make it possible to sc<strong>an</strong> <strong>the</strong> brain of subjects with facial makeup, we have developed a set of<br />
cosmetic materials which c<strong>an</strong> be used in fMRI by excluding some magnetized materials. Using<br />
<strong>the</strong> fMRI-ready cosmetic materials, we have conducted fMRI studies of <strong>the</strong> brain activities in<br />
subjects with facial makeup. The subjects were instructed to use <strong>the</strong> fMRI-ready cosmetics<br />
materials <strong>for</strong> 10 days be<strong>for</strong>e <strong>the</strong> experiments. After this preparatory period, <strong>the</strong> subjects applied<br />
facial makeup to <strong>the</strong>mselves in <strong>the</strong> laboratory using <strong>the</strong> fMRI-ready cosmetic materials. A<br />
questionnaire confirmed that <strong>the</strong> subjects could reproduce in essence <strong>the</strong> customary facial makeup.<br />
We compared <strong>the</strong> neural activities in <strong>the</strong> brain of <strong>the</strong> subjects sc<strong>an</strong>ned by fMRI with <strong>an</strong>d<br />
without makeup. We discuss <strong>the</strong> implications of <strong>the</strong> result <strong>for</strong> <strong>the</strong> social constructions of <strong>the</strong> self,<br />
in <strong>the</strong> context of brain circuits involved, including <strong>the</strong> default network (Buckner et al. 2008).<br />
Disclosures: A. Onzo, None; K. Saruwatari, None; Y. T<strong>an</strong>aka, None; S. Okazaki, None; A.<br />
Sasaki, None; K. Mogi, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.21/FF140<br />
Topic: F.01.l. Social cognition<br />
Support: Howard Hughes Medical Institute<br />
NDSEG Fellowship<br />
Ash<strong>for</strong>d Fellowship<br />
Title: Cl<strong>an</strong> mentality: Medial prefrontal cortex <strong>an</strong>d <strong>the</strong> representation of self <strong>an</strong>d o<strong>the</strong>r<br />
Authors: *F. M. KRIENEN 1,2 , P. TU 1,2,3,4 , R. L. BUCKNER 1,2,3,5 ;<br />
1 2<br />
Harvard Univ., Cambridge, MA; Athinoula A. Martinos Ctr. <strong>for</strong> Biomed. Imaging,<br />
Charlestown, MA; 3 Psychiatry <strong>an</strong>d Radiology, Massachusetts Gen. Hosp., Charlestown, MA;<br />
4 5<br />
Div. of Neurosci., Natl. Y<strong>an</strong>g-Ming Univ., Taipei, Taiw<strong>an</strong>; Howard Hughes Med. Inst. at<br />
Harvard Univ., Cambridge, MA<br />
Abstract: A ubiquitous finding in <strong>the</strong> field of social neuroscience is that medial prefrontal cortex<br />
(MPFC) is active when engaging in self-referential processing (Frith & Frith, 2006). It has<br />
fur<strong>the</strong>r been proposed that <strong>the</strong> representation in MPFC of <strong>the</strong> mental states of o<strong>the</strong>rs is<br />
modulated by whe<strong>the</strong>r <strong>the</strong>y are perceived to be similar to <strong>the</strong> self (Mitchell et al, 2006).
However, <strong>an</strong>o<strong>the</strong>r dimension of clear social import is <strong>the</strong> closeness or familiarity of o<strong>the</strong>rs<br />
(Fiske, 2004). O<strong>the</strong>rs who are members of <strong>the</strong> family or friendship groups may be perceived as<br />
being like oneself <strong>an</strong>d processed in a self-like fashion, despite having a propensity to view <strong>the</strong><br />
world quite differently. Five fMRI experiments (n = 133) sought to characterize <strong>the</strong> contributions<br />
of similarity <strong>an</strong>d closeness to mental state representation. In Experiment 1, healthy young adults<br />
(n = 26) were sc<strong>an</strong>ned while making attribute judgments about <strong>the</strong> self <strong>an</strong>d a famous but<br />
personally unknown o<strong>the</strong>r. Activity during self judgments was detected in MPFC, including a<br />
peak in <strong>the</strong> rostral <strong>an</strong>terior cingulate cortex consistent with previous findings. Functional<br />
connectivity <strong>an</strong>alysis of resting state sc<strong>an</strong>s in <strong>an</strong> independent dataset (n = 40) characterized <strong>the</strong><br />
brain network to which this region belongs (Experiment 2). In Experiment 3, <strong>the</strong> two dimensions<br />
(similarity, closeness) were crossed in a 2x2 factorial design (n = 28) to examine <strong>the</strong> differential<br />
contributions of each to activity in this region. Results show that thinking about <strong>the</strong> self <strong>an</strong>d<br />
about close o<strong>the</strong>rs drives <strong>the</strong> activity in this region, which was fur<strong>the</strong>r insensitive to similarity<br />
m<strong>an</strong>ipulations. Experiment 4 was carried out in <strong>an</strong> independent set of particip<strong>an</strong>ts (n = 21) who<br />
per<strong>for</strong>med a version of <strong>the</strong> task including only similar <strong>an</strong>d dissimilar unknown o<strong>the</strong>rs. Results of<br />
this experiment replicate Experiment 3, showing that unfamiliar o<strong>the</strong>rs do not robustly activate<br />
rACC. In Experiment 5 particip<strong>an</strong>ts (n=19) made inferences about a similar, unknown o<strong>the</strong>r <strong>an</strong>d<br />
a dissimilar friend in a task designed to directly contrast <strong>the</strong> extremes of <strong>the</strong> conditions presented<br />
in Experiment 3. Once again <strong>the</strong> close o<strong>the</strong>r (friend) robustly activates this region, regardless of<br />
perceived dissimilarity to <strong>the</strong> self. It appears that closeness or familiarity, but not similarity, is a<br />
fundamental dimension that dissociates MPFC activity in this region of rACC.<br />
Disclosures: F.M. Krienen, None; P. Tu, None; R.L. Buckner, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.22/GG1<br />
Topic: F.01.l. Social cognition<br />
Title: Olfactory signalling in <strong>the</strong> process of <strong>the</strong> self/non-self distinction in hum<strong>an</strong>s<br />
Authors: *E. MARXER-TOBLER;<br />
UCSD, La Jolla, CA<br />
Abstract: Since self/non-self recognition processes are evolved mech<strong>an</strong>isms lying at <strong>the</strong> core of<br />
development, immune defense <strong>an</strong>d socio-cultural behavior, it is likely that specific neural<br />
mech<strong>an</strong>isms evolved to encode <strong>an</strong>d process <strong>the</strong>m. Such self/non-self recognition processes are
phylogenetically highly conserved neural mech<strong>an</strong>isms serving <strong>for</strong> survival in <strong>the</strong> competition of<br />
<strong>the</strong> evolutionary process. In hum<strong>an</strong>s, <strong>the</strong> social self/non-self recognition revolves around <strong>an</strong><br />
ethnic distinction whereas ethnicity is defined as <strong>an</strong> identity of a group of hum<strong>an</strong> beings whose<br />
members identify with each o<strong>the</strong>r <strong>an</strong>d share true common <strong>an</strong>cestry. There<strong>for</strong>e, ethnicity is a<br />
genetically shared identification clue in <strong>the</strong> neural recognition process. These genetic-inherited<br />
clues serve as in<strong>for</strong>mation perceived <strong>an</strong>d understood by neural mech<strong>an</strong>isms. One such neural<br />
mech<strong>an</strong>ism is <strong>the</strong> olfactory sensory system, well established in <strong>an</strong>imals, which uses chemical<br />
signals encoded in body fluids to convey social in<strong>for</strong>mation. In <strong>an</strong> EEG study, we presented<br />
body odor stimuli to members of four different ethnic groups to test <strong>the</strong> hypo<strong>the</strong>sis that hum<strong>an</strong>s<br />
c<strong>an</strong> detect, via olfaction, <strong>the</strong> diversity in our own species. The study showed that hum<strong>an</strong>s are<br />
able to distinguish <strong>the</strong> different ethnic groups based on odor recognition conveyed by body scent.<br />
The EEG discloses that <strong>the</strong> neural system processes <strong>the</strong> self-odor <strong>an</strong>d <strong>the</strong> in-group odors in<br />
similar oscillation patterns, which reveals <strong>the</strong> genetic relatedness of <strong>the</strong> within group members.<br />
In <strong>the</strong> behavior study, particip<strong>an</strong>ts recognized <strong>the</strong> in-group as familiar but not as pleas<strong>an</strong>t or<br />
unpleas<strong>an</strong>t. Next to visual <strong>an</strong>d auditory clues, hum<strong>an</strong>s also use olfactory clues to distinguish self<br />
<strong>from</strong> non-self o<strong>the</strong>rs in order to protect <strong>an</strong>d maintain <strong>the</strong>ir social world.<br />
Disclosures: E. Marxer-Tobler, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.23/GG2<br />
Topic: F.01.l. Social cognition<br />
Support: C<strong>an</strong>adi<strong>an</strong> Institutes of Health Research Gr<strong>an</strong>t<br />
Heart <strong>an</strong>d Stroke Foundation Centre <strong>for</strong> Stroke Recovery Gr<strong>an</strong>t<br />
Title: The effects of extensive damage to a “<strong>the</strong>ory of mind” network on “<strong>the</strong>ory of mind”<br />
per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: L. ZACKHEIM 1 , D. T. STUSS 2,3 , S. GILLINGHAM 2 , D. REWILAK 4 , K. J.<br />
MURPHY 4 , *R. ROSENBAUM 5,2,1 ;<br />
1 Psychology, York Univ., Toronto, ON, C<strong>an</strong>ada; 2 Baycrest, Rotm<strong>an</strong> Res. Inst., Toronto, ON,<br />
C<strong>an</strong>ada; 3 Psychology <strong>an</strong>d Med., Univ. of Toronto, Toronto, ON, C<strong>an</strong>ada; 4 Psychology, Baycrest,<br />
Toronto, ON, C<strong>an</strong>ada; 5 Psychology, York Univ. <strong>an</strong>d Rotm<strong>an</strong> Res. Inst., Toronto, ON, C<strong>an</strong>ada
Abstract: There is debate in <strong>the</strong> literature concerning <strong>the</strong> relative contributions of <strong>the</strong><br />
ventromedial prefrontal cortex (VMPFC), temporoparietal junction/superior temporal sulcus<br />
(TPJ/STS), <strong>an</strong>d <strong>the</strong>ir subregions, to <strong>the</strong>ory of mind (ToM), <strong>the</strong> ability to ‘read’ o<strong>the</strong>r people’s<br />
mental states. Some view <strong>the</strong> VMPFC as necessary <strong>for</strong> ToM based on its <strong>an</strong>atomical relationship<br />
with <strong>the</strong> amygdala <strong>an</strong>d involvement in affective decision-making, whereas o<strong>the</strong>rs view <strong>the</strong><br />
TPJ/STS as <strong>the</strong> critical region based on its known roles in action representation <strong>an</strong>d processing<br />
eye gaze. Debate is complicated by <strong>the</strong> fact that m<strong>an</strong>y ToM tests used in past studies rely on<br />
varying degrees of cognitive processing/action representations (inferring o<strong>the</strong>rs’ false beliefs <strong>an</strong>d<br />
thoughts/intention <strong>from</strong> eye gaze <strong>an</strong>d motion cues) <strong>an</strong>d emotional <strong>an</strong>alysis (detecting faux pas,<br />
empathy, sarcasm, <strong>an</strong>d deception). To determine whe<strong>the</strong>r such tests differentially map onto<br />
VMPFC vs. TPJ/STS, <strong>the</strong> present study examined ToM in 25 healthy adults <strong>an</strong>d 5 cases with<br />
complementary lesion profiles: Patient AM with widespread damage to bilateral VMPFC that<br />
includes <strong>the</strong> frontal poles, a subregion previously implicated in ToM, amygdala, <strong>an</strong>d superior<br />
temporal gyrus located within TPJ; patient DA, with less extensive damage that includes bilateral<br />
VMPFC <strong>an</strong>d right amygdala but not <strong>the</strong> TPJ/STS; <strong>an</strong>d three cases with focal lesions to regions<br />
within right TPJ/STS. The results indicated that despite extensive damage, AM <strong>an</strong>d DA were<br />
able to make appropriate first- <strong>an</strong>d second-order inferences on a false belief test, but were<br />
impaired on a empathy/sarcasm test that has <strong>an</strong> additional affective component, whereas <strong>the</strong><br />
TPJ/STS patients were impaired regardless of dem<strong>an</strong>ds on affective processing. AM was <strong>the</strong><br />
only patient to show additional impairment on a faux pas test of affective ToM, <strong>an</strong>d toge<strong>the</strong>r<br />
with one TPJ/STS patient showed marginal impairment on a deception test known to be sensitive<br />
to frontal polar lesions. Finally, AM <strong>an</strong>d <strong>the</strong> three TPJ/STS patients, but not DA, were<br />
signific<strong>an</strong>tly impaired at inferring intentionality based on complex social interactions among<br />
moving shapes, though in AM, <strong>the</strong> difficulty was not restricted to ToM. Impaired per<strong>for</strong>m<strong>an</strong>ce<br />
was not due to difficulties in visual perspective-taking in <strong>an</strong>y patient or to recognizing complex<br />
social-cognitive emotions based on eye gaze in all but one TPJ/STS patient, even though <strong>the</strong><br />
patients had difficulty recognizing more basic emotional facial expressions. The results support<br />
<strong>the</strong> view that <strong>the</strong> VMPFC/amygdala is necessary <strong>for</strong> affective ToM, but that at least some<br />
regions within <strong>the</strong> right TPJ/STS play a domain-specific role in cognitive <strong>an</strong>alysis <strong>an</strong>d<br />
representing intention based on action.<br />
Disclosures: L. Zackheim, None; D.T. Stuss, None; S. Gillingham, None; D. Rewilak,<br />
None; K.J. Murphy, None; R. Rosenbaum, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.24/GG3
Topic: F.01.l. Social cognition<br />
Support: Ellison Medical Foundation<br />
John Merck Scholars Program<br />
Title: Developmental ch<strong>an</strong>ge in <strong>the</strong> neural mech<strong>an</strong>isms of <strong>the</strong>ory of mind<br />
Authors: *H. GWEON, D. DODELL-FEDER, M. M. BEDNY, E. OLSON-BROWN, R. R.<br />
SAXE;<br />
Brain <strong>an</strong>d Cognitive Sci., MIT, Cambridge, MA<br />
Abstract: Previous neuroimaging studies have found brain regions recruited <strong>for</strong> '<strong>the</strong>ory of mind',<br />
our ability to think about o<strong>the</strong>r people's mental states. These regions include <strong>the</strong> bilateral<br />
temporo-parietal junctions, posterior cingulate, <strong>an</strong>d medial prefrontal cortex. However, <strong>the</strong><br />
developmental trajectory of <strong>the</strong>se brain regions as well as behavioral correlates of <strong>the</strong> ch<strong>an</strong>ges<br />
remain unclear as most previous studies have been done with adults. Here we report fMRI results<br />
<strong>from</strong> children (5-12 years, n=18) <strong>an</strong>d adults (n=9) listening to stories that describe physical<br />
events, facts about people, <strong>an</strong>d mental states. The same children also participated in a behavioral<br />
task designed to provide multiple measures of <strong>the</strong> children's <strong>the</strong>ory of mind. In both adults <strong>an</strong>d<br />
children, we found a consistent set of brain regions that was recruited <strong>for</strong> stories about mental<br />
states compared to stories about physical events. These results converge with recent findings that<br />
<strong>the</strong>se regions are activated when people attribute not just false beliefs but <strong>an</strong>y mental states.<br />
Interestingly, we observed <strong>an</strong> increase in response selectivity of left <strong>an</strong>d right TPJ with age: <strong>the</strong>se<br />
regions were activated <strong>for</strong> both facts about people <strong>an</strong>d mental states in younger children (< 8.5<br />
yrs), but showed more selective response to mental states in older children (> 8.5 yrs), <strong>an</strong>d<br />
almost exclusive response to mental states in adults. Our findings provide insights on how this<br />
uniquely hum<strong>an</strong> ability to reason about o<strong>the</strong>r people's mental states develops <strong>from</strong> early to late<br />
childhood.<br />
Disclosures: H. Gweon, None; D. Dodell-Feder, None; M.M. Bedny, None; E. Olson-Brown,<br />
None; R.R. Saxe, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.25/GG4<br />
Topic: F.01.l. Social cognition
Support: John Merck Scholars Program<br />
Title: Is <strong>the</strong> right temporo-parietal junction selective <strong>for</strong> attention or <strong>the</strong>ory of mind? An fMRI<br />
investigation<br />
Authors: *D. DODELL-FEDER, L. YOUNG, R. SAXE;<br />
MIT, Cambridge, MA<br />
Abstract: Prior functional magnetic reson<strong>an</strong>ce imaging (fMRI) studies have demonstrated a<br />
critical role <strong>for</strong> a cortical region in <strong>the</strong> right temporo-parietal junction (RTPJ) in “<strong>the</strong>ory of<br />
mind,” <strong>the</strong> ability to represent <strong>an</strong>d reason about mental states (e.g., <strong>an</strong> agent’s thoughts <strong>an</strong>d<br />
beliefs). A separate body of work, employing fMRI <strong>an</strong>d neuropsychological methods, has<br />
demonstrated a role <strong>for</strong> a region near <strong>the</strong> RTPJ in exogenous attention, i.e. attentional reorienting<br />
to <strong>an</strong> unexpected stimulus. In this study, we used fMRI to address <strong>the</strong> following questions<br />
outside <strong>the</strong> visual-attentional domain, using verbal stimuli: Is <strong>the</strong> response in <strong>the</strong> RTPJ selective<br />
<strong>for</strong> mental state content? Is <strong>the</strong> RTPJ response to mental state content fur<strong>the</strong>r modulated by<br />
whe<strong>the</strong>r that content is expected or unexpected? Particip<strong>an</strong>ts read stories about mental<br />
representations (e.g., false beliefs) <strong>an</strong>d physical representations (e.g., outdated signs) to localize<br />
regions of interest <strong>for</strong> <strong>the</strong>ory of mind (e.g., RTPJ) <strong>an</strong>d a separate set of stories in a 2 x 2 design:<br />
mental stories that were expected (e.g., The Smiths have a pool in <strong>the</strong>ir yard. They think<br />
swimming is a good way to cool off.) or unexpected (e.g., The Smiths have a pool in <strong>the</strong>ir yard.<br />
They think swimming is a good way to grow fins.) or physical stories that were expected (e.g.,<br />
One of <strong>the</strong> sinks does not work very well. The water comes out in a very weak stream.) or<br />
unexpected (e.g., One of <strong>the</strong> sinks does not work very well. The water comes out tasting like<br />
milk chocolate.). The response in <strong>the</strong> RTPJ region of interest, as well as <strong>the</strong> LTPJ, PC, <strong>an</strong>d<br />
DMPFC, was greater <strong>for</strong> mental versus physical stories, in both expected <strong>an</strong>d unexpected<br />
conditions. The RTPJ response was additionally modulated by expectedness: <strong>the</strong> response was<br />
greater <strong>for</strong> unexpected th<strong>an</strong> expected mental stories; this difference did not obtain <strong>for</strong> <strong>the</strong><br />
physical stories. These results suggest that <strong>the</strong> RTPJ is selective <strong>for</strong> mental state content <strong>an</strong>d<br />
additionally sensitive to a dimension of <strong>the</strong> mental state, whe<strong>the</strong>r it is expected or unexpected.<br />
Disclosures: D. Dodell-Feder, None; L. Young, None; R. Saxe, None.<br />
Poster<br />
577. Communication <strong>an</strong>d Self: O<strong>the</strong>r Distinctions<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 577.26/GG5<br />
Topic: F.01.l. Social cognition
Support: CREST, 17022035<br />
KAKENHI, 20020026<br />
KAKENHI, 21700295<br />
Showa University Gr<strong>an</strong>t-in-Aid <strong>for</strong> Innovative Collaborative Research Projects<br />
Tamagawa University Center of Excellence<br />
Title: Inferring mental state <strong>from</strong> eye gaze in myotonic dystrophy type 1<br />
Authors: *N. TSURUYA 1,2 , M. KOBAYAKAWA 1,2 , A. TAKEDA 1 , M. KAWAMURA 1 ;<br />
1 Showa Univ., Tokyo, Jap<strong>an</strong>; 2 Tamagawa Univ., Tokyo, Jap<strong>an</strong><br />
Abstract: It has been noted that patients with myotonic dystrophy type 1 (DM1) exhibit social<br />
cognitive impairment. However, <strong>the</strong> mech<strong>an</strong>isms of social cognitive functions in DM1 have not<br />
been well examined. In our previous study, we reported that patients with DM1 were less<br />
sensitive to facial emotions (Takeda et. al., <strong>2009</strong>; Kawamura et. al., <strong>2009</strong>). The eyes are very<br />
import<strong>an</strong>t components of <strong>the</strong> face that c<strong>an</strong> provide social in<strong>for</strong>mation about emotional <strong>an</strong>d<br />
mental states. In order to investigate <strong>the</strong> cognitive basis of social cognitive impairment in DM1,<br />
we examined <strong>the</strong> ability to infer <strong>the</strong> mental states of o<strong>the</strong>rs <strong>from</strong> <strong>the</strong> eye region.<br />
We developed a Jap<strong>an</strong>ese adaptation of <strong>the</strong> revised version of <strong>the</strong> ‘Reading <strong>the</strong> Mind in <strong>the</strong> Eyes’<br />
Test (Baron-Cohen et. al., 2001). Seven DM1 patients (4men, 3women) <strong>an</strong>d 22 healthy control<br />
subjects (11 men, 11 women) participated in <strong>the</strong> experiment. The particip<strong>an</strong>ts were asked to<br />
judge what <strong>the</strong> person in <strong>the</strong> picture is thinking or feeling <strong>from</strong> only <strong>the</strong> eye region..<br />
As a result, per<strong>for</strong>m<strong>an</strong>ces on <strong>the</strong> Eyes test were signific<strong>an</strong>tly lower in <strong>the</strong> DM1 patients th<strong>an</strong> in<br />
<strong>the</strong> healthy control subjects. This could not be attributed to <strong>the</strong> perceptual problem, because<br />
DM1 patients could discriminate <strong>the</strong> gender as well as healthy controls.<br />
The present results indicated that DM1 patients had difficulties in inferring <strong>the</strong> mental state of<br />
o<strong>the</strong>rs <strong>from</strong> <strong>the</strong> eye region of <strong>the</strong> face. The Eyes test is thought to reflect <strong>the</strong> subcomponent of<br />
<strong>the</strong>ory of mind (ToM). Our results indicated that social cognitive dysfunction in patients with<br />
DM1 was associated with <strong>the</strong> ToM ability.<br />
Disclosures: N. Tsuruya, None; M. Kobayakawa, None; A. Takeda, None; M. Kawamura,<br />
None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 578.1/GG6<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t R03DC005938<br />
NIH Gr<strong>an</strong>t 5R01DC005779<br />
Title: Attention-driven memory <strong>for</strong> task-relev<strong>an</strong>t acoustic stimuli in <strong>the</strong> frontal cortex of <strong>the</strong><br />
ferret<br />
Authors: *J. B. FRITZ, S. V. DAVID, P. YIN, S. A. SHAMMA;<br />
Neural Systems Lab, Inst. Syst Res., Univ. Maryl<strong>an</strong>d, College Park, MD<br />
Abstract: One of <strong>the</strong> defining features of neurons in <strong>the</strong> prefrontal cortex (PFC) is selective<br />
encoding of task-relev<strong>an</strong>t in<strong>for</strong>mation. What is <strong>the</strong> time course of this representation in PFC? We<br />
investigated this question by studying responses in single units in <strong>the</strong> frontal cortex of <strong>the</strong><br />
behaving ferret. Guided by recent neuro<strong>an</strong>atomical studies (Duque et al., <strong>2009</strong>; Radtke-Schuller<br />
et al., <strong>2009</strong>) that have mapped out ferret PFC, <strong>an</strong>d its auditory inputs (Bizley et al., <strong>2009</strong>), we<br />
initiated neurophysiological studies in behaving <strong>an</strong>d quiescent ferrets. They were trained on<br />
multiple auditory detection <strong>an</strong>d discrimination tasks using ei<strong>the</strong>r conditioned avoid<strong>an</strong>ce or<br />
positive rein<strong>for</strong>cement paradigms. We recorded <strong>from</strong> single neurons (n=850) in dorsal PFC <strong>an</strong>d<br />
premotor cortex (PMC) in dorsal orbital gyrus <strong>an</strong>d <strong>an</strong>terior sigmoid gyrus of five head-fixed<br />
ferrets. M<strong>an</strong>y frontal cells (~50%) showed no response to acoustic stimuli in a pre-behavior<br />
quiescent “non-task” condition, but during behavior displayed striking enh<strong>an</strong>ced or suppressive<br />
"recognition" responses that were highly adaptive, selective, <strong>an</strong>d categorical <strong>for</strong> <strong>the</strong> class of<br />
target sounds. In some “tabula rasa” neurons, <strong>the</strong>se selective responses to recent task-relev<strong>an</strong>t<br />
target stimuli were strictly gated by behavior, <strong>an</strong>d v<strong>an</strong>ished in post-behavioral testing in a<br />
quiescent “non-task” condition. However, in “memory” neurons (~20%), <strong>the</strong>se selective<br />
responses to previously task-relev<strong>an</strong>t stimuli persisted after <strong>the</strong> behavioral epoch was over. In<br />
our task-conditions, memory was ev<strong>an</strong>escent. Selective target responses gradually decreased in<br />
amplitude <strong>an</strong>d eventually extinguished within 5-120 minutes. Our results suggest that <strong>the</strong> PFC<br />
rapidly resets responses in a state-dependent m<strong>an</strong>ner, tracking attended task-relev<strong>an</strong>t in<strong>for</strong>mation<br />
in ch<strong>an</strong>ging task conditions. However, in passive non-task conditions, responses to incoming<br />
in<strong>for</strong>mation may also be shaped by a fading memory of a behaviorally relev<strong>an</strong>t target <strong>from</strong> <strong>the</strong><br />
most recent task, which c<strong>an</strong> be swiftly erased if <strong>the</strong> stimulus is no longer behaviorally relev<strong>an</strong>t in<br />
a new task condition. We observed behaviorally-gated LFP coherence between PFC <strong>an</strong>d primary<br />
auditory cortex in simult<strong>an</strong>eous recordings, <strong>an</strong>d a time course <strong>for</strong> acquisition <strong>an</strong>d extinction of<br />
target representations in PFC that was similar to <strong>the</strong> temporal trajectory <strong>for</strong> task-related receptive<br />
field plasticity previously described in primary auditory cortex (Fritz et al., 2003; 2007). Based<br />
on <strong>the</strong>se findings, we conjecture that top-down signals <strong>from</strong> PFC may contribute to receptive<br />
field modulation in sensory cortex, not only during, but also following attentive goal-directed<br />
behavior.<br />
Disclosures: J.B. Fritz, None; S.V. David, None; P. Yin, None; S.A. Shamma, None.
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.2/GG7<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t R01DC005779<br />
Title: Anatomical <strong>an</strong>d functional connectivity of top-down inputs to <strong>the</strong> auditory cortex in <strong>the</strong><br />
mouse<br />
Authors: *D. E. WINKOWSKI, S. BANDYOPADHYAY, S. A. SHAMMA, P. O. KANOLD;<br />
Inst. Systems Res., Univ. Maryl<strong>an</strong>d, College Park, MD<br />
Abstract: Attention-induced ch<strong>an</strong>ges in auditory cortical response properties are likely to be <strong>the</strong><br />
consequence of top-down signals originating in higher order cortical areas <strong>an</strong>d, as previously<br />
demonstrated, mediated by neuromodulatory systems, such as those that use acetylcholine (i.e..<br />
nucleus basalis). At this time, <strong>the</strong> precise identity <strong>an</strong>d functional properties of <strong>the</strong> higher order<br />
regions that might orchestrate such ch<strong>an</strong>ges in <strong>the</strong> auditory cortex, particularly in smaller<br />
mammals, is largely unknown. Here, using st<strong>an</strong>dard <strong>an</strong>atomical tract tracing methods, we<br />
identified regions in <strong>the</strong> mouse prefrontal cortex (PFC) that project directly to <strong>the</strong> primary<br />
auditory cortex (A1) <strong>an</strong>d characterized <strong>the</strong> PFC projection patterns within A1. In addition, we<br />
found that this connection was indeed functional using electrical microstimulation in <strong>the</strong> PFC<br />
<strong>an</strong>d monitoring neural activity in A1. These data indicate that <strong>the</strong>re is a functional <strong>an</strong>d perhaps<br />
excitatory connection <strong>from</strong> <strong>the</strong> PFC to A1 in <strong>the</strong> mouse. These experiments lay <strong>the</strong> foundation<br />
<strong>for</strong> future experiments designed to provide a more complete picture of <strong>the</strong> mech<strong>an</strong>isms<br />
underlying top-down control of auditory processing <strong>an</strong>d may provide insight into <strong>the</strong> neural<br />
mech<strong>an</strong>isms underlying auditory cortical plasticity.<br />
Disclosures: D.E. Winkowski, None; S. B<strong>an</strong>dyopadhyay, None; S.A. Shamma, None; P.O.<br />
K<strong>an</strong>old, None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.3/GG8<br />
Topic: F.02.b. Executive function<br />
Support: NIH K99 DC009007<br />
NIH R01 DC005808<br />
Title: Behavior-depend<strong>an</strong>t modulation of primate prefrontal cortex neurons during natural<br />
behavior<br />
Authors: *C. T. MILLER, X. WANG;<br />
Biomed. Engin., Johns Hopkins Univ., Baltimore, MD<br />
Abstract: Primates evolved sophisticated behaviors in order to navigate <strong>the</strong>ir complex social<br />
systems. Although relatively little research has been directly aimed at examining <strong>the</strong> neural<br />
substrates underlying <strong>the</strong>se natural behaviors, it is clear that producing <strong>the</strong>se behaviors requires<br />
<strong>an</strong> integration of both <strong>the</strong> sensory <strong>an</strong>d motor in<strong>for</strong>mation. Amongst <strong>the</strong> most signific<strong>an</strong>t of <strong>the</strong>se<br />
behaviors are those involved in communicating with conspecifics. Here we recorded <strong>the</strong> singleunit<br />
activity of frontal cortex neurons in freely-moving common marmoset monkeys (Callithrix<br />
jacchus) engaged in a natural, species-typical behavior known as <strong>an</strong>tiphonal calling, a vocal<br />
behavior involving <strong>the</strong> reciprocal exch<strong>an</strong>ge of vocalizations between conspecifics. Producing <strong>an</strong><br />
<strong>an</strong>tiphonal call is dependent upon first hearing a particular vocalization, a phee call, <strong>an</strong>d<br />
producing <strong>the</strong> same call type in response. Import<strong>an</strong>tly, <strong>the</strong> sequence of sensory <strong>an</strong>d motor events<br />
in this behavior occurs over a 10-15s period of time. In our first set of <strong>an</strong>alyses, we compared<br />
neural activity during each of <strong>the</strong> three elements of this behavior: sensory stimulus (phee call<br />
stimulus), latency delay, <strong>an</strong>d vocal-motor response (<strong>an</strong>tiphonal call). The aim here was to<br />
determine whe<strong>the</strong>r neurons across <strong>the</strong> population showed firing rate ch<strong>an</strong>ges during <strong>an</strong>y of <strong>the</strong>se<br />
three periods individually or in combination. Analyses showed neuronal responses during each<br />
of <strong>the</strong>se behavioral periods, with a population showing ch<strong>an</strong>ges in neural activity over <strong>the</strong><br />
duration of <strong>the</strong> <strong>an</strong>tiphonal calling behavior. This population of neurons typically showed one of<br />
two responses. The first class showed <strong>an</strong> increased firing rate during <strong>the</strong> phee stimulus <strong>an</strong>d<br />
suppressed activity during <strong>the</strong> <strong>an</strong>tiphonal call. The second class showed <strong>the</strong> opposite response. In<br />
our second set of <strong>an</strong>alyses, we examined <strong>the</strong> effects of behavioral context of <strong>the</strong> neural response<br />
observed during <strong>an</strong>tiphonal calling. Specifically, during test sessions, we presented subjects with<br />
phee call stimuli that did not elicit <strong>an</strong>tiphonal callings <strong>an</strong>d subjects produced phee calls<br />
spont<strong>an</strong>eously. As such, we tested whe<strong>the</strong>r neurons responded similarly during ei<strong>the</strong>r <strong>the</strong> sensory<br />
stimulus or motor response alone compared to <strong>the</strong> same sensory or motor period during<br />
<strong>an</strong>tiphonal calls. Our initial <strong>an</strong>alysis suggests that <strong>the</strong> a<strong>for</strong>ementioned ‘<strong>an</strong>tiphonal calling’<br />
neurons show little response during <strong>the</strong> sensory stimulus <strong>an</strong>d vocal-motor response<br />
independently. Toge<strong>the</strong>r <strong>the</strong>se data suggest that it is <strong>the</strong> sensory-motor integration during <strong>the</strong><br />
<strong>an</strong>tiphonal calling behavior that is driving <strong>the</strong> neural response.
Disclosures: C.T. Miller, None; X. W<strong>an</strong>g, None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.4/GG9<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t DC04845<br />
Title: The role of primate ventrolateral prefrontal cortex during <strong>the</strong> evaluation of emotional faces<br />
<strong>an</strong>d vocalizations<br />
Authors: *M. M. DIEHL 1 , M. DILTZ 2 , L. M. ROMANSKI 2 ;<br />
1 Neurosci, 2 Neurobio. & Anat., Univ. Rochester SMD, Rochester, NY<br />
Abstract: Social communication requires <strong>the</strong> accurate integration of both auditory <strong>an</strong>d visual<br />
in<strong>for</strong>mation such as faces <strong>an</strong>d vocalizations in hum<strong>an</strong> <strong>an</strong>d non-hum<strong>an</strong> primates. We have begun<br />
to investigate <strong>the</strong> integration of face <strong>an</strong>d vocalization in<strong>for</strong>mation in <strong>the</strong> ventrolateral prefrontal<br />
cortex (VLPFC) of <strong>the</strong> rhesus macaque. Specifically, we are interested in <strong>the</strong> role of VLPFC<br />
with regard to <strong>the</strong> emotional content of vocalizations <strong>an</strong>d how this area encodes this aspect of<br />
vocal communication. In previous studies we have shown that single VLPFC cells are<br />
multisensory <strong>an</strong>d integrate face <strong>an</strong>d vocalization in<strong>for</strong>mation. We have also shown that neurons<br />
in VLPFC demonstrate context <strong>an</strong>d stimulus-related activity during audiovisual discrimination<br />
tasks involving movies of conspecifics vocalizing. To fur<strong>the</strong>r investigate <strong>the</strong> role of <strong>the</strong> VLPFC<br />
in social communication, we recorded single-cell activity while <strong>an</strong>imals per<strong>for</strong>med a similar task<br />
where <strong>the</strong> audiovisual vocalization stimuli were positive, negative, or neutral in valence <strong>an</strong>d<br />
presented in a non-match-to-sample paradigm. Stimuli included coos, lip-smacking, barks,<br />
growls, <strong>an</strong>d screams obtained <strong>from</strong> familiar <strong>an</strong>d unfamiliar callers. Recordings in <strong>the</strong> VLPFC<br />
were aimed at determining whe<strong>the</strong>r cells respond differentially to <strong>the</strong> types of face-vocalization<br />
stimuli based on emotional valence, identity of caller, or o<strong>the</strong>r features present in conspecific<br />
face <strong>an</strong>d vocalization stimuli. Comparison of neuronal activity in VLPFC <strong>an</strong>d orbitofrontal<br />
regions may yield different roles <strong>for</strong> <strong>the</strong>se two prefrontal regions in discriminating face <strong>an</strong>d<br />
vocalization in<strong>for</strong>mation. Continued <strong>an</strong>alysis <strong>an</strong>d recordings are aimed at determining <strong>the</strong> role of<br />
<strong>the</strong> VLPFC in sensory integration of communication in<strong>for</strong>mation.<br />
Disclosures: M.M. Diehl, None; M. Diltz, None; L.M. Rom<strong>an</strong>ski, None.
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.5/GG10<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t DC04845<br />
Title: Comparison of face <strong>an</strong>d non-face stimuli in <strong>an</strong> audio-visual discrimination task<br />
Authors: *J. HWANG 1 , L. M. ROMANSKI 2 ;<br />
1 2<br />
Brain & Cognitive Sci., Univ. Rochester, Rochester, NY; Neurobio. & Anat., Univ. of<br />
Rochester Med. Ctr., Rochester, NY<br />
Abstract: Communication is a multisensory process. In both hum<strong>an</strong>s <strong>an</strong>d non-hum<strong>an</strong> primates,<br />
in<strong>for</strong>mation <strong>from</strong> facial gestures is combined with vocal in<strong>for</strong>mation to enrich <strong>the</strong> me<strong>an</strong>ing of <strong>the</strong><br />
communicated message. Electrophysiological <strong>an</strong>d neuroimaging studies have shown that some<br />
cortical areas are specialized <strong>for</strong> <strong>the</strong> processing of faces <strong>an</strong>d fur<strong>the</strong>r, that some temporal <strong>an</strong>d<br />
prefrontal cortical regions are multisensory, responding to both faces <strong>an</strong>d vocalizations. In<br />
previous studies we have shown that single prefrontal neurons show enh<strong>an</strong>ced or suppressed<br />
responses to combined face <strong>an</strong>d vocalization stimuli compared to unimodal stimuli. In this study,<br />
we trained rhesus macaques to per<strong>for</strong>m <strong>an</strong> audio-visual discrimination task in which face <strong>an</strong>d<br />
non-face stimuli were presented in combination with conspecific vocalizations. The task was a<br />
non-match-to-sample task in which subjects were rewarded <strong>for</strong> detecting <strong>the</strong> non-match stimulus<br />
with a button press. Rapid detection was encouraged by delivering <strong>the</strong> reward immediately after<br />
<strong>the</strong> response button press. Preliminary <strong>an</strong>alysis of behavioral responses showed that response<br />
time in <strong>the</strong> audio-visual task differed depending on whe<strong>the</strong>r <strong>the</strong> face or <strong>the</strong> non-face visual<br />
stimuli were paired with <strong>the</strong> vocalizations, with faces eli<strong>citing</strong> <strong>the</strong> longest response time.<br />
Responses to unimodal auditory discrimination were <strong>the</strong> shortest by ~ 100 msec. This difference<br />
in response time may indicate that <strong>the</strong> processing of faces differs <strong>from</strong> <strong>the</strong> processing of non-face<br />
stimuli. Single unit recordings, per<strong>for</strong>med while subjects per<strong>for</strong>m this task, will target <strong>the</strong><br />
ventrolateral prefrontal cortex (VLPFC) since this area has been shown to be active in <strong>an</strong> audiovisual<br />
discrimination task <strong>an</strong>d has neurons that are responsive to face <strong>an</strong>d non-face visual<br />
stimuli. Our studies will elucidate <strong>the</strong> neuronal processes which underlie audio-visual integration<br />
of face <strong>an</strong>d non-face in<strong>for</strong>mation as well as <strong>the</strong> specific role of <strong>the</strong> VLPFC in <strong>the</strong>se processes.<br />
Disclosures: J. Hw<strong>an</strong>g, None; L.M. Rom<strong>an</strong>ski, None.
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.6/GG11<br />
Topic: F.02.b. Executive function<br />
Support: NIH DC007172<br />
NIH DC009224<br />
Title: Vocalization-context dependent neural representation of faces in primate ventrolateral<br />
prefrontal cortex<br />
Authors: *J. TSUNADA 1 , A. E. BAKER 2 , S. J. DAVIS 1 , A. A. GHAZANFAR 3 , Y. E.<br />
COHEN 1 ;<br />
1 Dept. of Otorhinolaryngology, Univ. of Pennsylv<strong>an</strong>ia Sch. of Med., Philadelphia, PA; 2 Dept. of<br />
Psychological <strong>an</strong>d Brain Sci. <strong>an</strong>d Ctr. <strong>for</strong> Cognitive Neurosci., Dartmouth Col., H<strong>an</strong>over, NH;<br />
3 Neurosci. Institute, Dept. of Psychology, Princeton Univ., Princeton, NJ<br />
Abstract: A neuron’s response to a stimulus is context dependent. For example, a neuron’s<br />
response to a stimulus c<strong>an</strong> be modulated as a function of <strong>the</strong> dem<strong>an</strong>ds of a going task. The type<br />
<strong>an</strong>d timing of preceding stimuli c<strong>an</strong> also modulate a neuron’s response to a stimulus. In this<br />
study, we tested how repeated presentation of a species-specific vocalization modulates neural<br />
responses to a silent movie of con-specific vocalizing. Since <strong>the</strong> ventrolateral prefrontal cortex<br />
(vPFC) processes both auditory <strong>an</strong>d visual communication signals, we hypo<strong>the</strong>sized that vPFC<br />
responses to a silent movie of vocalizing would be modulated by preceding vocalizations. To test<br />
this hypo<strong>the</strong>sis, we recorded local field potentials (LFPs) <strong>from</strong> <strong>the</strong> vPFC of rhesus monkeys<br />
while <strong>the</strong> monkeys listened to 3 - 5 repetitions of <strong>the</strong> same species-specific vocalization (a coo, a<br />
grunt, or a scream) <strong>an</strong>d <strong>the</strong>n viewed a silent movie of a monkey vocalizing. All of <strong>the</strong><br />
vocalization-movie stimuli came <strong>from</strong> <strong>the</strong> same monkey, eliminating <strong>an</strong>y individual-based<br />
factors. For each trial, <strong>the</strong> vocalization <strong>an</strong>d <strong>the</strong> movie were ei<strong>the</strong>r congruent (e.g., <strong>the</strong><br />
vocalization was a coo <strong>an</strong>d <strong>the</strong> movie showed a monkey cooing) or incongruent (e.g., <strong>the</strong><br />
vocalization was a coo <strong>an</strong>d <strong>the</strong> movie showed a monkey grunting). We recorded LFP <strong>from</strong> 168<br />
sites <strong>an</strong>d found that 93 sites had signific<strong>an</strong>t increases in LFP power during <strong>the</strong> presentation of <strong>the</strong><br />
movie (movie period). The increase in LFP power was most prominent in <strong>the</strong> frequency r<strong>an</strong>ge<br />
between 50 - 100 Hz. Interestingly, LFP power in this frequency r<strong>an</strong>ge was modulated by <strong>the</strong><br />
preceding vocalization but only when <strong>the</strong> vocalization had a lower putative affect th<strong>an</strong> <strong>the</strong><br />
putative affect of <strong>the</strong> movie. For example, LFP power was modulated when <strong>the</strong> vocalization was
a coo <strong>an</strong>d <strong>the</strong> movie was a scream but not when <strong>the</strong> vocalization was a scream <strong>an</strong>d <strong>the</strong> movie<br />
was coo. This affect-dependent result is reminiscent of habituation-discrimination studies using<br />
food calls of varying affect. These results also suggest that comparable affective in<strong>for</strong>mation<br />
may be carried in both auditory <strong>an</strong>d visual communication signals. Previous work <strong>from</strong> our<br />
group suggests that vPFC spiking activity is not modulated by contextual-affective in<strong>for</strong>mation.<br />
Since LFPs are thought to be inputs to a system, our results suggest that <strong>the</strong> computations<br />
represented by <strong>the</strong> vPFC spiking activity (<strong>the</strong> “output”) may be modulated by affective context<br />
but do not inherently represent such in<strong>for</strong>mation. Affective in<strong>for</strong>mation is most likely<br />
represented in o<strong>the</strong>r areas (e.g., medial PFC) <strong>an</strong>d integrated with o<strong>the</strong>r in<strong>for</strong>mation that guides <strong>an</strong><br />
<strong>an</strong>imal’s actions <strong>an</strong>d choices.<br />
Disclosures: J. Tsunada, None; A.E. Baker, None; S.J. Davis, None; A.A. Ghaz<strong>an</strong>far, None;<br />
Y.E. Cohen, NIH-NIDCD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t<br />
<strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.7/GG12<br />
Topic: F.02.b. Executive function<br />
Support: NIDCD Gr<strong>an</strong>t DC0007156<br />
Title: Task-related neuronal activity in primate prefrontal cortex during <strong>an</strong> auditory delayed<br />
matching-to-sample task<br />
Authors: *B. PLAKKE, C.-W. NG, R. OPHEIM, A. POREMBA;<br />
Psychology, Div. Behav & Cog Ne, Univ. Iowa, Iowa City, IA<br />
Abstract: Neuroimaging <strong>an</strong>d neurophysiological recording studies have demonstrated that <strong>the</strong><br />
prefrontal cortex (PFC) is responsive to auditory stimuli (e.g., Poremba et al., 2003; Rom<strong>an</strong>ski,<br />
2007). The PFC is involved in visual tasks requiring working or short-term memory (e.g.,<br />
Bodner, et al., 1996; Fuster, 2000; Warden & Miller 2007). Here, we record single-unit neuronal<br />
activity <strong>from</strong> <strong>the</strong> left ventral <strong>an</strong>d lateral b<strong>an</strong>k of <strong>the</strong> principal sulcus (area 46) in rhesus macaques<br />
on <strong>an</strong> auditory go/no go delayed matching-to-sample (DMTS) task wherein two acoustic stimuli<br />
(500 ms), separated by a fixed memory delay (5000 ms), were ei<strong>the</strong>r identical sound<br />
presentations, or two different sound presentations. A small c<strong>an</strong>dy reward was delivered after a<br />
correct button press on match trials. Each training session consisted of 200 trials, where half <strong>the</strong>
trials were match trials <strong>an</strong>d half were nonmatch trials. The eight sound stimuli <strong>for</strong> each recording<br />
day were chosen <strong>from</strong> a larger sound set with each sound stimulus repeated 8-12 times on both<br />
match <strong>an</strong>d nonmatch trials within a recording session. Sound stimuli included pure tones,<br />
frequency sweeps, m<strong>an</strong>-made environmental sounds, monkey vocalizations, hum<strong>an</strong><br />
vocalizations, natural sounds, syn<strong>the</strong>sized sounds, <strong>an</strong>d o<strong>the</strong>r <strong>an</strong>imal sounds. Assessment of<br />
neuronal firing rates <strong>an</strong>d wave<strong>for</strong>ms were completed <strong>for</strong> task related activity, as well as <strong>for</strong> <strong>the</strong><br />
same sound stimuli during passive listening. Preliminary results <strong>from</strong> two monkeys indicate that<br />
neurons in this area are responsive to complex auditory stimuli in a selective m<strong>an</strong>ner during <strong>the</strong><br />
memory task. Single-units also signific<strong>an</strong>tly increase or decrease <strong>the</strong>ir firing rate compared to<br />
baseline <strong>for</strong> individual events during <strong>the</strong> behavioral per<strong>for</strong>m<strong>an</strong>ce, such as <strong>the</strong> delay period, <strong>the</strong><br />
waiting to behaviorally respond period, <strong>an</strong>d/or during <strong>the</strong> response period. Comparing single-unit<br />
activity <strong>for</strong> correct match trials versus incorrect nonmatch trials, wherein behavioral responses<br />
are made on both trial types, revealed specific increases in firing rate during trials with correct<br />
match per<strong>for</strong>m<strong>an</strong>ce. Neurons in this prefrontal region, area 46, encode complex auditory signals<br />
<strong>an</strong>d also have firing patterns correlated with specific portions of <strong>the</strong> task in addition to its<br />
auditory-related encoding.<br />
Disclosures: B. Plakke, None; C. Ng, None; R. Opheim, None; A. Poremba, None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.8/GG13<br />
Topic: F.02.b. Executive function<br />
Support: NIH DC007172<br />
NIH DC009224<br />
Title: Context-dependent neural representation of vocalizations in primate ventrolateral<br />
prefrontal cortex<br />
Authors: *A. E. BAKER 1 , J. TSUNADA 2 , S. J. DAVIS 2 , Y. E. COHEN 2 , A. A.<br />
GHAZANFAR 3 ;<br />
1 Psychological <strong>an</strong>d Brain Sci., Dartmouth Coll, H<strong>an</strong>over, NH; 2 Dept. of Otorhinolaryngology,<br />
Univ. of Pennsylv<strong>an</strong>ia Sch. of Med., Philadelphia, PA; 3 Neurosci. Institute, Dept. of Psychology,<br />
Princeton Univ., Princeton, NJ
Abstract: A neuron’s response to a stimulus is context dependent. For example, a neuron’s<br />
response to a stimulus c<strong>an</strong> be modulated as a function of <strong>the</strong> dem<strong>an</strong>ds of a going task. The type<br />
<strong>an</strong>d timing of preceding stimuli c<strong>an</strong> also modulate a neuron’s response to a stimulus. In this<br />
study, we tested how repeated presentation of a species-specific vocalization modulates neural<br />
responses. Since <strong>the</strong> ventrolateral prefrontal cortex (vPFC) processes vocalizations, we<br />
hypo<strong>the</strong>sized that vPFC responses are sensitive to <strong>the</strong> occurrence of preceding vocalizations. To<br />
test this hypo<strong>the</strong>sis, we recorded local field potentials (LFPs) <strong>from</strong> <strong>the</strong> vPFC of rhesus monkeys<br />
while <strong>the</strong> monkeys listened to 3 - 5 repetitions of a vocalization. The repeated vocalization was<br />
one of three different rhesus vocalizations: a coo, a grunt, or a scream. We recorded LFP <strong>from</strong><br />
168 sites <strong>an</strong>d found that 83 sites had signific<strong>an</strong>t increases in LFP power during <strong>the</strong> presentation<br />
of <strong>the</strong> vocalization (vocalization period). The increase in LFP power was most prominent in <strong>the</strong><br />
frequency r<strong>an</strong>ge between 4 - 50 Hz. <strong>When</strong> we looked at how <strong>the</strong> LFP power ch<strong>an</strong>ged during <strong>the</strong><br />
vocalization period, we found that peak power decreased as <strong>the</strong> number of preceding<br />
vocalizations increased <strong>an</strong>d <strong>the</strong> latency (relative to vocalization onset) to peak increased. This<br />
context dependency is reminiscent of habituation <strong>an</strong>d stimulus-specific adaptation that are seen<br />
in o<strong>the</strong>r cortical regions. However, spiking activity in <strong>the</strong> vPFC does not appear to habituate<br />
when tested with paradigms similar to ours (Gif<strong>for</strong>d et al., 2005; Russ et al., 2008). Since LFPs<br />
are thought to be inputs to a system, our results suggest that <strong>the</strong> computations represented by <strong>the</strong><br />
vPFC spiking activity (<strong>the</strong> “output”) may be modulated by <strong>the</strong>se habituating processes but do not<br />
inherently represent such in<strong>for</strong>mation. Thus, this source of in<strong>for</strong>mation is most likely in o<strong>the</strong>r<br />
areas <strong>an</strong>d integrated with o<strong>the</strong>r in<strong>for</strong>mation that guides <strong>an</strong> <strong>an</strong>imal’s actions <strong>an</strong>d choices.<br />
Disclosures: A.E. Baker, None; J. Tsunada, None; S.J. Davis, None; Y.E. Cohen, NIH-<br />
NIDCD, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts<br />
as well as gr<strong>an</strong>ts already received); A.A. Ghaz<strong>an</strong>far, None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.9/GG14<br />
Topic: F.02.b. Executive function<br />
Support: PRESTO<br />
Title: Neuronal basis of socially oriented per<strong>for</strong>m<strong>an</strong>ce monitoring. I. Design of a behavioral<br />
paradigm <strong>for</strong> monkeys
Authors: *M. ISODA 1,2 , K. YAKO 1,3 , A. IRIKI 1 ;<br />
1 Lab. <strong>for</strong> Symbolic Cognitive Develop., RIKEN Brain Sci. Inst., Wako, Saitama, Jap<strong>an</strong>; 2 Presto,<br />
JST, Kawaguchi, Jap<strong>an</strong>; 3 Dept Neurosurg, Univ. Tokyo, Hongo, Jap<strong>an</strong><br />
Abstract: Successful behavior in <strong>the</strong> ch<strong>an</strong>ging environment requires continuous per<strong>for</strong>m<strong>an</strong>ce<br />
monitoring. An efficient way to find optimal behavior in social contexts is to monitor <strong>the</strong><br />
per<strong>for</strong>m<strong>an</strong>ce of o<strong>the</strong>rs as well as that of one’s own. This ability, which we will call “socially<br />
oriented per<strong>for</strong>m<strong>an</strong>ce monitoring,” facilitates adaptive decision making in social life. How <strong>the</strong><br />
brain implements this monitoring function remains unknown. To reveal its underlying<br />
mech<strong>an</strong>ism at <strong>the</strong> cellular level, we have devised a behavioral paradigm <strong>for</strong> monkeys, called<br />
“role-reversal choice task.” Two monkeys (Macaca fuscata) sat face to face across a table on<br />
which two sets of response buttons were placed, each set consisting of a start button <strong>an</strong>d two<br />
target buttons. On each trial one monkey was assigned <strong>the</strong> role of “actor,” whereas <strong>the</strong> o<strong>the</strong>r was<br />
<strong>the</strong> “observer.” The monkeys alternated roles every two trials. A trial started with <strong>the</strong><br />
illumination of <strong>the</strong> actor’s start button. Upon both monkeys pressing <strong>the</strong>ir start buttons <strong>for</strong> 1-1.5<br />
s with <strong>the</strong>ir right h<strong>an</strong>ds, <strong>the</strong> target buttons on <strong>the</strong> actor’s side turned on in two different colors,<br />
<strong>the</strong> positions of which were r<strong>an</strong>domized across trials. The actor <strong>the</strong>n was required to reach to one<br />
of <strong>the</strong>m. During a block of 5-17 trials, a reward was associated with one target color <strong>an</strong>d both<br />
monkeys were rewarded if <strong>the</strong> actor made a correct choice; if it made a wrong choice nei<strong>the</strong>r of<br />
<strong>the</strong>m was rewarded. The color-reward rule was reversed unpredictably in <strong>the</strong> next block. In this<br />
task <strong>the</strong> observer c<strong>an</strong> learn whe<strong>the</strong>r <strong>the</strong> color-reward rule has switched or not on <strong>the</strong> basis of<br />
integrated knowledge about <strong>the</strong> actor’s action <strong>an</strong>d its outcome. Specifically, <strong>the</strong> observer should<br />
switch <strong>the</strong> rule in <strong>the</strong> upcoming trial if <strong>the</strong> actor’s choice by following <strong>the</strong> preceding rule has<br />
resulted in no reward (this occurs in switch trials, i.e., <strong>the</strong> first trial in each block). In contrast,<br />
<strong>the</strong> observer should not switch <strong>the</strong> rule after <strong>the</strong> actor’s choice of nonrewarded target (this could<br />
occur in nonswitch trials). Indeed, behavioral <strong>an</strong>alyses revealed that <strong>the</strong> observer seemed to<br />
process <strong>the</strong> two types of errors differently as described above. These data suggest that macaque<br />
monkeys are capable of monitoring per<strong>for</strong>m<strong>an</strong>ce of o<strong>the</strong>rs to guide optimal behavior of one’s<br />
own. Our paradigm will offer a fruitful starting point <strong>for</strong> investigating <strong>the</strong> neuronal mech<strong>an</strong>isms<br />
of socially oriented per<strong>for</strong>m<strong>an</strong>ce monitoring in nonhum<strong>an</strong> primates.<br />
Disclosures: M. Isoda, None; K. Yako, None; A. Iriki, None.<br />
Poster<br />
578. Executive Function: Animal Models of Audition, Vocalization, <strong>an</strong>d Social Interaction<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 578.10/GG15<br />
Topic: F.02.b. Executive function
Support: PRESTO<br />
Title: Neuronal basis of socially oriented per<strong>for</strong>m<strong>an</strong>ce monitoring. II. Agent-related neuronal<br />
activity in <strong>the</strong> medial frontal cortex<br />
Authors: *K. YAKO 1,2 , A. IRIKI 1 , N. SAITO 2 , M. ISODA 1,3 ;<br />
1 RIKEN Brain Sci. Inst., Wako, Saitama, Jap<strong>an</strong>; 2 Dept Neurosurg, Univ. Tokyo, Hongo, Jap<strong>an</strong>;<br />
3 Presto, JST, Kawaguchi, Jap<strong>an</strong><br />
Abstract: In a comp<strong>an</strong>ion paper, we showed that monkeys successfully monitored <strong>the</strong><br />
per<strong>for</strong>m<strong>an</strong>ce of o<strong>the</strong>rs <strong>an</strong>d <strong>the</strong>reby guided <strong>the</strong>ir own behavior in a purposeful way. Although <strong>the</strong><br />
medial frontal cortex (MFC) has been implicated in monitoring one’s per<strong>for</strong>m<strong>an</strong>ce, whe<strong>the</strong>r <strong>an</strong>d<br />
how <strong>the</strong> MFC is involved in monitoring <strong>the</strong> behavior of o<strong>the</strong>rs remains uncertain. We<br />
hypo<strong>the</strong>sized that <strong>the</strong> function of <strong>the</strong> MFC in behavioral monitoring c<strong>an</strong> extend into <strong>the</strong> selfo<strong>the</strong>r<br />
framework. To test this hypo<strong>the</strong>sis, we recorded neuronal activity in <strong>the</strong> supplementary<br />
motor area (SMA) <strong>an</strong>d pre-SMA, which will be collectively called <strong>the</strong> supplementary motor<br />
complex (SMC), <strong>an</strong>d in <strong>the</strong> <strong>an</strong>terior cingulate cortex (ACC) of a monkey per<strong>for</strong>ming <strong>the</strong> rolereversal<br />
choice task with its partner. This study will focus on <strong>the</strong> activity around <strong>the</strong> <strong>an</strong>imals’<br />
reaching movement. We divided a movement-related period into “early” (50-250 ms after target<br />
onset) <strong>an</strong>d “late” (200-0 ms be<strong>for</strong>e movement offset). Of 441 single neurons (220 in <strong>the</strong> SMC<br />
<strong>an</strong>d 221 in <strong>the</strong> ACC), ~55 % of <strong>the</strong> neurons signific<strong>an</strong>tly ch<strong>an</strong>ged <strong>the</strong>ir activity in each of <strong>the</strong><br />
two periods. We applied a 4-way ANOVA (agent, target location, target color, <strong>an</strong>d trial order as<br />
factors) to <strong>the</strong> activity of <strong>the</strong>se neurons to examine <strong>the</strong> effect of <strong>the</strong> behavioral factors. Notably,<br />
<strong>the</strong> majority of MFC neurons was found to be selective <strong>for</strong>, or influenced by, <strong>the</strong> factor of<br />
response agent. Specifically, <strong>the</strong> first set of neurons (“partner type”) was signific<strong>an</strong>tly more<br />
active when <strong>the</strong> subject observed its partner executing <strong>the</strong> movement (observer condition) th<strong>an</strong><br />
when <strong>the</strong> subject executed <strong>the</strong> movement (actor condition), <strong>the</strong> second set (“self type”) was<br />
signific<strong>an</strong>tly more active in <strong>the</strong> actor condition th<strong>an</strong> in <strong>the</strong> observer condition, while <strong>the</strong> third set<br />
(“mirror type”) was equally active in both conditions. The proportion of partner-type neurons<br />
was signific<strong>an</strong>tly larger in <strong>the</strong> SMC th<strong>an</strong> in <strong>the</strong> ACC in both periods, whereas <strong>the</strong> proportion of<br />
self-type neurons was signific<strong>an</strong>tly larger in <strong>the</strong> ACC th<strong>an</strong> in <strong>the</strong> SMC during <strong>the</strong> late period.<br />
The three types of neuron were spatially intermingled in each area. Neurons selective <strong>for</strong> <strong>the</strong><br />
target location were less frequent (10-20 %) <strong>an</strong>d those selective <strong>for</strong> <strong>the</strong> target color or trial order<br />
were in <strong>the</strong> minority. These data suggest that <strong>the</strong> MFC plays a role in agent computation, a<br />
fundamental variable <strong>for</strong> decision-making in social contexts.<br />
Disclosures: K. Yako, None; A. Iriki, None; N. Saito, None; M. Isoda, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.1/GG16<br />
Topic: F.02.b. Executive function<br />
Support: MIUR 2005-2007 to AGS<br />
Young-Investigator project, coordinated by Walter Adri<strong>an</strong>i as PI, to LAR.<br />
Title: Prepuberal intr<strong>an</strong>asal dopamine differentially affects activity, non selective <strong>an</strong>d selective<br />
spatial attention, tyrosine hydroxylase <strong>an</strong>d DARPP32 in Naples high excitability rats<br />
Authors: *L. A. RUOCCO 1 , M. A. DE SOUZA SILVA 2 , D. VIGGIANO 3 , C. TRENO 1 , B.<br />
TOPIC 2 , C. MATTERN 4 , J. P. HUSTON 2 , A. G. SADILE 1 ;<br />
1 SECOND UNIV. OF NAPLES, NAPLES, Italy; 2 Inst. of Physiological Psychology, Univ. of<br />
Düsseldorf, Düsseldorf, Germ<strong>an</strong>y; 3 Dept. of Hlth. Sci., Univ. of Molise, Campobasso, Italy; 4 M<br />
& P Pharma AG, St<strong>an</strong>s, Switzerl<strong>an</strong>d<br />
Abstract: Intr<strong>an</strong>asal application of dopamine (IN-DA) has been shown to increase motor activity<br />
in different behavioral tasks via DA receptors on mesostriatal <strong>an</strong>d mesolimbic DA neurons.<br />
Neuropsychiatric problems such as Autism <strong>an</strong>d Attention Deficit/Hyperactivity Disorder<br />
(ADHD) are characterized by hyperactivity <strong>an</strong>d attention problems. We fur<strong>the</strong>r investigated <strong>the</strong><br />
effects of prepuberal IN-DA on activity, non selective (NSA) <strong>an</strong>d selective spatial attention<br />
(SSA), tyrosine hydroxylase <strong>an</strong>d DARPP-32 immunohistochemistry in prepuberal male rats of<br />
<strong>the</strong> Naples High Excitability line (NHE). These rats c<strong>an</strong> be considered as a genetic model <strong>for</strong> <strong>the</strong><br />
mesocortical vari<strong>an</strong>t of ADHD with altered executive functions. NHE rats received daily<br />
application of DA (0.0, 0.075, 0.15, 0.3 mg/kg) into both nostrils, <strong>for</strong> 15 days <strong>from</strong> day 28 to day<br />
42. One hour after treatment, <strong>the</strong>y were exposed to a Làt maze <strong>an</strong>d a radial maze on days 41 <strong>an</strong>d<br />
42, respectively. Activity was monitored by frequency of horizontal (HA) <strong>an</strong>d vertical activity<br />
(VA; rearings <strong>an</strong>d le<strong>an</strong>ings against <strong>the</strong> walls). NSA <strong>an</strong>d SSA were monitored by duration of<br />
individual rearing <strong>an</strong>d le<strong>an</strong>ing episodes in <strong>the</strong> Làt maze; in <strong>the</strong> radial maze SSA was assessed by<br />
position of <strong>the</strong> first repetitive arm entry. Behavioral data showed 1) a decreased HA with 0.3<br />
mg/kg; 2) increased VA by rearings with 0.075 mg/kg; 3) increased duration of le<strong>an</strong>ing with 0.3<br />
mg/kg; 4) increased working memory with 0.15 mg/kg. Histochemical data revealed 1) <strong>an</strong><br />
increase in TH expression in medial <strong>an</strong>d lateral mesencephalon along with dorsal <strong>an</strong>d ventral<br />
striatum with 0.3mg/kg. In summary, IN-DA reduced hyperactivity (0.3 mg/kg), improved nonselective<br />
(0.075 mg/kg) <strong>an</strong>d selective attention (0.15 mg/kg) <strong>an</strong>d increased <strong>the</strong> density of THpositive<br />
sites (0.3 mg/kg). While <strong>the</strong> <strong>for</strong>mer result may indicate a signal nature of DA itself <strong>for</strong><br />
pruning, <strong>the</strong> behavioral data suggest a potential application of IN-DA <strong>for</strong> <strong>the</strong>rapeutic<br />
m<strong>an</strong>agement of hum<strong>an</strong> ADHD.<br />
Disclosures: L.A. Ruocco, None; M.A. de Souza Silva, None; D. Viggi<strong>an</strong>o, None; C. Treno,<br />
None; B. Topic, None; C. Mattern, None; J.P. Huston, None; A.G. Sadile, None.
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.2/GG17<br />
Topic: F.02.b. Executive function<br />
Title: Does <strong>the</strong> prefrontal cortex per<strong>for</strong>m a behaviorally specific function? An <strong>an</strong>alysis of firing<br />
statistics across PFC-dependent <strong>an</strong>d -independent tasks<br />
Authors: *J. M. HYMAN 1 , E. BALAGUER-BALLESTER 2 , D. DURSTEWITZ 2 , C. C.<br />
LAPISH 1 , A. BLAIR 1 , J. K. SEAMANS 1 ;<br />
1 Dept Psych, Univ. British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada; 2 Central Inst. of Mental Health,<br />
Univ. of Heidelburg, Heidelburg, Germ<strong>an</strong>y<br />
Abstract: The traditional localization viewpoint in neuroscience is based on <strong>the</strong> assumption that<br />
damage or activation of a specific region provides in<strong>for</strong>mation about <strong>the</strong> normal function of <strong>the</strong><br />
region. Damage to <strong>the</strong> primate <strong>an</strong>d rodent prefrontal cortex (PFC) affects only a small number of<br />
complex tasks (e.g. working memory, set shifting, conflict <strong>an</strong>d response inhibition), while m<strong>an</strong>y<br />
simple tasks are unaffected (e.g. lever pressing, novelty exploration, single trial <strong>for</strong>aging <strong>for</strong> food<br />
<strong>an</strong>d discrimin<strong>an</strong>t responding) even though <strong>the</strong>se behaviors are a part of more complex tasks. As a<br />
result, <strong>the</strong> PFC has been ascribed various highly specific functions. Yet recording <strong>an</strong>d imaging<br />
studies have reported signific<strong>an</strong>t activity during tasks that are unaffected by PFC lesions. To<br />
investigate whe<strong>the</strong>r <strong>the</strong> neural coding schemes differ <strong>for</strong> ‘PFC-dependent’ <strong>an</strong>d ‘PFCindependent’<br />
tasks, we recorded <strong>from</strong> large PFC networks during a series of increasingly<br />
complex tasks that are unaffected by PFC lesions (exploration, <strong>for</strong>aging, continuous alternation),<br />
culminating with <strong>the</strong> PFC-dependent delayed alternation task. We used <strong>an</strong> array of statistical<br />
tests comparing firing both across <strong>an</strong>d within tasks global population activity (inter-spike interval<br />
<strong>an</strong>d power spectrum distributions), temporally discrete population activity (inst<strong>an</strong>t<strong>an</strong>eous firing<br />
rate (iFR) distributions), synchrony (cross-correlations among units both in terms of iFR <strong>an</strong>d<br />
precise spiking times) as well individual neuron correlates (firing rate comparisons of behavioral<br />
epochs). Though a small number of tests detected signific<strong>an</strong>t vari<strong>an</strong>ce across or within tasks, no<br />
consistent or clear task related patterns emerged over <strong>the</strong> different measures. Moreover, all tests<br />
of <strong>the</strong> most direct comparison between a PFC-dependent <strong>an</strong>d a PFC-independent task<br />
(continuous vs delayed alternation) found no differences. The percentage of behaviorally<br />
correlated neurons was also similar, though <strong>the</strong> allocation of correlates was slightly different.<br />
Adding a delay appeared to recruit <strong>an</strong> additional 5-10% of cells that showed no correlates on <strong>the</strong><br />
continuous alternation task <strong>an</strong>d more neurons differentiated lever press type (left vs right) in<br />
delayed alternation. These results suggest that neural activity does not appreciably differ between<br />
simple PFC independent tasks <strong>an</strong>d more complex PFC dependent tasks with delays. From a
neural coding perspective <strong>the</strong> function of <strong>the</strong> medial PFC seems to be tracking of behavioral<br />
events across a wide r<strong>an</strong>ge of tasks.<br />
Disclosures: J.M. Hym<strong>an</strong>, None; E. Balaguer-Ballester, None; D. Durstewitz, None; C.C.<br />
Lapish, None; A. Blair, None; J.K. Seam<strong>an</strong>s, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.3/GG18<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t MH083973<br />
NIH Gr<strong>an</strong>t NS048995<br />
NIH Gr<strong>an</strong>t NS41083<br />
DoD# W81XWH-07-1-0012<br />
Swiss Federal Institute of Technology Zürich<br />
Title: Selective enh<strong>an</strong>cement of spatial working memory <strong>an</strong>d reversal learning following<br />
adenosine A2A receptor knockout restricted to <strong>the</strong> <strong>for</strong>ebrain or striatum<br />
Authors: *C. J. WEI 1,2 , P. SINGER 4 , D. BOISON 5 , J. FELDON 4 , B. K. YEE 4 , J.-F. CHEN 1,3 ;<br />
1 Dept. of Pharmacol., 2 Program in Biomed. Neurosci., 3 Dept. of Neurol., Boston Univ. Sch. of<br />
Med., Boston, MA; 4 Lab. of Behavioural Neurobio., Swiss Federal Inst. of Technol. Zürich,<br />
Schwerzenbach, Switzerl<strong>an</strong>d; 5 R.S. Dow Neurobio. Labs., Legacy Res., Portl<strong>an</strong>d, OR<br />
Abstract: Adenosine is a CNS neuromodulator involved in a variety of critical brain processes<br />
including memory <strong>an</strong>d cognition. Recent studies have shown that <strong>the</strong> adenosine A2A receptor<br />
(A2AR) may regulate memory processes in <strong>the</strong> spatial domain including spatial recognition,<br />
reference, <strong>an</strong>d working memory. These effects likely reflect <strong>the</strong> contribution of A2ARs in<br />
multiple relev<strong>an</strong>t brain regions, namely <strong>the</strong> cortex, hippocampus, <strong>an</strong>d striatum. Regional specific<br />
m<strong>an</strong>ipulations are <strong>the</strong>re<strong>for</strong>e required to dissect <strong>the</strong> precise relative functional contributions of<br />
A2ARs to cognitive processing. Following our initial demonstration that striatal <strong>an</strong>d extra-striatal<br />
(cortical <strong>an</strong>d hippocampal) A2ARs c<strong>an</strong> be dissociated in terms of behavioral <strong>an</strong>d
neurotr<strong>an</strong>smitter modulation (Shen et al. 2008, J Neurosci., 2970-5), we compared <strong>the</strong> relative<br />
impact of <strong>for</strong>ebrain- (cortex, hippocampus, <strong>an</strong>d striatum) versus striatum-specific A2AR<br />
knockout (fb-KO vs str-KO) on spatial learning: spatial recognition memory in <strong>the</strong> Y-maze,<br />
followed by working memory, reference memory, <strong>an</strong>d reversal learning in <strong>the</strong> water maze.<br />
Spatial recognition requiring familiarity judgment of spatial cues was unaffected in both<br />
knockout mouse lines at retention dem<strong>an</strong>ds r<strong>an</strong>ging <strong>from</strong> 2min to 24h. Acquisition of a spatial<br />
reference memory requiring <strong>the</strong> <strong>an</strong>imals to learn <strong>the</strong> fixed position of <strong>an</strong> escape plat<strong>for</strong>m hidden<br />
in <strong>the</strong> water maze was also unaffected. In contrast, when <strong>the</strong> escape plat<strong>for</strong>m was moved to a<br />
new location after successful acquisition of <strong>the</strong> <strong>for</strong>mer location, both fb-KO <strong>an</strong>d str-KO mice<br />
demonstrated enh<strong>an</strong>ced learning compared to <strong>the</strong>ir wild-type littermate controls. This effect was<br />
also accomp<strong>an</strong>ied by enh<strong>an</strong>ced working memory function: when <strong>the</strong> escape plat<strong>for</strong>m position,<br />
which remained unch<strong>an</strong>ged between trials on a given day, was shifted daily to a new location, fb-<br />
KO <strong>an</strong>d str-KO mice showed greater improvement <strong>from</strong> trial 1 to 2 compared to controls. In<br />
conclusion, our results demonstrate that striatal A2ARs assume a critical role in learning <strong>an</strong>d that<br />
suppression of A2AR activity, particularly in <strong>the</strong> striatum, may facilitate <strong>the</strong> cognitive flexibility<br />
required <strong>for</strong> effective per<strong>for</strong>m<strong>an</strong>ce under conditions of high proactive interference.<br />
Disclosures: C.J. Wei, None; P. Singer, None; D. Boison, None; J. Feldon, None; B.K. Yee,<br />
None; J. Chen, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.4/GG19<br />
Topic: F.02.b. Executive function<br />
Support: DA023109<br />
Title: The effects of delta-9-tetrahydroc<strong>an</strong>nabinol on working memory in adolescent rhesus<br />
monkeys<br />
Authors: *C. VERRICO, C. W. BRADBERRY, D. A. LEWIS;<br />
Psychiatry, Univ. of Pittsburgh, Pittsburgh, PA<br />
Abstract: Chronic c<strong>an</strong>nabis use has been associated with impairments in executive functions<br />
that persist even after abstinence. Executive functions, such as working memory (WM), improve<br />
during adolescence when <strong>the</strong> circuitry of <strong>the</strong> dorsolateral prefrontal cortex matures, suggesting<br />
that adolescence might be a particularly sensitive period <strong>for</strong> <strong>the</strong> development of c<strong>an</strong>nabis-
induced cognitive impairments. Although few hum<strong>an</strong> studies have addressed this issue,<br />
accumulating evidence indicates that c<strong>an</strong>nabis exposure during adolescence may lead to lasting<br />
deficits in executive functions. In fact, c<strong>an</strong>nabis use during adolescence has been associated with<br />
<strong>an</strong> increased risk of developing schizophrenia, a disorder characterized by persistent WM<br />
impairments. However, hum<strong>an</strong> studies are confounded by several variables that make data<br />
interpretation difficult. For example, it is difficult to account <strong>for</strong> potential pre-exposure<br />
differences between users <strong>an</strong>d non-users in intellectual, cognitive, or psychological attributes. In<br />
addition, chronic c<strong>an</strong>nabis use has been hypo<strong>the</strong>sized to produce <strong>an</strong> amotivational syndrome<br />
such that users might become less engaged in activities that promote cognitive development<br />
during adolescence. Finally, qu<strong>an</strong>tifying <strong>the</strong> degree of exposure to c<strong>an</strong>nabis <strong>an</strong>d determining <strong>the</strong><br />
impact of comorbid factors, such as poly-drug use, is difficult. In order to directly address <strong>the</strong><br />
effects of c<strong>an</strong>nabis exposure during adolescence while controlling <strong>for</strong> <strong>the</strong>se confounds,<br />
adolescent (~ 24 months of age) rhesus monkeys are trained on WM [delayed match-to-sample<br />
(DMTS) <strong>an</strong>d spatial-delayed response (SDR)] <strong>an</strong>d control tasks prior to <strong>the</strong> onset of drug<br />
administration. Training on <strong>the</strong> WM tasks are considered complete when <strong>an</strong>imals achieve 70%<br />
correct responses in 96 consecutive trials at a 1 sec delay in 4 out of 5 consecutive sessions on<br />
both tasks. Animals <strong>the</strong>n begin one month of daily (Mon-Fri) baseline sessions. Delays of 1, 4, 8,<br />
<strong>an</strong>d 16 sec are presented in r<strong>an</strong>domized order, with 20 trials <strong>for</strong> each delay. Current data suggest<br />
that <strong>an</strong>imals c<strong>an</strong> be trained to criterion on <strong>the</strong> tasks in 4 months <strong>an</strong>d that administration of 40<br />
µg/kg (which approximates <strong>the</strong> intake of THC <strong>from</strong> a c<strong>an</strong>nabis cigarette), impairs per<strong>for</strong>m<strong>an</strong>ce<br />
on <strong>the</strong> DMTS task. Thus, after baseline training, <strong>an</strong>d as <strong>the</strong> initial phase of a long-term exposure<br />
study, 6 pairs of adolescent monkeys, in which each pair is matched <strong>for</strong> sex, age <strong>an</strong>d baseline<br />
per<strong>for</strong>m<strong>an</strong>ce, receive escalating doses (20 µg/kg to 60 µg/kg) of THC each week <strong>an</strong>d are tested<br />
30 minutes after THC exposure. Dose-response data on <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of each THC-exposed<br />
monkey, compared to <strong>the</strong>ir own baseline per<strong>for</strong>m<strong>an</strong>ce <strong>an</strong>d to <strong>the</strong>ir matched control partner, will<br />
be presented.<br />
Disclosures: C. Verrico, None; C.W. Bradberry, None; D.A. Lewis, Currently receive<br />
investigator-initiated research support <strong>from</strong> <strong>the</strong> BMS Foundation, Bristol-Myers Squibb,<br />
Curridium Ltd <strong>an</strong>d Pfizer, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or<br />
o<strong>the</strong>r in-kind support); in <strong>the</strong> areas of target identification <strong>an</strong>d validation <strong>an</strong>d new compound<br />
development to AstraZeneca, Bristol-Myers Squibb, Hoffm<strong>an</strong>-Roche, Lilly, Merck <strong>an</strong>d<br />
Neurogen., F. Consult<strong>an</strong>t/Advisory Board.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.5/GG20
Topic: F.02.b. Executive function<br />
Support: NIA Gr<strong>an</strong>t P01 AG024387<br />
NIENH Gr<strong>an</strong>t T32 ES007326<br />
Title: Impaired executive function in rodents consuming multiple daily genistein doses<br />
Authors: *S. L. NEESE 1 , S. B. BANDARA 1 , W. G. HELFERICH 1 , D. R. DOERGE 2 , D. L.<br />
KOROL 1 , S. L. SCHANTZ 1 ;<br />
1 Univ. Illinois, Champaign, IL; 2 Natl. Ctr. <strong>for</strong> Toxicological Res., Jefferson, AR<br />
Abstract: The use of extracts that are highly enriched in phytoestrogens, such as genistein, has<br />
become popular to promote healthy aging. These compounds are promoted as safe, natural<br />
alternatives to hormone replacement <strong>the</strong>rapy (HRT). Previous research in our lab found that once<br />
daily oral genistein treatment in ovariectomized (OVX) female Long-Ev<strong>an</strong>s (LE) rats resulted in<br />
subtle alterations in per<strong>for</strong>m<strong>an</strong>ce on tests of executive function. The present study fur<strong>the</strong>r<br />
modeled hum<strong>an</strong> exposure to genistein by dosing 12-month old OVX female LE rats three times<br />
daily at a dose resulting in serum concentrations similar to those that could be achieved in<br />
hum<strong>an</strong>s consuming commercially available soy isoflavone supplements. Sucrose pellets (control<br />
or 0.97 mg genistein) were orally administered to <strong>an</strong>imals daily, 30 minutes be<strong>for</strong>e behavioral<br />
testing, <strong>an</strong>d again both 4 <strong>an</strong>d 8 hours later. The test battery consisted of a delayed spatial<br />
alternation task (DSA) which tested working memory <strong>an</strong>d a differential rein<strong>for</strong>cement of low<br />
rates of responding (DRL) task that tested time estimation <strong>an</strong>d inhibitory control. Sucrose treated<br />
<strong>an</strong>imals per<strong>for</strong>med better th<strong>an</strong> genistein treated <strong>an</strong>imals on <strong>the</strong> DSA task, with <strong>the</strong> genistein<br />
treated <strong>an</strong>imals committing more lose-stay errors. Sucrose control rats also per<strong>for</strong>med better th<strong>an</strong><br />
genistein exposed rats on <strong>the</strong> DRL task, making fewer lever presses overall <strong>an</strong>d having a higher<br />
ratio of rein<strong>for</strong>ced to non-rein<strong>for</strong>ced lever presses th<strong>an</strong> <strong>the</strong> genistein treated group. Although<br />
<strong>the</strong>se effects were much clearer in rats dosed 3 times daily th<strong>an</strong> in rats given <strong>the</strong> same genistein<br />
dose only once daily, <strong>the</strong> effects of genistein on <strong>the</strong>se tasks were still less pronounced th<strong>an</strong> <strong>the</strong><br />
striking deficits we have observed following chronic estradiol exposure on <strong>the</strong> same tasks in<br />
previous studies.<br />
Disclosures: S.L. Neese, None; S.B. B<strong>an</strong>dara, None; W.G. Helferich, None; D.R. Doerge,<br />
None; D.L. Korol, None; S.L. Sch<strong>an</strong>tz, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 579.6/GG21<br />
Topic: F.02.b. Executive function<br />
Support: NIH AA007474<br />
Title: Chronic phencyclidine attenuates phase-locking of prefrontal neurons to hippocampal<br />
<strong>the</strong>ta during working memory task<br />
Authors: *K. E. ABERNATHY, L. J. CHANDLER;<br />
Med. Univ. of South Carolina, Charleston, SC<br />
Abstract: Executive functioning (EF) is a collection of processes involved in behavioral <strong>an</strong>d<br />
cognitive flexibility, rule acquisition, <strong>an</strong>d use of short-term memory <strong>for</strong> <strong>the</strong> pl<strong>an</strong>ning of future<br />
actions. The prefrontal cortex has long been known to orchestrate EF, <strong>an</strong>d impaired EF in<br />
phencyclidine (PCP) users may reflect disturb<strong>an</strong>ces in prefrontal activity. Working memory<br />
(WM) is one facet of EF <strong>an</strong>d involves <strong>the</strong> active m<strong>an</strong>ipulation <strong>an</strong>d monitoring of goal-related<br />
in<strong>for</strong>mation to derive predictions <strong>an</strong>d guide decisions. In behavioral tests of WM, one subset of<br />
tasks depends on <strong>the</strong> communication of spatial in<strong>for</strong>mation <strong>from</strong> <strong>the</strong> hippocampus to <strong>the</strong><br />
prefrontal cortex that is used to direct goal-oriented behavior. Previous work has demonstrated<br />
that chronic exposure to PCP results in spatial working memory deficits in rats. In <strong>the</strong> present<br />
study, we sought to determine if communication between <strong>the</strong> hippocampus <strong>an</strong>d prefrontal cortex<br />
is disrupted after chronic exposure to PCP during a working memory task. Multi-electrode arrays<br />
were impl<strong>an</strong>ted in dorsal hippocampus <strong>an</strong>d medial prefrontal cortex of Long-Ev<strong>an</strong>s rats, <strong>an</strong>d<br />
multiple single unit recordings obtained while <strong>the</strong> <strong>an</strong>imals per<strong>for</strong>med a spatial working memory<br />
task. Behavioral testing <strong>an</strong>d unit recordings were made both be<strong>for</strong>e <strong>an</strong>d after rats underwent<br />
chronic PCP exposure. A decrease in phase locking of prefrontal neurons to <strong>the</strong> hippocampal<br />
<strong>the</strong>ta rhythm was observed after PCP exposure, suggesting a disruption in communication<br />
between <strong>the</strong> two regions. This effect was partially ameliorated by <strong>the</strong> administration of clozapine<br />
be<strong>for</strong>e a testing session.<br />
Disclosures: K.E. Abernathy, None; L.J. Ch<strong>an</strong>dler, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.7/GG22<br />
Topic: F.02.b. Executive function
Support: PHS Gr<strong>an</strong>t AG022698<br />
PHS Gr<strong>an</strong>t AG029289<br />
Busch Foundation Gr<strong>an</strong>t<br />
Title: Selective attention training improves general learning abilities in CD-1 outbred mice<br />
Authors: *K. R. LIGHT, S. KOLATA, D. COLAS-ZELIN, C. WASS, A. WADDELL, L. D.<br />
MATZEL;<br />
Rutgers Univ., Piscataway, NJ<br />
Abstract: In hum<strong>an</strong>s, general intelligence, or "g," is a single domin<strong>an</strong>t factor that influences<br />
multiple domain-specific learning abilities. We have previously reported <strong>an</strong> <strong>an</strong>alogous latent<br />
factor in CD-1 outbred mice. The latent ability described by this factor has been shown to impact<br />
per<strong>for</strong>m<strong>an</strong>ce across diverse learning tasks including <strong>the</strong> Lashley III maze, Morris water maze,<br />
one-trial passive avoid<strong>an</strong>ce task, associative fear conditioning, radial arm maze, <strong>an</strong>d odor-guided<br />
discrimination. In both hum<strong>an</strong>s <strong>an</strong>d in mice, general intelligence is highly related to <strong>the</strong> efficacy<br />
of components of <strong>the</strong> working memory system, in particular selective attention. To assess <strong>the</strong><br />
possibility that a causal relationship exists between general intelligence <strong>an</strong>d working memory,<br />
we devised a working memory training exercise to improve not only working memory but, more<br />
specifically, selective attention abilities in our mice. To this end, we trained mice to asymptote<br />
on two radial arm mazes with overlapping extra-maze visual cues, after which <strong>the</strong>y were<br />
required to per<strong>for</strong>m in both mazes simult<strong>an</strong>eously. This procedure (conducted over 12 days) was<br />
presumed to tax selective attention. <strong>When</strong> compared to equivalently h<strong>an</strong>dled controls, trained<br />
mice had improved selective attention (as measured by a mouse-adapted Stroop task) as well as<br />
<strong>an</strong> increased capacity to learn across six different learning tasks (i.e. improved general learning<br />
abilities). To determine whe<strong>the</strong>r <strong>the</strong> selective attention load of <strong>the</strong> training was responsible <strong>for</strong><br />
increasing learning abilities, we devised a second training regimen which decreased <strong>the</strong> selective<br />
attention load of <strong>the</strong> training by using non-overlapping cues. Although mice trained on that<br />
modified regimen still demonstrated enh<strong>an</strong>ced general learning abilities (compared to<br />
equivalently h<strong>an</strong>dled, food deprived, <strong>an</strong>d rewarded controls), <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce was intermediate<br />
between controls <strong>an</strong>d mice trained with overlapping cues. Thus, reducing <strong>the</strong> selective attention<br />
load of <strong>the</strong> task reduced <strong>the</strong> efficacy of <strong>the</strong> “working memory exercise.” Lastly, we examined <strong>the</strong><br />
effect of <strong>the</strong> working memory training regimen with overlapping cues on mice at a younger age<br />
(starting training in pre-pubescence). Those mice trained starting in pre-pubescence showed a<br />
increase in general learning abilities compared to both equivalently h<strong>an</strong>dled, food deprived, <strong>an</strong>d<br />
rewarded controls <strong>an</strong>d compared to mice trained in young-adulthood. At present, differences in<br />
attention-mediating brain are undergoing histological <strong>an</strong>alysis to identify potential mediators of<br />
this robust behavioral influence on general learning abilities.<br />
Disclosures: K.R. Light, None; S. Kolata, None; D. Colas-Zelin, None; C. Wass, None; A.<br />
Waddell, None; L.D. Matzel, None.
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.8/GG23<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t MH069374<br />
Title: Dynamics of fronto-parietal coherence during visual working memory<br />
Authors: *N. M. DOTSON, R. F. SALAZAR, C. M. GRAY;<br />
Mont<strong>an</strong>a State Univ., Bozem<strong>an</strong>, MT<br />
Abstract: The fronto-parietal cortical network plays a key role in controlling working memory.<br />
These processes are thought to involve <strong>the</strong> temporal coordination of activity within <strong>an</strong>d between<br />
areas of <strong>the</strong> prefrontal(PFC) <strong>an</strong>d posterior parietal (PPC) cortical regions. Previously, we<br />
reported task-dependent fronto-parietal coherence, during <strong>the</strong> delay period of a working memory<br />
task, in <strong>the</strong> beta (15-25 Hz) frequency r<strong>an</strong>ge of <strong>the</strong> local field potential (LFP). Here we<br />
characterize <strong>the</strong> time-dependent dynamics of <strong>the</strong>se interactions. We recorded <strong>the</strong> LFP <strong>from</strong> 2-6<br />
sites simult<strong>an</strong>eously in both <strong>the</strong> PFC <strong>an</strong>d PPC of a macaque monkey per<strong>for</strong>ming a rule-based,<br />
delayed match-to-sample task in which <strong>the</strong> monkey was required to remember ei<strong>the</strong>r <strong>the</strong> location<br />
or <strong>the</strong> identity of <strong>the</strong> sample object. The LFP was b<strong>an</strong>dpass filtered (10-50 Hz) <strong>an</strong>d a moving<br />
window correlation <strong>an</strong>alysis (200 ms window, ±50 ms time lag) was per<strong>for</strong>med on all pairs of<br />
signals on each trial. The peak correlation coefficients were compared to shuffled surrogate<br />
distributions <strong>from</strong> <strong>the</strong> same data (p80% correct responses), <strong>the</strong> probability <strong>an</strong>d magnitude of correlation within <strong>an</strong>d<br />
between <strong>the</strong> PFC <strong>an</strong>d PPC was tr<strong>an</strong>siently suppressed during <strong>the</strong> sample stimulus, increased<br />
monotonically during <strong>the</strong> delay period, <strong>an</strong>d dropped again following <strong>the</strong> match stimulus. These<br />
ch<strong>an</strong>ges were accomp<strong>an</strong>ied by a decrease in <strong>the</strong> vari<strong>an</strong>ce of <strong>the</strong> correlation phase lag during <strong>the</strong><br />
delay that reached a minimum at match onset. These effects occurred in 18(16) of 85(85) PFC<br />
pairs, 58(46) of 102(102) PPC pairs, <strong>an</strong>d 87(82) of 243(243) PFC-PPC pairs during <strong>the</strong><br />
location(identity) task. In a subset of <strong>the</strong> pairs, correlation probability differed signific<strong>an</strong>tly<br />
across <strong>the</strong> set of 9 sample stimuli used in <strong>the</strong> task. These results demonstrate a time-dependent<br />
increase in <strong>the</strong> magnitude <strong>an</strong>d precision of neuronal synchrony within <strong>an</strong>d between <strong>the</strong> PFC <strong>an</strong>d<br />
PPC that is task specific <strong>an</strong>d peaks just prior to <strong>the</strong> match stimulus. Thus, synchronization of<br />
neural activity across <strong>the</strong> fronto-parietal network is likely to play <strong>an</strong> import<strong>an</strong>t role in <strong>the</strong> control<br />
of working memory <strong>an</strong>d visuospatial attention.<br />
Disclosures: N.M. Dotson, None; R.F. Salazar, None; C.M. Gray, None.
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.9/GG24<br />
Topic: F.02.b. Executive function<br />
Title: A prefrontal cortex network representation of mental set in rats<br />
Authors: *J. K. SEAMANS 1 , J. M. HYMAN 1 , C. C. LAPISH 1 , E. BALAGUER-<br />
BALLESTER 2 , L. MA 1 , D. DURSTEWITZ 2 ;<br />
1 2<br />
Psychiatry, UBC, V<strong>an</strong>couver, BC, C<strong>an</strong>ada; Central Inst. of Mental Health, Univ. of<br />
Heidelburg, Heidelburg, Germ<strong>an</strong>y<br />
Abstract: A multitude of <strong>the</strong>ories have attempted to describe <strong>the</strong> complex <strong>an</strong>d elusive function<br />
of <strong>the</strong> prefrontal cortex (PFC). Most accounts have been based on <strong>the</strong> behaviorally specific<br />
deficits that lesions cause. However imaging <strong>an</strong>d recording studies have revealed similar activity<br />
during PFC-dependent <strong>an</strong>d PFC-independent tasks (see Hym<strong>an</strong> et al. <strong>abstract</strong>). We recorded<br />
<strong>from</strong> large PFC networks in behaving <strong>an</strong>imals , revealing robust ch<strong>an</strong>ges that occur in <strong>the</strong><br />
activity <strong>an</strong>d org<strong>an</strong>ization of PFC networks in response to different neutral contexts <strong>an</strong>d stimuli.<br />
Utilizing powerful ma<strong>the</strong>matical techniques that allowed us to characterize <strong>the</strong> state of <strong>the</strong> entire<br />
recorded network through time, we reliably observed distinct processing states when <strong>an</strong>imals<br />
were in different contexts or presented with neutral objects in a context. This <strong>an</strong>alysis allows us<br />
to plot a point representing <strong>the</strong> firing rates of all simult<strong>an</strong>eously recorded neurons within a 5s bin<br />
(5s bins were used to capture more stable representations). <strong>When</strong> points cluster toge<strong>the</strong>r in this<br />
space, it me<strong>an</strong>s that activity across <strong>the</strong> network (i.e relative firing rates of all neurons) was<br />
similar <strong>for</strong> <strong>the</strong> respective time bins. Thus, across multiple subjects <strong>an</strong>d sessions, movement in <strong>the</strong><br />
space was mainly, <strong>an</strong>d consistently, influenced by three major factors: context, what action was<br />
taken in that context <strong>an</strong>d how long <strong>the</strong> <strong>an</strong>imal had been in a given context. Fur<strong>the</strong>rmore, when<br />
<strong>the</strong> same task (<strong>for</strong>aging or continuous alternation) was per<strong>for</strong>med in two distinct contexts, <strong>the</strong><br />
separation in activity states was much smaller, indicating that <strong>the</strong> PFC networks treated <strong>the</strong> two<br />
contexts as more similar when <strong>the</strong> rat was per<strong>for</strong>ming a task. In this way, <strong>the</strong> task itself beg<strong>an</strong> to<br />
<strong>for</strong>m <strong>the</strong> context ra<strong>the</strong>r th<strong>an</strong> simply <strong>the</strong> environmental cues. . Finally, <strong>the</strong> state of <strong>the</strong> system<br />
smoothly ch<strong>an</strong>ged with <strong>the</strong> passage of time. PFC networks <strong>the</strong>re<strong>for</strong>e track multiple aspects of <strong>the</strong><br />
rat’s experience, taking into account immediate sensory stimuli <strong>an</strong>d actions, task context <strong>an</strong>d<br />
time. We term <strong>the</strong> global representation of <strong>the</strong>se factors a mental set because it combines diverse<br />
in<strong>for</strong>mation into a behaviorally relev<strong>an</strong>t whole. The ability to <strong>for</strong>m such mental sets appears to<br />
occur automatically <strong>an</strong>d immediately in <strong>an</strong> array of situations but may only be necessary under<br />
certain circumst<strong>an</strong>ces. For inst<strong>an</strong>ce, without <strong>the</strong> ability to combine <strong>the</strong> current contextual,
ehavioral <strong>an</strong>d temporal in<strong>for</strong>mation into a task relev<strong>an</strong>t mental set, <strong>an</strong>imals would not be able to<br />
realize that task rules had ch<strong>an</strong>ged (set-shifting) or that in<strong>for</strong>mation obtained on a single trial is<br />
functionally different <strong>from</strong> in<strong>for</strong>mation obtained over successive previous trials (working<br />
memory).<br />
Disclosures: J.K. Seam<strong>an</strong>s, None; J.M. Hym<strong>an</strong>, None; C.C. Lapish, None; E. Balaguer-<br />
Ballester, None; L. Ma, None; D. Durstewitz, None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.10/GG25<br />
Topic: F.02.b. Executive function<br />
Support: MIUR 2005-2007 gr<strong>an</strong>t to AGS<br />
Young-Investigator project, coordinated by Walter Adri<strong>an</strong>i as PI, to LAR .<br />
Title: Prepuberal subchronic methylphenidate <strong>an</strong>d atomoxetine induce differential long-term<br />
effects on adult behaviour <strong>an</strong>d <strong>for</strong>ebrain dopamine, norepinephrine <strong>an</strong>d serotonin in Naples<br />
High-Excitability rats<br />
Authors: *A. G. SADILE 1 , L. A. RUOCCO 1 , U. A. GIRONI CARNEVALE 2 , C. TRENO 1 , R.<br />
CONTE 1 , C. ARRA 3 , M. IBBA 4 , C. SCHIRRU 4 , E. CARBONI 4 ;<br />
1 Second Univ. Naples (SUN), Naples, Italy; 2 Dept. Neurosci., Univ. Naples “Federico II”,<br />
Naples, Italy; 3 Animal Facility, Natl. C<strong>an</strong>cer Institute"G.Pascale", Naples, Italy; 4 Dept. Toxicol,<br />
Univ.Cagliari, Cagliari, Italy<br />
Abstract: Dopamine (DA) <strong>an</strong>d Norepinephrine (NE), brain pathways have been involved in<br />
different aspects of in<strong>for</strong>mation processing, cognition <strong>an</strong>d behaviour. Thus DA <strong>an</strong>d NE are<br />
involved in neuropsychiatric syndromes e.g. Schizophrenia, Autism <strong>an</strong>d Attention Deficit<br />
Hyperactivity Disorder (ADHD). The aim of this study was to investigate long-term effects of<br />
prepuberal methylphenidate (MPH) <strong>an</strong>d atomoxetine (ATX) on adult behaviour <strong>an</strong>d <strong>for</strong>ebrain<br />
DA, NE, Serotonin (5HT) <strong>an</strong>d <strong>the</strong>ir metabolite tissue content in Naples High-Excitability rats<br />
(NHE). They have been used as genetic model <strong>for</strong> mesocortical vari<strong>an</strong>t of ADHD. Male NHE<br />
rats were given <strong>an</strong> intra peritoneal injection of 1.0 mg/kg of MPH, ATX or vehicle, daily <strong>for</strong> 14<br />
days in <strong>the</strong> 5 th <strong>an</strong>d 6 th week of life. At <strong>the</strong> age of 70-75 days, rats were exposed to spatial novelty<br />
in <strong>the</strong> Làt <strong>an</strong>d in a radial maze (Olton type). Behaviour was <strong>an</strong>alyzed <strong>for</strong> indices of activity
horizontal (HA), vertical (VA), non selective (NSA) <strong>an</strong>d selective spatial attention (SSA).<br />
Results indicated that only MPH signific<strong>an</strong>tly reduced HA in both novelty paradigms in NHE<br />
rats only <strong>for</strong> MPH. No differences were found <strong>for</strong> frequency <strong>an</strong>d duration of rearings in both<br />
tests. Likewise, no treatment effect was observed <strong>for</strong> orientation in <strong>the</strong> radial maze. DA, NE, 5-<br />
HT <strong>an</strong>d <strong>the</strong>ir metabolites were assessed by HPLC in <strong>the</strong> prefrontal cortex (PFC), cortical motor<br />
area (MC), dorsal <strong>an</strong>d ventral striatum (DS,VS), hippocampus (HPC), <strong>an</strong>d mesencephalon<br />
(MES). Results indicated that MPH decreased i) DA, NE, 5-HT <strong>an</strong>d <strong>the</strong>ir metabolites in <strong>the</strong> PFC;<br />
ii) DA, DOPAC, HVA, 5-HT, 5-HIAA in DS iii) DA, DOPAC, HVA <strong>an</strong>d MHPG but increased<br />
NE in VS, <strong>an</strong>d iv) NE, 5HT <strong>an</strong>d <strong>the</strong>ir metabolites in HPC. ATX increased DA but decreased<br />
MHPG in <strong>the</strong> PFC <strong>an</strong>d decreased DA, DOPAC, HVA, 5HT,5HIAA in DS <strong>an</strong>d MHPG in VS.<br />
There<strong>for</strong>e MPH treatment in prepuberal NHE rats yielded long-lasting effects on a cognitive<br />
behavioural trait, i.e. HA, in two spatial novelty contexts. This in turn may be related to a<br />
differential rearr<strong>an</strong>gement of DA, NE <strong>an</strong>d 5HT circuitries in PFC, DS <strong>an</strong>d VS.<br />
Disclosures: A.G. Sadile, None; L.A. Ruocco, None; U.A. Gironi Carnevale, None; C.<br />
Treno, None; R. Conte, None; C. Arra, None; M. Ibba, None; C. Schirru, None; E. Carboni,<br />
None.<br />
Poster<br />
579. Executive Function: Rodent Models of Memory<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 579.11/GG26<br />
Topic: F.02.b. Executive function<br />
Support: KAKENHI 20020015<br />
KAKENHI 21240024<br />
Title: Task-related activity of neurons in <strong>the</strong> primate dorsolateral prefrontal cortex during a<br />
metamemory paradigm<br />
Authors: *A. TANAKA 1 , S. FUNAHASHI 1,2 ;<br />
1 Grad. Sch. of Hum<strong>an</strong> <strong>an</strong>d Envrn. Studies, 2 Kokoro Res. Ctr., Kyoto Univ., Kyoto, Jap<strong>an</strong><br />
Abstract: The ability to monitor one’s own memory processes is <strong>an</strong> import<strong>an</strong>t feature of hum<strong>an</strong><br />
cognition <strong>an</strong>d is referred to as metamemory. Hum<strong>an</strong> neuropsychological <strong>an</strong>d neuroimaging<br />
studies have demonstrated that <strong>the</strong> prefrontal cortex (PFC) plays a crucial role in metamemory<br />
processes. However, little is known about <strong>the</strong> neuronal mech<strong>an</strong>isms of metamemory, partly
ecause this ability in nonhum<strong>an</strong> <strong>an</strong>imals is difficult to assess objectively. To address this issue,<br />
we recorded single-neuron activity <strong>from</strong> <strong>the</strong> dorsolateral PFC while a rhesus monkey per<strong>for</strong>med<br />
a modified oculomotor working memory task. In this task, <strong>the</strong> monkey was required to remember<br />
<strong>the</strong> location of a visual cue toward which it had to make a saccade after a several-sec delay. Task<br />
difficulty was controlled by varying <strong>the</strong> number of distracters presented during <strong>the</strong> delay <strong>an</strong>d<br />
response periods. Prior to <strong>the</strong> response period, <strong>the</strong> monkey was sometimes allowed to choose<br />
whe<strong>the</strong>r to take or escape <strong>from</strong> a memory test (FrC condition) <strong>an</strong>d was sometimes <strong>for</strong>ced to take<br />
<strong>the</strong> test (FoT condition). The escape option led to <strong>an</strong> easy visually guided saccade task, but only<br />
30 to 50% of correct saccades were rewarded. The monkey chose <strong>the</strong> escape option more<br />
frequently as task difficulty increased. Moreover, <strong>the</strong> proportion of correct responses on <strong>the</strong><br />
memory tests in <strong>the</strong> FrC condition was higher th<strong>an</strong> that in <strong>the</strong> FoT condition. These results<br />
suggest that <strong>the</strong> monkey used <strong>the</strong> metamnemonic ability when deciding whe<strong>the</strong>r or not to take<br />
<strong>the</strong> tests. We observed task-related neuronal activities during <strong>the</strong> cue, delay, choice, <strong>an</strong>d response<br />
periods. Some of <strong>the</strong>se neurons were activated differentially between trials in which <strong>the</strong> monkey<br />
chose to take <strong>the</strong> memory test <strong>an</strong>d trials in which <strong>the</strong> monkey chose to escape. Detailed <strong>an</strong>alysis<br />
of <strong>the</strong>se activities may contribute to <strong>the</strong> underst<strong>an</strong>ding of <strong>the</strong> functional roles of prefrontal<br />
neurons in metamemory processes.<br />
Disclosures: A. T<strong>an</strong>aka, None; S. Funahashi, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.1/GG27<br />
Topic: F.02.b. Executive function<br />
Title: The effects of housing conditions <strong>an</strong>d methylphenidate on two volitional inhibition tasks<br />
Authors: *F. SANABRIA, J. HILL, J. TERRY, P. COVARRUBIAS;<br />
Arizona State Univ., Tempe, AZ<br />
Abstract: Volitional inhibition is <strong>the</strong> ability to withhold <strong>an</strong> oper<strong>an</strong>t response. We assessed<br />
volitional inhibition in rats using two variations of <strong>the</strong> differential rein<strong>for</strong>cement of low rates<br />
(DRL) schedule of rein<strong>for</strong>cement. In <strong>the</strong> Head-Exit variation, sucrose rewards were delivered <strong>for</strong><br />
leaving <strong>the</strong> food hopper (initial response) <strong>an</strong>d <strong>the</strong>n returning (terminal response) at least 6 s later.<br />
The Lever-Press variation was similar, except that <strong>the</strong> initial response was pressing a lever; <strong>the</strong><br />
terminal response was <strong>an</strong> entry to <strong>the</strong> food hopper. Inferences on volitional inhibition were<br />
drawn <strong>from</strong> <strong>the</strong> distribution of waiting times_those between initial <strong>an</strong>d terminal responses.
Yoked non-waiting controls were conducted to distinguish between ch<strong>an</strong>ges in volitional<br />
inhibition <strong>an</strong>d ch<strong>an</strong>ges in overall levels of activity. We evaluated <strong>the</strong> effects of st<strong>an</strong>dard- vs.<br />
enriched-environment (SE vs. EE) rearing <strong>an</strong>d of methylphenidate (0.5, 2, <strong>an</strong>d 8 mg/kg, i.p.) on<br />
completing Head-Exit <strong>an</strong>d Lever-Press sequences, with <strong>an</strong>d without waiting. Head-Exit waiting<br />
per<strong>for</strong>m<strong>an</strong>ce showed <strong>the</strong> typical DRL bimodal distribution; Lever-Press waiting per<strong>for</strong>m<strong>an</strong>ce<br />
dissociated iterated responding <strong>from</strong> timing. In both sequences EE rats waited less th<strong>an</strong> SE rats,<br />
which suggests a relative deficit in volitional inhibition in EE rats. Methylphenidate did not have<br />
a subst<strong>an</strong>tial effect on waiting, but generally decreased latency to sequence initiation, <strong>an</strong>d<br />
increased iterated lever pressing. We discuss <strong>the</strong> implications of <strong>the</strong>se results in light of a<br />
qu<strong>an</strong>titative model of volitional inhibition.<br />
Disclosures: F. S<strong>an</strong>abria, None; J. Hill, None; J. Terry, None; P. Covarrubias, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.2/GG28<br />
Topic: F.02.b. Executive function<br />
Title: An optimized mice training protocol suitable <strong>for</strong> CatWalk, a computer-assisted gait<br />
<strong>an</strong>alysis system<br />
Authors: *R. LODICO 1 , M. PELLITTERI 1 , A. CHAKIR 1 , S. BECCHI 1 , F. SCHIO 1 , R.<br />
MARIOTTI 1 , O. BORTOLAMI 2 , G. VERLATO 2 , A. SBARBATI 1 , P. FABENE 1 ;<br />
1 Dept. of Morphological <strong>an</strong>d Biomed. Sci., Univ. of Verona, Verona, Italy; 2 Epidemiology <strong>an</strong>d<br />
Statistics, Univ. of Verona (Italy), Verona, Italy<br />
Abstract: Movement abnormalities are import<strong>an</strong>t symptoms in clinical neurology. Neurological<br />
diseases such as Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis<br />
(ALS), stroke<br />
<strong>an</strong>d pain present with abnormalities of motor function <strong>an</strong>d severe locomotor deficits. Over <strong>the</strong><br />
years, m<strong>an</strong>y methods have been developed to <strong>an</strong>alyze locomotor behavior in small laboratory<br />
<strong>an</strong>imals, particularly in rat.<br />
The CatWalk, a computer-assisted automated qu<strong>an</strong>titative gait <strong>an</strong>alysis system, allows rapid <strong>an</strong>d<br />
objective qu<strong>an</strong>tification of a large number of gait parameters. In comparison to ink-test or similar<br />
approaches, previously used to assess static locomotor alterations, CatWalk c<strong>an</strong> also obtain<br />
dynamic parameters. Despite <strong>the</strong> extensive use of <strong>the</strong> mouse in experimental models <strong>for</strong><br />
neurological disease <strong>the</strong>re have been relatively few studies on CatWalk in mouse. Fur<strong>the</strong>rmore,
few in<strong>for</strong>mation are available regarding methodological aspects on training prior to test.<br />
The aims of <strong>the</strong> present study are: (1) to provide reliable <strong>an</strong>d reproducible results on over-ground<br />
locomotion in intact mouse using three different training protocol <strong>an</strong>d, (2) to <strong>an</strong>alyze <strong>the</strong> effect of<br />
training protocol on over-ground locomotion.<br />
In this study we tested three different experimental training, in order to get <strong>the</strong> best protocol <strong>for</strong><br />
<strong>the</strong> use of <strong>the</strong> CatWalk in mice models. Animals were trained to run <strong>the</strong> walkway <strong>for</strong> different<br />
duration, ei<strong>the</strong>r 11, 4 or 3 days, providing groups A, B <strong>an</strong>d C respectively; mice of groups A <strong>an</strong>d<br />
B were left to cross freely <strong>the</strong> walkway, while group C was <strong>for</strong>ced to run only in one direction.<br />
Mice of groups A <strong>an</strong>d B were subjected to a food deprivation (diet of 75% <strong>an</strong>d <strong>the</strong>n of 30%),<br />
while ad libitum food was available <strong>for</strong> mice of group C. Fur<strong>the</strong>rmore, mice of groups B <strong>an</strong>d C<br />
were rewarded with high palatable food placed in a cage at <strong>the</strong> end of <strong>the</strong> runway, <strong>for</strong> motivating<br />
<strong>the</strong>m.<br />
We objectively qu<strong>an</strong>tified a large number of gait parameters during over-ground locomotion <strong>an</strong>d<br />
we focused <strong>for</strong> <strong>the</strong> first time on relation between <strong>an</strong>imal weight <strong>an</strong>d basic gait parameters<br />
because different training protocols c<strong>an</strong> act as confounder <strong>for</strong> <strong>the</strong> interpretation of <strong>the</strong> results.<br />
The obtained results c<strong>an</strong> thus serve as a template <strong>for</strong> studying ch<strong>an</strong>ges in locomotor per<strong>for</strong>m<strong>an</strong>ce<br />
in mouse due to neurological disease or trauma to <strong>the</strong> CNS <strong>an</strong>d may provide relev<strong>an</strong>t in<strong>for</strong>mation<br />
that c<strong>an</strong> be adv<strong>an</strong>tageous to select a particular experimental protocol to be used in different<br />
models of movement disorders.<br />
Disclosures: R. Lodico, None; M. Pellitteri, None; A. Chakir, None; S. Becchi, None; F.<br />
Schio, None; R. Mariotti, None; O. Bortolami, None; G. Verlato, None; A. Sbarbati,<br />
None; P. Fabene, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.3/GG29<br />
Topic: F.02.b. Executive function<br />
Support: MH074811<br />
Title: The effects of excitotoxic lesions of <strong>the</strong> <strong>an</strong>terior cingulate cortex in rats on conflict<br />
monitoring<br />
Authors: *L. A. NEWMAN, E. S. CARTER, J. A. MCGAUGHY;<br />
Psychol, Univ. New Hampshire, Durham, NH
Abstract: The <strong>an</strong>terior cingulate cortex (ACC) has repeatedly been shown to be active in<br />
paradigms that require conflict monitoring. Activation of <strong>the</strong> ACC occurs when errors are made<br />
<strong>an</strong>d when trials present conflicting in<strong>for</strong>mation. Dysfunction of <strong>the</strong> ACC is hypo<strong>the</strong>sized to<br />
underlie deficits in conflict monitoring shown by patients with attention deficit hyperactivity<br />
disorder (ADHD) <strong>an</strong>d schizophrenia. These subjects tend to emit more errors th<strong>an</strong> controls <strong>an</strong>d<br />
have longer latencies when simult<strong>an</strong>eously presented with stimuli previously paired with<br />
conflicting response rules. Though some studies have found no behavioral differences between<br />
controls <strong>an</strong>d ADHD or schizophrenic patient populations, <strong>the</strong>se studies have found that <strong>the</strong>se<br />
patients have increased activation of <strong>the</strong> ACC during conflict trials. The current study assesses<br />
<strong>the</strong> effects of ibotenic acid lesions of <strong>the</strong> ACC on a novel conflict monitoring paradigm <strong>for</strong> rats.<br />
Male, Long Ev<strong>an</strong>s rats were trained to discriminate between two stimuli in <strong>an</strong> auditory modality<br />
(const<strong>an</strong>t tone vs. pulsing tone) <strong>an</strong>d two stimuli in a visual modality (const<strong>an</strong>t light vs. pulsing<br />
light). Animals were rein<strong>for</strong>ced <strong>for</strong> responding with a left lever press to const<strong>an</strong>t stimuli <strong>an</strong>d a<br />
right lever press to pulsing stimuli. During training, all stimuli were presented in <strong>the</strong> same<br />
modality within one session. During <strong>the</strong> testing phase, stimuli with conflicting stimulus-response<br />
rules were presented simult<strong>an</strong>eously (e.g. const<strong>an</strong>t tone <strong>an</strong>d flashing light) <strong>an</strong>d <strong>an</strong>imals were<br />
rein<strong>for</strong>ced <strong>for</strong> responding to one set of response rules. Rules were primed by presenting rats with<br />
a block of single modality trials in <strong>the</strong> beginning of <strong>the</strong> session <strong>an</strong>d r<strong>an</strong>domly throughout <strong>the</strong> rest<br />
of <strong>the</strong> session to rein<strong>for</strong>ce <strong>the</strong> target modality. All <strong>an</strong>imals showed signific<strong>an</strong>t accuracy<br />
impairments on conflicting trials <strong>an</strong>d longer incorrect latencies to conflicting trials. Sham-Lx<br />
<strong>an</strong>imals also demonstrated accuracy improvements on conflict trials that were preceded<br />
immediately by a conflict trial, <strong>an</strong> effect that has previously been demonstrated in hum<strong>an</strong>s <strong>an</strong>d<br />
has been suggested to be dependent upon increased activation of <strong>the</strong> ACC by <strong>the</strong> first conflict<br />
trial. ACC-Lx <strong>an</strong>imals had signific<strong>an</strong>t longer latencies on correct <strong>an</strong>d incorrect responses to<br />
conflicting trials, <strong>an</strong> effect that increased with time on task. This study suggests that <strong>the</strong> ACC is<br />
involved in <strong>the</strong> efficient processing of conflicting in<strong>for</strong>mation.<br />
Disclosures: L.A. Newm<strong>an</strong>, None; E.S. Carter, None; J.A. McGaughy, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.4/GG30<br />
Topic: F.02.b. Executive function<br />
Title: Working memory opposite phenotypes between COMT female <strong>an</strong>d male null mut<strong>an</strong>t<br />
mice: Potential implication of PFC dopamine <strong>an</strong>d CaMK pathways
Authors: *S. GARCIA, F. PAPALEO, L. ERICKSON, Q. TIAN, J. CHEN, J. CRAWLEY, D.<br />
WEINBERGER;<br />
Natl. Inst. of Mental Hlth., Be<strong>the</strong>sda, MD<br />
Abstract: Catechol-O-methyltr<strong>an</strong>sferase (COMT) plays a major role in <strong>the</strong> metabolism of<br />
dopamine in <strong>the</strong> prefrontal cortex (PFC). Common polymorphisms ch<strong>an</strong>ging COMT activity<br />
impact several domains of hum<strong>an</strong> behavior. Evidence also suggests interactions between COMT<br />
polymorphisms <strong>an</strong>d sex in <strong>the</strong> predisposition to psychiatric disorders. However, despite critical<br />
broad interest, it is not yet known how <strong>an</strong>d why COMT polymorphisms interact with sex in <strong>the</strong><br />
development of abnormal behaviors. Here we contrasted behavioral <strong>an</strong>d molecular ch<strong>an</strong>ges in<br />
COMT knockout female mice with <strong>the</strong>ir male counterparts. In striking contrast to what has been<br />
found in males, decreased COMT activity in females produces working memory impairments<br />
<strong>an</strong>d different PFC molecular ch<strong>an</strong>ges in dopamine <strong>an</strong>d CaMK pathways. No COMT genotype x<br />
sex interaction was found <strong>for</strong> stress reactivity or reference memory. The present findings unravel<br />
a key role <strong>for</strong> <strong>the</strong> COMT gene in <strong>the</strong> modulation of working memory <strong>an</strong>d PFC molecular<br />
org<strong>an</strong>ization in a sex dependent way.<br />
Disclosures: S. Garcia, None; F. Papaleo, None; L. Erickson, None; Q. Ti<strong>an</strong>, None; J. Chen,<br />
None; J. Crawley, None; D. Weinberger, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.5/GG31<br />
Topic: F.02.b. Executive function<br />
Support: NSF IOS-0642951<br />
Title: Motor areas of frontal cortex <strong>an</strong>d <strong>the</strong> top-down control of action<br />
Authors: *N. J. SMITH 1,2 , M. LAUBACH 1 ;<br />
1 2<br />
John B Pierce Lab., New Haven, CT; Interdepartmental Neurosci. Program, Yale Univ. Sch. of<br />
Med., New Haven, CT<br />
Abstract: Medial areas of <strong>the</strong> frontal cortex have been shown to influence neuronal activity in<br />
motor cortex <strong>an</strong>d to mediate top-down control of action (Naray<strong>an</strong><strong>an</strong> <strong>an</strong>d Laubach, 2006).<br />
However, <strong>the</strong>re are no direct <strong>an</strong>atomical connections between medial frontal areas <strong>an</strong>d primary<br />
(caudal) motor cortex in rats (Donoghue & Wise, 1983). In this study, we used tract-tracing
experiments to identify a brain region that may mediate top-down control <strong>an</strong>d <strong>the</strong>n investigated<br />
its role in guiding behavior in a simple reaction time task (Laubach et al., 2000). Intracortical<br />
microstimulation was used to locate <strong>the</strong> rostral <strong>an</strong>d caudal <strong>for</strong>elimb areas (Donoghue & Wise,<br />
1982; Neafsey & Sievert, 1982), located in <strong>the</strong> medial <strong>an</strong>d lateral agr<strong>an</strong>ular areas, respectively.<br />
Biotinylated dextr<strong>an</strong> amine (BDA) was injected at sites that controlled <strong>the</strong> <strong>for</strong>elimbs. These<br />
injections revealed dense projections between <strong>the</strong> rostral <strong>an</strong>d caudal motor areas. Rostral<br />
injections also revealed connections with orbital <strong>an</strong>d medial frontal areas as well as frontal <strong>an</strong>d<br />
sensorimotor nuclei of <strong>the</strong> thalamus. These <strong>an</strong>atomical data suggest that medial frontal areas<br />
exert top-down control of action by acting on neurons in <strong>the</strong> rostral motor cortex. We <strong>the</strong>n<br />
studied <strong>the</strong> role of <strong>the</strong>se cortical areas during a simple RT task by inactivating <strong>the</strong> rostral <strong>an</strong>d<br />
caudal <strong>for</strong>elimb areas using infusions of muscimol. Inactivations of both motor areas resulted in<br />
slowing of responses to <strong>the</strong> trigger stimulus. Rostral, but not caudal, infusions also resulted in<br />
altered behavior after errors (Naray<strong>an</strong><strong>an</strong> <strong>an</strong>d Laubach, 2008): increased lever pressing during<br />
time-outs (lack of context control) <strong>an</strong>d repeated errors (lack of per<strong>for</strong>m<strong>an</strong>ce adjustment). These<br />
results suggest that <strong>the</strong> rostral <strong>an</strong>d caudal <strong>for</strong>elimb representations in <strong>the</strong> rat motor cortex are<br />
dissociable based on <strong>an</strong>atomical <strong>an</strong>d behavioral criteria <strong>an</strong>d that <strong>the</strong> rostral part of motor cortex<br />
may enable top-down control of action through its connections with <strong>the</strong> medial frontal cortex.<br />
Disclosures: N.J. Smith, None; M. Laubach, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.6/GG32<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t MH48404<br />
Pittsburgh Life sciences Greenhouse<br />
Title: Medial prefrontal cortex encoding of oper<strong>an</strong>t set-shifting is selectively impaired by<br />
NMDA receptor <strong>an</strong>tagonist<br />
Authors: *H. HOMAYOUN 1 , B. MOGHADDAM 2 ;<br />
1 Univ. Pittsburgh, Pittsburgh, PA; 2 Univ. of Pittsburgh, Pittsburgh, PA<br />
Abstract: The ability to shift between distinct sets of rules is one of <strong>the</strong> key mech<strong>an</strong>isms through<br />
which prefrontal cortex (PFC) exerts control over behavior. Selective impairment of set-shifting
<strong>an</strong>d cognitive flexibility occurs in schizophrenia <strong>an</strong>d o<strong>the</strong>r disorders associated with PFC<br />
malfunction. We hypo<strong>the</strong>sized that neurons in medial PFC (mPFC) participate in encoding <strong>for</strong><br />
set-shifting in <strong>an</strong> oper<strong>an</strong>t paradigm consisting of two perceptual discrimination rules (side versus<br />
light). Rats were trained to achieve a stable level of per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> set-shifting task <strong>an</strong>d <strong>the</strong>n<br />
were treated with vehicle, <strong>the</strong> psychotomimetic NMDA <strong>an</strong>tagonist MK801 or <strong>the</strong> combination of<br />
MK801 <strong>an</strong>d <strong>the</strong> <strong>an</strong>tipsychotic agent clozapine. Using in vivo extracellular recording, we<br />
identified subsets of mPFC neurons that selectively encoded <strong>for</strong> instrumental responses only<br />
during a distinct rule (rule encoder) or only immediately after a shift (shift encoder). Toge<strong>the</strong>r,<br />
<strong>the</strong>se flexible responder cells <strong>for</strong>med <strong>the</strong> majority of recorded mPFC neurons. In addition,<br />
<strong>an</strong>o<strong>the</strong>r subset of cells selectively encoded <strong>for</strong> <strong>the</strong> perseverative errors. As previously reported,<br />
MK801 impaired set-shifting ability <strong>an</strong>d increased perseverative errors (Darrah et al, <strong>2009</strong>). This<br />
impairment was associated with a selective decrease in <strong>the</strong> proportion of neurons with a flexible<br />
response, specially <strong>the</strong> shift encoder neurons, <strong>an</strong>d neurons encoding <strong>for</strong> perseverative errors. Cotreatment<br />
with clozapine ameliorated <strong>the</strong> impairment in behavioral set-shifting <strong>an</strong>d maintained<br />
encoding <strong>for</strong> both shifts <strong>an</strong>d perseverative errors. Thus, <strong>the</strong> mPFC control of cognitive flexibility<br />
involves distinct neuronal ensembles dedicated to supervision of shifts between rules <strong>an</strong>d<br />
monitoring of perseverative errors. While NMDA <strong>an</strong>tagonist selectively disrupts <strong>the</strong>se shiftrelated<br />
neuronal ensembles in association with behavioral impairment, clozapine c<strong>an</strong> restore <strong>the</strong><br />
cognitive flexibility with preserving <strong>the</strong> mPFC encoding of set-shifting.<br />
Disclosures: H. Homayoun, None; B. Moghaddam, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.7/GG33<br />
Topic: F.02.b. Executive function<br />
Support: Wellcome Trust<br />
Title: Sex differences in novelty-preference <strong>an</strong>d behavioural measures <strong>from</strong> adolescence to<br />
adulthood: The influence of gonadal hormones <strong>an</strong>d dopamine<br />
Authors: *D. M. CYRENNE, G. R. BROWN;<br />
Sch. Psychol, Univ. of St Andrews, St Andrews, United Kingdom<br />
Abstract: In hum<strong>an</strong> beings, novelty-seeking behaviour is high during adolescence, particularly<br />
in males, <strong>an</strong>d has been associated with dopaminergic neurotr<strong>an</strong>smitter system. Rodent studies
have shown that <strong>the</strong> dopamine system ch<strong>an</strong>ges across adolescence <strong>an</strong>d differs in function<br />
between males <strong>an</strong>d females. Additionally, gonadal hormone production increases during<br />
adolescence in both hum<strong>an</strong>s <strong>an</strong>d rodents. However, few studies have investigated ch<strong>an</strong>ges in<br />
novelty-seeking behaviour across adolescence in male <strong>an</strong>d female rodents. In our study, male<br />
<strong>an</strong>d female Lister-hooded rats were tested on <strong>the</strong> novel object recognition task at ei<strong>the</strong>r postnatal<br />
day 21, 28, 40 or 80 (we<strong>an</strong>ing to adulthood). The test consisted of a 10-minute familiarisation<br />
trial, in which <strong>the</strong> <strong>an</strong>imal was placed in a novel box (68 x 68cm), followed by i) a 5-minute trial<br />
with 2 novel objects <strong>an</strong>d ii) a 5-minute trial prior to which one of <strong>the</strong> objects had been replaced<br />
with a different novel object. As females showed a stronger side bias th<strong>an</strong> males, side<br />
preferences in each trial were calculated <strong>an</strong>d adjusted to reflect Novelty Preference Ch<strong>an</strong>ge<br />
between <strong>the</strong> 2 trials, with movement more towards <strong>the</strong> novel object indicating novelty<br />
preference. The results showed that males displayed a preference <strong>for</strong> novelty increasing with age,<br />
with a peak at PD 40, followed by a decline at PD 80. Females, however, did not show noveltypreference<br />
until PD 80. The results also showed that <strong>the</strong> total amount of contact with <strong>the</strong> objects,<br />
<strong>the</strong> duration of locomotion, <strong>an</strong>d time spent in <strong>the</strong> centre of <strong>the</strong> apparatus increased with age <strong>for</strong><br />
both males <strong>an</strong>d females, with males peaking at PD 40, <strong>an</strong>d females not until PD 80. Although<br />
<strong>the</strong>se measures increase linearly due to development, <strong>the</strong>y do so at different rates <strong>for</strong> males <strong>an</strong>d<br />
females. With novelty-preference, males exhibit a greater degree th<strong>an</strong> females during<br />
adolescence. These results are consistent with <strong>the</strong> literature where males have <strong>an</strong> increase in<br />
dopamine D4 receptors through PD 40. D4 receptors have not yet been examined <strong>for</strong> sex<br />
differences in development, unlike D1 <strong>an</strong>d D2 receptors which do show <strong>an</strong> increase in males, but<br />
not females. Although differences in striatal D1 <strong>an</strong>d D2 were found to be unrelated to gonadal<br />
hormones production in adolescence, D4 is primarily found in <strong>the</strong> cortex where hormonal<br />
influences were not examined. Studies are in progress to examine <strong>the</strong> influence of gonadal<br />
hormones <strong>an</strong>d D4 on sex differences in novelty-seeking behaviour at PD 40 <strong>an</strong>d adulthood. This<br />
study supports <strong>the</strong> hypo<strong>the</strong>sis that response to novelty ch<strong>an</strong>ges across adolescence in male <strong>an</strong>d<br />
female rats, which might be linked to developmental ch<strong>an</strong>ges in <strong>the</strong> dopamine system, <strong>an</strong>d may<br />
be influenced by <strong>the</strong> ch<strong>an</strong>ges in hormone production.<br />
Disclosures: D.M. Cyrenne, None; G.R. Brown, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.8/GG34<br />
Topic: F.02.b. Executive function<br />
Support: MH074811
Title: Prenatal protein malnutrition in male rats alters susceptibility to distraction in a test of<br />
visual sustained attention<br />
Authors: *D. J. MOKLER, Ph.D. 1 , P. J. MORGANE 1 , A. BATES 1 , J. R. GALLER 2 , J. A.<br />
MCGAUGHY 3 ;<br />
1 Dept Pharmacol., Univ. New Engl<strong>an</strong>d, Bidde<strong>for</strong>d, ME; 2 Psychiatry, Judge Baker Children's<br />
Center, Harvard Med. Sch., Boston, MA; 3 Psychology, Univ. of New Hampshire, Durham, NH<br />
Abstract: Exposure to prenatal malnutrition has been shown in hum<strong>an</strong>s to increase <strong>the</strong> incidence<br />
of disorders of executive function including schizophrenia, depression <strong>an</strong>d attention deficit<br />
hyperactivity disorder (ADHD). The use of <strong>an</strong> <strong>an</strong>imal model of prenatal protein malnutrition<br />
allows <strong>the</strong> determination of <strong>the</strong> neurobiological basis of <strong>the</strong>se deficits in executive function. In<br />
<strong>the</strong> present study we tested male, Long-Ev<strong>an</strong>s rats on a test of visual, sustained attention. In<br />
addition to assessing per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> st<strong>an</strong>dard version of <strong>the</strong> task, we assessed <strong>the</strong> effects of<br />
visual <strong>an</strong>d auditory distractors on <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce. We found that prenatal protein malnutrition<br />
produces a decrease in susceptibility to visual distractors as evidenced by <strong>the</strong> superior<br />
per<strong>for</strong>m<strong>an</strong>ce of malnourished (6% protein prenatally) rats to well-nourished rats (25% protein<br />
prenatally) during sessions with <strong>the</strong> houselight flashing. There was no difference in <strong>the</strong><br />
per<strong>for</strong>m<strong>an</strong>ce between groups when <strong>the</strong> distractor was in a different modality th<strong>an</strong> <strong>the</strong> target<br />
stimuli. In contrast to <strong>the</strong> effects of distraction, rats previously exposed to 6% prenatal protein<br />
showed greater accuracy impairments on <strong>the</strong> st<strong>an</strong>dard sustained attention task th<strong>an</strong> 25% rats if<br />
correct responses were no longer rein<strong>for</strong>ced with food pellets. Per<strong>for</strong>m<strong>an</strong>ce did not differ<br />
between <strong>the</strong> groups when a variable or consistent delay was introduced between correct<br />
responses <strong>an</strong>d rein<strong>for</strong>cement suggesting that delays in rein<strong>for</strong>cement are equally well tolerated<br />
by both 6% <strong>an</strong>d 25% rats. These data suggest that prefrontal systems critical to processing taskirrelev<strong>an</strong>t<br />
stimuli <strong>an</strong>d ch<strong>an</strong>ges in rein<strong>for</strong>cement contingencies are compromised by prenatal<br />
malnutrition. Additionally, <strong>the</strong> persistence of <strong>the</strong>se deficits in adult rats exposed to prenatal<br />
protein malnutrition in utero without additional insult post-natally will be discussed in terms of<br />
its implications <strong>for</strong> adults with executive function disorders.<br />
Disclosures: D.J. Mokler, None; P.J. Morg<strong>an</strong>e, None; A. Bates, None; J.R. Galler,<br />
None; J.A. McGaughy, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.9/GG35<br />
Topic: F.02.b. Executive function
Title: Characterization of <strong>the</strong> pro-cognitive effect of <strong>the</strong> mGlu5 positive allosteric modulators<br />
(PAM) ADX47273 <strong>an</strong>d CDPPB<br />
Authors: *L. LERDRUP 1 , N. PLATH 1 , C. MURPHY 2 , J. ALFRED 2 , M. UBERTI 2 , C.<br />
BONVICINO 2 , M. CAJINA 2 , D. SMITH 2 , L. MELTZER 2 , R. BRODBECK 2 ;<br />
1 H. Lundbeck A/S, Valby, Denmark; 2 Lundbeck Res. USA, Inc., Paramus, NJ<br />
Abstract: Psychiatric (schizophrenia) <strong>an</strong>d neurological (Alzheimer’s disease (AD)) disorders are<br />
characterized by deficits in temporal <strong>an</strong>d frontal lobe mediated cognitive behaviors. Activation<br />
of mGlu5 receptors enh<strong>an</strong>ces NMDA-induced neurotr<strong>an</strong>smission which is known to be impaired<br />
in schizophrenia (<strong>an</strong>d AD) <strong>an</strong>d linked to cognitive processing. Acute <strong>an</strong>d sub-chronic<br />
administration of <strong>the</strong> NMDA receptor <strong>an</strong>tagonist phencyclidine (PCP) induces schizophrenialike<br />
symptoms in healthy subjects <strong>an</strong>d exacerbates <strong>the</strong>se symptoms in patients with<br />
schizophrenia. In <strong>the</strong> present study, ADX47273 <strong>an</strong>d CDPPB, which have been determined to be<br />
mGlu5 PAMs with in vitro binding <strong>an</strong>d functional activity assays, were assessed <strong>for</strong> <strong>the</strong>ir procognitive<br />
properties in <strong>the</strong> attentional set-shifting <strong>an</strong>d novel object recognition tasks in rats <strong>an</strong>d<br />
<strong>the</strong> continuous alternation task in mice.<br />
In <strong>the</strong> attentional set-shifting (intra-/extradimensional; ID/ED) executive function task, adult rats<br />
were administered PCP (5 mg/kg, b.i.d.) <strong>for</strong> 7 days. After a 7 day washout period, <strong>the</strong>y were<br />
trained to dig in bowls baited with a food reward <strong>an</strong>d to discriminate <strong>the</strong> bowls based on odour or<br />
digging media. On test day, rats per<strong>for</strong>med a series of discriminations following acute<br />
administration of ei<strong>the</strong>r vehicle, CDPPB (80 mg/kg, p.o.) or ADX47273 (0.1-10 mg/kg, i.p.).<br />
Impaired executive function (ED function) was observed in PCP-treated rats (extra-dimensional<br />
or ED function), <strong>an</strong>d this was signific<strong>an</strong>tly reversed by mGlu5 PAM treatment.<br />
The T-maze continuous alternation task assesses working memory <strong>an</strong>d, like attentional setshifting,<br />
is a measure of pre-frontal cortex functioning. ADX47273 (0.1-30 mg/kg, i.p.) was<br />
shown to signific<strong>an</strong>tly reverse a scopolamine (1 mg/kg, i.p.) induced deficit in alternation<br />
behaviour in mice. The mGlu5 PAM reversal of <strong>the</strong> scopolamine-induced cognitive deficit was<br />
equally as signific<strong>an</strong>t as that of donepezil (0.3 mg/kg, i.p.).<br />
Finally, a novel object recognition task was used to assess <strong>the</strong> pro-cognitive potential of <strong>an</strong><br />
mGlu5 PAM. ADX47273 (0.1-30 mg/kg, i.p.) signific<strong>an</strong>tly increased time spent exploring a<br />
novel object in <strong>the</strong> rat visual episodic memory task object recognition. In this, time delayinduced<br />
deficit, novel object recognition task, ADX47273 was equally as efficacious a procognitive<br />
as donepezil (0.5 mg/kg, i.p.).<br />
Taken toge<strong>the</strong>r, <strong>the</strong>se data imply that positive modulation of <strong>the</strong> mGlu5 receptor may have procognitive<br />
efficacy which might be beneficial <strong>for</strong> both schizophrenic <strong>an</strong>d Alzheimer’s patients.<br />
Disclosures: L. Lerdrup, H. Lundbeck A/S, Otilliavej 9, 2500 Valby, Denmark, A.<br />
Employment (full or part-time); N. Plath, H. Lundbeck A/S, Otilliavej 9, 2500 Valby, Denmark,<br />
A. Employment (full or part-time); C. Murphy, Lundbeck Research USA, Inc, Paramus, USA,<br />
A. Employment (full or part-time); J. Alfred, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time); M. Uberti, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time); C. Bonvicino, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time); M. Cajina, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time); D. Smith, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time); L. Meltzer, Lundbeck Research USA, Inc, Paramus, USA, A.
Employment (full or part-time); R. Brodbeck, Lundbeck Research USA, Inc, Paramus, USA, A.<br />
Employment (full or part-time).<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.10/GG36<br />
Topic: F.02.b. Executive function<br />
Support: NIMH Gr<strong>an</strong>t MH48404<br />
Pittsburgh Life Sciences Greenhouse<br />
Title: The effects of methylphenidate on single unit activity <strong>an</strong>d local field potential oscillations<br />
in <strong>an</strong>terior cingulate <strong>an</strong>d medial prefrontal cortex during preparatory attention<br />
Authors: *N. K. B. TOTAH, B. MOGHADDAM;<br />
Neurosci., Univ. of Pittsburgh, Pittsburgh, PA<br />
Abstract: Attention-deficit hyperactivity disorder (ADHD) <strong>an</strong>d schizophrenia are both<br />
associated with prefrontal cortex (PFC) dysfunctions, such as alterations of neural activity <strong>an</strong>d<br />
reduced dopaminergic tr<strong>an</strong>smission. Fur<strong>the</strong>rmore, low dose methylphenidate (Ritalin), which is<br />
commonly used to treat ADHD, may improve cognition by increasing dopamine<br />
neurotr<strong>an</strong>smission in <strong>the</strong> PFC. We have recently demonstrated that putative pyramidal neurons in<br />
<strong>the</strong> rat <strong>an</strong>terior cingulate cortex (ACC) <strong>an</strong>d <strong>the</strong> medial PFC (mPFC) exhibit phasic excitatory <strong>an</strong>d<br />
inhibitory responses prior to <strong>an</strong> attended cue in <strong>the</strong> 3-choice serial reaction time task (Totah et al.<br />
J Neurosci.20: 6418. <strong>2009</strong>). These responses are related to <strong>the</strong> level of preparatory (precue)<br />
attention <strong>an</strong>d subsequent correct or incorrect choices, in that <strong>the</strong> magnitude of <strong>the</strong> single units’<br />
response to <strong>the</strong> cue was lower on incorrect trials <strong>an</strong>d was not different th<strong>an</strong> baseline on<br />
unattended trials. In order to assess <strong>the</strong> effects of methylphenidate on attention-related neural<br />
activity, we recorded both local field potential (LFP) <strong>an</strong>d single unit firing patterns during <strong>the</strong> 3choice<br />
serial reaction time task be<strong>for</strong>e <strong>an</strong>d after acute treatment of methylphenidate or vehicle.<br />
The 3-choice task requires a rat to orient toward <strong>an</strong>d divide attention between 3 brief (300 msec<br />
duration) light stimuli presented in r<strong>an</strong>dom order across nose poke holes in <strong>an</strong> oper<strong>an</strong>t<br />
conditioning chamber. We recorded neural activity during 10 min of task per<strong>for</strong>m<strong>an</strong>ce, followed<br />
by i.p. administration of vehicle, 0.25 mg/kg methylphenidate, or 1.0 mg/kg methylphenidate,<br />
<strong>an</strong>d <strong>the</strong>n recorded <strong>an</strong>o<strong>the</strong>r 30 min of neural activity during <strong>the</strong> task. We found that low dose<br />
(0.25 mg/kg) methylphenidate increased delta <strong>an</strong>d <strong>the</strong>ta oscillations in LFP during preparatory
attention compared to vehicle. The <strong>an</strong>alysis of ch<strong>an</strong>ges in <strong>the</strong> number of responsive single units<br />
<strong>an</strong>d attention-related neural activity is ongoing. Given that individuals with ADHD exhibit<br />
abnormal LFP oscillatory activity in <strong>the</strong> PFC that may be corrected by methylphenidate, <strong>the</strong>se<br />
studies provide import<strong>an</strong>t tr<strong>an</strong>slational in<strong>for</strong>mation about neuronal alterations associated with<br />
ADHD. By studying ch<strong>an</strong>ges in neural activity at a “multi-scale” level (both LFP <strong>an</strong>d single<br />
units), we c<strong>an</strong> assess <strong>the</strong> discrete <strong>an</strong>d distributed networks that are implicated in <strong>the</strong> mech<strong>an</strong>ism<br />
of action of methylphenidate.<br />
Disclosures: N.K.B. Totah, None; B. Moghaddam, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.11/GG37<br />
Topic: F.02.b. Executive function<br />
Support: National Institute on Alcohol Abuse <strong>an</strong>d Alcoholism Intramural Research Program<br />
Title: Corticostriatal mediation of over-learning <strong>an</strong>d cognitive flexibility in a mouse touchscreen<br />
apparatus<br />
Authors: *J. L. BRIGMAN 1 , C. GRAYBEAL 1 , T. WRIGHT 1 , L. M. SAKSIDA 2 , T. J.<br />
BUSSEY 2 , S. JINDE 3 , M. I. DAVIS 1 , K. NAKAZAWA 3 , E. DELPIRE 4 , D. M. LOVINGER 1 , A.<br />
HOLMES 1 ;<br />
1 NIAAA/NIH, Rockville, MD; 2 Univ. of Cambridge, Cambridge, United Kingdom;<br />
3 NIMH/NIH, Be<strong>the</strong>sda, MD; 4 V<strong>an</strong>derbilt Univ., Nashville, TN<br />
Abstract: The prefrontal cortex (PFC) <strong>an</strong>d dorsal striatum (DS) are thought to mediate <strong>the</strong><br />
learning <strong>an</strong>d expression of efficient, automatized behavior (e.g., habit) <strong>an</strong>d cognitive flexibility<br />
in <strong>the</strong> face of shifting environmental conditions, respectively. Functional alterations that favor<br />
<strong>the</strong> <strong>for</strong>mer are associated with neuropsychiatric disorders such as OCD <strong>an</strong>d addiction. However,<br />
<strong>the</strong> precise neural circuitry <strong>an</strong>d molecular basis of <strong>the</strong>se functions are still not fully understood.<br />
Here we employed a number of approaches to examine corticostriatal function in a mouse<br />
touchscreen-based oper<strong>an</strong>t procedure <strong>for</strong> assessing rewarded pairwise visual discrimination<br />
learning <strong>an</strong>d reversal (a test of cognitive flexibility). We first mapped patterns of corticostriatal<br />
functional activation by qu<strong>an</strong>tifying immunoreactivity of <strong>the</strong> immediate-early gene c-Fos in<br />
C57BL/6J mice per<strong>for</strong>ming at various stages of discrimination (ch<strong>an</strong>ce, well-learned) <strong>an</strong>d<br />
reversal (early/high perseveration, ch<strong>an</strong>ce, well-learned). To test <strong>for</strong> synaptic physiological
correlates of behavioral per<strong>for</strong>m<strong>an</strong>ce, we per<strong>for</strong>med ex vivo recordings of DS medium spiny<br />
neurons. Next, to determine whe<strong>the</strong>r PFC <strong>an</strong>d DS were necessary <strong>for</strong> discrimination learning<br />
<strong>an</strong>d/or reversal, we assessed <strong>the</strong>se behaviors following pre-training excitotoxic lesions of <strong>the</strong><br />
ventrolateral orbital or infralimbic/prelimbic subregions of PFC, or lateral or medial divisions of<br />
<strong>the</strong> DS. Finally, to test <strong>for</strong> a potential molecular mech<strong>an</strong>ism subserving corticostriatal regulation<br />
of <strong>the</strong>se behaviors, we phenotyped mut<strong>an</strong>t mice in which <strong>the</strong> NMDA receptor NR2B subunit was<br />
deleted in ei<strong>the</strong>r PFC but not DS, or PFC <strong>an</strong>d DS. Results showed that c-Fos activity in <strong>the</strong> DS<br />
increased as mice learned <strong>the</strong> discrimination <strong>an</strong>d increased in <strong>the</strong> PFC during reversal. Synaptic<br />
plasticity in <strong>the</strong> DS also ch<strong>an</strong>ged with behavioral per<strong>for</strong>m<strong>an</strong>ce. Preliminary data suggest <strong>the</strong> loss<br />
of NR2B in PFC but not DS predomin<strong>an</strong>tly impairs reversal, while loss of NR2B in both<br />
structures impairs discrimination. These findings provide new insight into <strong>the</strong> role of<br />
corticostriatal circuitry in <strong>the</strong> mediation of well-learned behavior <strong>an</strong>d cognitive flexibility. This<br />
could ultimately have implications <strong>for</strong> elucidating <strong>the</strong> pathophysiology <strong>an</strong>d treatment of various<br />
neuropsychiatric disorders. Research supported by <strong>the</strong> National Institute on Alcohol Abuse <strong>an</strong>d<br />
Alcoholism Intramural Research Program.<br />
Disclosures: J.L. Brigm<strong>an</strong>, None; C. Graybeal, None; T. Wright, None; L.M. Saksida,<br />
None; T.J. Bussey, None; S. Jinde, None; M.I. Davis, None; K. Nakazawa, None; E. Delpire,<br />
None; D.M. Lovinger, None; A. Holmes, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.12/GG38<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t 1R01NS063009<br />
Autism Speaks Gr<strong>an</strong>t<br />
Title: Impaired reversal learning in Lc/+ mice with complete cerebellar Purkinje cell loss:<br />
Relev<strong>an</strong>ce to autism<br />
Authors: *M. M. MILLER 1 , P. E. DICKSON 1 , E. L. CLARDY 1 , N. DEL MAR 2 , D. H.<br />
HECK 2 , D. GOLDOWITZ 3 , C. D. BLAHA 1 , G. MITTLEMAN 1 ;<br />
1 Dept Psychology, Univ. Memphis, Memphis, TN; 2 Dept. of Anat. & Neurobio., Univ. of<br />
Tennessee Hlth. Sci. Ctr., Memphis, TN; 3 Child <strong>an</strong>d Family Res. Inst., Ctr. <strong>for</strong> Mol. Med. <strong>an</strong>d<br />
Therapeutics, Dept Med. Genet., Univ. of British Columbia, V<strong>an</strong>couver, BC, C<strong>an</strong>ada
Abstract: Autism spectrum disorders are characterized by impairments in social interaction <strong>an</strong>d<br />
communication, as well as restricted, repetitive, stereotyped patterns of behavior.<br />
Neuropsychological studies suggest that this r<strong>an</strong>ge of abnormal behaviors is secondary to more<br />
fundamental cognitive impairments in attention, memory, <strong>an</strong>d executive function. Post mortem<br />
<strong>an</strong>d imaging studies of autistic brains show neuropathology of <strong>the</strong> cerebellum, with <strong>the</strong> most<br />
consistent finding being a reduction in <strong>the</strong> number of cerebellar Purkinje cells, <strong>the</strong> only efferent<br />
projections arising <strong>from</strong> <strong>the</strong> cerebellar cortex. In order to explore <strong>the</strong> role of <strong>the</strong> cerebellum in<br />
executive function, we used a mouse model of developmental Purkinje cell loss. Specifically, we<br />
made aggregation chimeras (Lc/+↔+/+) between lurcher (Lc/+) mut<strong>an</strong>t embryos <strong>an</strong>d wildtype<br />
control (+/+) embryos. Lurcher mice lose 100% of <strong>the</strong>ir Purkinje cells postnatally due to a cellintrinsic<br />
gain-of-function mutation. Chimeric mice have varying numbers of Purkinje cells,<br />
depending on <strong>the</strong> degree of incorporation of <strong>the</strong> wildtype lineage. In this study, we tested lurcher,<br />
wildtype, <strong>an</strong>d chimeric mice in a visual serial reversal learning task. Mice were trained to press<br />
one of two levers <strong>for</strong> a liquid reward in response to <strong>the</strong> illumination of a stimulus light displaying<br />
ei<strong>the</strong>r a horizontal or vertical bar. After acquiring <strong>the</strong> task, all mice completed at least two serial<br />
reversals in which <strong>the</strong> stimulus-reward contingencies were reversed. We found that lurcher mice<br />
committed signific<strong>an</strong>tly more errors on <strong>the</strong> first <strong>an</strong>d second serial reversals relative to chimeric<br />
<strong>an</strong>d wildtype mice, indicating a tendency by lurcher mice to perseverate following a reversal of<br />
<strong>the</strong> stimulus-reward contingencies. Acquisition of <strong>the</strong> visual discrimination was comparable<br />
across <strong>the</strong>se groups. These results are consistent with recent findings indicating that <strong>an</strong>imals with<br />
cerebellar damage show impaired per<strong>for</strong>m<strong>an</strong>ce on multiple tests of executive function (Bartolo<br />
<strong>2009</strong>; Lalonde 2008).<br />
Disclosures: M.M. Miller, None; P.E. Dickson, None; E.L. Clardy, None; N. Del Mar,<br />
None; D.H. Heck, None; D. Goldowitz, None; C.D. Blaha, None; G. Mittlem<strong>an</strong>, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.13/GG39<br />
Topic: F.02.b. Executive function<br />
Title: Mech<strong>an</strong>isms of plasticity in simple taxis behaviors in Drosophila<br />
Authors: *M.- . RENTINCK, B. BEUSTER, B. BREMBS;<br />
Freie Univ. Berlin/ Neurobiologie, Berlin, Germ<strong>an</strong>y
Abstract: Like <strong>the</strong> proverbial moth drawn to <strong>the</strong> c<strong>an</strong>dle flame, <strong>the</strong> fruit fly Drosophila also<br />
stereotypically approaches light sources. This positive phototaxis is <strong>the</strong> archetypal example of<br />
hard-wired input-output behaviors. However, it has long been known that defects to <strong>the</strong> wings of<br />
<strong>the</strong> fly, ei<strong>the</strong>r by mutation or by damage, reduce not only phototaxis but also geotaxis in walking<br />
Drosophila. If <strong>the</strong>se behaviors are so hard-wired, how c<strong>an</strong> m<strong>an</strong>ipulating <strong>an</strong> unrelated org<strong>an</strong> affect<br />
<strong>the</strong>m? Using <strong>the</strong> classic countercurrent photo-/geotaxis essay developed by Seymour Benzer, we<br />
tested <strong>the</strong> hypo<strong>the</strong>sis that instead of taxis being a simple matter of stimulus <strong>an</strong>d response, <strong>the</strong>re<br />
may be a central decision-making stage which is influenced by <strong>the</strong> wing m<strong>an</strong>ipulations. We<br />
discovered that <strong>the</strong> phenomenon of reduced taxis in flies with m<strong>an</strong>ipulated wings is very robust.<br />
The reduction in photo-/geotaxis c<strong>an</strong> be observed not only in several different strains of wild<br />
type flies, but also in flies with white eyes, in strains which fail in multiple learning paradigms<br />
<strong>an</strong>d in flies with impaired mushroom-body function. However, flies with wings de<strong>for</strong>med by<br />
mutation (<strong>an</strong>d thus unable to fly) did not reduce <strong>the</strong>ir taxis <strong>an</strong>y fur<strong>the</strong>r. This results suggests that<br />
<strong>the</strong> ability to fly <strong>an</strong>d not injury or pain determines <strong>the</strong> reduction in photo-/geotaxis. Experiments<br />
without guiding stimuli (walking horizontally in <strong>the</strong> dark) showed that <strong>the</strong> decrease is due to<br />
central processes <strong>an</strong>d exclusively observed in response to <strong>the</strong> eli<strong>citing</strong> stimuli (see figure, wild<br />
type strain C<strong>an</strong>ton S, N=6). Consistent with our hypo<strong>the</strong>sis, our results suggest that <strong>the</strong>re are<br />
dedicated circuits in <strong>the</strong> nervous system of <strong>the</strong> fly which monitor <strong>the</strong> fly’s ability to fly <strong>an</strong>d<br />
modulate its walking activity depending on this status.<br />
Disclosures: M. Rentinck, None; B. Beuster, None; B. Brembs, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 580.14/GG40<br />
Topic: F.02.b. Executive function<br />
Title: Gene expression profiling in <strong>the</strong> frontal cortex of catechol-o-methyltr<strong>an</strong>sferase gene<br />
knockout <strong>an</strong>d tr<strong>an</strong>sgenic overexpressing COMT-Val mouse models <strong>for</strong> schizophrenia<br />
Authors: *P. GLINEBURG, J. CHEN, Q. TIAN, F. PAPALEO, B. K. LIPSKA, J. PICKEL, T.<br />
YE, D. R. WEINBERGER;<br />
Natl. Inst. of Mental Hlth., Be<strong>the</strong>sda, MD<br />
Abstract: The catechol-o-methyltr<strong>an</strong>sferase (COMT) Val(158)Met polymorphism is associated<br />
with schizophrenia susceptibility <strong>an</strong>d dopamine signaling in prefrontal cortex. Enh<strong>an</strong>ced<br />
dopamine metabolism by COMT-Val allele or limited metabolism by COMT-Met allele could<br />
have widespread influence on tr<strong>an</strong>scription of various genes. Utilizing tr<strong>an</strong>sgenic mice<br />
overexpressing hum<strong>an</strong> COMT-Val <strong>an</strong>d traditional knockout mice <strong>for</strong> COMT gene, we<br />
comprehensively measured mRNA tr<strong>an</strong>script expression in response to variations in COMT<br />
enzyme levels <strong>an</strong>d activity. Expression profiling was per<strong>for</strong>med by microarray <strong>an</strong>alysis of frontal<br />
cortex using Affymetrics 430 2.0 Arrays. Differential expression of regulated genes in COMT<br />
knockout or tr<strong>an</strong>sgenic mice will be verified by qu<strong>an</strong>titative polymerase chain reaction <strong>an</strong>d<br />
western blotting. In COMT-Val <strong>an</strong>d COMT deficient mice, reciprocal mRNA levels were<br />
apparent in genes related to myelination, synapse <strong>for</strong>mation, <strong>an</strong>d cAMP signaling, including<br />
myelin basic protein, activity regulated cytoskeletal protein, <strong>an</strong>d adenylate cyclase. We fur<strong>the</strong>r<br />
found increased mRNA associated with neuronal migration <strong>an</strong>d development, glutamate<br />
neurotr<strong>an</strong>smission, <strong>an</strong>d ubiquitination in COMT deficient mice compared to controls. Our<br />
findings suggest extensive COMT regulation of genes required <strong>for</strong> neuronal architectural<br />
complexity, excitatory neurotr<strong>an</strong>smission, secondary messenger signaling, <strong>an</strong>d neuronal signal<br />
tr<strong>an</strong>sduction. A COMT schizophrenia relation could <strong>the</strong>re<strong>for</strong>e extend far beyond dopamine levels<br />
<strong>an</strong>d include multiple cellular network mech<strong>an</strong>isms.<br />
Disclosures: P. Glineburg, None; J. Chen, None; Q. Ti<strong>an</strong>, None; F. Papaleo, None; B.K.<br />
Lipska, None; J. Pickel, None; T. Ye, None; D.R. Weinberger, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.15/GG41<br />
Topic: F.02.b. Executive function
Support: Roche-Palo Alto<br />
Department of Psychology, Florida State University<br />
Title: Evaluation of dopamine involvement in PCP-induced cognitive deficits in <strong>the</strong> rodent<br />
attentional set-shifting task<br />
Authors: *S. K. SALAND 1,2 , J. S. RODEFER 2 ;<br />
1 Iowa City, IA; 2 Psychology & Neurosci., Florida State Univ., Tallahassee, FL<br />
Abstract: Currently accepted treatments <strong>for</strong> schizophrenia have shown effectiveness in treating<br />
some, but not all, of <strong>the</strong> core deficits associated with <strong>the</strong> disease with <strong>the</strong> improvement of<br />
cognitive dysfunction being a critical unmet need. Cognitive improvement by pharmacological<br />
intervention is often difficult to demonstrate in healthy <strong>an</strong>imals <strong>an</strong>d may have little predictive<br />
validity <strong>for</strong> efficacy in disease models of schizophrenia. A more desirable starting point<br />
methodologically is one where normal cognitive function is disrupted <strong>an</strong>d <strong>the</strong> effect of observed<br />
treatments in ameliorating deficits c<strong>an</strong> be observed. In <strong>the</strong> current series of experiments we<br />
utilized a rodent model of executive function that is sensitive to <strong>the</strong> effects of lesions, natural<br />
aging <strong>an</strong>d pharmacological m<strong>an</strong>ipulations. In addition, we employed a well-validated subchronic<br />
phencyclidine (PCP) administration treatment paradigm to produce enduring cognitive deficits<br />
similar to those observed in schizophrenia. Given <strong>the</strong> well-documented involvement of<br />
dopaminergic mech<strong>an</strong>isms in working memory <strong>an</strong>d aspects of schizophrenia, we investigated <strong>the</strong><br />
acute administration of different dopamine (DA) receptor agonists <strong>an</strong>d <strong>an</strong>tagonists on cognitive<br />
per<strong>for</strong>m<strong>an</strong>ce in <strong>the</strong> set-shifting task. Administration of <strong>the</strong> DA receptor <strong>an</strong>tagonists SCH23390<br />
(DA-D1) <strong>an</strong>d raclopride (DA-D2) failed to improve set-shifting per<strong>for</strong>m<strong>an</strong>ce in rats, whereas <strong>the</strong><br />
effects of DA receptor agonist administration were mixed: SKF81297 (DA-D1) produced modest<br />
improvements in cognitive flexibility whereas cognitive effects resulting <strong>from</strong> quinpirole (DA-<br />
D2) administration were inconsistent. These data suggest that dopamine receptor subtypes in<br />
PCP-treated <strong>an</strong>imals may contribute differentially to aspects of cognitive <strong>an</strong>d behavioral<br />
flexibility in rats that mirrors those core deficits associated with schizophrenia.<br />
Disclosures: S.K. Sal<strong>an</strong>d, None; J.S. Rodefer, Roche-Palo Alto, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.16/GG42
Topic: F.02.b. Executive function<br />
Support: Smoking Resarch Foundation Gr<strong>an</strong>t<br />
Title: The effects of milnacipr<strong>an</strong> on impulsive-like action assessed by <strong>the</strong> 3-choice serial<br />
reaction time task: A simple <strong>an</strong>d valid model of impulsive action using rats<br />
Authors: I. TSUTSUI-KIMURA, Y. OHMURA, *T. IZUMI, T. YAMAGUCHI, Y. KUBO, T.<br />
YOSHIDA, M. YOSHIOKA;<br />
Dept Neuropharmacol, Hokkaido Univ. Sch. Med., Sapporo 060-8638, Jap<strong>an</strong><br />
Abstract: Impulsive acts are often viewed as everyday normal behavior; however, excessive<br />
levels of impulsivity are associated with several psychiatric disorders, underscoring <strong>the</strong> need <strong>for</strong><br />
<strong>an</strong>imal models of impulsive action to develop a brief screening method <strong>for</strong> novel <strong>the</strong>rapeutic<br />
agents of impulsive action. The 5-choice serial reaction time task (5-CSRTT) is one of <strong>the</strong> most<br />
prevalent <strong>an</strong>imal models of impulsive action. Although this task is useful, it takes a long time to<br />
train <strong>the</strong> <strong>an</strong>imals involved.<br />
Excessive levels of impulsivity could also be a risk factor <strong>for</strong> suicide. Suicide in depressed<br />
patients is a major problem of public health. A recent study reported <strong>the</strong> relationship between<br />
impulsive action <strong>an</strong>d suicide attempts in bipolar disorder patients. If some <strong>an</strong>tidepress<strong>an</strong>ts have<br />
beneficial effects on impulsive action, <strong>the</strong>y may not only treat impulsivity-related disorders but<br />
also lower <strong>the</strong> risk of suicide in depressed patients.<br />
Our goals were (1) to validate <strong>the</strong> 3-choice serial reaction time task (3-CSRTT) as a simplified<br />
version of <strong>the</strong> 5-CSRTT <strong>for</strong> approaching impulsive-like action by using nicotine <strong>an</strong>d<br />
atomoxetine that were well characterized in previous studies using <strong>the</strong> 5-CSRTT <strong>an</strong>d (2) to<br />
assess <strong>the</strong> effects of two <strong>an</strong>tidepress<strong>an</strong>t drugs, fluvoxamine (a selective serotonin reuptake<br />
inhibitor) <strong>an</strong>d milnacipr<strong>an</strong> (a serotonin/noradrenaline reuptake inhibitor), in <strong>the</strong> 3-CSRTT.<br />
Following <strong>the</strong> training in <strong>the</strong> 3-CSRTT, rats were administered nicotine (0, 0.1, 0.2, <strong>an</strong>d 0.4<br />
mg/kg, salt, s.c.) or atomoxetine (0, 0.01, 0.1, <strong>an</strong>d, 1.0 mg/kg, i.p.) to validate <strong>the</strong> 3-CSRTT as<br />
compared with <strong>the</strong> previous studies using <strong>the</strong> 5-CSRTT. Rats were administered fluvoxamine (0,<br />
2, 4, <strong>an</strong>d 8 mg/kg, i.p.) or milnacipr<strong>an</strong> (0, 3, <strong>an</strong>d 10 mg/kg, i.p.) to assess <strong>the</strong> effects of <strong>the</strong>se<br />
drugs on impulsive action using <strong>the</strong> 3-CSRTT. The training time <strong>for</strong> <strong>the</strong> 3-CSRTT (<strong>for</strong> about 6<br />
weeks) was signific<strong>an</strong>tly shorter th<strong>an</strong> that <strong>for</strong> <strong>the</strong> 5-CSRTT (<strong>for</strong> about 10 weeks). A comparison<br />
of <strong>the</strong> behavioral parameters in <strong>the</strong> 3-CSRTT with those in <strong>the</strong> 5-CSRTT revealed that data<br />
variability did not differ between two tasks. Nicotine increased, while atomoxetine decreased <strong>the</strong><br />
number of premature responses, <strong>an</strong> index of impulsive action, which is consistent with previous<br />
studies. Moreover, we found that milnacipr<strong>an</strong>, but not fluvoxamine, dose-dependently decreased<br />
premature responses without ch<strong>an</strong>ging <strong>an</strong>y o<strong>the</strong>r behavioral parameters. These results indicate<br />
that <strong>the</strong> 3-CSRTT could provide <strong>an</strong> appropriate <strong>an</strong>d simple rodent model of impulsive action <strong>an</strong>d<br />
that milnacipr<strong>an</strong> could have some beneficial effects on impulsivity-related disorders <strong>an</strong>d might<br />
lower <strong>the</strong> risk of suicide in depressed patients.<br />
Disclosures: I. Tsutsui-Kimura, None; Y. Ohmura, None; T. Izumi, Smoking Resarch<br />
Foundation, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); T. Yamaguchi, None; Y. Kubo, None; T. Yoshida,<br />
None; M. Yoshioka, None.
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.17/GG43<br />
Topic: F.02.b. Executive function<br />
Title: Cognitive bias in <strong>the</strong> chick <strong>an</strong>xiety-depression model<br />
Authors: *A. L. SALMETO, K. A. HYMEL, E. C. CAPENTER, K. J. SUFKA;<br />
Psychology, Univ. Mississippi, Ox<strong>for</strong>d, MS<br />
Abstract: Cognitive disturb<strong>an</strong>ces are core symptoms of <strong>an</strong>xiety <strong>an</strong>d depressive disorders.<br />
Anxious individuals tend to adopt a more pessimistic outlook on life <strong>an</strong>d depressed individuals<br />
tend to adopt a less optimistic outlook on life. In <strong>an</strong> <strong>an</strong>imal stress model, <strong>the</strong> thought is that<br />
“more pessimism” is reflected by increased reactivity to threatening stimuli while “less<br />
optimism” is reflected by decreased approach behaviors to appetitive stimuli, particularly when<br />
<strong>the</strong>se stimuli are somewhat ambiguous. Avi<strong>an</strong> species have been shown to be influenced by a<br />
variety of factors in runway-type tasks, as well as to express cognitive bias in appetitive tasks.<br />
The present series of experiments involve <strong>the</strong> evaluation of cognitive bias in chicks using a<br />
straight alley maze with varying degrees of chick/owl morphed images at <strong>the</strong> end of <strong>the</strong> runway<br />
(mirror, 75% chick image, 50/50 morph of chick <strong>an</strong>d owl, 75% owl image <strong>an</strong>d <strong>an</strong> owl image).<br />
The first experiment involved only <strong>the</strong> mirror stimulus <strong>an</strong>d 95% of chicks reached <strong>the</strong> goal box<br />
within 1 min, with similar results in all fur<strong>the</strong>r experiments. In <strong>the</strong> second experiment, a second<br />
trial was added with <strong>an</strong> owl stimulus; results showed a marked decline in goal latency. In <strong>the</strong><br />
third experiment, chicks were assessed on responses to <strong>the</strong> five stimulus categories <strong>an</strong>d <strong>an</strong><br />
incremental increase in goal latency was observed to images with increasingly threatening<br />
stimulus features. In <strong>the</strong> fourth experiment chicks were tested in <strong>the</strong> maze following exposure to<br />
a social condition, a 5 min isolation period (<strong>an</strong>xiety-like phase, more pessimism) or a 60 min<br />
isolation period (depression-like phase, less optimism) of <strong>the</strong> chick <strong>an</strong>xiety-depression model.<br />
Chicks within <strong>the</strong> social condition did not display differences in start latency <strong>for</strong> <strong>the</strong> stimulus<br />
categories, but did show increases in goal latencies relative to <strong>the</strong> mirror condition <strong>for</strong> all o<strong>the</strong>r<br />
stimulus conditions. Those exposed to <strong>the</strong> <strong>an</strong>xiety-like phase of <strong>the</strong> model displayed increases in<br />
start <strong>an</strong>d goal latencies <strong>for</strong> morphs with greater owl characteristics. Chicks exposed to <strong>the</strong><br />
depression-like phase of <strong>the</strong> model displayed <strong>an</strong> increase in start latencies to all stimuli,<br />
including stimuli with signific<strong>an</strong>t chick characteristics, including <strong>the</strong> mirror. A similar pattern<br />
was seen with goal latency data. These findings demonstrate a pattern of cognitive bias in chicks,<br />
where more pessimistic behavior is present in <strong>the</strong> <strong>an</strong>xiety phase <strong>an</strong>d both more pessimism <strong>an</strong>d<br />
less optimism is present in <strong>the</strong> depression phase.
Disclosures: A.L. Salmeto, None; K.A. Hymel, None; E.C. Capenter, None; K.J. Sufka,<br />
None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.18/GG44<br />
Topic: F.02.b. Executive function<br />
Support: MH074811<br />
Title: Prenatal protein malnutrition produces impairments in attentional set shifting in adult rats<br />
Authors: *J. R. GALLER 1 , A. BATES 2 , D. J. MOKLER 2 , P. MORGANE 2 , J. A.<br />
MCGAUGHY 3 ;<br />
1 Judge Baker Children's Ctr., Boston, MA; 2 Pharmacol., Univ. of New Engl<strong>an</strong>d, Bidde<strong>for</strong>d, ME;<br />
3 Psychology, Univ. of New Hampshire, Durham, NH<br />
Abstract: Malnutrition afflicts 14% of <strong>the</strong> global population <strong>an</strong>d 26% of children under <strong>the</strong> age<br />
of five. Whereas it has long been appreciated that malnutrition impacts health <strong>an</strong>d physical<br />
growth, <strong>the</strong>re is increasing evidence that brain <strong>an</strong>d behavioral development are also adversely<br />
impacted specifically in <strong>the</strong> area of executive function as indicated by higher rates of ADHD,<br />
depression <strong>an</strong>d schizophrenia in <strong>the</strong>se subjects. Toge<strong>the</strong>r <strong>the</strong>se data support <strong>the</strong> hypo<strong>the</strong>sis that<br />
prenatal protein malnutrition may produce dysfunction in <strong>the</strong> prefrontal cortices. To better<br />
determine <strong>the</strong> precise dysfunction in prefrontal cortices, we assessed <strong>the</strong> effects of prenatal<br />
malnutrition in <strong>an</strong> <strong>an</strong>imal model.In <strong>the</strong> present study we tested adult, male rats in <strong>an</strong> intradimensional/<br />
extra-dimensional (ID/ED) set shifting task after prenatal exposure to protein<br />
malnutrition (6% casein diet) or adequate prenatal protein (25%). This task allows independent<br />
assessments of affective <strong>an</strong>d attentional-set shifting abilities. Preliminary data support <strong>the</strong><br />
hypo<strong>the</strong>sis that prenatal protein malnutrition impairs <strong>the</strong> ability of rats to shift attentional set<br />
(ED) with no impact on <strong>the</strong> ability of rats to <strong>for</strong>m <strong>an</strong> attentional set, per<strong>for</strong>m simple<br />
discriminations or affective shifts. As per<strong>for</strong>m<strong>an</strong>ce on <strong>the</strong> ED has been shown to depend upon<br />
noradrenergic afferents to <strong>the</strong> prelimbic cortex, <strong>the</strong>se data suggest that prenatal protein<br />
malnutrition produces impairments in <strong>the</strong> functional integrity of this system.<br />
Disclosures: J.R. Galler, None; A. Bates, None; D.J. Mokler, None; P. Morg<strong>an</strong>e, None; J.A.<br />
McGaughy, None.
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.19/GG45<br />
Topic: F.02.b. Executive function<br />
Support: National Institute on Alcohol Abuse <strong>an</strong>d Alcoholism Intramural Research Program<br />
Title: Stress-induced impairments of cognitive flexibility in a mouse touchscreen-based assay<br />
Authors: *C. GRAYBEAL 1 , M. FEYDER 1 , L. M. SAKSIDA 2 , T. J. BUSSEY 2 , A. HOLMES 1 ;<br />
1 Natl. Inst. on Alcohol Abuse <strong>an</strong>d Alcoholism, NIH, Rockville, MD; 2 Dept. of Exptl.<br />
Psychology, Univ. of Cambridge, Cambridge, United Kingdom<br />
Abstract: Cognitive flexibility is <strong>the</strong> ability to alter behavioral strategies based on ch<strong>an</strong>ging<br />
circumst<strong>an</strong>ces <strong>an</strong>d has been shown to be a prefrontal cortex (PFC)-mediated behavior. A simple<br />
yet effective tool <strong>for</strong> assessing cognitive flexibility across species is reversal learning, in which<br />
previously learned stimulus-reward pairings are reversed. Impairments in cognitive flexibility are<br />
m<strong>an</strong>ifested in various neuropsychiatric disorders <strong>for</strong> which stress c<strong>an</strong> be a risk factor. Here we<br />
examined whe<strong>the</strong>r reversal learning was altered by exposure to a relatively modest stress regime<br />
previously shown to impair o<strong>the</strong>r PFC-mediated executive-like behaviors. C57BL/6J mice<br />
learned a pairwise visual discrimination on a touchscreen-based oper<strong>an</strong>t apparatus, were exposed<br />
to 3 consecutive days of 10-min <strong>for</strong>ced swim stress, <strong>an</strong>d tested <strong>for</strong> reversal of <strong>the</strong> discrimination<br />
1 day later. Results showed that mice exposed to stress made fewer correct responses during <strong>the</strong><br />
first reversal session th<strong>an</strong> non-stressed controls, but did not differ in control measure <strong>for</strong><br />
locomotion (total trials per<strong>for</strong>med) or motivation (choice reaction times <strong>an</strong>d reward retrieval<br />
latency). In separate experiments, <strong>the</strong> same stress regimen did not affect <strong>the</strong> acquisition or<br />
extinction of a simple touchscreen oper<strong>an</strong>t response or sensorimotor gating; demonstrating <strong>the</strong><br />
specificity of <strong>the</strong> stress effects to reversal. Ongoing experiments will examine whe<strong>the</strong>r <strong>the</strong> effects<br />
of stress are mimicked by lesion of <strong>the</strong> orbitofrontal cortex in C57BL/6J mice, <strong>an</strong>d whe<strong>the</strong>r<br />
stress-induced reversal deficits are associated with altered levels of brain-derived neurotrophic<br />
factor in this brain region. Taken toge<strong>the</strong>r <strong>the</strong>se data suggest that cognitive flexibility may be<br />
more sensitive to <strong>the</strong> effects of modest stress exposure th<strong>an</strong> certain o<strong>the</strong>r <strong>for</strong>ms of cognitive<br />
function. Our findings provide a basis <strong>for</strong> elucidating <strong>the</strong> neural <strong>an</strong>d molecular mech<strong>an</strong>isms<br />
underlying <strong>the</strong>se effects. Research supported by <strong>the</strong> National Institute on Alcohol Abuse <strong>an</strong>d<br />
Alcoholism Intramural Research Program.
Disclosures: C. Graybeal, None; M. Feyder, None; L.M. Saksida, None; T.J. Bussey,<br />
None; A. Holmes, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.20/GG46<br />
Topic: F.02.b. Executive function<br />
Support: NARSAD Young Investigator Award<br />
NIH MH063266<br />
Title: Distinct effects of modulating cortical GABA tr<strong>an</strong>smission on attention, locomotor<br />
activity, <strong>an</strong>d reward<br />
Authors: *T. A. PAINE, L. E. SLIPP, W. A. CARLEZON, Jr;<br />
Psychiatry, Harvard Med. School-McLe<strong>an</strong>, Belmont, MA<br />
Abstract: Background: Attention is dysregulated in a number of psychiatric conditions<br />
including attention deficit hyperactivity disorder (ADHD), schizophrenia, <strong>an</strong>d bipolar disorder.<br />
The medial prefrontal cortex (mPFC) is critical <strong>for</strong> optimal attentional per<strong>for</strong>m<strong>an</strong>ce in rats.<br />
Because GABAA receptors regulate <strong>the</strong> activity of cortical pyramidal neurons, we investigated<br />
how altering GABAA receptor activity within <strong>the</strong> mPFC affects attention in <strong>the</strong> 5-choice serial<br />
reaction time task (5CSRTT). Alterations in motivation <strong>an</strong>d locomotor activity c<strong>an</strong> have nonspecific<br />
effects on 5CSRTT per<strong>for</strong>m<strong>an</strong>ce, so we also examined <strong>the</strong> effects of m<strong>an</strong>ipulating<br />
cortical GABAA receptor activity on intracr<strong>an</strong>ial self-stimulation (ICSS) <strong>an</strong>d behavior in <strong>an</strong> open<br />
field.<br />
Methods: Male Sprague-Dawley rats were trained on <strong>the</strong> 5CSRTT until <strong>the</strong>y reached criterion<br />
per<strong>for</strong>m<strong>an</strong>ce (>60% accuracy,
eward sensitivity.<br />
Results: BMI impaired attention (decreased accuracy, increased omissions) without affecting<br />
locomotor activity. Although BMI increased <strong>the</strong> latency to retrieve <strong>the</strong> reward in <strong>the</strong> 5CSRTT<br />
(which often reflects reduced motivation), it decreased ICSS thresholds (which often reflects<br />
increased activity of brain reward systems). In contrast, MUS increased impulsivity (i.e.,<br />
increased premature responses), but decreased locomotor activity <strong>an</strong>d did not affect ICSS.<br />
Summary: Altering GABAA receptor activation within <strong>the</strong> mPFC had distinct effects on<br />
attention, impulsivity, locomotor activity, <strong>an</strong>d reward sensitivity. Interestingly, inattention <strong>an</strong>d<br />
impulsivity (key symptoms of ADHD) <strong>an</strong>d were produced separately by treatments with opposite<br />
effects on GABAA activity in <strong>the</strong> mPFC. These studies indicate that alterations in GABAergic<br />
signaling within <strong>the</strong> mPFC directly affect attention, <strong>an</strong>d raise <strong>the</strong> possibility that disorders such<br />
as ADHD might involve multiple cellular mech<strong>an</strong>isms.<br />
Disclosures: T.A. Paine, None; L.E. Slipp, None; W.A. Carlezon, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.21/GG47<br />
Topic: F.02.b. Executive function<br />
Support: MH065658<br />
MH073689<br />
MH084436<br />
Title: Unit activity in <strong>the</strong> orbital prefrontal cortex of <strong>the</strong> rat: Reversal learning <strong>an</strong>d strategy<br />
switching<br />
Authors: *J. J. YOUNG, M. L. SHAPRIO;<br />
Mount Sinai Sch. Med., New York, NY<br />
Abstract: Damage to <strong>the</strong> orbitofrontal cortex (OFC) in primates <strong>an</strong>d rodents is associated with<br />
deficits in behavioral flexibility, specifically in reversal learning. In rats, OFC neurons<br />
specifically encode stimulus-response pairings, <strong>an</strong>d <strong>the</strong>se pairings ch<strong>an</strong>ge during reversal.<br />
However, it is still unknown whe<strong>the</strong>r such OFC encoding ch<strong>an</strong>ges are restricted to reversal<br />
learning or whe<strong>the</strong>r <strong>the</strong>y reflect a more general representation related to reward. Recent evidence
<strong>from</strong> our lab suggests that OFC activity during o<strong>the</strong>r types of flexible responding tasks - namely,<br />
strategy switching - c<strong>an</strong> improve per<strong>for</strong>m<strong>an</strong>ce in subsequent reversals, suggesting that import<strong>an</strong>t<br />
encoding ch<strong>an</strong>ges may occur in OFC during strategy switches. We are now using high density,<br />
single-unit recording methods to investigate coding dynamics in <strong>the</strong> rat OFC in <strong>the</strong> context of<br />
repeated reversals <strong>an</strong>d strategy switches. Single unit activity ch<strong>an</strong>ges during <strong>the</strong>se tasks will be<br />
compared to determine whe<strong>the</strong>r OFC encoding best correlated with strategy-, task-, or spatial<br />
paths.<br />
Disclosures: J.J. Young, None; M.L. Shaprio, None.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.22/GG48<br />
Topic: F.02.b. Executive function<br />
Support: NIH Gr<strong>an</strong>t MH052885<br />
Veter<strong>an</strong>’s Administration VISN22 MIRECC<br />
Title: The ontogeny of behavioral flexibility impairments in isolation-reared rats<br />
Authors: *S. B. POWELL, J. W. YOUNG, M. R. BUELL, C. N. SCOTT, S. CALDWELL, E.<br />
TSAN, M. A. GEYER;<br />
UCSD, La Jolla, CA<br />
Abstract: Emotional <strong>an</strong>d cognitive impairments appear early in <strong>the</strong> progression of<br />
schizophrenia, often preceding <strong>the</strong> symptoms of psychosis. Thus <strong>the</strong> systems subserving <strong>the</strong>se<br />
functions may be more vulnerable to, <strong>an</strong>d mech<strong>an</strong>istically linked with, initial pathology.<br />
Underst<strong>an</strong>ding <strong>the</strong> trajectory of behavioral <strong>an</strong>d <strong>an</strong>atomical abnormalities relev<strong>an</strong>t to <strong>the</strong><br />
schizophrenia prodrome <strong>an</strong>d <strong>the</strong>ir sensitivity to interventions in <strong>an</strong>imal models will be critical to<br />
identifying potentially prophylactic <strong>the</strong>rapeutic strategies. Isolation rearing of rats is <strong>an</strong><br />
environmental perturbation that deprives rodents of social contact <strong>from</strong> we<strong>an</strong>ing through<br />
adulthood <strong>an</strong>d produces behavioral <strong>an</strong>d neuronal abnormalities that mirror some pathophysiology<br />
associated with schizophrenia, including deficits in prepulse inhibition of startle (PPI) <strong>an</strong>d<br />
abnormalities in frontal cortex. Previous studies in our laboratory have shown that PPI deficits in<br />
isolation-reared rats emerge around <strong>the</strong> time of puberty (4 weeks post-we<strong>an</strong>ing) but are not<br />
present when tested at 2 weeks post-we<strong>an</strong>ing (Bakshi & Geyer, Physiol Behav 67: 385-392,
1999). Here, we examined <strong>the</strong> time course of behavioral impairments in reversal learning <strong>an</strong>d set<br />
shifting in isolation-reared rats. Separate groups of male Sprague Dawley rats were tested in <strong>the</strong><br />
attentional set shifting task (ASST) at 2 <strong>an</strong>d 8 weeks post-we<strong>an</strong>ing. Isolation-reared rats<br />
displayed impaired reversal learning at <strong>the</strong> 2-week <strong>an</strong>d 8-week time points. Per<strong>for</strong>m<strong>an</strong>ce<br />
differences between intra-dimensional (ID) shifts <strong>an</strong>d extra-dimensional (ED) shifts were not<br />
detected, thus we were unable to confirm <strong>the</strong> <strong>for</strong>mation of <strong>an</strong> attentional set. Isolation rearing<br />
was also associated with deficits in PPI at 4 weeks post-we<strong>an</strong>ing. These data corroborate<br />
previous studies showing reversal-learning deficits in isolation-reared rats (Schrijver et al.,<br />
Behav Brain Res 152: 307-314, 2004) <strong>an</strong>d suggest that <strong>the</strong>se deficits emerge early in <strong>the</strong> course<br />
of isolation. Hence, isolation rearing of rats may offer a unique model to examine <strong>the</strong> ontogeny<br />
of behavioral <strong>an</strong>d neurobiological alterations which may be relev<strong>an</strong>t to preclinical models of<br />
prodromal psychosis.<br />
Disclosures: S.B. Powell, None; J.W. Young, None; M.R. Buell, None; C.N. Scott, None; S.<br />
Caldwell, None; E. Ts<strong>an</strong>, None; M.A. Geyer, Equity interest in S<strong>an</strong> Diego Instruments, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property).<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.23/GG49<br />
Topic: F.02.b. Executive function<br />
Support: Sophia University ORC Gr<strong>an</strong>t HISRP<br />
Title: Factor <strong>an</strong>alysis of attentional set-shifting task per<strong>for</strong>m<strong>an</strong>ce of aged <strong>an</strong>d young mice<br />
Authors: *S. TANAKA 1 , V. RISBROUGH 2 , J. YOUNG 2 , J. GRESACK 2 , M. GEYER 2 ;<br />
1 Dept In<strong>for</strong>mation Sci., Sophia Univ., Tokyo, Jap<strong>an</strong>; 2 Univ. of Cali<strong>for</strong>nia, S<strong>an</strong> Diego, CA<br />
Abstract: Background:<br />
There seems to be no fixed course of age-related ch<strong>an</strong>ges in cognitive functions <strong>an</strong>d <strong>the</strong><br />
differences tend to increase with aging. Cognitive aging might have not only negative but<br />
positive aspects. Large individual differences in cognitive function in senescence raise <strong>the</strong><br />
question as to how we c<strong>an</strong> live better lives with aging. It would, <strong>the</strong>re<strong>for</strong>e, be me<strong>an</strong>ingful to<br />
study individual differences in cognitive functions in aged <strong>an</strong>d young subjects.<br />
Methods:<br />
Young (4 months <strong>an</strong>d n = 16) <strong>an</strong>d aged (23 months <strong>an</strong>d n = 18) C57BL/6N mice per<strong>for</strong>med <strong>the</strong>
attentional set-shifting task (ASST), which includes intra- <strong>an</strong>d extradimensional set shifting <strong>an</strong>d<br />
reversals as well as simple <strong>an</strong>d compound discrimination. A factor <strong>an</strong>alysis was used to uncover<br />
<strong>the</strong> latent structure of variables <strong>from</strong> <strong>the</strong>se data.<br />
Results:<br />
The factor <strong>an</strong>alysis has extracted a factor with high loadings on intradimensional shift <strong>an</strong>d<br />
extradimensional reversal in both <strong>the</strong> young <strong>an</strong>d aged mice in <strong>the</strong> 3-, 4- <strong>an</strong>d 5-factor models. The<br />
factor loading on extradimensional shift is not consistent. There were no differences between <strong>the</strong><br />
age groups. The <strong>an</strong>alysis has extracted <strong>an</strong>o<strong>the</strong>r factor that increases <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of most of<br />
<strong>the</strong> ASST items but decreases <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of simple discrimination in <strong>the</strong> 3-, 4- <strong>an</strong>d 5-factor<br />
models. This factor is seen in both <strong>the</strong> young <strong>an</strong>d aged mice groups. In <strong>the</strong> young mice, this<br />
factor decreases <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of simple <strong>an</strong>d compound discrimination <strong>an</strong>d increases <strong>the</strong><br />
per<strong>for</strong>m<strong>an</strong>ce of all <strong>the</strong> o<strong>the</strong>r ASST items. This pattern is clearly seen in <strong>the</strong> 3- <strong>an</strong>d 4-factor<br />
models. The results of <strong>the</strong> aged mice are less consistent.<br />
Conclusions:<br />
A factor <strong>an</strong>alysis of <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong> attentional set-shifting task extracted factors that<br />
account <strong>for</strong> <strong>the</strong> variation of <strong>the</strong> per<strong>for</strong>m<strong>an</strong>ce of <strong>the</strong> ASST items distinctively, seemingly<br />
depending on <strong>the</strong> difficulty of each task item. There are commonalities <strong>an</strong>d differences in <strong>the</strong><br />
structure between <strong>the</strong> young <strong>an</strong>d aged mice groups. It seems to be still unclear whe<strong>the</strong>r <strong>the</strong><br />
structure of <strong>the</strong> variables of <strong>the</strong> aged mice is me<strong>an</strong>ingfully different <strong>from</strong> that of <strong>the</strong> young mice.<br />
Fur<strong>the</strong>r <strong>an</strong>alyses with <strong>the</strong> combination of o<strong>the</strong>r multivariate <strong>an</strong>alyses will explore <strong>the</strong> structure of<br />
<strong>the</strong> data of cognitive functions of young <strong>an</strong>d aged mice.<br />
Disclosures: S. T<strong>an</strong>aka, None; V. Risbrough, NIMH gr<strong>an</strong>t MH52885-12, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); Stein Institute on Research on Aging, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); J. Young,<br />
Stein Institute on Research on Aging, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Wyeth Nutritional Inc., F.<br />
Consult<strong>an</strong>t/Advisory Board; J. Gresack, None; M. Geyer, Wyeth, Scientific Advisory Board, F.<br />
Consult<strong>an</strong>t/Advisory Board; Omeros Pharma, Scientific Advisory Board, F. Consult<strong>an</strong>t/Advisory<br />
Board.<br />
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.24/GG50<br />
Topic: F.02.b. Executive function
Support: NIH Gr<strong>an</strong>t MH48404<br />
Pittsburgh Life Sciences Greenhouse<br />
Title: Inhibition of NMDA or GABA-A receptors but not reduction of GAD activity in <strong>the</strong><br />
prefrontal cortex impairs preparatory attention<br />
Authors: *A. L. PEHRSON, N. K. B. TOTAH, B. MOGHADDAM;<br />
Dept Neurosci, Univ. Pittsburgh, Pittsburgh, PA<br />
Abstract: Schizophrenia is associated with cognitive dysfunction, including deficits in attention.<br />
The severity of cognitive deficits may be <strong>the</strong> best predictor of functional outcome among this<br />
patient population. Postmortem studies have reported altered cortical GABA neurotr<strong>an</strong>smission<br />
in schizophrenia. For example, in <strong>the</strong> dorsolateral prefrontal cortex <strong>an</strong>d <strong>an</strong>terior cingulate cortex<br />
(ACC), <strong>the</strong>re is reduced density of GABA neurons, reduced expression of <strong>the</strong> GABA<br />
syn<strong>the</strong>sizing enzyme glutamic acid decarboxylase (GAD), <strong>an</strong>d increased expression of <strong>the</strong><br />
GABAA receptor. GABA interneurons in <strong>the</strong> prefrontal cortex are critical <strong>for</strong> proper functioning<br />
of this region <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e most recent <strong>the</strong>ories on cognitive deficits incorporate GABA<br />
deficiency in <strong>the</strong> microcircuits that subserve <strong>the</strong>se deficits. Among <strong>the</strong>se, two leading <strong>the</strong>ories<br />
are that: 1) reduced GABA activity is <strong>an</strong> indirect consequence of NMDA deficiency whereby<br />
reduced NMDA-mediated glutamatergic drive of <strong>the</strong>se neurons lowers <strong>the</strong>ir normal activity<br />
during task relev<strong>an</strong>t events, <strong>an</strong>d 2) reduced GAD expression is a primary pathology in<br />
schizophrenia leading to reduced GABA availability. One concern with <strong>the</strong> latter <strong>the</strong>ory is that<br />
GABA availability is governed by <strong>the</strong> GABA shunt, which serves not only to produce GABA,<br />
but also to conserve <strong>the</strong> available supply. Thus, it may be <strong>the</strong> case that reduced GAD expression<br />
in schizophrenia is a compensatory mech<strong>an</strong>ism that is not coupled with reductions in GABA<br />
availability, <strong>an</strong>d may have little effect on cognition. In this study, we compared <strong>the</strong> effect of<br />
GAD inhibition with that of NMDA <strong>an</strong>d GABA-A receptor inhibition in <strong>the</strong> ACC on<br />
per<strong>for</strong>m<strong>an</strong>ce of <strong>an</strong> attention task (3 choice serial reaction time task - 3CSRT) that is dependent<br />
on <strong>the</strong> ACC. This task requires a rat to attend to <strong>an</strong> array of three nose poke holes until a short<br />
(300ms) cue is lit in one of <strong>the</strong> holes, <strong>an</strong>d <strong>the</strong>n to poke in that hole to obtain a reward. Drugs<br />
were given ei<strong>the</strong>r systemically or micro-injected into <strong>the</strong> ACC <strong>an</strong>d included <strong>the</strong> GAD inhibitor 3mercaptopropionic<br />
acid (3MPA), <strong>the</strong> GABAA <strong>an</strong>tagonist SR95531, <strong>an</strong>d <strong>the</strong> NMDA <strong>an</strong>tagonist<br />
MK801. We find that that systemic 3MPA does not signific<strong>an</strong>tly affect 3CSRT per<strong>for</strong>m<strong>an</strong>ce at<br />
sub seizure-mimetic doses. Microinjection of 3MPA into <strong>the</strong> ACC at concentrations of 1-10 mM<br />
also fails to impair per<strong>for</strong>m<strong>an</strong>ce. In contrast, local injection of SR95531 impaired 3CSRT<br />
per<strong>for</strong>m<strong>an</strong>ce in a dose-dependent m<strong>an</strong>ner. Consistent with previous findings, NMDA receptor<br />
inhibition also affected per<strong>for</strong>m<strong>an</strong>ce. Collectively <strong>the</strong>se findings suggest that while inhibition of<br />
GABA-A <strong>an</strong>d NMDA receptors impair a cognitive function that is relev<strong>an</strong>t to schizophrenia,<br />
inhibition of GAD, at least tr<strong>an</strong>siently, does not.<br />
Disclosures: A.L. Pehrson, None; N.K.B. Totah, None; B. Moghaddam, None.<br />
Poster
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.25/GG51<br />
Topic: F.02.b. Executive function<br />
Support: MH074811<br />
Title: The effects of excitotoxic lesions of <strong>the</strong> <strong>an</strong>terior cingulate cortex in rats on attentional setshifting<br />
Authors: *J. A. MCGAUGHY, E. S. CARTER, L. A. NEWMAN;<br />
Dept Psych, Univ. New Hampshire, Durham, NH<br />
Abstract: The <strong>an</strong>terior cingulate cortex (ACC) has been shown to be active in paradigms that<br />
require executive control. Patients with schizophrenia have previously been found to have<br />
functional <strong>an</strong>d morphological deficits in <strong>the</strong> ACC. Previous studies have also demonstrated<br />
patients with schizophrenia have deficits in attentional set-shifting using ei<strong>the</strong>r <strong>the</strong> Wisconsin<br />
card sorting task or <strong>the</strong> intradimensional/extradimensional set-shifting task. While it has been<br />
hypo<strong>the</strong>sized that dysfunction in <strong>the</strong> dorsolateral prefrontal cortex (dlPFC) underlies <strong>the</strong> deficits<br />
seen in attentional set-shifting, patients with schizophrenia also have signific<strong>an</strong>t deficits on tests<br />
where <strong>an</strong> novel irrelev<strong>an</strong>t stimulus dimension is introduced. This type of deficit does not occur<br />
after lesions to dlPFC or its homolog, <strong>the</strong> prelimbic cortex in rats. The current study examines<br />
<strong>the</strong> effects of ibotenic acid lesions of <strong>the</strong> <strong>an</strong>terior cingulate cortex (ACC) in rats on repeated<br />
testing of <strong>the</strong> attentional set-shifting task. While <strong>the</strong>re were no impairments in <strong>the</strong> ability to <strong>for</strong>m<br />
or shift attentional set, ACC-Lx rats required more trials to reach criterion per<strong>for</strong>m<strong>an</strong>ce on <strong>the</strong><br />
compound discrimination th<strong>an</strong> Sham-Lx rats. This finding suggests dysfunction of <strong>the</strong> ACC may<br />
lead to impairments in filtering irrelev<strong>an</strong>t in<strong>for</strong>mation <strong>an</strong>d underlie <strong>the</strong> impairments seen at <strong>the</strong><br />
compound discrimination in patients with schizophrenia. ACC-Lx rats were also found to have<br />
impairments on <strong>the</strong> first reversal in a session when compared to Sham-Lx rats. Previous research<br />
has demonstrated impairments on reversal learning with lesions of <strong>the</strong> orbitofrontal cortex. The<br />
similar effect of ACC lesions may result <strong>from</strong> dense interconnections between ACC <strong>an</strong>d <strong>the</strong><br />
orbitofrontal as this region has previously been implicated in reversal learning. Additional<br />
studies were per<strong>for</strong>med to determine if <strong>the</strong> presence of irrelev<strong>an</strong>t stimuli contributed to <strong>the</strong><br />
deficits in reversal learning. Simple serial reversals were conducted in order to assess two-choice<br />
discrimination reversal learning. ACC-Lx <strong>an</strong>imals were not signific<strong>an</strong>tly impaired compared to<br />
Sham-Lx <strong>an</strong>imals in serial reversal tests. These data support <strong>the</strong> hypo<strong>the</strong>sis that <strong>the</strong> ACC is<br />
critical <strong>for</strong> discrimination <strong>an</strong>d reversal learning in <strong>the</strong> presence of distracting stimuli.<br />
Disclosures: J.A. McGaughy, None; E.S. Carter, None; L.A. Newm<strong>an</strong>, None.
Poster<br />
580. Executive Function: Rodent Models of Flexible Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 580.26/GG52<br />
Topic: F.02.b. Executive function<br />
Title: Genetic interaction between COMT <strong>an</strong>d Dysbindin produce schizophrenia-like phenotypes<br />
Authors: *F. PAPALEO 1 , J. CHEN 1 , B. LU 2 , J. N. CRAWLEY 1 , D. R. WEINBERGER 1 ;<br />
1 NIMH, Be<strong>the</strong>sda, MD; 2 NIH, Be<strong>the</strong>sda, MD<br />
Abstract: The etiology of schizophrenia is complex <strong>an</strong>d largely unknown, with both genetic <strong>an</strong>d<br />
environmental contributing factors. Genetic association studies have indicated several potential<br />
schizophrenia-susceptibility genes (such as COMT, Dysbindin, DISC1, KCNH2, Neuregulin<br />
etc). Since schizophrenia is most likely related to <strong>the</strong> combination of malfunction of multiple<br />
genes, ra<strong>the</strong>r th<strong>an</strong> mutation of only a single gene; producing COMT*dysbindin double knockout<br />
mice, we investigated <strong>the</strong> interacting effects between COMT <strong>an</strong>d dysbindin created in <strong>the</strong> same<br />
mouse line. Based on prior evidence that decreased COMT increases cortical dopamine <strong>an</strong>d<br />
decreased dysbindin increases D2 receptor signaling, we predicted synergistic interacting effects<br />
of reduced COMT <strong>an</strong>d dysbindin in <strong>the</strong> same subject leading to excessive cortical dopamine<br />
signaling.<br />
We demonstrate that genetic modifications resulting in combined decrease activity of <strong>the</strong> COMT<br />
<strong>an</strong>d dysbindin genes produce working memory deficits, hyperactivity, amphetamine<br />
supersensitivity <strong>an</strong>d abnormal response to acoustic stimuli: all parameters used as rodent<br />
correlates of schizophrenia-like core symptoms. These findings illustrate <strong>the</strong> first experimental<br />
<strong>an</strong>imal model based on a predicted epistatic interaction of two putative schizophreniasusceptibility<br />
genes <strong>an</strong>d unravel interesting genetic mech<strong>an</strong>isms in <strong>the</strong> etiology of this<br />
devastating mental illness.<br />
Disclosures: F. Papaleo, None; J. Chen, None; B. Lu, None; J.N. Crawley, None; D.R.<br />
Weinberger, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.1/GG53<br />
Topic: F.02.e. Associative, nonassociative, <strong>an</strong>d skill learning<br />
Support: ANR<br />
FRM<br />
Title: Spatial learning sculpts <strong>the</strong> dendritic arbor of adult-born hippocampal neurons<br />
Authors: *S. TRONEL 1 , A. FABRE 1 , V. CHARRIER 1 , S. H. R. OLIET 1 , F. H. GAGE 2 , D. N.<br />
ABROUS 1 ;<br />
1 INSERM U862, Bordeaux, Fr<strong>an</strong>ce; 2 Lab. of Genet., Salk Inst., La Jolla, CA<br />
Abstract: Neurogenesis in <strong>the</strong> dentate gyrus of <strong>the</strong> hippocampus is characterized by <strong>the</strong> birth of<br />
thous<strong>an</strong>d of cells that generate neurons throughout life. The fate of <strong>the</strong>se adult newborn neurons<br />
is dependent upon life experiences. In particular, spatial learning promotes <strong>the</strong> survival <strong>an</strong>d death<br />
of new neurons. One of <strong>the</strong> requirements <strong>for</strong> <strong>the</strong> new neurons to process in<strong>for</strong>mation is <strong>the</strong><br />
development of extensive dendritic arbors, a key parameter <strong>for</strong> synaptic integration <strong>an</strong>d signal<br />
processing. Whe<strong>the</strong>r learning influences <strong>the</strong> development of <strong>the</strong> dendritic tree of <strong>the</strong> surviving<br />
neurons is unknown.<br />
To address this issue, we used doublecortin <strong>an</strong>d retroviral labeling to examine to what extent<br />
spatial learning in <strong>the</strong> water maze affects <strong>the</strong> dendritic morphology of adult-born neurons.<br />
Fur<strong>the</strong>rmore, we investigated whe<strong>the</strong>r <strong>the</strong> level of cognitive dem<strong>an</strong>d influences <strong>the</strong> development<br />
of new neurons <strong>an</strong>d <strong>the</strong> mech<strong>an</strong>isms underlying <strong>the</strong>se learning-induced ch<strong>an</strong>ges in neurogenesis.<br />
We found that spatial learning sculpts neo-networks not only by regulating <strong>the</strong> number of<br />
surviving adult-born neurons but also by shaping <strong>the</strong>ir dendritic arbor. Spatial learning increases<br />
<strong>the</strong> complexity of <strong>the</strong> dendritic arbor <strong>an</strong>d accelerates <strong>the</strong> differentiation of stabilized neurons<br />
toward a neuronal phenotype. These effects are lasting <strong>for</strong> several months <strong>an</strong>d are specific to<br />
neurons born during adulthood. Fur<strong>the</strong>rmore, learning-induced shaping is a function of <strong>the</strong><br />
cognitive dem<strong>an</strong>d of <strong>the</strong> task <strong>an</strong>d depends on NMDA receptors. In <strong>the</strong> search <strong>for</strong> <strong>the</strong> structural<br />
ch<strong>an</strong>ges underlying long-term memory, <strong>the</strong>se observations lead to <strong>the</strong> conclusion that shaping<br />
neo-networks is a novel key player in memory.<br />
Disclosures: S. Tronel, None; A. Fabre, None; V. Charrier, None; S.H.R. Oliet, None; F.H.<br />
Gage, None; D.N. Abrous, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.2/GG54<br />
Topic: F.02.e. Associative, nonassociative, <strong>an</strong>d skill learning<br />
Support: CIHR Gr<strong>an</strong>t to JMW<br />
Title: Effects of adult neurogenesis on episodic memory interference<br />
Authors: P. LUU 1 , I. HUTCHINGS 2 , D. M. SMITH 2 , *J. M. WOJTOWICZ 1 ;<br />
1 Dept Physiol, Univ. Toronto, Toronto, ON, C<strong>an</strong>ada; 2 Psychology, Cornell Univ., Ithaca, NY<br />
Abstract: Adult neurogenesis is restricted to <strong>the</strong> Dentate Gyrus (DG) <strong>an</strong>d <strong>the</strong> sub-ventricular<br />
zone (SVZ). These areas continuously supply new neurons to <strong>the</strong> hippocampus <strong>an</strong>d olfactory<br />
bulb respectively. Newly generated neurons <strong>from</strong> <strong>the</strong> SVZ migrate to <strong>the</strong> olfactory bulb where<br />
<strong>the</strong>y have been shown to fulfil a regenerative role in <strong>the</strong> olfactory bulb, <strong>an</strong>d may play a role in<br />
boosting <strong>the</strong> ability to discriminate between odors in rats. New neurons within <strong>the</strong> DG have been<br />
shown to play a role in learning <strong>an</strong>d memory; however <strong>the</strong>ir specific functional signific<strong>an</strong>ce has<br />
been not well understood. Recent computational <strong>an</strong>d behavioural work on adult neurogenesis has<br />
begun to implicate <strong>the</strong> presence of new hippocampal neurons in <strong>the</strong> reduction of memory<br />
interference. The presence of young neurons may play a role in allowing additional<br />
representation of contextual in<strong>for</strong>mation within two interfering memory traces, allowing <strong>for</strong><br />
orthogonalization <strong>an</strong>d reduction of interference. We postulate that by selectively disrupting <strong>the</strong><br />
proliferation of new neurons in <strong>the</strong> DG, while sparing neurogenesis in <strong>the</strong> SVZ, <strong>the</strong> ability to<br />
learn highly interfering pieces of in<strong>for</strong>mation will be impaired while <strong>the</strong> ability to learn noninterfering<br />
in<strong>for</strong>mation will be spared. In order to address this hypo<strong>the</strong>sis, rats were trained on a<br />
novel memory interference task in which two interfering lists of odours were taught to irradiated<br />
(8 Gy) <strong>an</strong>d non-irradiated (0 Gy) rats in <strong>the</strong> same or different contexts. Sections of <strong>the</strong><br />
Hippocampus, SVZ, Rostral Migratory Stream (RMS) <strong>an</strong>d Olfactory Bulb were stained using<br />
immunohistochemistry <strong>for</strong> doublecortin (DCX), <strong>an</strong>d Bromodeoxyuridine (BrdU) to characterize<br />
<strong>the</strong> effect of irradiation on <strong>the</strong> brain. Preliminary <strong>an</strong>alysis of <strong>the</strong> RMS revealed no signific<strong>an</strong>t<br />
reduction in neurogenesis. Preliminary <strong>an</strong>alysis of <strong>the</strong> DG reveal that presence of new neurons<br />
was reduced by approximately 64±33% (n=3) in irradiated rats in comparison to <strong>the</strong> control<br />
group. Initial behavioural results show no effect of irradiation on learning <strong>the</strong> first list, but reveal<br />
impairment in learning <strong>the</strong> second list which involves subst<strong>an</strong>tial interference. Data suggest that<br />
that <strong>the</strong> neurogenesis may have a specific effect on learning in high interference situations.<br />
Disclosures: P. Luu, None; I. Hutchings, None; D.M. Smith, None; J.M. Wojtowicz, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.3/GG55<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: Gatsby Initiative<br />
Title: A model of hippocampal neurogenesis based on metaplastic synapses<br />
Authors: *A. J. DECOSTANZO, A. SAHAY, R. HEN, S. FUSI;<br />
Neurosci., Columbia Univ., New York, NY<br />
Abstract: Newly adult-born gr<strong>an</strong>ule cells of <strong>the</strong> dentate gyrus display novel physiological<br />
properties during <strong>the</strong> first three weeks of maturation, <strong>the</strong>n gradually become indistinguishable<br />
<strong>from</strong> mature dentate gr<strong>an</strong>ule cells (after around two months). New neurons are both more<br />
excitable <strong>an</strong>d more plastic th<strong>an</strong> <strong>the</strong>ir mature counterparts, having a more depolarized resting<br />
potential <strong>an</strong>d increased long-term potentiation (LTP) of synaptic strength. For this reason<br />
neurogenesis c<strong>an</strong> be seen as a <strong>for</strong>m of metaplasticity. It has been demonstrated that metaplastic<br />
synapses c<strong>an</strong> yield long memory lifetimes with a power-law decay of memories (Fusi et al,<br />
2005). Here we employ a model of synaptic metaplasticity that represents neurogenesis as a<br />
metaplastic tr<strong>an</strong>sition between synaptic states. In this model synapses are represented as having<br />
two “visible” states, ei<strong>the</strong>r depressed or potentiated, each with a cascade of “hidden” states as<br />
described in Fusi et al, 2005. Into <strong>the</strong> model of Fusi et al, we introduce a “reservoir” state that<br />
allows us to control <strong>the</strong> synaptic state into which new synapses are born, <strong>an</strong>d <strong>the</strong> birth <strong>an</strong>d death<br />
rate of synapses independently. Birth of new neurons <strong>from</strong> <strong>the</strong> reservoir state is represented as a<br />
tr<strong>an</strong>sition into <strong>the</strong> highly plastic state, <strong>an</strong>d, over time, or with increasing activity, <strong>the</strong>se synapses<br />
tr<strong>an</strong>sition into less plastic states, indicative of <strong>the</strong>ir recruitment into a memory trace. Death<br />
occurs as a tr<strong>an</strong>sition into <strong>the</strong> reservoir state at a rate varying according to <strong>the</strong> metaplastic state of<br />
<strong>the</strong> synapse, representing <strong>the</strong> preferential death of young versus mature neurons. Experiments<br />
have revealed that decreased neurogenesis impairs contextual fear learning, me<strong>an</strong>while enh<strong>an</strong>ced<br />
neurogenesis improves <strong>the</strong> ability to distinguish between similar contexts or environments. Our<br />
computational model reproduces <strong>the</strong>se effects <strong>an</strong>d makes several predictions. 1. During<br />
contextual discrimination, <strong>the</strong> degree of similarity should determine <strong>the</strong> magnitude of <strong>the</strong>se<br />
effects, 2. Retention <strong>for</strong> learned contexts should follow <strong>the</strong> time course of <strong>the</strong> S/N ratio, ie with<br />
more neurogenesis, memories will be improved at short retention intervals, <strong>an</strong>d worse at longer<br />
retention intervals (<strong>the</strong> retention curves will cross), <strong>the</strong>re<strong>for</strong>e, 3. The inter-trial interval during<br />
training will determine whe<strong>the</strong>r increased neurogenesis will result in faster or slower learning.<br />
Disclosures: A.J. DeCost<strong>an</strong>zo, None; A. Sahay, None; R. Hen, None; S. Fusi, None.<br />
Poster
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.4/GG56<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: NIH Gr<strong>an</strong>t 1F31NS060517-01A1<br />
NIH Gr<strong>an</strong>t R01 HD045224-03<br />
NIH Gr<strong>an</strong>t P30 HD04024-36<br />
NIH Gr<strong>an</strong>t R01 HD45224-02S1<br />
Anna <strong>an</strong>d John J. Sie Foundation<br />
National Down Syndrome <strong>Society</strong><br />
Title: Long term behavioral effects of postnatal neural progenitor cell impl<strong>an</strong>tation in Ts65Dn<br />
mice<br />
Authors: *A. RACHUBINSKI, S. K. CORNELIUS, K. N. MACLEAN, K. B. BJUGSTAD;<br />
Dept Pediatrics, Univ. Colorado Denver, Aurora, CO<br />
Abstract: While showing some ch<strong>an</strong>ges <strong>from</strong> birth, much of <strong>the</strong> cognitive decline associated<br />
with Down syndrome (DS) occurs postnatally. Especially compromised is spatial <strong>an</strong>d association<br />
task per<strong>for</strong>m<strong>an</strong>ce. While current DS treatments are directed at restoring already impaired<br />
cognitive functions, a treatment used earlier in development may postpone or prevent <strong>the</strong><br />
emerging impairments. Neural progenitor cells (NPC) have been investigated <strong>for</strong> <strong>the</strong>ir<br />
<strong>the</strong>rapeutic effects in a wide r<strong>an</strong>ge of neurodegenerative disorders with promising results. The<br />
present study sought to determine if NPC could be used as a treatment <strong>for</strong> DS when impl<strong>an</strong>ted<br />
neonatally, be<strong>for</strong>e <strong>the</strong> DS brain undergoes postnatal degenerative ch<strong>an</strong>ges. The Ts65Dn mouse<br />
model of DS, which has delayed development <strong>an</strong>d cognitive impairments, was used. On<br />
postnatal day 2, trisomic <strong>an</strong>d disomic Ts65Dn littermates were bilaterally impl<strong>an</strong>ted into <strong>the</strong><br />
hippocampus with 100,000 C17.2 murine NPC (mNPC), saline, or remained untr<strong>an</strong>spl<strong>an</strong>ted. At<br />
14 weeks, mice were tested to assess <strong>the</strong>ir function in cognitive tasks in which trisomic mice<br />
have previously been deemed deficient. The Plus maze, a spont<strong>an</strong>eous alternation task that used<br />
novel alternations as a gauge of learning, <strong>an</strong>d <strong>the</strong> Morris water maze (MWM), which required<br />
mice to remember <strong>the</strong> location of a hidden plat<strong>for</strong>m in a pool, were used as a measure of spatial<br />
working memory. Conditioned Taste Avoid<strong>an</strong>ce (CTA), used to evaluate association learning, is<br />
a classical conditioning task that relies on making <strong>an</strong> association between a novel flavor <strong>an</strong>d a<br />
feeling of nausea. In <strong>the</strong> Plus maze, trisomic mice were signific<strong>an</strong>tly impaired (p
nei<strong>the</strong>r saline nor mNPC improved <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce. In addition, <strong>the</strong> MWM found no<br />
signific<strong>an</strong>t effect of mNPC or saline, regardless of karyotype. In CTA, trisomic mice<br />
tr<strong>an</strong>spl<strong>an</strong>ted ei<strong>the</strong>r with mNPC or saline had a signific<strong>an</strong>tly improved measure of learning when<br />
compared to trisomic mice that did not receive <strong>an</strong>y treatment (p
During neurogenesis, immature dentate gr<strong>an</strong>ule cells (DGCs) display distinctive physiological<br />
characteristics while undergoing morphological maturation be<strong>for</strong>e final integration into <strong>the</strong><br />
neural circuits. We developed a tr<strong>an</strong>sgenic mouse model that allowed us to tr<strong>an</strong>siently reduce<br />
adult-born DGCs at particular maturation stages in a regulatable m<strong>an</strong>ner. We found that mice<br />
with a reduction in <strong>the</strong> population of adult-born DGCs at <strong>the</strong> immature stage were defective in<br />
<strong>for</strong>ming robust, long-term spatial memory <strong>an</strong>d displayed impaired per<strong>for</strong>m<strong>an</strong>ce in a contextual<br />
fear extinction task. These results suggest that immature DGCs make import<strong>an</strong>t contributions to<br />
learning <strong>an</strong>d memory.<br />
Disclosures: W. Deng, None; M. Saxe, None; I. Gallina, None; F.H. Gage, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.6/GG58<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: R01 DC009239-01A1 NIDCD<br />
Title: Ablation of subventricular zone neurogenesis in adult mice reduces <strong>the</strong> conditioned<br />
response in <strong>an</strong> odor-cued fear conditioning model<br />
Authors: *T. R. MULLEN, M. T. VALLEY, S. FIRESTEIN;<br />
Biol. Sci., Columbia Univ., New York, NY<br />
Abstract: In <strong>the</strong> adult mouse a vast number of newly born neurons perpetually populate <strong>the</strong><br />
olfactory bulb after migrating <strong>from</strong> <strong>the</strong> subventricular zone neurogenic niche. Focal X-irradiation<br />
of <strong>the</strong> subventricular zone almost entirely eliminates this population of neurons, but no clear<br />
ch<strong>an</strong>ge in olfactory function has been observed in this model. In a cued fear conditioning<br />
paradigm irradiated mice displayed diminished freezing in response to a conditioned odor as<br />
compared to non-irradiated mice; this reduction in <strong>the</strong> conditioned response was not observed<br />
when <strong>an</strong> audio tone was used as <strong>the</strong> conditioned stimulus. This observation indicates that, in<br />
mice without adult born olfactory neurons, <strong>the</strong> association of <strong>the</strong> odor with <strong>the</strong> foot shock was<br />
diminished, or that <strong>the</strong> capacity <strong>for</strong> <strong>the</strong> odor to elicit <strong>the</strong> freezing response was diminished. Our<br />
conclusion is that adult born neurons of <strong>the</strong> olfactory bulb have a complex role in olfactory<br />
processing that involves centrifugal connectivity to association areas of <strong>the</strong> brain. Here we<br />
present behavioral data <strong>from</strong> <strong>the</strong> fear conditioning studies as well as c-fos labeling to illuminate<br />
<strong>the</strong> <strong>an</strong>atomical structures involved in odor cued fear conditioning.
Disclosures: T.R. Mullen, None; M.T. Valley, None; S. Firestein, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.7/GG59<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: NIH Gr<strong>an</strong>t 59970<br />
NSF Gr<strong>an</strong>t 0444364<br />
Title: Learning a difficult trace memory but not <strong>an</strong> easy trace memory rescues new neurons <strong>from</strong><br />
death<br />
Authors: *M. L. ANDERSON 1 , J. WADDELL 2 , T. J. SHORS 1 ;<br />
1 Rutgers Univ., Piscataway, NJ; 2 Univ. of Maryl<strong>an</strong>d Sch. of Med., Baltimore, MD<br />
Abstract: The hippocampus produces thous<strong>an</strong>ds of new cells each day, but m<strong>an</strong>y die within just<br />
weeks of birth. However, if <strong>an</strong>imals are trained on <strong>an</strong> eyeblink conditioning task that requires <strong>the</strong><br />
hippocampus, m<strong>an</strong>y of <strong>the</strong> new cells survive <strong>an</strong>d differentiate into neurons (e.g., Gould et al.,<br />
1999; Leuner et al. 2004; 2006; 2007; Waddell & Shors, 2008). Trace eyeblink conditioning in<br />
which a conditioned stimulus (CS) <strong>an</strong>d unconditioned stimulus (US) are separated by a 500 msec<br />
gap, is hippocampal dependent <strong>an</strong>d rescues new neurons <strong>from</strong> death (e.g., Beylin et al., 2001;<br />
Gould et al., 2001). These data also suggest that tasks that are more difficult to learn are more<br />
effective in saving <strong>the</strong> new neurons. To assess <strong>the</strong> contribution of task difficulty to neurogenesis,<br />
we rendered <strong>the</strong> st<strong>an</strong>dard trace task easier to learn by reducing <strong>the</strong> temporal gap. Trace<br />
conditioning procedure with a shorter trace interval (250 msec; Trace 250) requires <strong>an</strong> intact<br />
hippocampus, <strong>an</strong>d acquisition is faster relative to rats trained with <strong>the</strong> 500 msec trace interval<br />
(e.g., Weiss, et al., 1999). In <strong>an</strong>o<strong>the</strong>r group, we facilitate acquisition of <strong>the</strong> 500 msec trace<br />
interval by inserting a third cue just after termination of <strong>the</strong> US. This cue served to signal <strong>the</strong><br />
onset of <strong>the</strong> ITI. Groups of rats were injected once with BrdU (200mg/kg) <strong>an</strong>d trained one week<br />
later. The percentage of conditioned responses (CRs) during <strong>the</strong> 250 msec period prior to <strong>the</strong> US<br />
was assessed using electromyography (EMG) to detect eyeblinks. Of <strong>the</strong> training procedures,<br />
only Trace 500 effectively enh<strong>an</strong>ced survival of new cells, <strong>an</strong>d acquisition of this task proceeded<br />
slowly relative to <strong>the</strong> Trace 250 <strong>an</strong>d Trace 500 Cue conditions. A second experiment using<br />
excitotoxic lesions of <strong>the</strong> hippocampus confirmed that eyeblink conditioning with <strong>the</strong> 250 msec<br />
trace interval is hippocampal dependent, as is training with <strong>the</strong> 500 msec interval. However,
training with <strong>the</strong> post-US cue was not hippocampal dependent, with <strong>the</strong> majority of lesion rats in<br />
this condition reaching a predetermined criterion of conditioned responding. These data<br />
demonstrate that <strong>the</strong> rate of acquisition <strong>an</strong>d not hippocampal dependence determine <strong>the</strong> influence<br />
of learning on adult neurogenesis, <strong>an</strong>d that trace eyeblink conditioning may require <strong>the</strong><br />
hippocampus to discriminate between <strong>the</strong> trace interval <strong>an</strong>d <strong>the</strong> ITI.<br />
Disclosures: M.L. Anderson, None; J. Waddell, None; T.J. Shors, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.8/GG60<br />
Topic: F.02.e. Associative, nonassociative, <strong>an</strong>d skill learning<br />
Support: NIH (NIMH 59970 to TJS<br />
NSF (IOB-0444364) to TJS<br />
Title: Learning increases neurogenesis in <strong>the</strong> hippocampus provided that learning is both<br />
difficult to achieve <strong>an</strong>d successful<br />
Authors: *D. M. CURLIK, II, D. A. GRAY, T. J. SHORS;<br />
Rutgers Univ., Piscataway, NJ<br />
Abstract: A decade ago, it was reported that learning could increase <strong>the</strong> survival of new neurons<br />
generated in <strong>the</strong> adult hippocampal <strong>for</strong>mation (Gould et al., 1999). More recent studies indicate<br />
that learning <strong>an</strong>d not merely training is critical (Sisti <strong>an</strong>d Shors, 207, <strong>2009</strong>). Moreover, <strong>an</strong>imals<br />
that learn <strong>the</strong> conditioned response (CR), but take longer to do so, rescue more of <strong>the</strong>se newborn<br />
cells th<strong>an</strong> <strong>an</strong>imals that quickly acquire <strong>the</strong> CR (Waddell <strong>an</strong>d Shors, 2008; Dalla et al., <strong>2009</strong>). In<br />
total, <strong>the</strong>se studies suggest that <strong>an</strong>imals that fail to acquire <strong>the</strong> CR <strong>an</strong>d <strong>an</strong>imals that very rapidly<br />
acquire <strong>the</strong> CR will have fewer surviving newborn neurons th<strong>an</strong> <strong>an</strong>imals that take m<strong>an</strong>y trials to,<br />
but do successfully, acquire <strong>the</strong> CR. To date, all of our previous studies have m<strong>an</strong>ipulated rate of<br />
learning by altering <strong>the</strong> task parameters. The question arises: would pharmacological<br />
m<strong>an</strong>ipulations of learning produce similar effects on neuronal survival? Specifically, would<br />
learning that was facilitated by a cognitive enh<strong>an</strong>cer fur<strong>the</strong>r increase <strong>the</strong> number of surviving<br />
cells <strong>an</strong>d conversely, would prevention of learning prevent <strong>the</strong> increase? To <strong>an</strong>swer <strong>the</strong>se two<br />
questions we enh<strong>an</strong>ced learning with <strong>the</strong> partial glycine agonist d-cycloserine(DCS) <strong>an</strong>d<br />
prevented learning with <strong>the</strong> competitive NMDA receptor <strong>an</strong>tagonist, CPP.
Adult male rats underwent stereotaxic surgery <strong>for</strong> eyeblink conditioning. At least five days after<br />
surgery, one week be<strong>for</strong>e training, <strong>an</strong>imals were injected once with BrdU (200mg/kg). They<br />
were <strong>the</strong>n trained with 800 trials of eyeblink conditioning over four days. Each day be<strong>for</strong>e<br />
training <strong>an</strong>imals were injected with ei<strong>the</strong>r DCS, CPP, or saline. During training <strong>an</strong>imals were<br />
exposed to ei<strong>the</strong>r paired trace conditioning, or explicitly unpaired conditioning. All <strong>an</strong>imals were<br />
sacrificed twenty-one days after BrdU injection.<br />
Overall, <strong>the</strong> cognitive enh<strong>an</strong>cer DCS increased <strong>the</strong> rate of acquisition of <strong>the</strong> CR during trace<br />
paired conditioning when compared to <strong>an</strong>imals that were trained with trace conditioning in <strong>the</strong><br />
absence of <strong>the</strong> drug (p
gross structural neurological ch<strong>an</strong>ges or peripheral tissue damage, which suggests a more subtle<br />
neural process may be altered. In rodents, IRR causes a long-lasting decrease in adult<br />
hippocampal neurogenesis, reduces hippocampal growth factor content, <strong>an</strong>d impairs spatial<br />
memory. Our study was designed to characterize short- <strong>an</strong>d long-term effects of whole-brain (5<br />
Gy) radiation on hippocampal neurogenesis, cognitive function, <strong>an</strong>d depressive-like behavior of<br />
adult female C57Bl6 mice, <strong>an</strong>d to determine whe<strong>the</strong>r exercise following IRR aids recovery of<br />
brain <strong>an</strong>d behavior. We found that spatial learning on <strong>the</strong> Barnes maze was slightly impaired 2-3<br />
weeks after IRR, <strong>an</strong>d that large memory deficits were observed at 3 months post-IRR. To<br />
examine depressive-like behavior, we recorded <strong>the</strong> latency to become immobile <strong>an</strong>d immobility<br />
time in <strong>the</strong> <strong>for</strong>ced swim <strong>an</strong>d tail suspension tests, <strong>an</strong>d found that IRR increased depressive-like<br />
behavior at 2 weeks <strong>an</strong>d at 3 mo post-IRR. Following <strong>the</strong> initial behavioral assessment at 2-3<br />
weeks after IRR, half of <strong>the</strong> sham IRR <strong>an</strong>d IRR mice were exposed to daily voluntary exercise<br />
on running wheels <strong>an</strong>d <strong>the</strong>n behaviorally characterized a second time prior to sacrifice. To<br />
examine <strong>the</strong> recovery of hippocampal-dependent spatial learning <strong>an</strong>d memory, we trained mice<br />
to learn <strong>the</strong> spatial location of <strong>an</strong> escape hole on <strong>the</strong> Barnes maze <strong>an</strong>d probed <strong>the</strong>ir spatial<br />
memory at 1 hr, 4 d, <strong>an</strong>d 18 d after training. We found that 8 weeks of daily voluntary wheel<br />
running enh<strong>an</strong>ced spatial memory of sham IRR mice <strong>an</strong>d completely rescued <strong>the</strong> long-term<br />
spatial memory deficit elicited by IRR at all spatial memory retention delays. Exercise also aided<br />
recovery <strong>from</strong> increased depressive-like behavior. To assess recovery of hippocampal<br />
neurogenesis, we qu<strong>an</strong>tified <strong>the</strong> number of newborn neurons immunolabeled with immature<br />
neuronal marker doublecortin (DCX) in <strong>the</strong> dentate gyrus. We also assessed protein levels of<br />
VEGF as a proximate mech<strong>an</strong>ism <strong>for</strong> ch<strong>an</strong>ges in neurogenesis. We found that voluntary wheel<br />
running partially rescued <strong>the</strong> IRR-induced decline in hippocampal neurogenesis <strong>an</strong>d VEGF<br />
protein. These findings reveal that cognitive deficits <strong>an</strong>d increased depressive-like behavior are<br />
evident as early as 2 weeks post-IRR, but worsen months after IRR. These findings also suggest<br />
that daily exercise is <strong>an</strong> effective me<strong>an</strong>s of partially rescuing hippocampal neurogenesis,<br />
improving spatial memory, <strong>an</strong>d reducing depressive-like behavior following a clinically relev<strong>an</strong>t<br />
dose of whole-brain radiation.<br />
Disclosures: S.J. Wong-Goodrich, None; M.L. Pfau, None; C.T. Flores, None; L.W. Jones,<br />
None; C.L. Williams, None.<br />
Poster<br />
581. Adult Neurogenesis <strong>an</strong>d Cognition<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 581.10/GG62<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems
Title: The timing of differentiation of adult hippocampal neurons is crucial <strong>for</strong> spatial memory<br />
Authors: D. SARAULLI 1,2 , S. FARIOLI-VECCHIOLI 3 , M. COSTANZI 1 , S. PACIONI 4 , I.<br />
CINÀ 3 , M. ACETI 1 , L. MICHELI 3 , A. BACCI 4 , V. CESTARI 1,2 , *F. TIRONE 5 ;<br />
1 CNR - Inst. <strong>for</strong> Neurosci., Rome, Italy; 2 LUMSA Univ., Rome, Italy; 3 Inst. di Neurobiologia e<br />
Medicina Molecolare, Rome, Italy; 4 Europe<strong>an</strong> Brain Res. Inst., Rome, Italy; 5 Inst. Di<br />
Neurobiologia E Medicina Molecolare, Rome, Italy<br />
Abstract: Adult neurogenesis in <strong>the</strong> dentate gyrus plays a critical role in hippocampusdependent<br />
spatial learning. It remains unknown, however, how new neurons become functionally<br />
integrated into spatial circuits <strong>an</strong>d contribute to hippocampus-mediated <strong>for</strong>ms of learning <strong>an</strong>d<br />
memory. To investigate <strong>the</strong>se issues, we used a mouse model in which <strong>the</strong> differentiation of<br />
adult-generated dentate gyrus neurons c<strong>an</strong> be <strong>an</strong>ticipated by conditionally expressing <strong>the</strong> prodifferentiative<br />
gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. This strategy<br />
selectively ch<strong>an</strong>ges <strong>the</strong> timing of differentiation of newly generated neurons, without affecting<br />
<strong>the</strong>ir number. New, adult-generated dentate gyrus progenitors, in which <strong>the</strong> PC3 tr<strong>an</strong>sgene was<br />
expressed, showed accelerated differentiation <strong>an</strong>d signific<strong>an</strong>tly reduced dendritic arborization<br />
<strong>an</strong>d spine density. Functionally, this genetic m<strong>an</strong>ipulation specifically affected different<br />
hippocampus-dependent learning <strong>an</strong>d memory tasks, <strong>an</strong>d selectively reduced synaptic plasticity<br />
in <strong>the</strong> dentate gyrus. Thus, <strong>the</strong> correct unwinding of memory functions by newly generated<br />
dentate gyrus neurons is critically dependent on <strong>the</strong> correct timing of <strong>the</strong> initial stages of neuron<br />
maturation <strong>an</strong>d connection to existing circuits.<br />
Disclosures: D. Saraulli, None; S. Farioli-Vecchioli, None; M. Cost<strong>an</strong>zi, None; S. Pacioni,<br />
None; I. Cinà, None; M. Aceti, None; L. Micheli, None; A. Bacci, None; V. Cestari, None; F.<br />
Tirone, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.1/GG63<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: MH050479<br />
Title: Blockade of <strong>the</strong> behavioral effects of social defeat by prior controllable stress requires <strong>the</strong><br />
ventral-medial prefrontal cortex
Authors: R. M. ALEKSEJEV 1 , *J. AMAT 2 , D. F. BIANCHI 1 , K. R. HOYT 1 , K. M.<br />
SEATON 1 , L. R. WATKINS 1 , S. F. MAIER 1 ;<br />
1 Psychology <strong>an</strong>d Ctr. <strong>for</strong> Neurosci., 2 Univ. Colorado, Boulder, CO<br />
Abstract: Experiencing behavioral control over a stressor ch<strong>an</strong>ges <strong>the</strong> immediate behavioral <strong>an</strong>d<br />
physiological impact of <strong>the</strong> stressor, as well as how <strong>the</strong> org<strong>an</strong>ism reacts to future stressful events.<br />
We have shown that exposure to <strong>an</strong> escapable stressor (ES, tailshocks each of which c<strong>an</strong> be<br />
terminated by turning a wheel) c<strong>an</strong> block or blunt reactions to a later inescapable stressor (IS,<br />
tailshocks that are yoked to ES subjects <strong>an</strong>d c<strong>an</strong>not be terminated by <strong>the</strong> stressed subject).<br />
Fur<strong>the</strong>r, we have found that this "immunization" effect c<strong>an</strong> be blocked by inactivating <strong>the</strong> ventral<br />
medial prefrontal cortex (vmPFC) during <strong>the</strong> initial ES or during <strong>the</strong> later IS experience, which<br />
suggests that <strong>the</strong> vmPFC plays a crucial role in behavioral immunization. The present<br />
experiments sought to determine <strong>the</strong> generality of that finding using controllable tailshock as <strong>the</strong><br />
immunizing experience <strong>an</strong>d social defeat (SD) as <strong>the</strong> subsequent stressful event. Adult male<br />
Sprague-Dawley rats (280-350 g) were subjected to ES or maintained in <strong>the</strong>ir home cage as<br />
controls (HCC). Seven days later 50 ng of muscimol in 0.5 µL of saline was injected bilaterally<br />
in <strong>the</strong> vmPFC, <strong>an</strong>d 1 hr later SD was conducted by exposure to a larger Long Ev<strong>an</strong>s male (600-<br />
800 g). Level of social interaction with a juvenile Sprague Dawley male 24 h after SD was used<br />
as a measure of <strong>the</strong> behavioral effects of SD. SD reduced interaction with a juvenile, <strong>an</strong>d prior<br />
exposure to ES blocked this effect of SD. Import<strong>an</strong>tly, inactivation of <strong>the</strong> vmPFC eliminated <strong>the</strong><br />
protective effects of prior ES. Thus, <strong>the</strong> prefrontal cortex is involved in <strong>the</strong> behavioral<br />
immunization to stressful experience elicited by a totally unrelated controllable stressful event.<br />
Disclosures: R.M. Aleksejev, None; J. Amat, None; D.F. Bi<strong>an</strong>chi, None; K.R. Hoyt,<br />
None; K.M. Seaton, None; L.R. Watkins, None; S.F. Maier, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.2/GG64<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: V98C1-076 <strong>from</strong> Taipei Veter<strong>an</strong>s General Hospital, Taipei, Taiw<strong>an</strong><br />
Title: The role of sympa<strong>the</strong>tic nervous system in learned helplessness
Authors: *K. CHUNG-HSUN 1,2 , C.-J. HONG 2,3 , T. B. KUO 2 , C. C. YANG 2 ;<br />
1 B<strong>an</strong>ciao City, Taiw<strong>an</strong>; 2 Inst. of Brain Science, Natl. Y<strong>an</strong>g-Ming Univ., Taipei, Taiw<strong>an</strong>; 3 Dept.<br />
of Psychiatry, Taipei Veter<strong>an</strong>s Gen. Hosp., Taipei, Taiw<strong>an</strong><br />
Abstract: The effects of sympa<strong>the</strong>tic nervous system in learned helplessness<br />
Abstract:<br />
Background: Major depression is usually associated with physical symptoms, such as<br />
palpitation, dyspnea, sweating, tremor <strong>an</strong>d dyspepsia, which are similar to autonomic nervous<br />
dysregulation. Some scholars suggest that depressive mood results in autonomic nervous<br />
dysregulation. However, some physici<strong>an</strong>s believe that depression is secondary to autonomic<br />
nervous dysregulation <strong>an</strong>d this kind of expl<strong>an</strong>ation has been widely accepted by patients <strong>an</strong>d<br />
<strong>the</strong>ir families. Question/Hypo<strong>the</strong>sis: Depression is secondary to autonomic nervous<br />
dysregulation. Specific aims: Study <strong>the</strong> causal relationship between automatic nervous<br />
dysregulation <strong>an</strong>d learned helplessness as <strong>an</strong> <strong>an</strong>imal model of depression. Experimental design:<br />
We used inescapable stress to induce learned helplessness in rats, <strong>an</strong>d evaluated autonomic<br />
nervous system function by measuring <strong>the</strong> parameters of heart rate variability. Different kind of<br />
drugs that regulate <strong>the</strong> autonomic nervous system will be used to test <strong>the</strong>ir effect on learned<br />
helplessness. Preliminarily, we found that propr<strong>an</strong>olol (a beta blocker) reduced <strong>the</strong> rate of<br />
learned helplessness when it was administered during inescapable stress, but learned helplessness<br />
could not be reversed when propr<strong>an</strong>olol was given afterward. Signific<strong>an</strong>ce: Clarifying <strong>the</strong><br />
pathophysiology of depression <strong>an</strong>d helping <strong>the</strong> development of new drugs/methods to treat<br />
depression.<br />
Disclosures: K. Chung-Hsun, None; C. Hong, None; T.B. Kuo, None; C.C. Y<strong>an</strong>g, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.3/GG65<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: NIDA Intramural Research Program<br />
NIAAA Intramural Research Program<br />
Title: Role of neuropeptide Y, but not corticotropin-releasing factor (CRF), in <strong>the</strong> expression of<br />
conditioned fear incubation in rats
Authors: *C. L. PICKENS 1 , T. ADAMS-DEUTSCH 1 , S. G. NAIR 1 , M. HEILIG 2 , Y.<br />
SHAHAM 1 ;<br />
1 Behav Neurosci RB, NIDA IRP, Baltimore, MD; 2 NIAAA IRP, Be<strong>the</strong>sda, MD<br />
Abstract: Background: We recently developed a procedure to study incubation of fear<br />
conditioning in rats (Pickens et al. Biol Psychiatry <strong>2009</strong>). In this procedure, rats given 100 toneshock<br />
pairings over 10 days show low fear 2 days after <strong>the</strong> end of conditioned fear training,<br />
moderate fear after 15 days, <strong>an</strong>d high fear after 30 or 60 days. Thus, under our experimental<br />
conditions, fear progressively increases or incubates over time. Here, we studied <strong>the</strong> role of<br />
corticotrophin-releasing factor (CRF) <strong>an</strong>d neuropeptide Y (NPY) in <strong>the</strong> incubated fear seen 30<br />
days after <strong>the</strong> last day of training.<br />
Methods: We trained food-restricted rats to lever-press <strong>for</strong> food pellets in daily 90-min sessions.<br />
We <strong>the</strong>n gave each rat one-hundred 30-s tones co-terminating with a 0.5-s, 0.5-mA footshock<br />
over 10 days (10 pairings per day). Thirty days after training, we examined <strong>the</strong> expression of<br />
conditioned fear, as measured by conditioned suppression of lever-pressing, after injections of<br />
<strong>the</strong> nonspecific CRF receptor <strong>an</strong>tagonist d-Phe-CRF12-41 (0 or 10 µg, i.c.v.), <strong>the</strong> CRF1 receptor<br />
<strong>an</strong>tagonist MTIP (0, 10 or 20 mg/kg, s.c.), or NPY (0, 5 or 10 µg, i.c.v.).<br />
Results: We found that expression of conditioned fear was strongly reduced by NPY, but not by<br />
d-Phe-CRF12-41 or MTIP. The pharmacological m<strong>an</strong>ipulations had no effect on baseline foodrein<strong>for</strong>ced<br />
lever responding.<br />
Conclusions: Our data suggest a role <strong>for</strong> NPY, but not CRF, in incubation of fear conditioning,<br />
as assessed by our procedure.<br />
Disclosures: C.L. Pickens , None; T. Adams-Deutsch, None; S.G. Nair, None; M. Heilig,<br />
None; Y. Shaham, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.4/GG66<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: CAPES<br />
CNPq
FAPESP<br />
HHMI<br />
Title: Stress-inducible protein 1 peptide promotes memory mainten<strong>an</strong>ce in nude mice bearing<br />
glioblastoma xenograft<br />
Authors: *A. WASILEWSKA-SAMPAIO, M. LOPES, N. QUEIROZ, V. MARTINS;<br />
Ludwig Inst. of C<strong>an</strong>cer Res., São Paulo, Brazil<br />
Abstract: The cellular iso<strong>for</strong>m of prion protein (PrP C ), normal iso<strong>for</strong>m of its infectious<br />
counterparts (PrP SC ) is a cell membr<strong>an</strong>e protein constitutively express in m<strong>an</strong>y cells, mainly in<br />
neurons. Our group has shown that PrP C interacts with <strong>the</strong> 66 kDa protein stress-inducible<br />
protein 1 (STI1), a heat shock protein, also referred as Hop (Hsp70/Hsp90 org<strong>an</strong>izing protein).<br />
Remarkably, a STI1/Hop peptide (STI1/Hop230-245), corresponding to PrP C binding site, c<strong>an</strong><br />
modulate neural plasticity mech<strong>an</strong>isms including neuroprotection, neuritogenesis <strong>an</strong>d memory<br />
<strong>for</strong>mation. Interestingly, we have shown that STI1 secreted by hum<strong>an</strong> glioblastoma cell line is<br />
able to increase proliferation rate in a PrP C -dependent m<strong>an</strong>ner. Interestingly, <strong>the</strong> engagement<br />
between PrPC-STI1 could be abolished by STI1/Hop230-245 peptide, inhibiting glioma cell<br />
lineages proliferation. Recent results pointed out to a potent blockage effect of STI1/Hop230-245 in<br />
glioblastoma proliferation in vitro <strong>an</strong>d in vivo. Consequently, <strong>the</strong> STI1/Hop230-245 as emerged as a<br />
potential <strong>the</strong>rapeutic target presenting two import<strong>an</strong>t features: <strong>the</strong> ability to improve cognitive<br />
activity <strong>an</strong>d its ability to block <strong>the</strong> proliferative action of STI1/Hop230-245 on glioma cell line.<br />
Herein, we have evaluated <strong>the</strong> cognition effects of STI1/Hop230-245 peptide object recognition<br />
(OR) tasks in nude mice with orthotopic xenografts of hum<strong>an</strong> glioblastoma cell line (U87) into<br />
(striatum). After 9 days post cell injection, mice bearing xenograft were treated with<br />
STI1/Hop230-245 three times a week, during 25 days. In parallel, <strong>an</strong> irrelev<strong>an</strong>t N-terminal peptide<br />
(STI1/Hop62-76) was administered as a control group. During this period, <strong>an</strong>imals have been<br />
trained <strong>for</strong> OR. In OR task, <strong>the</strong> <strong>an</strong>imals are trained in a cage containing two identical objects <strong>an</strong>d<br />
<strong>the</strong> exploration time is measured. During <strong>the</strong> test phase, <strong>an</strong> object is replaced by a novel object<br />
<strong>an</strong>d <strong>the</strong> exploration time is determined. Our results showed that STI1/Hop230-245 treated-mice<br />
spent more time exploring <strong>the</strong> novel object, indicating that <strong>the</strong> mice were able to recognize <strong>the</strong><br />
familiar object demonstrating <strong>the</strong>ir learning ability. On <strong>the</strong> o<strong>the</strong>r h<strong>an</strong>d, control group, spend <strong>the</strong><br />
same exploration period <strong>for</strong> both objects (novel <strong>an</strong>d familial), exhibiting memory deficits which<br />
might be related to glioblastoma development. Taken toge<strong>the</strong>r, <strong>the</strong>se results indicate that<br />
STI1/Hop230-245 peptide could act in tumor progression <strong>an</strong>d, consequently, in <strong>the</strong> memory status<br />
mainten<strong>an</strong>ce. Finally, <strong>an</strong>ti-tumoral action of STI1/Hop230-245 peptide underlies its potential as a<br />
leading compound <strong>for</strong> <strong>the</strong> development of novel <strong>the</strong>rapeutic approaches <strong>for</strong> brain c<strong>an</strong>cer, such as,<br />
glioblastoma.<br />
Disclosures: A. Wasilewska-Sampaio, None; M. Lopes, None; N. Queiroz, None; V.<br />
Martins, None.<br />
Poster
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.5/GG67<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: Korea Gvernment KRF-7-2008-0409<br />
Title: Electroencephalogram (EEG) patterns to cope with repetitive restraint stress<br />
Authors: Y. YIM 1 , G. KIM 1 , C. KIM 1 , *D. KIM 2 ;<br />
1 Yonsei Univ. Coll Med., Seoul, Republic of Korea; 2 Yonsei Univ. Coll Med., Seoul.<br />
Abstract: There is <strong>an</strong> individual variation in stress response to <strong>the</strong> same stressor. Individual<br />
differences in <strong>the</strong> stress response could come <strong>from</strong> a different memory of <strong>the</strong> same stress<br />
episode. The neurobiological mech<strong>an</strong>ism to be responsible <strong>for</strong> different memory <strong>for</strong>mation to <strong>the</strong><br />
same stressor has not been clear yet. We aimed to investigate <strong>the</strong> neurobiological substrate to<br />
determine <strong>the</strong> unique stress response to <strong>the</strong> same stressor. In <strong>the</strong> first step, we w<strong>an</strong>ted to know if<br />
<strong>the</strong>re are different general brain functions in accord<strong>an</strong>ce with different stress responses to <strong>the</strong><br />
same stressor. We chose electroencephalogram (EEG) to determine general brain function<br />
because EEG c<strong>an</strong> measure general brain activity of relatively large area in <strong>the</strong> behaving <strong>an</strong>imal.<br />
The EEG of <strong>the</strong> Sprague-Dawley rat was measured by radiotelemetry <strong>an</strong>d <strong>the</strong> data was <strong>an</strong>alyzed<br />
with <strong>the</strong> software Dataquest (Data Sciences International, USA). Two weeks after recovery of<br />
surgery, rats were subjected to <strong>the</strong> restraint stress <strong>for</strong> 1 hour/day, <strong>an</strong>d this stress-episode repeated<br />
<strong>for</strong> 6 days. EEG ch<strong>an</strong>ges during <strong>the</strong> stress episode were measured in <strong>the</strong> first <strong>an</strong>d <strong>the</strong> sixth day of<br />
stress exposure. In <strong>the</strong> spectral <strong>an</strong>alyses, <strong>the</strong> frequency of EEG was divided into 4 b<strong>an</strong>d<br />
categories: Delta (1-4Hz), Theta (4-8Hz), Alpha (8-12Hz), <strong>an</strong>d Beta (12-32Hz) waves <strong>an</strong>d <strong>the</strong><br />
power of each wave b<strong>an</strong>d was <strong>an</strong>alyzed as each wave portion (%) of total power. EEG <strong>an</strong>alysis<br />
enabled <strong>the</strong> pattern to be grouped into 3 different coping strategies in Theta, Alpha <strong>an</strong>d Beta<br />
waves except Delta wave that is believed to confound with muscle movement. Groups are <strong>the</strong><br />
high sensitive, low sensitive <strong>an</strong>d <strong>the</strong> extremely low sensitive groups. The high sensitive group<br />
showed low b<strong>an</strong>d power of EEG just after restraint, <strong>an</strong>d <strong>the</strong>n <strong>the</strong> power gradually increased<br />
throughout <strong>the</strong> period of restraint at <strong>the</strong> 1 st day. The low sensitive group showed <strong>the</strong> lowest<br />
power in <strong>the</strong> middle of <strong>the</strong> restraint at <strong>the</strong> 1 st day. At <strong>the</strong> 6 th day, <strong>the</strong> power of <strong>the</strong> high sensitive<br />
group fairly reached <strong>the</strong> level of <strong>the</strong> power of <strong>the</strong> 1 st day in <strong>the</strong> pre-restraint period, <strong>an</strong>d <strong>the</strong><br />
power was higher th<strong>an</strong> that of <strong>the</strong> low sensitive group. A recovery of <strong>the</strong> power at pre-restraint<br />
period in <strong>the</strong> high sensitive group seems to be a result of adaptation to repetitive exposure to <strong>the</strong><br />
same stressor. The extremely low sensitive group showed almost no ch<strong>an</strong>ges upon repeating<br />
sessions. These results suggest that different traits <strong>for</strong> coping with <strong>the</strong> same stressor determine <strong>an</strong><br />
adaptation pattern to <strong>the</strong> repetitive exposure to that stressor. This work was supported by <strong>the</strong><br />
Korea Research Foundation Gr<strong>an</strong>t funded by <strong>the</strong> Korea Gvernment (KRF-7-2008-0409).
Disclosures: Y. Yim, None; G. Kim, None; C. Kim, None; D. Kim, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.6/GG68<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: Swiss National Science Foundation Gr<strong>an</strong>t 3100A0-108102<br />
Title: Stress, individual differences, <strong>an</strong>d norepinephrine in prediction <strong>an</strong>d optimization of mouse<br />
behavior using rein<strong>for</strong>cement learning<br />
Authors: *G. LUKSYS, C. SANDI, W. GERSTNER;<br />
EPFL, Laus<strong>an</strong>ne, Switzerl<strong>an</strong>d<br />
Abstract: Behavioral per<strong>for</strong>m<strong>an</strong>ce during learning is known to be affected by modulatory<br />
factors such as stress <strong>an</strong>d motivation, as well as by individual differences that influence<br />
sensitivity to <strong>the</strong>se factors. Despite numerous studies, no integrative framework is available that<br />
could predict how a particular <strong>an</strong>imal would per<strong>for</strong>m a certain learning task in a realistic<br />
situation. Here, we show that a simple rein<strong>for</strong>cement learning model c<strong>an</strong> predict mouse behavior<br />
in a hole-box conditioning task, if model meta-parameters (<strong>the</strong> learning rate, explorationexploitation<br />
bal<strong>an</strong>ce, <strong>an</strong>d future reward discounting) are dynamically controlled based on <strong>the</strong><br />
in<strong>for</strong>mation about <strong>the</strong> <strong>an</strong>imal’s genotype <strong>an</strong>d phenotype, stress conditions, recent per<strong>for</strong>m<strong>an</strong>ce<br />
feedback, <strong>an</strong>d pharmacological m<strong>an</strong>ipulations of adrenergic alpha-2 receptors. We find that<br />
stress <strong>an</strong>d motivation affect behavioral per<strong>for</strong>m<strong>an</strong>ce by altering <strong>the</strong> exploration-exploitation<br />
bal<strong>an</strong>ce in a genotype dependent m<strong>an</strong>ner. Our results also provide computational insights into<br />
how <strong>an</strong> inverted-U-shape relation between stress/arousal/norepinephrine levels <strong>an</strong>d focused<br />
per<strong>for</strong>m<strong>an</strong>ce could be explained through ch<strong>an</strong>ges in per<strong>for</strong>m<strong>an</strong>ce accuracy <strong>an</strong>d future reward<br />
discounting. Finally, we <strong>an</strong>alyze under which meta-parameter settings <strong>the</strong> modeled <strong>an</strong>imal c<strong>an</strong><br />
acquire <strong>the</strong> maximal reward during <strong>the</strong> experimental sessions, <strong>an</strong>d which experimental<br />
m<strong>an</strong>ipulations should be applied <strong>for</strong> different mice to help <strong>the</strong>m improve <strong>the</strong>ir per<strong>for</strong>m<strong>an</strong>ce.<br />
Disclosures: G. Luksys, None; C. S<strong>an</strong>di, None; W. Gerstner, None.<br />
Poster
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.7/GG69<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Title: Early life stress blocks renewal of fear in adolescent <strong>an</strong>d adult rats <strong>an</strong>d facilitates spatial<br />
water maze per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: *N. C. HUFF 1 , V. TSANG 2 , K. S. LABAR 1 , S. D. BILBO 2 ;<br />
1 Ctr. Cognitive Neurosci, 2 Psychology & Neurosci., Duke Univ., Durham, NC<br />
Abstract: Early-life stress has been shown to alter brain neurochemistry <strong>an</strong>d behavior in rodents.<br />
In particular, hippocampal function is often disrupted. We investigated whe<strong>the</strong>r removing cage<br />
bedding during postnatal days (P) 2-14, which results in fragmented maternal care (after Ivy et<br />
al., 2008), alters hippocampus-dependent behaviors in both adolescence <strong>an</strong>d adulthood. At P23<br />
rats were trained on two consecutive days on a spatial water maze task <strong>an</strong>d probe tested 1 hour<br />
later. Analysis of time spent in <strong>the</strong> target quadr<strong>an</strong>t revealed that <strong>the</strong> Early Stress (ES) <strong>an</strong>imals<br />
per<strong>for</strong>med superior to <strong>the</strong> controls. To investigate context-dependent memory recall, fear<br />
renewal was investigated. Rats were trained in <strong>an</strong> auditory cue fear conditioning paradigm (P30)<br />
followed by two days of extinction training in a novel context with tone-cue but no shock<br />
present. On <strong>the</strong> fourth day, fear renewal to <strong>the</strong> cue was assessed in <strong>the</strong> extinction context <strong>an</strong>d <strong>an</strong><br />
alternate context. Consistent with <strong>the</strong> literature (Bouton, 2004) controls displayed strong fear to<br />
<strong>the</strong> extinguished cue when it was presented outside of <strong>the</strong> extinction context (fear renewal),<br />
whereas ES <strong>an</strong>imals did not display fear to <strong>the</strong> tone when it was presented outside <strong>the</strong> extinction<br />
context. An absence of fear renewal was also found in adult <strong>an</strong>imals (P60) that had experienced<br />
early stress. Toge<strong>the</strong>r <strong>the</strong> findings suggest that early stress alters <strong>the</strong> limbic system circuitry to<br />
render <strong>an</strong>imals more impulsive <strong>an</strong>d less responsive to previous learning experiences, <strong>an</strong>d<br />
moreover <strong>the</strong> effect appears to be long lasting into adulthood. These findings suggest that <strong>the</strong><br />
impact of early maternal neglect on brain <strong>an</strong>d cognitive outcomes c<strong>an</strong> be investigated with this<br />
particular model of early-life stress.<br />
Supported by: NIH F32 MH078471 to NCH<br />
Disclosures: N.C. Huff, None; V. Ts<strong>an</strong>g, None; K.S. LaBar, None; S.D. Bilbo, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.8/GG70<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Title: Post-training <strong>an</strong>d pre-training chronic stressors have dramatically different effects on<br />
Pavlovi<strong>an</strong> fear in mice<br />
Authors: *M. J. SANDERS;<br />
Marquette Univ., Milwaukee, WI<br />
Abstract: Prior work in this laboratory, comparing chronic variable stress <strong>an</strong>d chronic restraint<br />
stress, had indicated that chronic variable stress inflates context fear in mice when stress<br />
exposure occurs prior to fear conditioning. The current set of studies was undertaken to<br />
distinguish stress effects in acquisition <strong>from</strong> stress effects in memory consolidation <strong>an</strong>d<br />
per<strong>for</strong>m<strong>an</strong>ce. Here, mice were trained in a typical delay conditioning procedure wherein <strong>the</strong>y<br />
were exposed to a series of three tone-shock pairings. For 7 d <strong>the</strong>reafter, <strong>the</strong>y were exposed to<br />
chronic variable stress or chronic restraint stress (or control procedures). Following stress<br />
treatment, mice were tested <strong>for</strong> fear of <strong>the</strong> contextual <strong>an</strong>d auditory stimuli that predicted shock<br />
during training (with <strong>the</strong> freezing response serving as <strong>the</strong> dependent measure of fear). The effects<br />
of post-training stress st<strong>an</strong>d in marked contrast to those of pre-training stress: Chronic variable<br />
stress inflated <strong>the</strong> tone response without affecting <strong>the</strong> context response. More remarkable,<br />
however, restraint stress impaired context fear. That is to say, 7 d of restraint stress has no effect<br />
when administered prior to acquisition but causes a dramatic reduction in context fear when<br />
administered after acquisition. Restraint stress did not have a similar effect on tone fear. The<br />
stimulus-specificity of <strong>the</strong> effect would suggest that restraint stress specifically alters<br />
consolidation processes necessary <strong>for</strong> contextual memory, ra<strong>the</strong>r th<strong>an</strong> altering defensive<br />
responses per se.<br />
Disclosures: M.J. S<strong>an</strong>ders, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.9/GG71<br />
Topic: F.02.h. Memory modulation, consolidation, <strong>an</strong>d reconsolidation
Support: HHMI (AMS)<br />
NIH Gr<strong>an</strong>t DA20126 (LGK)<br />
Title: Stress-dependent enh<strong>an</strong>cement <strong>an</strong>d impairment of retention by naloxone: Evidence <strong>for</strong> <strong>an</strong><br />
endogenous opioid-based system protective of reactivated fear memory<br />
Authors: *A. M. SCHNEIDER 1 , A. BELMONT 1 , W. DARBY 1 , J. KEEFE 1 , K. WALTON 1 , L.<br />
WALKER 1 , N. FREIER 1 , C. MAYER-BACON 1 , L. G. KIRBY 2 , P. E. SIMSON 3 ;<br />
1 Dept Psychol, Swarthmore Coll, Swarthmore, PA; 2 Dept. Anat. & Cell Biol. <strong>an</strong>d Ctr. <strong>for</strong><br />
Subst<strong>an</strong>ce Abuse Res., Temple Univ. Sch. of Med., Philadelphia, PA; 3 Dept. Psychology, Miami<br />
Univ., Ox<strong>for</strong>d, OH<br />
Abstract: The effect of stress on memory storage is mediated via concomit<strong>an</strong>t activation of<br />
opposing modulatory systems: excitatory systems that stimulate memory-related brain sites, <strong>an</strong>d<br />
inhibitory systems that protect against over-stimulation of <strong>the</strong>se sites. Evidence that <strong>an</strong> inhibitory<br />
opioid-based system is protective of memory storage comes <strong>from</strong> <strong>an</strong> earlier study in which we<br />
found that pharmacological blockade of <strong>the</strong> opioid system, under conditions of mild or intense<br />
stress, enh<strong>an</strong>ced or impaired retention, respectively. In <strong>the</strong> present study we tested <strong>the</strong> hypo<strong>the</strong>sis<br />
that a similar opioid-inhibitory system, activated under conditions of mild or intense stress, is<br />
also protective of newly-retrieved (reactivated) fear memory. In <strong>the</strong> two experiments conducted,<br />
male, Long Ev<strong>an</strong>s rats underwent passive-avoid<strong>an</strong>ce training (shock <strong>for</strong> stepping <strong>from</strong> a lighted<br />
to dark compartment) on day 1, reactivation of fear memory (direct placement in <strong>the</strong> dark<br />
compartment in <strong>the</strong> absence of shock) on day 2, <strong>an</strong>d a retention test (<strong>an</strong> opportunity to step <strong>from</strong><br />
<strong>the</strong> lighted to dark compartment in <strong>the</strong> absence of shock) on day 3. The two experiments differed<br />
with regard to <strong>the</strong> level of stress that accomp<strong>an</strong>ied <strong>the</strong> reactivated fear memory. In Experiment 1<br />
(mild stress) <strong>the</strong> <strong>an</strong>imals received <strong>the</strong> reactivation (direct placement) experience alone; in<br />
Experiment 2 (intense stress) <strong>the</strong> <strong>an</strong>imals received <strong>the</strong> reactivation (direct placement) experience<br />
followed by swim stress (15 min). In both experiments <strong>an</strong>imals received <strong>the</strong> opioid <strong>an</strong>tagonist<br />
naloxone (3 mg/kg, i.p.) or vehicle immediately after <strong>the</strong> reactivation experience, that is,<br />
immediately after direct placement alone (Experiment 1) or direct placement followed by swim<br />
stress (Experiment 2). The results indicated that naloxone, administered after conditions of mild<br />
or intense stress, enh<strong>an</strong>ced or impaired <strong>the</strong> newly-retrieved (reactivated) fear memory as<br />
measured by increased or decreased step-through latencies during <strong>the</strong> retention test. These<br />
findings provide evidence <strong>for</strong> a protective endogenous opioid-based system that, when not<br />
blocked pharmacologically, limits enh<strong>an</strong>cement or impairment of newly-retrieved (reactivated)<br />
fear memory under conditions of mild <strong>an</strong>d intense stress, respectively.<br />
Disclosures: A.M. Schneider, None; A. Belmont, None; W. Darby, None; J. Keefe, None; K.<br />
Walton, None; L. Walker, None; N. Freier, None; C. Mayer-Bacon, None; L.G. Kirby,<br />
None; P.E. Simson, None.<br />
Poster
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.10/GG72<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: internal funds <strong>from</strong> UNH<br />
Title: Intermittent swim stress interferes with spatial learning over time in rats<br />
Authors: *T. A. WARNER, R. C. DRUGAN;<br />
Psychology, Univ. of New Hampshire|910006291|0, Durham, NH<br />
Abstract: Intermittent swim stress (ISS) is a new <strong>an</strong>imal model of depression that is used to<br />
investigate <strong>the</strong> role of stress in subsequent learning <strong>an</strong>d memory. ISS exposes a rat to cold water<br />
(15°C) <strong>an</strong>d <strong>the</strong> effects of <strong>the</strong> procedure produce detrimental effects in instrumental learning <strong>an</strong>d<br />
activity measures 24 hours later. The ISS model c<strong>an</strong> be used with <strong>the</strong> Morris Water Maze<br />
(MWM) to investigate <strong>the</strong> impact of stress on spatial learning known to involve <strong>the</strong><br />
hippocampus. The current study evaluated <strong>the</strong> effects of ISS on spatial learning over a number of<br />
days. 32 Sprague Dawley rats were divided into ISS, who experienced 100 5s exposures to cold<br />
water, <strong>an</strong>d Confined Control (CC), who went through <strong>the</strong> same procedure, but in <strong>the</strong> absence of<br />
water (n = 16/per group). The rats were exposed to a spaced learning procedure where all rats<br />
received 20 training trials across 4 days. The first day involved <strong>the</strong> ISS treatment followed by 8<br />
learning trials in <strong>the</strong> MWM on days 2 <strong>an</strong>d 3, <strong>an</strong>d 4 trials on day 4. Rats were placed in different<br />
quadr<strong>an</strong>ts of <strong>the</strong> pool (N, E, S, W) in a semi-r<strong>an</strong>dom order. An hour following <strong>the</strong> last training<br />
trial on Day 4, <strong>the</strong> plat<strong>for</strong>m was removed <strong>from</strong> <strong>the</strong> pool <strong>an</strong>d <strong>the</strong> rats were placed in <strong>the</strong> pool to<br />
see if <strong>the</strong>re was a preference <strong>for</strong> <strong>the</strong> previous location of <strong>the</strong> plat<strong>for</strong>m. Results indicated a main<br />
effect of stress across <strong>the</strong> 3 day MWM learning test <strong>an</strong>d confirmed original findings that <strong>the</strong> ISS<br />
model affects hippocampally based spatial learning.<br />
Disclosures: T.A. Warner, None; R.C. Drug<strong>an</strong>, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 582.11/GG73<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Support: NIMH MH073994<br />
Title: Effects of propr<strong>an</strong>olol on CREB phosphorylation in <strong>the</strong> amygdala <strong>an</strong>d hippocampus of<br />
juvenile rats exposed to predator odor<br />
Authors: *P. A. KABITZKE 1,2 , L. K. SILVA 3 , C. P. WIEDENMAYER 1,4 ;<br />
1 Developmental Psychobiol, NY State Psychiatric Inst., New York, NY; 2 Biopsychology &<br />
Behavioral Neurosci., The Grad. Ctr. CUNY, New York, NY; 3 Psychology, Hunter Col. CUNY,<br />
New York, NY; 4 Psychiatry, Columbia Univ., New York, NY<br />
Abstract: The classic fear-conditioning paradigm employs adult <strong>an</strong>imals <strong>an</strong>d electric shock.<br />
Although much has been learned <strong>from</strong> such experiments, relatively little is known about fear<br />
learning in ecologically-relev<strong>an</strong>t situations or <strong>the</strong> development of fear learning in early life.<br />
Previously, we demonstrated that predator odors could be used to induce fear responses <strong>an</strong>d<br />
contextual fear learning in 26-day-old juvenile rats. The aim of <strong>the</strong> present study was to<br />
investigate <strong>the</strong> role of <strong>the</strong> noradrenergic system in fear conditioning. Indeed, m<strong>an</strong>ipulation of <strong>the</strong><br />
norepinephrine (NE) system has been promising in treating fear- <strong>an</strong>d <strong>an</strong>xiety-related disorders.<br />
In particular, we hypo<strong>the</strong>sized that NE is involved in <strong>the</strong> <strong>for</strong>mation of long-term fear memories.<br />
We found that systemic injections of <strong>the</strong> beta-adrenergic blocker propr<strong>an</strong>olol after exposure to<br />
cat odor inhibited <strong>the</strong> <strong>for</strong>mation of contextual fear memories in juvenile rats. Next, we examined<br />
<strong>the</strong> effects of NE m<strong>an</strong>ipulation on <strong>the</strong> expression of CREB, a tr<strong>an</strong>scription factor involved in <strong>the</strong><br />
<strong>for</strong>mation of long-term fear memories. It was expected that fear learning increases levels of<br />
phosphorylated CREB (pCREB) in <strong>the</strong> amygdala <strong>an</strong>d hippocampus <strong>an</strong>d that a NE <strong>an</strong>tagonist<br />
would inhibit this phosphorylation. We per<strong>for</strong>med western blot <strong>an</strong>alysis to measure levels of<br />
pCREB in <strong>the</strong> hippocampus <strong>an</strong>d amygdala after exposure to cat odor <strong>an</strong>d found differences in<br />
pCREB expression in <strong>the</strong> dorsal hippocampus, <strong>the</strong> ventral hippocampus, <strong>an</strong>d amygdala in<br />
<strong>an</strong>imals that had been exposed to cat odor compared to <strong>an</strong>imals that had been exposed to control<br />
odor. Differences in pCREB expression were also seen when <strong>an</strong>imals were injected systemically<br />
with ei<strong>the</strong>r saline or propr<strong>an</strong>olol after cat odor exposure. These results support our central<br />
hypo<strong>the</strong>sis that NE mediates <strong>the</strong> <strong>for</strong>mation of contextual fear memories by activation of <strong>the</strong><br />
tr<strong>an</strong>scription factor CREB. The same mech<strong>an</strong>isms that operate in <strong>the</strong> adult <strong>an</strong>imal are thus<br />
functional in juveniles but likely not in inf<strong>an</strong>ts, who are not able to acquire contextual fear<br />
memories.<br />
Disclosures: P.A. Kabitzke, None; L.K. Silva, None; C.P. Wiedenmayer, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.12/GG74<br />
Topic: F.02.f. Fear <strong>an</strong>d aversive learning <strong>an</strong>d memory<br />
Title: Online observation of distress: A rat-empathy model<br />
Authors: *P. ATSAK 1,2 , M. ORRE 2 , L. CERLIANI 3 , P. BAKKER 2 , M. MOITA 4 , B.<br />
ROOZENDAAL 2 , V. GAZZOLA 3 , C. KEYSERS 3 ;<br />
1 BCN, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 2 Dept. of Neuroscience, Univ. Med. Ctr. Groningen, Univ. of<br />
Groningen, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 3 Dept. of Neurosci., BCN Neuroimaging Center, Univ. of<br />
Groningen, Univ. Med. Ctr. Groningen, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 Champalimaud Neurosci.<br />
Programme, Inst. Gulbenki<strong>an</strong> de Ciência, Oeiras, Portugal<br />
Abstract: Although empathy is considered as a unique social capacity of primates; recent studies<br />
showed that this ability is also present in o<strong>the</strong>r species. Establishing a simple <strong>an</strong>imal model <strong>for</strong><br />
empathy <strong>an</strong>d its basic <strong>for</strong>ms would be a valuable tool to investigate <strong>the</strong> neuronal mech<strong>an</strong>isms<br />
that underlie empathy <strong>an</strong>d social cognition. Previously we found evidence that rats show<br />
behavioral reactions to <strong>the</strong> observation of shock-exposed conspecifics. In <strong>the</strong> current study, we<br />
investigated social modulation of distress in rats using a distress observation paradigm <strong>an</strong>d<br />
explored <strong>the</strong> effect of previous distress experience on social modulation. Briefly, observer <strong>an</strong>d<br />
demonstrator pairs were placed in adjacent foot-shock cages separated by Plexiglas allowing <strong>for</strong><br />
visual, olfactory <strong>an</strong>d auditory contact. Observer groups were ei<strong>the</strong>r naïve to footshock (naïve<br />
observer) or experienced with footshock (experienced observer). Demonstrator pairs were<br />
always naïve. Observer rats observed <strong>the</strong>ir paired demonstrator receiving a series of<br />
electroshocks (shock observation condition) or not (control observation condition). We<br />
qu<strong>an</strong>tified a number of distress responses in <strong>the</strong> rats (freezing, ultrasonic vocalizations <strong>an</strong>d<br />
corticosterone levels). In <strong>the</strong> shock observation condition, we found a robust increase in freezing<br />
behavior <strong>an</strong>d emitted ultrasounds in experienced observer-demonstrator pairs compared to <strong>the</strong><br />
naïve observer-demonstrator pairs. In <strong>the</strong> group <strong>an</strong>alysis, experienced observers showed<br />
signific<strong>an</strong>tly more freezing th<strong>an</strong> naïve ones. This finding was consistent with <strong>the</strong> hum<strong>an</strong><br />
literature in which experience enh<strong>an</strong>ces empathic response. Moreover, demonstrators paired with<br />
experienced <strong>an</strong>d naïve observer also signific<strong>an</strong>tly differed in <strong>the</strong>ir freezing <strong>an</strong>d corticosterone<br />
levels. Observer’ freezing behavior strongly correlated with demonstrator freezing, suggesting a<br />
reciprocal social modulation between pairs: If observers freeze less, its demonstrator does so as<br />
well <strong>an</strong>d vice versa. Considering <strong>the</strong>se findings, we propose <strong>the</strong> online observation of a<br />
distressed conspecific is a suitable rat model <strong>for</strong> empathy.<br />
Disclosures: P. Atsak, None; M. Orre, None; L. Cerli<strong>an</strong>i, None; P. Bakker, None; M. Moita,<br />
None; B. Roozendaal, None; V. Gazzola, None; C. Keysers, None.
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.13/GG75<br />
Topic: F.02.d. Cognitive learning <strong>an</strong>d memory systems<br />
Support: NIH gr<strong>an</strong>t P20 RR15567<br />
Title: Stress coping via aggression, submission <strong>an</strong>d learned escape: 3-D behavioral <strong>an</strong>alyses<br />
Authors: *D. H. ARENDT, J. P. SMITH, T. R. SUMMERS, R. E. CARPENTER, C. H.<br />
SUMMERS;<br />
Univ. South Dakota, Vermillion, SD<br />
Abstract: We propose a new method of <strong>an</strong>alysis, which integrates adaptive behavioral responses<br />
to stressful conditions. This model of social stress coping recognizes that aggression, submission<br />
<strong>an</strong>d “learned escape” are not expressed in isolation, <strong>an</strong>d considers <strong>the</strong> temporal framework in<br />
which <strong>the</strong>y coexist. The adv<strong>an</strong>tage of this <strong>an</strong>alytical method is a unique single vision of<br />
ecologically <strong>an</strong>d evolutionarily relev<strong>an</strong>t behaviors in three contextual dimensions. Be<strong>for</strong>e <strong>an</strong>y<br />
one adaptive behavioral goal c<strong>an</strong> be committed to, <strong>the</strong> three contexts (r<strong>an</strong>k acquisition, social<br />
subordination, <strong>an</strong>d learned escape) must be appraised by <strong>an</strong>imals during a social interaction. The<br />
benefit of this scale is that it takes into account multiple behaviors, each designed to realize a<br />
separate goal. The proposed three dimensional scale allows <strong>the</strong> ability to distinguish patterns<br />
during concurrent behaviors. Each leg of <strong>the</strong> scale starts at 0 <strong>an</strong>d increases to 5 with a threshold<br />
value at 2.5 <strong>for</strong> each of <strong>the</strong> three behavioral dimensions, where 2.5 represents a point of<br />
commitment towards a behavioral goal. For example, a submissive <strong>an</strong>imal may start showing<br />
early submissive behaviors (0 - 2.5 on <strong>the</strong> scale), later more committed submissive behaviors<br />
(2.5 - 5) may follow. Long-Ev<strong>an</strong>s male rats were paired with a larger conspecific male <strong>for</strong> up to<br />
15 minutes. At <strong>the</strong> time <strong>the</strong> divider is removed to allow social interaction, <strong>an</strong>o<strong>the</strong>r divider is<br />
removed to reveal <strong>an</strong> escape hole large enough only <strong>for</strong> <strong>the</strong> smaller test rat. As in a previous<br />
version of this model using trout, test <strong>an</strong>imals separated into two populations: Escapers <strong>an</strong>d nonescapers.<br />
In trials with sufficient US stimulation (elevated aggression), ~44% became submissive<br />
<strong>an</strong>d ~56% escaped. In trials that produce submission, <strong>the</strong> first third of <strong>the</strong> interaction is devoted<br />
to appraisal of all behavioral options, including aggression <strong>an</strong>d escape. <strong>When</strong> <strong>the</strong> threshold value<br />
is reached <strong>for</strong> commitment to submission, aggressiveness <strong>an</strong>d escape behaviors do not persist. In<br />
repeated trials, escaping rats appeared to exhibit “learned escape”, finding <strong>an</strong>d using <strong>the</strong> escape<br />
hole signific<strong>an</strong>tly quicker with each trial. “Learned Escape” appears to be <strong>an</strong> evolutionarily<br />
conserved attribute that requires gaining proficiency through a series of steps: 1) finding <strong>the</strong><br />
escape route, 2) using <strong>the</strong> escape route <strong>for</strong> <strong>the</strong> first time, 3) remembering <strong>the</strong> location of <strong>the</strong><br />
escape route, 4) minimizing vulnerability <strong>from</strong> attack during escape, 5) developing ballistic or
stealthy movements to safely accomplish escape, <strong>an</strong>d 6) assessing <strong>the</strong> safety of <strong>the</strong> novel<br />
environment on <strong>the</strong> first escape, plus remembering this security in future attempts.<br />
Disclosures: D.H. Arendt, None; J.P. Smith, None; T.R. Summers, None; R.E. Carpenter,<br />
None; C.H. Summers, None.<br />
Poster<br />
582. Stress <strong>an</strong>d Behavior<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 582.14/GG76<br />
Topic: F.02.j. Learning <strong>an</strong>d memory: Pharmacology<br />
Support: internal funds <strong>from</strong> UNH<br />
Title: Does desipramine reverse <strong>the</strong> intermittent swim stress-induced immobility in <strong>the</strong> <strong>for</strong>ced<br />
swim test?<br />
Authors: *R. C. DRUGAN, H. A. MACOMBER;<br />
Dept Psychol, Univ. New Hampshire, Durham, NH<br />
Abstract: Intermittent swim stress (ISS) causes <strong>an</strong> increase in immobility in <strong>the</strong> <strong>for</strong>ced swim test<br />
(FST) in comparison to confined controls. This ISS-induced immobility is not reversed by<br />
subacute (3 injections) <strong>the</strong>rapeutic administration of <strong>the</strong> Selective Serotonin Reuptake Inhibitor<br />
(SSRI), fluoxetine. In <strong>the</strong> present study, we sought to determine if this immobility could be<br />
reversed by <strong>the</strong> tricyclic <strong>an</strong>tidepress<strong>an</strong>t, desipramine. Rats were r<strong>an</strong>domly assigned to one of four<br />
groups, Saline-ISS, Saline-confined control (CC), desipramine (DES) (20 mg/kg, s.c.)-ISS, <strong>an</strong>d<br />
DES-CC. ISS rats were exposed to 100-5 second exposures to 15oC water on a variable time 60<br />
sec schedule, while <strong>the</strong> CC group was exposed to <strong>the</strong> same procedure in <strong>the</strong> absence of water.<br />
All rats were injected with ei<strong>the</strong>r DES or saline 23.5, 5 <strong>an</strong>d 1 hour prior to a FST <strong>the</strong> following<br />
day. All rats were videotaped during <strong>the</strong> FST <strong>an</strong>d <strong>the</strong>ir behaviors were categorized every 5<br />
seconds as climbing, swimming, or floating by investigators blind to group membership.<br />
Preliminary results suggest that DES reduces <strong>the</strong> immobility <strong>an</strong>d increases climbing behavior in<br />
<strong>the</strong> ISS, but not in <strong>the</strong> CC group. No effect was observed in swimming behavior. These findings<br />
suggest that ISS-induced immobility is mediated by norepinephrine systems. Research was<br />
supported by <strong>the</strong> undergraduate research opportunities program at UNH.<br />
Disclosures: R.C. Drug<strong>an</strong>, None; H.A. Macomber, None.
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.1/GG78<br />
Topic: F.03.a. Reward<br />
Support: FAPESP Gr<strong>an</strong>t 04/13849-5<br />
FAPESP 04/14312-5<br />
FAPESP 05/59286-4<br />
FAPESP 06/57647-2<br />
Title: The role of rostrolateral periaqueductal gray (rlPAG) in a motivational drive <strong>for</strong> food<br />
seeking behavior<br />
Authors: *M. H. SUKIKARA 1 , M. V. BALDO 3 , L. F. FELICIO 2 , C. F. ELIAS 4 , J. C.<br />
BITTENCOURT 4 , N. S. CANTERAS 4 , S. R. MOTA-ORTIZ 4,5 ;<br />
1 2 3<br />
Pathology, Dept. of Pathology, Univ. de Sao Paulo, Sao Paulo, Brazil; Dept. of Physiol.,<br />
4 5<br />
Anat., Univ. of São Paulo, São Paulo, Brazil; Lab. of Neurosciences, City Univ. of São Paulo,<br />
São Paulo, Brazil<br />
Abstract: The periaqueductal gray (PAG) have been considered a final common pathway <strong>for</strong> a<br />
number of behavioral responses, including defensive, sexual <strong>an</strong>d maternal behaviors. Previous<br />
studies <strong>from</strong> our lab have challenged this view <strong>an</strong>d suggested <strong>an</strong> additional ra<strong>the</strong>r integrative<br />
PAG function. In particular, we have shown that a specific site in <strong>the</strong> PAG, at <strong>the</strong> level of <strong>the</strong><br />
oculomotor nucleus, located in <strong>the</strong> outer half of <strong>the</strong> lateral column <strong>an</strong>d characterized by a heavy<br />
acetylcholinesterase staining, have been shown to influence <strong>the</strong> motivation drive to <strong>for</strong>age or<br />
hunt. To start unraveling how this specific neural site that is referred to as <strong>the</strong> rostrolateral PAG<br />
(rlPAG) should be influence <strong>the</strong> motivational drive <strong>for</strong> food seeking behavior, we first <strong>an</strong>alyzed<br />
systematically using <strong>the</strong> <strong>an</strong>terograde tracer Phaseolus vulgaris-leucoaglutinin (PHAL) <strong>the</strong><br />
overall pattern of rlPAG axonal ascending projections. The results indicated that <strong>the</strong> major<br />
terminal field was labeled in <strong>the</strong> lateral hypothalamic area (LHA), in particular <strong>the</strong> dorsal,<br />
supra<strong>for</strong>nical, justadorsomedial, <strong>an</strong>d justaventromedial areas, which containing orexin neurons.<br />
By double-labeled immunohistochemistry, we identified that <strong>the</strong> <strong>an</strong>terogradelly labeled fibers are<br />
largely overlapped with orexin neurons in LHA. In a second step, we <strong>an</strong>alyzed <strong>the</strong> orexin cells<br />
pattern of Fos expression in <strong>the</strong> LHA, after <strong>the</strong> <strong>an</strong>imals per<strong>for</strong>ming predatory hunting. The
findings showed a stronger increase in activation of orexin cells located in <strong>the</strong> lateral region of<br />
LHA. Interesting, <strong>the</strong> orexin neurons located in lateral region of <strong>the</strong> LHA are primarily involved<br />
in reward processing <strong>for</strong> food or drug. Finally, we tested <strong>the</strong> behavioral ch<strong>an</strong>ges in rlPAGlesioned<br />
<strong>an</strong>imals during predatory hunting <strong>an</strong>d <strong>an</strong>alyzed <strong>the</strong> orexin cells pattern of Fos<br />
expression after NMDA lesions of rlPAG. Bilateral lesions of <strong>the</strong> rlPAG produced dramatic<br />
effects in motivation <strong>for</strong> hunting <strong>the</strong> prey <strong>an</strong>d strongly reduced <strong>the</strong> activation of orexin neurons<br />
in <strong>the</strong> lateral part of <strong>the</strong> LHA. Overall, <strong>the</strong> present findings support <strong>the</strong> idea that <strong>the</strong> rlPAG<br />
modulates <strong>the</strong> motivational drive <strong>for</strong> food seeking behaviors, probably influencing <strong>the</strong> lateral<br />
hypothalamic orexin neurons which are critically involved in reward processing <strong>for</strong> food through<br />
<strong>the</strong> modulation of <strong>the</strong> ventral tegmental area <strong>an</strong>d <strong>the</strong> nucleus accumbens.<br />
Disclosures: M.H. Sukikara, None; M.V. Baldo, None; L.F. Felicio, None; C.F. Elias,<br />
None; J.C. Bittencourt, None; N.S. C<strong>an</strong>teras, None; S.R. Mota-Ortiz, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.2/GG79<br />
Topic: F.03.a. Reward<br />
Support: Academy of Finl<strong>an</strong>d Gr<strong>an</strong>t 119088<br />
Title: Combined 18F-FDG PET <strong>an</strong>d fMRI reveals <strong>the</strong> role of caudate nucleus in food craving<br />
Authors: *L. NUMMENMAA 1,2 , J. HIRVONEN 3 , A. KARMI 3 , J. C. HANNUKAINEN 3 , H.<br />
LEPPÄNEN 3 , M. M. LINDROOS 3,4 , A. HEINO 3 , P. NUUTILA 3,5 ;<br />
1 Dept. of Psychology, Univ. of Turku, Turku, Finl<strong>an</strong>d; 2 Dept. of Psychology, Univ. of Tampere,<br />
Tampere, Finl<strong>an</strong>d; 3 Turku PET Centre, Univ. of Turku <strong>an</strong>d Turku Univ. Hosp., Turku, Finl<strong>an</strong>d;<br />
4 Dept. of Neurol., 5 Dept. of Med., Univ. of Turku <strong>an</strong>d Turku Univ. Hosp., Turku, Finl<strong>an</strong>d<br />
Abstract: Recently <strong>the</strong>re has been increased interest in <strong>the</strong> possible parallels between drug<br />
craving in drug addiction <strong>an</strong>d food craving in obesity. The most clearly established commonality<br />
of <strong>the</strong> mech<strong>an</strong>isms of drug <strong>an</strong>d food intake is <strong>the</strong>ir ability to activate <strong>the</strong> dopamine containing<br />
link in <strong>the</strong> brain’s reward systems. Drug-related sensory cues may trigger drug seeking behavior<br />
by eli<strong>citing</strong> hyperactivity in <strong>the</strong> brain’s reward circuit, similarly, food-related cues may trigger<br />
food-seeking behavior via <strong>the</strong> same system. Hence, identifying <strong>the</strong> neural correlates of food<br />
craving would be crucial <strong>for</strong> underst<strong>an</strong>ding <strong>the</strong> current high prevalence of obesity. In this study<br />
we examined how individual differences in food craving map to <strong>the</strong> functioning of <strong>the</strong> reward
system.<br />
Ten healthy, normal-weight subjects participated this combined fMRI-PET study. In <strong>the</strong> fMRI<br />
experiment, <strong>the</strong> particip<strong>an</strong>ts passively viewed images of appetizing, high-calorie <strong>an</strong>d bl<strong>an</strong>d, lowcalorie<br />
foods. Insulin stimulated cerebral glucose uptake was measured during normoglycemia<br />
with 2-[18F]fluoro-2-deoxyglucose PET. Self-reported food craving was measured with <strong>the</strong> State<br />
Food Craving Questionnaire (FCQ) prior to imaging. Both PET <strong>an</strong>d fMRI data were <strong>an</strong>alyzed<br />
with statistical parametric mapping (SPM) approach with FCQ scores as a covariate.<br />
The fMRI data showed that viewing appetizing vs. bl<strong>an</strong>d foods activated components of <strong>the</strong><br />
reward circuit including amygdala, medial prefrontal cortex <strong>an</strong>d ventral striatum. Fur<strong>the</strong>r, <strong>the</strong><br />
responses in <strong>the</strong> left dorsal caudate nucleus were positively associated with self-reported food<br />
craving while viewing appetizing vs. bl<strong>an</strong>d foods. The PET data revealed that glucose uptake in<br />
<strong>the</strong> left ventral caudate nucleus was negatively associated with self-reported food craving.<br />
In sum, our study shows that individual differences in food craving predict both glucose<br />
metabolism in caudate nucleus, as well as tr<strong>an</strong>sient caudate responses to <strong>the</strong> sight of appetitive<br />
foods. This confirms that <strong>the</strong> caudate nucleus - which has previously been associated with habit<strong>for</strong>ming<br />
as well as drug craving - also mediates food craving, <strong>an</strong>d may serve as a crucial link<br />
when <strong>for</strong>ming addictions to foods.<br />
Disclosures: L. Nummenmaa, None; J. Hirvonen, None; A. Karmi, None; J.C.<br />
H<strong>an</strong>nukainen, None; H. Leppänen, None; M.M. Lindroos, None; A. Heino, None; P.<br />
Nuutila, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.3/GG80<br />
Topic: F.03.a. Reward<br />
Support: CONACyT 82462<br />
PAPIIT UNAM IN-203-907<br />
Title: Behavioural modification <strong>an</strong>d neuronal activation in limbic structures of rats exposed to a<br />
daily palatable food. Building up <strong>an</strong> addiction?<br />
Authors: A. S. BLANCAS, K. RODRIGUEZ, *C. B. ESCOBAR;<br />
Fac de Medicina UNAM, Mexico, Mexico
Abstract: Palatable food causes behavioural <strong>an</strong>d neuronal ch<strong>an</strong>ges similar to those associated<br />
with drugs of abuse. Daily access to a palatable snack, like a cube of sugar or chocolate, causes<br />
<strong>an</strong>ticipatory expectation <strong>an</strong>d increased c-Fos in <strong>the</strong> Nucleus Accumbens (NAcc) <strong>an</strong>d Prefrontal<br />
Cortex (PFCx). <strong>When</strong> <strong>the</strong> stimulus is withdrawn this behavioural <strong>an</strong>d neuronal activation persists<br />
<strong>for</strong> several days. The aim of this study was to determine <strong>the</strong> process <strong>for</strong> building up <strong>the</strong><br />
<strong>an</strong>ticipatory activation of <strong>the</strong> NAcc <strong>an</strong>d PFCx under <strong>the</strong> paradigm of palatable food entrainment<br />
in rats. We explored day by day <strong>the</strong> pattern of behavioural <strong>an</strong>d c-Fos activation in rats receiving<br />
or expecting <strong>the</strong> palatable snack.<br />
Male Wistar rats (250-300gr) were divided in two groups, those who were given <strong>an</strong>d those who<br />
were expecting a palatable stimulus <strong>the</strong> day of sacrifice. Sacifice days were: 1, 2, 3, 5 <strong>an</strong>d 8 of<br />
chocolate exposure. Rats were perfused whit 4% para<strong>for</strong>maldehyde, brains were processed <strong>for</strong> c-<br />
Fos immunohistochemistry. Positive cells were counted with a semiautomatic image <strong>an</strong>alysis<br />
system.<br />
Rats after receiving a palatable snack showed increased c-Fos positive cells in <strong>the</strong> NAcc Core,<br />
Shell <strong>an</strong>d PFCx, with similar values among days, indicating a reactive activation to <strong>the</strong> palatable<br />
stimulus. The “expect<strong>an</strong>t” rats showed a progressive increase of c-Fos <strong>an</strong>d behavioural<br />
activation.suggesting <strong>the</strong> building up of <strong>the</strong> expect<strong>an</strong>cy mech<strong>an</strong>ism.Conclusions: The<br />
acquisition of a persever<strong>an</strong>t behaviour directed to a palatable stimulus involves <strong>the</strong> Nucleus<br />
Accumbens <strong>an</strong>d <strong>the</strong> PFCx. The absence of <strong>the</strong> palatable stimulus produces a reaction of both<br />
subnuclei of <strong>the</strong> Nucleus Acumbens similar to that observed after ingestion of palatable food.<br />
Disclosures: A.S. Bl<strong>an</strong>cas, None; K. Rodriguez, None; C.B. Escobar, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.4/GG81<br />
Topic: F.03.a. Reward<br />
Title: Hotspots <strong>for</strong> hedonic “liking” <strong>an</strong>d aversive “disliking” in ventral pallidum <strong>an</strong>d nucleus<br />
accumbens<br />
Authors: *C.-Y. HO, K. BERRIDGE;<br />
Univ. Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Hedonic hotspots <strong>for</strong> amplifying taste pleasure have been previously identified in<br />
nucleus accumbens (NAc) <strong>an</strong>d ventral pallidum (VP). For example, microinjections of u-opioid<br />
agonist (DAMGO) or endoc<strong>an</strong>nabinoid (<strong>an</strong><strong>an</strong>damide) in a 1 cubic-millimeter hotspot in NAc
medial shell amplify ‘liking’ reactions to sucrose taste. Similarly, microinjection of DAMGO in<br />
a hotspot in posterior VP amplifies hedonic “liking” reactions. Conversely, separate studies have<br />
found that excitotoxin lesions near posterior VP block sucrose ‘liking’ reactions <strong>an</strong>d replace<br />
<strong>the</strong>m with ‘disliking’ reactions to sucrose, suggesting this region may also be necessary <strong>for</strong><br />
normal sweet pleasure, <strong>an</strong>d in absence cause taste aversion. However, no study has yet explicitly<br />
compared <strong>the</strong> hedonic hotspots that enh<strong>an</strong>ce positive liking to <strong>the</strong> aversion hotspots, where<br />
lesions disrupt normal pleasure. The goal of <strong>the</strong> present study is to map <strong>an</strong>d compare <strong>the</strong> specific<br />
“liking” <strong>an</strong>d “disliking” hotspots of ventral pallidum <strong>an</strong>d nucleus accumbens. In <strong>the</strong> same rats,<br />
we have compared sucrose ‘liking’ enh<strong>an</strong>cements caused by microinjection of u-opioid agonist<br />
or orexin to ‘disliking’ caused by reversible inactivation via GABA agonists<br />
(muscimol/baclofen) or excitotoxin lesion (quinolinic acid or ibotenic acid). We <strong>the</strong>n<br />
functionally mapped <strong>the</strong> responsible sites using a Fos plume mapping technique or a similar<br />
lesion mapping technique. Our preliminary data suggest that VP contains overlapping hotspots<br />
sufficient <strong>for</strong> enh<strong>an</strong>cing ’liking’, necessary <strong>for</strong> normal pleasure, <strong>an</strong>d where neuronal dysfunction<br />
causes ‘disliking’. By comparison, <strong>the</strong> NAc hotspot may be sufficient to enh<strong>an</strong>ce ‘liking’ but not<br />
needed <strong>for</strong> normal ‘liking’, <strong>an</strong>d lesions may not cause aversion. This finding may extend our<br />
underst<strong>an</strong>ding of <strong>the</strong> role of accumbens-pallidal circuits in reward processing <strong>an</strong>d carries<br />
implications <strong>for</strong> underst<strong>an</strong>ding hedonic dysfunction in drug addiction, obesity <strong>an</strong>d mood<br />
disorders.<br />
Disclosures: C. Ho, None; K. Berridge, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.5/GG82<br />
Topic: F.03.a. Reward<br />
Support: NIH Gr<strong>an</strong>t 5R01DA017752-05<br />
Title: Dopaminergic modulation of neural coding of sweet <strong>an</strong>d bitter tastes in <strong>the</strong> ventral<br />
pallidum<br />
Authors: *C. A. ITOGA 1 , K. C. BERRIDGE 2 , J. W. ALDRIDGE 2 ;<br />
1 Neurosci., Univ. Of Michig<strong>an</strong>, Ann Arbor, MI; 2 Psychology, Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Dopaminergic drugs modulate <strong>the</strong> incentive motivation <strong>for</strong> sweet rewards<br />
(“w<strong>an</strong>ting”), even when not similarly altering <strong>the</strong> hedonic impact of <strong>the</strong> same rewards (“liking”).
Here we investigated systemic drug effects on neural coding of taste stimuli in <strong>the</strong> ventral<br />
pallidum (VP), a critical output structure in mesolimbic reward circuits. Unconditioned tastes<br />
have been posited to ‘reboost’ incentive salience attributed to associated cues, which may<br />
involve dopamine <strong>an</strong>d be suppressed by neuroleptics, but <strong>the</strong> neural basis of reboosting has not<br />
yet been identified. Amphetamine (1 mg/kg; sc), which promotes monoamine release, or<br />
haloperidol, a D2 receptor blocker, (1 mg/kg; sc) were used to increase <strong>an</strong>d decrease dopamine<br />
levels. Three tastes of different hedonic valence were delivered via oral c<strong>an</strong>nula in 0.1 ml pulses:<br />
neutral (distilled water), hedonic (9% sucrose solution), <strong>an</strong>d aversive (0.01% quinine). Neural<br />
activity was recorded with multi-wire moveable electrodes impl<strong>an</strong>ted bilaterally in VP. A neural<br />
response to a stimulus was classified as excitation if <strong>the</strong> rate in 1 or more 50 ms bins were ≥ 3<br />
SD above me<strong>an</strong> baseline rate or 2 or more bins were ≥ 2 SD above me<strong>an</strong> baseline rate. Neurons<br />
were classified as inhibited if <strong>the</strong>re were 1 or more bins ≥ 2 SD below me<strong>an</strong> baseline rate or 2 or<br />
more bins ≥ 1 SD below me<strong>an</strong> baseline rate.<br />
We assessed 91 neural units in 4 rats <strong>for</strong> population coding of tastes. Systemic haloperidol<br />
suppressed <strong>the</strong> population of excitations to all 3 tastes compared to vehicle control trials (Hal:<br />
36-41%; Veh: 81-58%; p < 0.05 chi square) <strong>an</strong>d simult<strong>an</strong>eously increased <strong>the</strong> population of<br />
inhibitions <strong>for</strong> sucrose, water <strong>an</strong>d quinine (Hal: 32-36%; Veh: 8-15%; p
Authors: *S. E. ROSS 1 , J. W. ALDRIDGE 2 ;<br />
1 Biomed. Engin., 2 Psychology, Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Continuous high frequency stimulation (HFS) in <strong>the</strong> brain is a successful treatment <strong>for</strong><br />
Parkinson’s disease <strong>an</strong>d o<strong>the</strong>r motor disorders, <strong>an</strong>d it has spawned interest in treating o<strong>the</strong>r<br />
neurological disorders (e.g., depression, obsessive compulsive disorder, morbid obesity, etc.).<br />
High frequency (> 100 Hz) stimulation may activate <strong>an</strong>d entrain neurons effectively creating <strong>an</strong><br />
“in<strong>for</strong>mation lesion” (Grill et al., 2004). We are testing this idea by attempting to diminish<br />
“w<strong>an</strong>ting” <strong>for</strong> food rewards by HFS in <strong>the</strong> caudal ventral pallidum, <strong>an</strong> import<strong>an</strong>t output node in<br />
mesolimbic reward circuits that is modulated by <strong>the</strong> incentive value of rewards (Tindell et al.,<br />
2005).<br />
We first tested <strong>the</strong> effect of stimulation on free consumption of M&Ms (chocolate c<strong>an</strong>dies - a<br />
highly palatable food reward). The number of M&Ms consumed in 30 mins was measured under<br />
continuous const<strong>an</strong>t current biphasic stimulation at low (20 Hz or 25 Hz) or high (130 Hz or 200<br />
Hz) frequencies, <strong>an</strong>d under no stimulation. Overall M&M consumption decreased on average<br />
<strong>from</strong> 7.5 M&Ms with no stimulation to 4.3 M&Ms during HFS <strong>an</strong>d to only 6.3 M&Ms during<br />
LFS (N = 3). A second test used a Pavlovi<strong>an</strong> association task with a predictive cue (10 s<br />
presentation of a lever <strong>an</strong>d light) followed by a feeder-click <strong>an</strong>d <strong>the</strong> delivery of a sucrose pellet.<br />
Pellet-delivery was not contingent on responses on <strong>the</strong> lever. The same stimulation protocol<br />
described above was used. The latency to approach <strong>the</strong> lever cue or food cup <strong>an</strong>d <strong>the</strong> latency to<br />
consume <strong>the</strong> pellet were measured. Number of contacts with <strong>the</strong> lever cue <strong>an</strong>d <strong>the</strong> pellet-delivery<br />
chute were also calculated. Rats took longer to approach <strong>the</strong> pellet-delivery chute with<br />
continuous stimulation (none: 9.9 s; HFS: 17.9 s; LFS: 21.3 s, N = 3). Contact with <strong>the</strong> lever did<br />
not differ between stimulation <strong>an</strong>d no stimulation (none: 4.8; HFS: 3.5; LFS: 4.6, N =3). There<br />
was also no difference in latency to approach <strong>the</strong> lever between stimulation <strong>an</strong>d no stimulation<br />
(none: 4.2 s; HFS: 4.4 s; LFS: 5 s, N = 3). The results suggest that stimulation in <strong>the</strong> ventral<br />
pallidum may decrease food intake, perhaps by decreasing incentive motivation (longer latency<br />
to approach <strong>an</strong>d consume food) but difference between high <strong>an</strong>d low rates of stimulation was not<br />
apparent.<br />
Grill, W. M., Snyder, A. N., & Miocinovic, S. (2004). Deep brain stimulation creates <strong>an</strong><br />
in<strong>for</strong>mational lesion of <strong>the</strong> stimulated nucleus. Neuroreport, 15, 1137-1140.<br />
Tindell, A. J., Berridge, K. C., Zh<strong>an</strong>g, J., Pecina, S., & Aldridge, J. W. (2005). Ventral pallidal<br />
neurons code incentive motivation: amplification by mesolimbic sensitization <strong>an</strong>d amphetamine.<br />
Europe<strong>an</strong> Journal of Neuroscience, 22, 2617-2634.<br />
Disclosures: S.E. Ross, None; J.W. Aldridge, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon
Program#/Poster#: 583.7/GG84<br />
Topic: F.03.a. Reward<br />
Support: NIH Gr<strong>an</strong>t DA015188<br />
NIH Gr<strong>an</strong>t MH63649<br />
NIDA Gr<strong>an</strong>t T32 DA007281<br />
Title: Which cue to ‘w<strong>an</strong>t?’ Amygdala opioid control of incentive salience on a prepotent<br />
sucrose goal-cue<br />
Authors: *A. G. DIFELICEANTONIO, K. C. BERRIDGE;<br />
Psychology, Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: The amygdala is implicated in appetitive motivation <strong>an</strong>d learning, as well as fear. The<br />
central nucleus of <strong>the</strong> amygdala (CeA) in particular participates in generating <strong>an</strong>d focusing<br />
enh<strong>an</strong>ced incentive salience on a learned Pavlovi<strong>an</strong> cue <strong>for</strong> sucrose reward (Mahler & Berridge,<br />
in press). Mu opioid stimulation of CeA by DAMGO microinjection produces <strong>an</strong> increase in <strong>the</strong><br />
“motivational magnet” properties of a lever CS+ that predicts sucrose as UCS, enh<strong>an</strong>cing<br />
frenzies of nibbles <strong>an</strong>d sniffs <strong>for</strong> rats that preferred it, in <strong>an</strong> autoshaping paradigm. This led to <strong>the</strong><br />
hypo<strong>the</strong>sis that CeA enh<strong>an</strong>ces ‘w<strong>an</strong>ting’ of a rats prepotent CS. Here we examined rats that<br />
prefer to approach <strong>the</strong> cup (CScup) that delivers sucrose during <strong>the</strong> CS+ (ra<strong>the</strong>r th<strong>an</strong> approach<br />
<strong>the</strong> lever). Does DAMGO in CeA increase <strong>the</strong> frenzy of nibble <strong>an</strong>d sniff behaviors directed<br />
toward <strong>the</strong> CScup? Rats were given microinjections of ei<strong>the</strong>r vehicle or DAMGO after 5<br />
autoshaping training sessions. We found that DAMGO selectively increased nibble/sniff type<br />
behaviors on <strong>the</strong> CScup in rats that preferred to approach that stimulus. We also replicated<br />
enh<strong>an</strong>ced nibbles <strong>an</strong>d sniffs toward <strong>the</strong> lever <strong>for</strong> rats preferred <strong>the</strong> CS+. DAMGO also increased<br />
intake of a food UCS <strong>for</strong> all rats. Opioid activation of <strong>the</strong> CeA <strong>the</strong>re<strong>for</strong>e selectively increases<br />
appetitive <strong>an</strong>d consummatory motivation <strong>for</strong> <strong>the</strong> most preferred CS in a winner take all fashion.<br />
In this way, CeA helps determine which cue to ‘w<strong>an</strong>t,’ as well as <strong>the</strong> intensity of ‘w<strong>an</strong>ting.’<br />
Disclosures: A.G. DiFelice<strong>an</strong>tonio, None; K.C. Berridge, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.8/GG85
Topic: F.03.a. Reward<br />
Support: NIH Gr<strong>an</strong>t DA015188<br />
NIH Gr<strong>an</strong>t MH63649<br />
Title: Prefrontal cortical control of nucleus accumbens-generated fear <strong>an</strong>d feeding<br />
Authors: *J. M. RICHARD, K. C. BERRIDGE;<br />
Psychology, Univ. of Michig<strong>an</strong>, Ann Arbor, MI<br />
Abstract: Disruption of glutamate signals at specific locations in <strong>the</strong> medial shell of <strong>the</strong> nucleus<br />
accumbens generates ei<strong>the</strong>r appetitive or defensive motivated behaviors. Blockade of AMPA<br />
receptors by DNQX microinjections in rostral medial shell generates appetitive eating behavior,<br />
whereas AMPA blockade in caudal shell generates defensive behaviors such as defensive<br />
treading, distress vocalizations, escape attempts <strong>an</strong>d bites. Here we tested top-down cortical<br />
modulation of accumbens-generated motivation. We ei<strong>the</strong>r inactivated regions of prefrontal<br />
cortex using microinjections of combined GABA agonists, baclofen <strong>an</strong>d muscimol, or activated<br />
prefrontal cortex via disinhibition with microinjections of <strong>the</strong> GABA <strong>an</strong>tagonist, bicuculline.<br />
Simult<strong>an</strong>eously we stimulated motivated behaviors by microinjections of <strong>the</strong> AMPA <strong>an</strong>tagonist<br />
DNQX into accumbens shell. Inactivation of infralimbic (IL) <strong>an</strong>d medial orbitofrontal (OFC)<br />
cortex enh<strong>an</strong>ced <strong>the</strong> hyperphagia normally caused by DNQX into some sites of shell. Activation<br />
of OFC conversely suppressed DNQX-induced feeding <strong>an</strong>d defensive behaviors. Activation of<br />
IL suppressed accumbens generation of defensive behaviors but not appetitive eating. Thus<br />
glutamatergic projections <strong>from</strong> OFC <strong>an</strong>d IL regions of prefrontal cortex into nucleus accumbens<br />
may modulate glutamatergic generation of appetitive <strong>an</strong>d fearful behaviors by medial shell.<br />
Disclosures: J.M. Richard, None; K.C. Berridge, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.9/GG86<br />
Topic: F.03.a. Reward<br />
Support: NIH Gr<strong>an</strong>t DA015188<br />
NIH Gr<strong>an</strong>t MH63649
NIH Gr<strong>an</strong>t DA021481<br />
Title: Mu makes me 'w<strong>an</strong>t' you: Central amygdala µ opioid stimulation enh<strong>an</strong>ces <strong>the</strong> incentive<br />
salience of sexual stimuli<br />
Authors: *S. V. MAHLER 1,2 , B. E. DZWONEK 2 , K. C. BERRIDGE 2 ;<br />
1 2<br />
Neurosciences, Med. Univ. of South Carolina, Charleston, SC; Psychology, Univ. of Michig<strong>an</strong>,<br />
Ann Arbor, MI<br />
Abstract: Stimulating µ opioid receptors in <strong>the</strong> central nucleus of <strong>the</strong> amygdala (CeA) increases<br />
‘w<strong>an</strong>ting’ of food rewards <strong>an</strong>d <strong>the</strong>ir associated cues, but is amygdala part of a common neural<br />
circuit <strong>for</strong> motivation shared by all incentives (food, sex, drugs, etc.)? Here we asked if CeA<br />
opioid stimulation in male rats increases <strong>the</strong> incentive salience of sexual stimuli. An estrous<br />
female rat <strong>an</strong>d a non-estrous female were enclosed in small per<strong>for</strong>ated chambers on opposite<br />
sides of a larger testing environment, allowing a male to approach <strong>an</strong>d sniff both females, but not<br />
to copulate. Males received bilateral microinjections in CeA of DAMGO (0.1µg/0.2µl) or<br />
vehicle (on different days in counter-bal<strong>an</strong>ced order <strong>for</strong> within-subject comparison), <strong>an</strong>d <strong>the</strong>n<br />
were allowed to freely investigate <strong>an</strong>d approach both females during a 30 min test. Overall,<br />
males preferred to approach <strong>an</strong>d sniff <strong>the</strong> estrous female more th<strong>an</strong> <strong>the</strong> non-estrous female.<br />
Opioid stimulation of CeA fur<strong>the</strong>r enh<strong>an</strong>ced this preference <strong>for</strong> <strong>the</strong> estrous female, <strong>an</strong>d did not<br />
ch<strong>an</strong>ge interactions with or proximity to <strong>the</strong> non-estrous female. These results indicate that CeA<br />
opioid stimulation enh<strong>an</strong>ces <strong>an</strong>d focuses ‘w<strong>an</strong>ting’ <strong>for</strong> a preferred sexual incentive stimulus, as<br />
we have previously reported it does <strong>for</strong> a food incentive stimulus. This supports <strong>the</strong> hypo<strong>the</strong>sis<br />
CeA opioid circuits are part of common neural substrates <strong>for</strong> directing incentive salience toward<br />
a prepotent reward in a “winner take all” fashion.<br />
Disclosures: S.V. Mahler, None; B.E. Dzwonek, None; K.C. Berridge, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.10/GG87<br />
Topic: F.03.a. Reward<br />
Support: Cali<strong>for</strong>nia State Funds<br />
National Alli<strong>an</strong>ce <strong>for</strong> Research on Schizophrenia <strong>an</strong>d Depression
Title: Activation of nucleus accumbens neurons produces a pause in sucrose consumption<br />
Authors: *M. KRAUSE 1 , P. W. GERMAN 1 , S. A. TAHA 2 , H. L. FIELDS 1 ;<br />
1 2<br />
UCSF - Ernest Gallo Clin. <strong>an</strong>d Res. Ctr., Emeryville, CA; Physiol., Univ. Sch. of Med., Salt<br />
Lake City, UT<br />
Abstract: Activation of GABA receptors, or blocking AMPA receptors in <strong>the</strong> medial part of <strong>the</strong><br />
rat nucleus accumbens (NAc) shell signific<strong>an</strong>tly increases food intake. This indicates that<br />
inhibition of NAc neurons c<strong>an</strong> inhibit feeding. Previously, our lab found that a subpopulation of<br />
neurons (~30%) in <strong>the</strong> rat NAc showed a pause in <strong>the</strong>ir firing rate beginning ~1 s prior to <strong>the</strong><br />
onset of sucrose licking. These pauses continued <strong>for</strong> <strong>the</strong> entire period of consumption,<br />
independent of body movement (Taha et al., J.Neurosci., 2005 & 2006). Based on <strong>the</strong>se results<br />
we proposed that firing in a subpopulation of NAc neurons inhibited licking so that a pause in<br />
<strong>the</strong>ir activity is required to release (or gate) feeding behavior. To fur<strong>the</strong>r test this hypo<strong>the</strong>sis we<br />
recorded single unit activity in <strong>the</strong> NAc toge<strong>the</strong>r with licking during a sucrose licking task.<br />
Following recording <strong>the</strong> same electrodes were used <strong>for</strong> local stimulation at <strong>the</strong> recording site. In<br />
agreement with our previous results we found that about one third of neurons recorded in <strong>the</strong><br />
nucleus accumbens decreased <strong>the</strong>ir firing rate prior to <strong>the</strong> onset of sucrose consumption. In a<br />
given trial sucrose was available <strong>for</strong> 3 s, <strong>an</strong>d rats routinely licked beyond that time period. Local<br />
stimulation (15 or 40 Hz, 100 or 200 µs per pulse, <strong>for</strong> 1 s) in <strong>the</strong> medial NAc shell, starting 1 s<br />
after lick onset, disrupted licking <strong>for</strong> about 1 s. Licking resumed immediately after <strong>the</strong> end of <strong>the</strong><br />
stimulation. The disruptive effect of <strong>the</strong> stimulation on licking was persistent, <strong>an</strong>d depended on<br />
<strong>the</strong> pulse train frequency, <strong>the</strong> pulse duration, <strong>an</strong>d <strong>the</strong> stimulus intensity (up to 600 µA). We<br />
assume that stimulation excites neurons in <strong>the</strong> vicinity of <strong>the</strong> electrodes <strong>an</strong>d prevents <strong>the</strong> pause<br />
required <strong>for</strong> licking to occur. These data support <strong>the</strong> hypo<strong>the</strong>sis that a firing pause is required in a<br />
subpopulation of NAc neurons to permit sucrose consumption.<br />
Disclosures: M. Krause, None; P.W. Germ<strong>an</strong>, None; S.A. Taha, None; H.L. Fields, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.11/GG88<br />
Topic: F.03.a. Reward<br />
Support: NIH DA021280<br />
NIH DA015267
NIH DA003240<br />
NIH DA019942<br />
NIH DA024196<br />
NIH DA023301<br />
Title: Oper<strong>an</strong>t responding <strong>for</strong> sucrose by rats selectively bred <strong>for</strong> high or low saccharin<br />
consumption<br />
Authors: *B. A. GOSNELL 1 , A. MITRA 1 , R. A. AVANT 1 , J. J. ANKER 2 , M. E. CARROLL 2 ,<br />
A. S. LEVINE 1 ;<br />
1 Food Sci, Nutrit, Univ. Minnesota, St. Paul, MN; 2 Psychiatry, Univ. Minnesota, Minneapolis,<br />
MN<br />
Abstract: The use of rats differing in preferences or intake of sweet subst<strong>an</strong>ces has highlighted<br />
some interesting parallels between taste preferences <strong>an</strong>d drug self-administration. For example,<br />
<strong>the</strong> HiS <strong>an</strong>d LoS rat strains, selectively bred to have a high or low consumption of saccharin<br />
solution (when compared to water consumption), show corresponding differences in <strong>the</strong><br />
acquisition, mainten<strong>an</strong>ce, escalation <strong>an</strong>d reinstatement of cocaine-seeking behavior. The<br />
development of <strong>the</strong> HiS <strong>an</strong>d LoS phenotypes was based on consumption when water or saccharin<br />
solutions were available ad libitum. In this study, we measured whe<strong>the</strong>r <strong>the</strong> two strains also<br />
differ when response requirements are imposed <strong>for</strong> obtaining a sweet-tasting rein<strong>for</strong>cer. Male<br />
HiS <strong>an</strong>d LoS were measured <strong>for</strong> oper<strong>an</strong>t responding <strong>for</strong> 45 mg sucrose pellets. They were<br />
maintained on ad libitum food <strong>an</strong>d water with <strong>the</strong> exception of 1 - 2 overnight food deprivation<br />
periods to facilitate learning <strong>the</strong> task. They were tested under fixed ratio (FR) schedules of 1, 3, 5<br />
<strong>an</strong>d 10. Following <strong>the</strong>se, <strong>the</strong>y were tested under a progressive ratio (PR) schedule, during which<br />
<strong>the</strong> response requirement <strong>for</strong> each successive pellet increased according to <strong>an</strong> exponential<br />
schedule. The breakpoint was defined as <strong>the</strong> highest ratio completed when one hour elapsed<br />
without completing <strong>the</strong> next ratio. The effects of naltrexone (0.3. 1 <strong>an</strong>d 3 mg/kg) on PR<br />
responding were also tested. Under FR 1, 3, 5 <strong>an</strong>d 10 schedules, <strong>the</strong> me<strong>an</strong> numbers of pellets<br />
obtained by <strong>the</strong> LoS rats were 56, 52, 46 <strong>an</strong>d 35%, respectively, of <strong>the</strong> me<strong>an</strong>s of <strong>the</strong> HiS rats.<br />
Under <strong>the</strong> PR schedule, HiS rats earned 8.0 + 0.5 pellets vs. 6.3 + 0.4 pellets earned by <strong>the</strong> LoS<br />
rats. These correspond to breakpoints of approximately 20 <strong>an</strong>d 12, respectively. No phenotype<br />
differences in <strong>the</strong> effects of naltrexone were observed. Measures of locomotor activity taken<br />
prior to oper<strong>an</strong>t trials suggest that differences in FR <strong>an</strong>d PR responding are not due to differences<br />
in general activity levels. These results extend <strong>the</strong> characteristics of <strong>the</strong> HiS <strong>an</strong>d LoS phenotypes<br />
to include differences in sucrose intake when a work requirement is imposed, indicating a<br />
difference in <strong>the</strong> motivation to acquire <strong>an</strong>d consume sweet subst<strong>an</strong>ces.<br />
Disclosures: B.A. Gosnell, None; A. Mitra, None; R.A. Av<strong>an</strong>t, None; J.J. Anker, None; M.E.<br />
Carroll, None; A.S. Levine, None.
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.12/GG89<br />
Topic: F.03.a. Reward<br />
Support: NIDA gr<strong>an</strong>t DA 09311<br />
Title: Mapping subregion-specific contributions of frontal cortex to opiate-mediated 'bingeing'<br />
on sweetened fat<br />
Authors: *R. C. TWINING, A. E. KELLEY, B. A. BALDO;<br />
UW - Madison, Madison, WI<br />
Abstract: Stimulation of µ-opioid receptors in <strong>the</strong> nucleus accumbens (Acb) produces <strong>an</strong> intense<br />
hyperphagia; food intake after this m<strong>an</strong>ipulation equals or exceeds that seen after moderate to<br />
severe food deprivation. Moreover, feeding responses induced by intra-Acb m<strong>an</strong>ipulation of µopioid<br />
receptors exhibit some degree of specificity <strong>for</strong> palatable foods <strong>an</strong>d tast<strong>an</strong>ts such as<br />
sucrose, saccharin, palatable saline solutions, <strong>an</strong>d sweetened lard. In<strong>for</strong>med in part by <strong>the</strong>se<br />
observations, we have hypo<strong>the</strong>sized that opioid tr<strong>an</strong>smission in <strong>the</strong> Acb ‘magnifies’ <strong>the</strong> reward<br />
valuation of foods that <strong>the</strong> <strong>an</strong>imal finds intrinsically palatable. This could occur through several<br />
mech<strong>an</strong>isms such as <strong>the</strong> modulation of incoming corticolimbic in<strong>for</strong>mation regarding taste or <strong>the</strong><br />
initial affective response to food. Accordingly, it has previously been shown that inactivation of<br />
<strong>the</strong> basolateral amygdala, <strong>an</strong> import<strong>an</strong>t source of glutamate input to <strong>the</strong> Acb, reduces opioiddriven<br />
‘binging’ on sweetened lard (Will et al., 2004). In <strong>the</strong> present study, we aimed to explore<br />
<strong>the</strong> contribution of frontal cortical regions. Four groups of rats were prepared with two sets of<br />
bilateral infusion c<strong>an</strong>nulae. One set was aimed at <strong>the</strong> Acb shell in all groups. The o<strong>the</strong>r set was<br />
aimed at agr<strong>an</strong>ular insular cortex (i.e., gustatory cortex, N=7), rostral lateral orbital cortex (N=7),<br />
ventral medial prefrontal cortex (vmPFC, at <strong>the</strong> dorsal boundary of infralimbic cortex, N=8), <strong>an</strong>d<br />
dorsal medial prefrontal cortex (Cg1, or <strong>an</strong>terior cingulate cortex; N=8). Rats received infusions<br />
of saline or <strong>the</strong> µ-opioid agonist, DAMGO (0.25 µg), into <strong>the</strong> Acb shell, <strong>an</strong>d infusions of saline,<br />
or <strong>the</strong> GABA-A agonist muscimol (75 ng) into <strong>the</strong> cortical site under study. Muscimol is widely<br />
used to produce temporary inactivation in cortical sites. We found a selective potentiation of<br />
DAMGO-induced sweetened-fat intake after inactivation of vmPFC. No effects were observed<br />
on sweetened-fat intake after inactivation of <strong>the</strong> o<strong>the</strong>r sites. These results suggest that vmPFC<br />
may serve as a ‘check’ on opiate-driven reward-related behaviors, <strong>an</strong>d perhaps counterbal<strong>an</strong>ce<br />
<strong>the</strong> effect of limbic input in <strong>the</strong> amygdala-Acb pathway. Results will be discussed in <strong>the</strong> context<br />
of vmPFC’s role in <strong>the</strong> context-appropriate gating of ‘prepotent’ appetitive behaviors.<br />
Disclosures: R.C. Twining, None; A.E. Kelley, None; B.A. Baldo, None.
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.13/GG90<br />
Topic: F.03.a. Reward<br />
Support: RO1DA017676 (R.J.D)<br />
F32DA023739 (R.S.)<br />
RL1AA017537 (J.R.T. <strong>an</strong>d R.J.D.)<br />
Title: Orexin signaling via <strong>the</strong> orexin 1 receptor mediates food oper<strong>an</strong>t responding<br />
Authors: *R. SHARF, M. SARHAN, D. J. GUARNIERI, C. E. BRAYTON, J. R. TAYLOR, R.<br />
J. DILEONE;<br />
Dept Psych, Yale Univ., New Haven, CT<br />
Abstract: Orexin signaling is implicated in drug addiction <strong>an</strong>d reward, but its role in feeding <strong>an</strong>d<br />
food-motivated behavior remains unclear. Here, we investigated orexin's contribution to foodrein<strong>for</strong>ced<br />
instrumental responding using <strong>an</strong> orexin 1 receptor (Ox1r) <strong>an</strong>tagonist, orexin -/-<br />
(OKO) mice, <strong>an</strong>d RNAi-mediated knockdown of orexin. Mice were trained to nose poke <strong>for</strong> food<br />
under a variable ratio (VR) schedule of rein<strong>for</strong>cement <strong>an</strong>d once stable responding was<br />
demonstrated, a progressive ratio (PR) schedule was initiated to evaluate motivation to obtain<br />
food reward. Blockade of Ox1r in C57BL/6J mice (n=26) resulted in normal acquisition of <strong>the</strong><br />
nose poking task, but signific<strong>an</strong>tly reduced responding under both <strong>the</strong> VR <strong>an</strong>d PR schedules of<br />
rein<strong>for</strong>cement. In contrast, OKO mice (n=12) initially demonstrated a delay in acquisition, but<br />
eventually achieved levels of responding similar to those observed in wild-type (WT) <strong>an</strong>imals<br />
<strong>an</strong>d did not differ <strong>from</strong> WT mice under a PR schedule. RNAi mediated knockdown of orexin<br />
was <strong>the</strong>n used to eliminate <strong>an</strong>y compensation in OKO mice due to <strong>the</strong> absence of orexin<br />
throughout development. Orexin gene knockdown in <strong>the</strong> lateral hypothalamus (LH) in C57BL/6J<br />
mice (n=22) resulted in normal acquisition, but blunted per<strong>for</strong>m<strong>an</strong>ce under both <strong>the</strong> VR <strong>an</strong>d PR<br />
schedules, resembling data obtained following Ox1r <strong>an</strong>tagonism. These data suggest that<br />
activation of <strong>the</strong> Ox1r is a necessary component of food reward <strong>an</strong>d/or motivation in normal<br />
mice. Fur<strong>the</strong>r, <strong>the</strong> opposite patterns of behavior seen in OKO mice is likely to reflect<br />
developmental compensation commonly seen in mut<strong>an</strong>t <strong>an</strong>imals.
Disclosures: R. Sharf, None; M. Sarh<strong>an</strong>, None; D.J. Guarnieri, None; C.E. Brayton,<br />
None; J.R. Taylor, None; R.J. DiLeone, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.14/GG91<br />
Topic: F.03.a. Reward<br />
Support: NIDA Gr<strong>an</strong>t 2T32DA007255-16<br />
Title: To seek or not to seek: Leptin's role in feeding as resources become scarce<br />
Authors: *C. R. FRAZIER 1 , X. ZHUANG 2 ;<br />
1 Committee on Neurobio., 2 Dept. of Neurobio., Univ. of Chicago, Chicago, IL<br />
Abstract: A hallmark of obesity is elevated plasma leptin concentrations but a resist<strong>an</strong>ce to<br />
leptin signaling in <strong>the</strong> brain. <strong>When</strong> food is readily-available, leptin signaling reduces feeding <strong>an</strong>d<br />
promotes energy expenditure through metabolic <strong>an</strong>d behavioral ch<strong>an</strong>ges. However, leptin is not<br />
considered a satiety signal since circulating leptin concentrations do not increase immediately<br />
after a meal. Instead leptin is a signal representing <strong>the</strong> availability of internal energy stores <strong>an</strong>d<br />
its production <strong>an</strong>d secretion <strong>from</strong> fat is a dynamic process. While <strong>the</strong> effects of leptin on feeding<br />
in <strong>an</strong> ad libitum environment have been studied extensively, <strong>the</strong> role of leptin signaling in food<br />
seeking behavior when food is scarce has received little attention. Using homecage oper<strong>an</strong>t<br />
boxes, in which a mouse must lever press <strong>for</strong> all of its food, we evaluated <strong>the</strong> <strong>for</strong>aging strategies<br />
of le<strong>an</strong> <strong>an</strong>d leptin-deficient obese mice (ob/ob) as <strong>the</strong> scarcity of food increased, i.e., <strong>the</strong> lever<br />
pressing requirement increased every four days over <strong>the</strong> course of two weeks. As expected, we<br />
found that when <strong>the</strong> cost of food was relatively low, ob/ob mice lever pressed more <strong>for</strong> food th<strong>an</strong><br />
le<strong>an</strong> controls <strong>an</strong>d as a consequence earned more food, in agreement with <strong>the</strong>ir hyperphagic<br />
phenotype when food is freely available. However, when <strong>the</strong> cost of food was relatively high,<br />
ob/ob mice lever pressed less <strong>for</strong> food th<strong>an</strong> le<strong>an</strong> mice <strong>an</strong>d earned less food th<strong>an</strong> le<strong>an</strong> controls.<br />
These data suggest that leptin may have dual functions. In addition to its well-established ability<br />
to reduce feeding <strong>an</strong>d promote metabolic energy expenditure during periods of plenty, leptin<br />
signaling may maintain or promote goal-directed energy expenditure <strong>an</strong>d food seeking as<br />
resources become scarce. To test this hypo<strong>the</strong>sis, we are investigating whe<strong>the</strong>r acute leptin c<strong>an</strong><br />
promote food-seeking when <strong>the</strong> cost of food is relatively high, as well as <strong>the</strong> neural substrates<br />
that might mediate this effect. We will discuss how leptin’s dual function may affect <strong>an</strong> <strong>an</strong>imal's<br />
fitness during periods of food scarcity <strong>an</strong>d <strong>the</strong> implications of leptin-resist<strong>an</strong>ce in obesity.
Disclosures: C.R. Frazier, None; X. Zhu<strong>an</strong>g, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.15/GG92<br />
Topic: F.03.a. Reward<br />
Title: Drug <strong>an</strong>d food rewards differentially modulate learning strategies <strong>an</strong>d brain regional p-<br />
CREB expression<br />
Authors: M. BAUDONNAT, J.-L. GUILLOU, M. CORIO, N. MONS, Y. PORTE, Q.<br />
BARRAUD, *V. DAVID;<br />
Ctr. De Neurosci. Integrative & Cognitive CNRS Umr5228/Univ. Bordeaux 1-2, Talence, Fr<strong>an</strong>ce<br />
Abstract: Two <strong>for</strong>ms of memory are proposed to be mediated by independent brain systems: <strong>the</strong><br />
“declarative / cognitive” memory relying on hippocampal circuitry, whereas <strong>the</strong> “habitual /<br />
procedural / response” memory is associated to <strong>the</strong> dorsal striatal system. Although distinct,<br />
<strong>the</strong>se memory systems may operate ei<strong>the</strong>r cooperatively (McDonald et al., 2004; Voerm<strong>an</strong>s et<br />
al., 2004) or competitively (Poldrack <strong>an</strong>d Packard, 2003). We have compared <strong>the</strong> effects of a<br />
food vs drug rein<strong>for</strong>cement on <strong>the</strong> selection of memory systems during learning. Mice were<br />
trained <strong>for</strong> 10 days to learn a discrimination task in a Y-maze using ei<strong>the</strong>r a cued or spatial<br />
protocol, <strong>an</strong>d were rein<strong>for</strong>ced with ei<strong>the</strong>r a palatable food (crisps) or drug-induced activation of<br />
<strong>the</strong> reward system (intra-VTA morphine injection). Mice trained under <strong>the</strong> cue-guided protocol<br />
learned <strong>the</strong> Y-maze arm discrimination task faster, whe<strong>the</strong>r <strong>the</strong>y were drug or food-rein<strong>for</strong>ced.<br />
Surprisingly, food-rein<strong>for</strong>ced mice show good per<strong>for</strong>m<strong>an</strong>ces with <strong>the</strong> spatial protocol, whereas<br />
drug-rein<strong>for</strong>ced <strong>an</strong>imals did not exhibit <strong>an</strong>y arm discrimination. All subjects were submitted 48<br />
hrs later to a water-maze competition task allowing <strong>the</strong> use of ei<strong>the</strong>r cued or spatial strategies.<br />
We found that mice previously drug-rein<strong>for</strong>ced tend to use <strong>the</strong> strategy previously learned in <strong>the</strong><br />
Y-maze task to resolve <strong>the</strong> water-maze competition task. P-CREB <strong>an</strong>alysis per<strong>for</strong>med 15 min<br />
after <strong>the</strong> last training in <strong>the</strong> Y-maze revealed that mice trained under <strong>the</strong> cued-strategy exhibited<br />
a decrease in p-CREB expression within <strong>the</strong> dorsal hippocampus <strong>an</strong>d <strong>an</strong> increase within <strong>the</strong><br />
dorsomedial striatum, independently of <strong>the</strong> type of reward. In contrast, p-CREB <strong>an</strong>alysis<br />
per<strong>for</strong>med on <strong>an</strong>imals trained under <strong>the</strong> spatial condition revealed that food-, but not drugrewarded<br />
<strong>an</strong>imals show a strategy-dependent activation of <strong>the</strong> CA1 field of <strong>the</strong> dorsal<br />
hippocampus. We conclude that drug could modify <strong>the</strong> relative contribution of <strong>the</strong> different brain<br />
memory systems used during <strong>the</strong> Y-maze task <strong>an</strong>d influence subsequent learning by promoting a<br />
learning strategy based on implicit memory.
Disclosures: M. Baudonnat, None; J. guillou, None; M. Corio, None; N. Mons, None; Y.<br />
porte, None; Q. barraud, None; V. David, Baudonnat M., Guillou J-L, Corio M., Mons N.,<br />
Porte Y., Barraud Q., David V., A. Employment (full or part-time).<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.16/GG93<br />
Topic: F.03.a. Reward<br />
Support: DFG Schw 559/10-1<br />
Title: Ultrasonic vocalizations during a runway task: 50-kHz calls as <strong>an</strong> index of incentive<br />
motivation?<br />
Authors: *R. K. SCHWARTING, J. C. BRENES;<br />
Philipps-University of Marburg, Marburg, Germ<strong>an</strong>y<br />
Abstract: Adolescent <strong>an</strong>d adult rats emit at least two different types of ultrasonic vocalizations<br />
(USV), which are thought to represent <strong>an</strong> index of <strong>the</strong>ir subjective emotional state <strong>an</strong>d/or a social<br />
communication signal. Low-frequency (22-kHz) calls are displayed when <strong>an</strong>imals are exposed to<br />
predators, foot-shocks, during inter-male aggression, <strong>an</strong>d drug withdrawal. In general, <strong>the</strong>se calls<br />
usually appear in <strong>an</strong>ticipation of, or during aversive environmental or internal situations. Highfrequency<br />
(50-kHz) calls, in contrast, have been observed in response to or expect<strong>an</strong>cy of<br />
positive affective stimuli such as sex, food, drug of abuse, electrical brain stimulation, play, <strong>an</strong>d<br />
tickling. Although 50-kHz USV could address a positive emotional state, <strong>the</strong> role of <strong>the</strong>se calls<br />
in incentive motivation tasks has not been systematically investigated. Traditionally, <strong>the</strong> speed<br />
with which a trained <strong>an</strong>imal traverses a runway (long straight alley) <strong>for</strong> positive stimuli, like food<br />
or water has been taken as a reliable index of <strong>the</strong> subject's incentive motivation. Here, a modified<br />
runway is used to <strong>an</strong>alyze simult<strong>an</strong>eously goal-oriented behavior (i.e. <strong>an</strong>ticipation, approach, <strong>an</strong>d<br />
consumption) <strong>an</strong>d USV in rats with repeated reward experience. Several variations of runway<br />
parameters such as reward size, waiting time, <strong>an</strong>d contrast effects are tested in male Wistar rats<br />
trained to earn a high palatable food. Evidence will be presented, which shows whe<strong>the</strong>r <strong>an</strong>d how<br />
<strong>the</strong> occurrence of 50-kHz calls is dependent on factors such as experience, phases of <strong>an</strong>ticipation,<br />
<strong>an</strong>d individual predispositions.<br />
Disclosures: R.K. Schwarting, None; J.C. Brenes, None.
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.17/GG94<br />
Topic: F.03.a. Reward<br />
Support: KAKENHI 854029<br />
KAKENHI 17022027<br />
KAKENHI 18020019<br />
KAKENHI 20033013<br />
KAKENHI 20300139<br />
Title: Analyses of <strong>the</strong> time course of neuronal activity of <strong>the</strong> pedunculopontine tegmental<br />
nucleus in monkeys <strong>for</strong> reward conditioned saccade task<br />
Authors: *K.-I. OKADA 1 , Y. KOBAYASHI 1,2 ;<br />
1 Osaka Univ., Toyonaka, Jap<strong>an</strong>; 2 ATR, Kyoto, Jap<strong>an</strong><br />
Abstract: M<strong>an</strong>y physiological studies have suggested a role of <strong>the</strong> pedunculopontine tegmental<br />
nucleus (PPTN) in controlling associative learning <strong>an</strong>d <strong>the</strong> conditioned behavior. We previously<br />
examined neuronal activity of <strong>the</strong> PPTN during a reward conditioned visually guided saccade<br />
task, where <strong>the</strong> task was started <strong>from</strong> <strong>the</strong> fixation target (FT) presentation <strong>an</strong>d terminated by<br />
reward delivery (RD) after <strong>the</strong> saccade target (ST) offset, <strong>an</strong>d <strong>the</strong> magnitude of given reward was<br />
cued by <strong>the</strong> shape of <strong>the</strong> FT, <strong>an</strong>d reported that a population of <strong>the</strong> neurons (FT neurons) showed<br />
sustained response persisted between <strong>the</strong> FT presentation <strong>an</strong>d RD, while o<strong>the</strong>rs (RD neurons)<br />
showed phasic response after <strong>the</strong> RD. The FT <strong>an</strong>d RD neurons carried predicted <strong>an</strong>d actual<br />
reward value in<strong>for</strong>mation, respectively, both of which are essential <strong>for</strong> <strong>the</strong> computation of reward<br />
prediction error in rein<strong>for</strong>cement learning.<br />
To fur<strong>the</strong>r investigate <strong>the</strong> FT <strong>an</strong>d RD responses, we examined time course of <strong>the</strong> neuronal<br />
activity in response to <strong>the</strong> task stimuli, RD <strong>an</strong>d <strong>an</strong>imal’s task behavior. A population of <strong>the</strong> FT<br />
neurons gradually started <strong>the</strong>ir sustained activity be<strong>for</strong>e <strong>the</strong> task start (FT presentation), <strong>an</strong>d <strong>the</strong><br />
activity was correlated with <strong>the</strong> time of <strong>the</strong> monkey's preparatory centering gaze shift waiting <strong>for</strong><br />
<strong>the</strong> time of FT presentation. To examine shutdown timing of <strong>the</strong> sustained FT response around<br />
termination of a trial, we reversed <strong>the</strong> order of <strong>the</strong> task sequence in between <strong>the</strong> ST offset <strong>an</strong>d
RD <strong>for</strong> a block of trials. In <strong>the</strong> reversed trial, <strong>the</strong> ST remained visible after <strong>the</strong> RD <strong>an</strong>d was<br />
turned off at <strong>the</strong> end of trial, <strong>an</strong>d <strong>the</strong> monkey kept fixation to <strong>the</strong> ST even after <strong>the</strong> RD. A<br />
population of <strong>the</strong> sustained FT responses continued until <strong>the</strong> ST offset in <strong>the</strong> reversed trial. Thus,<br />
<strong>the</strong> sustained activity was modulated <strong>for</strong> <strong>the</strong> time prediction of task start (FT presentation) <strong>an</strong>d<br />
actual time of task end (<strong>the</strong> RD in <strong>the</strong> normal trial <strong>an</strong>d <strong>the</strong> ST offset in <strong>the</strong> reversed trial).<br />
Ano<strong>the</strong>r population of <strong>the</strong> FT neurons ceased <strong>the</strong> sustained activity after <strong>the</strong> RD independent of<br />
<strong>the</strong> task sequence, <strong>an</strong>d thus <strong>the</strong>se neurons send <strong>the</strong> working memory of <strong>the</strong> reward prediction<br />
until <strong>the</strong> time of RD. For <strong>the</strong> RD neurons, <strong>the</strong> task sequence reversal did not influence <strong>the</strong> RD<br />
response consistent with <strong>the</strong> idea that <strong>the</strong>se neurons send <strong>the</strong> actual reward signal. However, a<br />
population of <strong>the</strong> RD neurons showed weak but signific<strong>an</strong>t time prediction activity be<strong>for</strong>e <strong>the</strong><br />
task reward.<br />
These results suggest that, in addition to <strong>the</strong> predicted <strong>an</strong>d actual reward value signals <strong>for</strong><br />
rein<strong>for</strong>cement learning, <strong>the</strong> PPTN neurons carry time prediction <strong>an</strong>d actual time signals of <strong>the</strong><br />
task events <strong>an</strong>d would contribute to execution of <strong>the</strong> conditioned task behavior.<br />
Disclosures: K. Okada, None; Y. Kobayashi, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.18/GG95<br />
Topic: F.03.a. Reward<br />
Support: NSERC Discovery Gr<strong>an</strong>t<br />
OMHF<br />
Title: C<strong>an</strong> over-eating induce a conditioned taste avoid<strong>an</strong>ce in previously food restricted rats?<br />
Authors: *A. HERTEL, R. EIKELBOOM;<br />
Dept. of Psychology, Wilfrid Laurier Univ., Waterloo, ON, C<strong>an</strong>ada<br />
Abstract: While feeding is rewarding, <strong>the</strong> feeling of satiation has been <strong>the</strong>orized to have a mixed<br />
affect (Woods, 1991). Using a food-restriction model of bingeing developed in our laboratory, it<br />
was found that overeating is capable of supporting a conditioned taste avoid<strong>an</strong>ce. Young, adult<br />
male, Sprague-Dawley rats had ei<strong>the</strong>r ad lib (n=8) or restricted (receiving 50% of <strong>the</strong> food eaten<br />
by ad lib fed rats; n=24) food access <strong>for</strong> 20 days. On days 9, 14, <strong>an</strong>d 19 all rats were <strong>the</strong>n given<br />
24 hr access to a 0.1% saccharin solution, <strong>an</strong>d two groups of food-restricted rats were given
access to ei<strong>the</strong>r 100% of <strong>the</strong> food eaten by <strong>the</strong> ad lib rats, or ad lib food. Ad lib access in <strong>the</strong><br />
restricted rats induced <strong>an</strong> overeating of 125% on each of <strong>the</strong> three exposures. All groups were<br />
returned to ad lib feeding <strong>an</strong>d tested, on day 24, <strong>for</strong> saccharin consumption in a two-bottle choice<br />
test of saccharin <strong>an</strong>d water. In <strong>the</strong> restriction-overeating group, <strong>the</strong> two-bottle test showed<br />
signific<strong>an</strong>tly reduced saccharin consumption compared to rats in <strong>the</strong> o<strong>the</strong>r three groups.<br />
A latent inhibition paradigm was used to examine whe<strong>the</strong>r <strong>the</strong> overeating-induced avoid<strong>an</strong>ce was<br />
<strong>the</strong> result of a learned association. Only continuously ad lib (n=32) <strong>an</strong>d restricted-overeating<br />
(n=32) groups were used in this experiment. Half <strong>the</strong> rats in each group received eight days of<br />
pre-exposure to <strong>the</strong> saccharin solution, after which <strong>the</strong> experimental procedure used in<br />
Experiment 1 was repeated. At <strong>the</strong> two-bottle choice test, <strong>the</strong> restriction-overeating rats without<br />
prior exposure to <strong>the</strong> saccharin displayed <strong>an</strong> avoid<strong>an</strong>ce equivalent to that seen in Experiment 1.<br />
However, <strong>the</strong> avoid<strong>an</strong>ce was not evident in <strong>the</strong> restriction-overeating rats pre-exposed to <strong>the</strong><br />
saccharin solution. Prior experience with <strong>the</strong> saccharin did not affect consumption in <strong>the</strong><br />
continuously ad lib fed rats. These results suggest that self-induced overeating is capable of<br />
supporting a conditioned taste avoid<strong>an</strong>ce to a novel solution <strong>an</strong>d that this avoid<strong>an</strong>ce is not<br />
evident if <strong>the</strong> <strong>an</strong>imals are made familiar with <strong>the</strong> solution.<br />
Disclosures: A. Hertel, None; R. Eikelboom, NSERC, B. Research Gr<strong>an</strong>t (principal<br />
investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received);<br />
OMHF, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts<br />
as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.19/GG96<br />
Topic: F.03.a. Reward<br />
Support: NIH gr<strong>an</strong>t MH061933 (KW)<br />
NIH gr<strong>an</strong>t DA011806 (KW)<br />
Title: Food-maintained oper<strong>an</strong>t responding in mice: Variables influencing per<strong>for</strong>m<strong>an</strong>ce<br />
Authors: *D. M. HALUK, K. WICKMAN;<br />
Dept. of Pharmacol., Univ. of Minnesota, Minneapolis, MN
Abstract: Oper<strong>an</strong>t tasks have been used extensively in studies involving <strong>the</strong> rat <strong>an</strong>d represent a<br />
powerful me<strong>an</strong>s to study reward. The increasing availability of genetically-m<strong>an</strong>ipulated mice<br />
af<strong>for</strong>ds <strong>an</strong> unprecedented opportunity to probe <strong>the</strong> molecular <strong>an</strong>d cellular bases of reward.<br />
Relatively few oper<strong>an</strong>t-based studies involving mice have been published, <strong>an</strong>d <strong>the</strong> impact of<br />
study design on per<strong>for</strong>m<strong>an</strong>ce in such tasks has not been evaluated systematically. Here, we<br />
evaluated <strong>the</strong> impact of four variables (m<strong>an</strong>ipul<strong>an</strong>da, session duration, housing conditions, <strong>an</strong>d<br />
strain) on oper<strong>an</strong>t per<strong>for</strong>m<strong>an</strong>ce <strong>for</strong> food under both fixed ratio 1 (FR1) <strong>an</strong>d progressive ratio (PR)<br />
schedules of rein<strong>for</strong>cement. We found that acquisition rate, discrimination, <strong>an</strong>d response levels<br />
during FR1 test sessions were largely similar <strong>for</strong> lever-press <strong>an</strong>d nose-poke designs, over a broad<br />
r<strong>an</strong>ge (30-90 min) of FR1 session durations, <strong>an</strong>d <strong>for</strong> two divergent mouse strains (C57BL/6 <strong>an</strong>d<br />
129/Sv). Group-housed mice exposed to <strong>an</strong> enriched environment, however, did exhibit<br />
signific<strong>an</strong>tly higher levels of responding th<strong>an</strong> ei<strong>the</strong>r single- or group-housed mice exposed to<br />
st<strong>an</strong>dard housing environments. PR per<strong>for</strong>m<strong>an</strong>ce was unaffected by FR1 session duration <strong>an</strong>d<br />
housing status, though group-housed mice exposed to environmental enrichment tended to<br />
exhibit less responding. Elevated responding during <strong>the</strong> PR test was observed <strong>for</strong> <strong>an</strong>imals in<br />
lever-press as compared to nose-poke designs, <strong>an</strong>d <strong>for</strong> C57BL/6 as compared to 129/SvJ mice.<br />
Altoge<strong>the</strong>r, <strong>the</strong>se findings confirm that mice are suitable subjects <strong>for</strong> oper<strong>an</strong>t studies involving<br />
food as <strong>the</strong> rein<strong>for</strong>cing agent. Moreover, experimental outcomes are largely insensitive to key<br />
design variables, facilitating me<strong>an</strong>ingful comparisons between studies <strong>an</strong>d across research<br />
groups.<br />
Disclosures: D.M. Haluk, None; K. Wickm<strong>an</strong>, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.20/GG97<br />
Topic: F.03.a. Reward<br />
Support: DHHS/NIH/NIDA/IRP<br />
Title: Activation of <strong>the</strong> ventral tegmental area afferents during <strong>an</strong>ticipation of a food reward in<br />
<strong>the</strong> rat<br />
Authors: *S. GEISLER, M. F. BARBANO, E. A. DUNCAN-VAIDYA, C. E. LANDA, Z.-B.<br />
YOU, R. A. WISE;<br />
Behavioral Neurosci. Br., NIH/NIDA, Baltimore, MD
Abstract: The ventral tegmental area (VTA) detects primary rewards <strong>an</strong>d reward-predicting<br />
stimuli <strong>an</strong>d enlists adaptive behavioral responses. How, remains unclear, since, among its<br />
subst<strong>an</strong>tial inputs <strong>from</strong> m<strong>an</strong>y sources, <strong>the</strong> VTA receives none <strong>from</strong> sensory structures reporting<br />
on <strong>the</strong> internal milieu or external environment.<br />
To address this, we used <strong>an</strong> <strong>an</strong>imal model of food expectation to investigate whe<strong>the</strong>r <strong>an</strong>d, if so,<br />
which, afferents of <strong>the</strong> VTA are activated when rats <strong>an</strong>ticipate a meal. To visualize VTA<br />
afferents, retrogradely tr<strong>an</strong>sported Fluoro-Gold was injected into <strong>the</strong> VTA of rats. After recovery<br />
<strong>from</strong> <strong>the</strong> surgery, rats in two groups (n=7/group) were trained to expect a meal each day during a<br />
fixed four hour time period. A third group (n=7) was fed ad libitum. After training, <strong>the</strong> rats in<br />
one meal-fed group were sacrificed just be<strong>for</strong>e <strong>the</strong> normal daily meal, i.e., during food<br />
<strong>an</strong>ticipation. Ad libitum fed rats were sacrificed 20 hours after removal of <strong>the</strong>ir food, such that<br />
<strong>the</strong>y <strong>an</strong>d <strong>the</strong> <strong>an</strong>ticipatory rats were without food <strong>for</strong> <strong>the</strong> same length of time. The o<strong>the</strong>r meal fed<br />
group was sacrificed 4 hours after beginning of food intake. Prior to sacrifice, blood samples<br />
were taken <strong>from</strong> all <strong>an</strong>imals <strong>for</strong> measurement of hormone levels. The brains were processed<br />
immunohistochemically <strong>for</strong> retrogradely tr<strong>an</strong>sported tracer <strong>an</strong>d neuronal activation was assessed<br />
by immunohistochemical detection of Fos protein.<br />
Me<strong>an</strong> plasma levels of ghrelin were increased in both groups of hungry rats, but signific<strong>an</strong>tly<br />
more in those expecting a scheduled meal. The recently fed rats had increased plasma levels of<br />
leptin, insulin <strong>an</strong>d adiponectin.<br />
In general, hungry rats not expecting a scheduled meal exhibited less activation of VTAprojecting<br />
neurons th<strong>an</strong> ei<strong>the</strong>r those expecting a meal or those recently fed. Rats expecting a<br />
meal showed a disproportionate increase of Fos label in VTA-projecting neurons in <strong>the</strong> ventral<br />
pallidum, whereas <strong>the</strong> o<strong>the</strong>r two groups had negligible Fos expression in this structure. O<strong>the</strong>r<br />
structures containing m<strong>an</strong>y Fos-positive VTA-projecting neurons in rats <strong>an</strong>ticipating a meal<br />
included <strong>the</strong> lateral hypothalamus, lateral preoptic area <strong>an</strong>d accumbens. In hungry rats not<br />
expecting a meal, elevated numbers of Fos-positive, VTA-projecting neurons were observed in<br />
<strong>the</strong> lateral hypothalamus, periaqueductal <strong>an</strong>d central gray <strong>an</strong>d lateral preoptic area. In fed rats,<br />
conspicuous numbers of Fos-expressing neurons projecting to <strong>the</strong> VTA were present in<br />
parabrachial nucleus, dorsal raphe <strong>an</strong>d lateral preoptic area. The data suggest that in<strong>for</strong>mation is<br />
conveyed to <strong>the</strong> VTA via m<strong>an</strong>y structures <strong>an</strong>d that <strong>the</strong> ventral pallidum plays <strong>an</strong> import<strong>an</strong>t role in<br />
conveying signals about upcoming rewards.<br />
Disclosures: S. Geisler, None; M.F. Barb<strong>an</strong>o, None; E.A. Dunc<strong>an</strong>-Vaidya, None; C.E.<br />
L<strong>an</strong>da, None; Z. You, None; R.A. Wise, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.21/GG98
Topic: F.03.a. Reward<br />
Title: The different effects of dopaminergic lesions in <strong>the</strong> nucleus accumbens shell <strong>an</strong>d core on<br />
tastes preference in rats<br />
Authors: *R. SHIBATA, M. KAMEISHI, H. UNEYAMA, K. TORII;<br />
Inst. Life Sci., Ajinomoto Co. Inc, Kawasaki, Jap<strong>an</strong><br />
Abstract: M<strong>an</strong>y studies support that <strong>the</strong> limbic dopaminergic system relates to <strong>the</strong> motivational<br />
<strong>an</strong>d reward processes. Previously we have reported that rats with bilateral dopaminergic lesions<br />
in <strong>the</strong> ventral tegmental areas (VTA) decreased <strong>the</strong>ir sugar consumption, however <strong>the</strong> preference<br />
<strong>for</strong> sugar of <strong>the</strong> rats with bilateral dopaminergic lesions in <strong>the</strong> nucleus accumbens core (NAC)<br />
was <strong>the</strong> same as <strong>the</strong> control rats. Dopaminergic neurons in <strong>the</strong> VTA project to <strong>the</strong> nucleus<br />
accumbens (NA), especially to <strong>the</strong> medial shell (NAS). In our present study, we have<br />
investigated <strong>the</strong> differences of taste preferences <strong>an</strong>d <strong>the</strong> feeding patterns between NAC lesioned<br />
rats <strong>an</strong>d NAS lesioned rats.<br />
After bilateral injections of 6-hydroxydopamine into <strong>the</strong> NA core (NAC rats) or <strong>the</strong> NA shell<br />
(NAS rats), we tested <strong>the</strong> difference of preferences among <strong>the</strong> NAC rats, <strong>the</strong> NAS rats <strong>an</strong>d sham<br />
surgically treated rats (sham rats) using a two-bottle choice test over 2 days with <strong>the</strong> following<br />
taste fluids: saline (0.9 % of NaCl) as a salty taste, monosodium glutamate (MSG, 30 <strong>an</strong>d 60<br />
mM) <strong>an</strong>d inosine monophosphate (IMP, 15 <strong>an</strong>d 30 mM) as umami tastes, sucrose (60 <strong>an</strong>d 120<br />
mM), Al<strong>an</strong>ine (Ala, 60 mM, sweet amino acid) <strong>an</strong>d saccharin (0.2 %) as sweet tastes. There was<br />
no signific<strong>an</strong>t difference among <strong>the</strong> consumption of <strong>the</strong>se tast<strong>an</strong>ts between <strong>the</strong> NAC rats <strong>an</strong>d <strong>the</strong><br />
sham rats. Regarding <strong>the</strong> consumption of salty or umami tast<strong>an</strong>ts, <strong>the</strong>re were no signific<strong>an</strong>t<br />
differences among all three groups of rats. However, while <strong>the</strong> consumption of sweet tast<strong>an</strong>ts did<br />
not ch<strong>an</strong>ge signific<strong>an</strong>tly <strong>for</strong> <strong>the</strong> NAC rats <strong>an</strong>d <strong>the</strong> sham rats, <strong>the</strong> NAS rats signific<strong>an</strong>tly decreased<br />
<strong>the</strong>ir sugar consumption. For example, consumptions of 120mM sucrose in <strong>the</strong> sham rats, NAC<br />
rats <strong>an</strong>d NAS rats were 16.7 ± 3.0 ml/100g body weight (BW), 15.7 ± 4.6 ml/100g BW <strong>an</strong>d 11.0<br />
± 2.3 ml/100g BW, respectively (me<strong>an</strong>s ± SD).<br />
Fur<strong>the</strong>rmore, all three groups of rats showed <strong>the</strong> same levels of <strong>the</strong> food intake/100g BW/day.<br />
The sham rats <strong>an</strong>d <strong>the</strong> NAC rats had 6.75 ± 0.9 feeding episodes in <strong>the</strong> dark phase, but <strong>the</strong><br />
number of feeding episodes <strong>for</strong> <strong>the</strong> NAS rats was 9.0 ± 1.9 <strong>an</strong>d <strong>the</strong>ir food consumption during<br />
each episode was less th<strong>an</strong> <strong>the</strong> o<strong>the</strong>r two groups of rats.<br />
These results suggest that <strong>the</strong> dopaminergic innervation in <strong>the</strong> NA shell is more strongly related<br />
to <strong>the</strong> consumption of sapid fluids <strong>an</strong>d meal size th<strong>an</strong> in <strong>the</strong> NA core.<br />
Disclosures: R. Shibata, Institute of Life Sciences, Ajinomoto Co., Inc., A. Employment (full<br />
or part-time); M. Kameishi, Institute of Life Sciences, Ajinomoto Co., Inc., A. Employment<br />
(full or part-time); H. Uneyama, Institute of Life Sciences, Ajinomoto Co., Inc., A. Employment<br />
(full or part-time); K. Torii, Institute of Life Sciences, Ajinomoto Co., Inc., A. Employment (full<br />
or part-time).<br />
Poster
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.22/GG99<br />
Topic: F.03.a. Reward<br />
Support: NSERC<br />
Title: Lesions of <strong>the</strong> caudal (but not rostral) dorsolateral septum reduce rats’ latency to initiate<br />
consumption of a palatable snack<br />
Authors: *S.-S. A. CHEE 1,2 , J. MENARD 2 ;<br />
1 Kingston, ON, C<strong>an</strong>ada; 2 Queen's Univ., Kingston, ON, C<strong>an</strong>ada<br />
Abstract: Prior studies have shown that lesions of <strong>the</strong> caudal (but not rostral) aspects of <strong>the</strong><br />
septal nucleus reduce rat’s <strong>an</strong>xiety-related responses. Our current interest was to determine<br />
whe<strong>the</strong>r this rostral/caudal distinction also applies when lesions are restricted to <strong>the</strong> dorsolateral<br />
septum (dLS). As <strong>the</strong> caudal aspects of <strong>the</strong> dLS have been implicated in reward <strong>an</strong>d motivation,<br />
it was import<strong>an</strong>t to utilize a test that c<strong>an</strong> dissociate between <strong>the</strong> effects of <strong>an</strong>xiety <strong>an</strong>d those of<br />
motivation; i.e., <strong>the</strong> novelty-induced suppression of feeding paradigm. In this paradigm, rodents<br />
readily approach <strong>an</strong>d consume a palatable snack when it is offered in <strong>the</strong> home-cage but show<br />
signific<strong>an</strong>tly increased latencies to initiate consumption of <strong>the</strong> same snack in <strong>an</strong> unfamiliar, novel<br />
cage. Anxiolytic effects in this test are indexed by reductions in <strong>the</strong> latency to initiate snack<br />
consumption in <strong>the</strong> novel-cage test without ch<strong>an</strong>ging response latencies in <strong>the</strong> home-cage test. In<br />
contrast, treatment-induced ch<strong>an</strong>ges in appetitive motivation would alter <strong>the</strong> latency to initiate<br />
snack consumption in both <strong>the</strong> home- <strong>an</strong>d novel-cage tests. Rats received ibotenic acid lesions to<br />
ei<strong>the</strong>r <strong>the</strong> rostral or caudal aspects of <strong>the</strong> dLS. Following recovery, rats were offered a palatable<br />
snack <strong>for</strong> 10 min on each of six consecutive days in <strong>the</strong>ir home cage; Days 1-5 served to<br />
habituate <strong>the</strong> rats to <strong>the</strong> snack, while <strong>the</strong> home-cage test occurred on Day 6. On Day 7, rats were<br />
given <strong>the</strong> same snack in a novel cage. Rats with lesions of <strong>the</strong> caudal (but not rostral) dLS took<br />
signific<strong>an</strong>tly less time to initiate snack consumption when <strong>the</strong> snack was first introduced in <strong>the</strong><br />
home-cage on Day 1. By Day 3, all rats achieved stable baseline response latency scores that did<br />
not differ ei<strong>the</strong>r within subjects or between groups (lesion type) across <strong>the</strong> remaining habituation<br />
trials (Days 4-5) or <strong>the</strong> home-cage test on Day 6. The latency to initiate feeding in <strong>the</strong> novel-cage<br />
test (Day 7) was signific<strong>an</strong>tly increased relative to <strong>the</strong> home-cage test (Day 6). This increase was<br />
markedly reduced in <strong>the</strong> caudal dLS lesioned rats compared to both <strong>the</strong> rostral dLS lesioned rats<br />
<strong>an</strong>d sham lesioned controls. In contrast, <strong>the</strong> latter two groups did not differ <strong>from</strong> each o<strong>the</strong>r.<br />
These results suggest that lesions of <strong>the</strong> caudal dLS do not affect motivation but instead have<br />
<strong>an</strong>xiolytic-like effects in <strong>the</strong> novelty-induced suppression of feeding paradigm.<br />
Disclosures: S.A. Chee, None; J. Menard, None.
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.23/GG100<br />
Topic: F.03.a. Reward<br />
Support: NHLBI Gr<strong>an</strong>t HL086465<br />
Title: Sleep loss, food intake <strong>an</strong>d reward systems: Behavioral <strong>an</strong>d molecular findings in sleep<br />
deprived rats<br />
Authors: *E. M. PALETZ, E. C. HANLON, Q. Z. BREMER, W. H. OBERMEYER, B. A.<br />
BALDO, R. M. BENCA;<br />
Psychiatry, Univ. Wisconsin, Madison, WI<br />
Abstract: It is well documented that total sleep deprivation (TSD) robustly increases baseline<br />
food intake in rats, but <strong>the</strong> reasons underlying this phenomenon are uncertain. Two hypo<strong>the</strong>ses<br />
come to mind that are not mutually exclusive: (1) sleep loss increases <strong>the</strong> reward value of food,<br />
<strong>an</strong>d (2) sleep loss affects systems that regulate energy homeostasis. To investigate <strong>the</strong>se<br />
hypo<strong>the</strong>ses, rats were ei<strong>the</strong>r housed on a conveyor-over-water (COW) apparatus that was used to<br />
induce TSD across 10 consecutive days or selectively deprived of REM sleep (REMSD) across 5<br />
days using <strong>the</strong> plat<strong>for</strong>m method. Some rats were sacrificed on <strong>the</strong> final day of sleep deprivation<br />
(or control) <strong>an</strong>d <strong>the</strong>ir brains <strong>an</strong>d blood were collected. To evaluate <strong>the</strong> reward hypo<strong>the</strong>sis,<br />
REMSD rats were tested on a progressive-ratio schedule of food rein<strong>for</strong>cement often used to<br />
assess motivation or reward value. REMSD had <strong>the</strong> effect of lowering response output, but it<br />
was not clear whe<strong>the</strong>r sleep loss reduced efficacy of reward per se because per<strong>for</strong>m<strong>an</strong>ce deficits<br />
were also observed, likely due to <strong>the</strong> large response costs involved. It was noteworthy, however,<br />
that REMSD rats displayed normal levels of responding in <strong>the</strong> initial portion of <strong>the</strong> session. To<br />
follow up, a brain stimulation reward (BSR) threshold procedure was used that required TSD rats<br />
to respond one time per trial, thus having <strong>the</strong> potential to minimize per<strong>for</strong>m<strong>an</strong>ce deficits. BSR<br />
thresholds were not altered after TSD during <strong>the</strong> initial portion of <strong>the</strong> session. We also per<strong>for</strong>med<br />
in situ hybridization to look at expression in <strong>the</strong> striatum. *** Toge<strong>the</strong>r, <strong>the</strong>se findings suggest<br />
that sleep loss does not modify reward systems. To address <strong>the</strong> energy homeostasis hypo<strong>the</strong>sis,<br />
in situ hybridization was per<strong>for</strong>med to evaluate expression of arousal <strong>an</strong>d feeding related<br />
peptides in <strong>the</strong> lateral hypothalamus, including NPY, MCH, <strong>an</strong>d orexin. In addition, circulating<br />
levels of insulin, leptin, <strong>an</strong>d ghrelin were measured. Results indicated that TSD produced<br />
signific<strong>an</strong>t differences in hormone levels compared to controls; insulin <strong>an</strong>d ghrelin levels were<br />
lower <strong>an</strong>d leptin levels higher in TSD rats. Taken toge<strong>the</strong>r, results best support a mech<strong>an</strong>ism in
which sleep deprivation augments feeding via <strong>an</strong> upregulation of orexigenic hormonal signals<br />
acting through hypothalamic peptide systems.<br />
Disclosures: E.M. Paletz, None; E.C. H<strong>an</strong>lon, None; Q.Z. Bremer, None; W.H. Obermeyer,<br />
None; B.A. Baldo, None; R.M. Benca, None.<br />
Poster<br />
583. Reward: Motivated Behaviors - Food, Taste, etc<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 583.24/GG101<br />
Topic: F.03.a. Reward<br />
Support: NSERC Discovery Gr<strong>an</strong>t<br />
Ontario Mental Health Foundation<br />
Title: Intermittent exposure to sweet solutions produces elevated intake <strong>the</strong> rat<br />
Authors: A. J. CELEJEWSKI, *R. EIKELBOOM;<br />
Wilfrid Laurier Univ., Waterloo, ON, C<strong>an</strong>ada<br />
Abstract: Access conditions are crucial to <strong>the</strong> development of excessive consumption in<br />
addiction. For example, 1 h of daily access to self-administered cocaine produces stable, low<br />
levels of consumption, while 6 h leads to <strong>an</strong> escalation in consumption (Ahmed <strong>an</strong>d Koob,<br />
1998). Our previous work with young adult male Sprague Dawley rats has indicated that<br />
discontinuous exposures (24 h once every 3 days) to a 4% sucrose solution induces a drastic,<br />
addiction-like elevation in <strong>the</strong> solution’s consumption relative to continuous access to <strong>the</strong> same<br />
solution. Fur<strong>the</strong>rmore, this consumption difference persists <strong>an</strong>d remains stable after <strong>the</strong> access<br />
schedule is switched to alternate day exposures <strong>for</strong> both groups. Similar elevations in<br />
consumption however, were not observed with discontinuous access to higher (8% <strong>an</strong>d 16%) <strong>an</strong>d<br />
lower (1%) sucrose concentrations. That <strong>the</strong> observed increase was elicited by <strong>the</strong> 4% solution<br />
only, may be due to some “ceiling effect” such as satiety occurring at higher concentrations.<br />
Alternatively, it is possible that <strong>the</strong> elevation is a “mid-r<strong>an</strong>ge” phenomenon apparent only at<br />
intermediate concentrations. We <strong>the</strong>re<strong>for</strong>e replicated <strong>the</strong> sucrose experiment with saccharin. Four<br />
groups (n = 16 each) of rats were provided concurrently with water <strong>an</strong>d one of 4 different<br />
saccharin solutions, 0.125%, 0.25%, 0.5% <strong>an</strong>d 1%, which were chosen to parallel <strong>the</strong> higher<br />
sucrose concentrations. For every concentration, each group was subdivided into discontinuous<br />
(24 h once every three days) <strong>an</strong>d continuous access conditions (n = 8 each). After consumption
levels stabilized (13 cycles), both groups were moved to alternate day access to assess <strong>an</strong>y<br />
enduring effects of <strong>the</strong> previous access conditions. In contrast to our findings with various<br />
concentrations of sucrose, consumption increases were observed at all concentrations in <strong>the</strong><br />
discontinuous access condition (55% to 81%) relative to <strong>the</strong> continuous access condition. Our<br />
results suggest strongly that: 1) <strong>the</strong> hedonic quality of <strong>the</strong> sweet taste alone is sufficient <strong>for</strong><br />
increased consumption <strong>an</strong>d 2) saccharin consumption increases were not affected by satiety<br />
which may have limited consummatory increases at higher sucrose concentrations. Our results<br />
underscore <strong>the</strong> import<strong>an</strong>ce of access schedules to <strong>the</strong> consumption of sweet solutions while<br />
drawing parallels to a large body literature implicating access conditions in a tr<strong>an</strong>sition to<br />
addiction.<br />
Disclosures: A.J. Celejewski, None; R. Eikelboom, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.1/GG102<br />
Topic: F.04.e. O<strong>the</strong>r<br />
Title: Profiling miRNA expression in <strong>the</strong> zebra finch brain<br />
Authors: *X.-C. LI 1 , G. LUO 2 , M. HAFNER 3 , T. TUSCHL 3 , X. WANG 2 ;<br />
1 2 3<br />
LSU, New Orle<strong>an</strong>s, LA; Inst. of Genet. <strong>an</strong>d Developmental Biol., BeiJing, China; The<br />
Rockefeller Univ., New York City, NY<br />
Abstract: As a first step to underst<strong>an</strong>ding <strong>the</strong> functions of miRNAs in neural development <strong>an</strong>d<br />
vocal learning in songbirds, we characterized miRNA expression in <strong>the</strong> zebra finch brain. A liver<br />
sample was also included as a comparison. We made two small RNA libraries <strong>from</strong> brain <strong>an</strong>d<br />
liver tissues of adult male zebra finches. The two libraries were sequenced using <strong>the</strong> high<br />
throughput 454 sequencing technology. After trimming, we obtained 21619 <strong>an</strong>d 18594 high<br />
quality small RNA sequence reads in <strong>the</strong> brain <strong>an</strong>d liver samples, which represent 4042 <strong>an</strong>d 4786<br />
unique sequences, respectively. Of <strong>the</strong> 8444 total unique sequences (brain <strong>an</strong>d liver samples<br />
combined), 2288 c<strong>an</strong> be mapped to <strong>the</strong> zebra finch genome with perfect matches. For <strong>the</strong>se<br />
sequences with genomic loci, we extracted <strong>the</strong>ir fl<strong>an</strong>king sequences <strong>from</strong> genome data <strong>an</strong>d<br />
per<strong>for</strong>med miRNA prediction by a comprehensive computational pipeline we developed. One<br />
hundred <strong>an</strong>d eighty three sequences passed our stringent criteria. Of <strong>the</strong>se, 74 are identical to<br />
known miRNAs recorded in mirBase, <strong>an</strong>d 75 of <strong>the</strong>m show sequence similarity to known<br />
miRNAs. O<strong>the</strong>r 35 sequences could be considered as novel miRNAs specific in zebra finches.
Sequence <strong>an</strong>alysis shows that <strong>the</strong> 6156 sequence reads without genomic loci generally originate<br />
<strong>from</strong> miRNAs (44%) <strong>an</strong>d tRNA/rRNA (13%). We <strong>the</strong>n compared this group of sequence reads<br />
to all <strong>the</strong> known miRNAs in mirBase. We identified 178 sequences that are identical to known<br />
miRNAs, <strong>an</strong>d thus c<strong>an</strong> be <strong>an</strong>notated as miRNAs as well. Altoge<strong>the</strong>r, we have identified 361<br />
zebra finch miRNAs. The overall contents of <strong>the</strong> miRNA in <strong>the</strong> sequencing samples are about<br />
50% <strong>for</strong> both libraries. Moreover, we also found evidence that some of <strong>the</strong>se putative miRNAs<br />
are edited at specific positions. A comparison between <strong>the</strong> miRNA profiles of <strong>the</strong> brain <strong>an</strong>d <strong>the</strong><br />
liver libraries indicates that m<strong>an</strong>y miRNAs show tissue specific expression patterns between <strong>the</strong><br />
two tissues. The brain specific expressions of some of <strong>the</strong> miRNAs are also conserved in hum<strong>an</strong>s<br />
<strong>an</strong>d mice.<br />
Disclosures: X. Li, None; G. Luo, None; M. Hafner, None; T. Tuschl, None; X. W<strong>an</strong>g, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.2/GG103<br />
Topic: F.04.c. Vocal/social communication<br />
Support: KAKENHI 21700294 <strong>from</strong> Ministry of Education, Culture, Sports, Science <strong>an</strong>d<br />
Technology of Jap<strong>an</strong><br />
Title: Gene expression ch<strong>an</strong>ges during song development in <strong>the</strong> songbird brain<br />
Authors: *M. KATO 1,2 , K. OKANOYA 2 ;<br />
1 Grad. Sch. of Hum<strong>an</strong> Relations, Keio Univ., Minato-Ku, Tokyo, Jap<strong>an</strong>; 2 Lab. <strong>for</strong><br />
Biolinguisitics, RIKEN BSI, Wako, Jap<strong>an</strong><br />
Abstract: Birdsong has been studied as a biological model of hum<strong>an</strong> l<strong>an</strong>guage because <strong>the</strong><br />
process of song learning is similar to that of l<strong>an</strong>guage development. Usually, male songbirds<br />
learn <strong>the</strong>ir songs <strong>from</strong> <strong>an</strong> adult-male tutor when <strong>the</strong>y are young. Song learning consists of two<br />
phases: a memorization phase during <strong>the</strong> early developmental stage <strong>an</strong>d a sensorimotor phase<br />
after memorization. In contrast, <strong>the</strong> development of vocalization consists of three phases:<br />
subsong, plastic song, <strong>an</strong>d crystallized song. Both vocalization <strong>an</strong>d song learning depend on <strong>the</strong><br />
structure of <strong>the</strong> songbird brain, i.e., a set of neuronal nuclei.<br />
We sought to isolate song nuclei-specific genes using <strong>an</strong> in-house cDNA microarray <strong>an</strong>d<br />
identified several genes that were specifically expressed or repressed in <strong>the</strong> HVC compared to<br />
neighboring areas. Then, we examined <strong>the</strong> expression ch<strong>an</strong>ges in microarray-selected genes
during development in <strong>the</strong> Bengalese finch brain using in situ hybridization.<br />
We found that <strong>the</strong> expression patterns of calcium/calmodulin-dependent protein kinase II alpha<br />
(CaMKIIa) <strong>an</strong>d mitogen-activated protein kinase kinase 1 (MAP2K1) ch<strong>an</strong>ged dynamically with<br />
development. CaMKIIa is a major postsynaptic density protein that plays a critical role in longterm<br />
potentiation (LTP) <strong>an</strong>d memory <strong>for</strong>mation. MAP2K1 (also called MEK1) is a component of<br />
<strong>the</strong> MAPK signaling pathway. MAP kinases are also extracellular signal-regulated kinases<br />
(ERKs) <strong>an</strong>d act as <strong>an</strong> integration point <strong>for</strong> multiple biochemical signals, including LTP, which is<br />
<strong>the</strong> cellular basis of learning <strong>an</strong>d memory. We found that during development, both CaMKIIa<br />
<strong>an</strong>d MAP2K1 were expressed ubiquitously in <strong>the</strong> juvenile brain, but <strong>the</strong> expression had<br />
decreased in <strong>the</strong> HVC 60 days after hatching, <strong>the</strong> time when a juvenile begins to switch <strong>from</strong> <strong>the</strong><br />
sensory learning phase to <strong>the</strong> sensory motor learning phase.<br />
These data suggest that sensory learning <strong>from</strong> <strong>the</strong> tutor’s song is based on <strong>the</strong> expression of<br />
CaMKIIa <strong>an</strong>d MAP2K1 in <strong>the</strong> HVC. Moreover, a decreased expression of <strong>the</strong>se genes in <strong>the</strong><br />
HVC may represent <strong>the</strong> stabilization of in<strong>for</strong>mation regarding <strong>the</strong> song template achieved<br />
through sensory learning. The expression ch<strong>an</strong>ges in <strong>the</strong>se genes might regulate <strong>the</strong> phase of<br />
sensory learning as a critical period in songbirds.<br />
Disclosures: M. Kato, None; K. Ok<strong>an</strong>oya, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.3/GG104<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NIH Gr<strong>an</strong>t NS059755<br />
Title: ZEBrA: A zebra finch gene expression brain atlas<br />
Authors: C. V. MELLO, *P. V. LOVELL, K. L. HORBACK;<br />
Dept. of Behavioral Neurosci., Oregon Hlth. & Sci. Univ., Beaverton, OR<br />
Abstract: Songbirds have become a major model org<strong>an</strong>ism in neurobiology, providing novel<br />
<strong>an</strong>d fundamental insights into processes such as <strong>the</strong> neural encoding of vocalizations, brain sex<br />
dimorphisms, seasonal <strong>an</strong>d photoperiodicity effects on brain <strong>an</strong>d behavior, <strong>an</strong>d adult<br />
neurogenesis <strong>an</strong>d neuronal replacement. However, <strong>the</strong>re remains a signific<strong>an</strong>t gap in our<br />
underst<strong>an</strong>ding of what genes, neurotr<strong>an</strong>smitter systems <strong>an</strong>d receptors, <strong>an</strong>d signaling molecules<br />
are expressed in <strong>the</strong> songbird brain <strong>an</strong>d how <strong>the</strong>se relate to <strong>the</strong> unique properties of <strong>the</strong> song
control system. To address <strong>the</strong>se gaps, we have recently launched a new web-based resource,<br />
ZEBrA, a Gene Expression Atlas that will provide researchers with on-line public access to highresolution<br />
in situ hybridization images documenting <strong>the</strong> brain expression of a large set of mRNA<br />
tr<strong>an</strong>scripts in <strong>the</strong> adult male zebra finch (Taeniopygia guttata). To construct this resource, we<br />
used high-throughput non-radioactive in situ protocols to hybridize ~50 cDNA-derived<br />
riboprobes to a series of parasagittal cryostat brain sections (10 µm) covering 9-10 medial-tolateral<br />
brain levels encompassing all of <strong>the</strong> song nuclei, as well as major pallial <strong>an</strong>d subpallial<br />
brain subdivisions. The Atlas, which c<strong>an</strong> be accessed through a st<strong>an</strong>dard web-browser, allows<br />
users to quickly search <strong>for</strong> genes by executing keyword/ gene name searches, or by sorting genes<br />
according to attributes such as inclusion within a gene family or relationship to a biological<br />
process, regulation by a specific physiological, environmental or behavioral state as established<br />
by songbird microarray studies, pattern of expression in song nuclei <strong>an</strong>d/or o<strong>the</strong>r brain regions,<br />
or expression in specific mammali<strong>an</strong> brain structures or regions. Each gene is also linked to<br />
several bioin<strong>for</strong>matics <strong>an</strong>d genomics resources (e.g. Entrez Gene, Ensembl, etc.), providing upto-date<br />
in<strong>for</strong>mation about functional attributes, genomic structure, <strong>an</strong>d expression in o<strong>the</strong>r model<br />
systems (e.g. chicken, mouse). Finally, researchers c<strong>an</strong> easily p<strong>an</strong>-<strong>an</strong>d-zoom through a series of<br />
images down to a cellular level of <strong>an</strong>alysis, each presented in registration with <strong>an</strong> <strong>an</strong>notated<br />
Nissl-stained section set. To fur<strong>the</strong>r develop this resource <strong>for</strong> <strong>the</strong> songbird <strong>an</strong>d avi<strong>an</strong> brain<br />
research communities, we pl<strong>an</strong> on adding data <strong>for</strong> ~2,500 genes. Thus, ZEBrA will represent one<br />
of <strong>the</strong> most comprehensive resources available <strong>for</strong> characterizing <strong>the</strong> brain distribution of genes<br />
<strong>an</strong>d molecules involved in neuronal physiology <strong>an</strong>d/or <strong>the</strong> development <strong>an</strong>d mainten<strong>an</strong>ce of<br />
functional circuits, <strong>an</strong>d identifying novel pharmacological tools <strong>an</strong>d targets <strong>for</strong> future genetic<br />
m<strong>an</strong>ipulations.<br />
Disclosures: C.V. Mello, None; P.V. Lovell, None; K.L. Horback, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.4/GG105<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NIH Gr<strong>an</strong>t NS059755<br />
NIH Gr<strong>an</strong>t NS045264<br />
Title: Cholecystokinin expression <strong>an</strong>d song regulation in <strong>the</strong> zebra finch brain
Authors: P. V. LOVELL, *C. V. MELLO;<br />
Dept. of Behavioral Neurosci., Oregon Hlth. Sci. Univ., Beaverton, OR<br />
Abstract: The gene encoding <strong>the</strong> gut peptide cholecystokinin (cck) is abund<strong>an</strong>tly expressed in<br />
<strong>the</strong> mammali<strong>an</strong> brain <strong>an</strong>d has been associated with a broad r<strong>an</strong>ge of functions, including feeding<br />
termination <strong>an</strong>d satiety, locomotion <strong>an</strong>d self-stimulation, <strong>the</strong> modulation of <strong>an</strong>xiety-like<br />
behaviors, <strong>an</strong>d learning <strong>an</strong>d memory. Here we describe <strong>the</strong> brain expression <strong>an</strong>d song regulation<br />
of <strong>the</strong> cck gene in <strong>the</strong> brain of adult male zebra finches (Taeniopygia guttata), a songbird species.<br />
Using in situ hybridization, we show that <strong>the</strong> cck mRNA is highly expressed in several discrete<br />
brain regions, most prominently <strong>the</strong> caudal-most portion of <strong>the</strong> hippocampal <strong>for</strong>mation, <strong>the</strong><br />
caudo-dorsal nidopallial shelf region <strong>an</strong>d <strong>the</strong> caudo-medial nidopallium (NCM), <strong>the</strong> caudo-dorsal<br />
mesopallium, <strong>an</strong> arc-shaped region in <strong>the</strong> dorsal arcopallium, <strong>the</strong> core or shell regions of various<br />
dorsal thalamic nuclei, some dopaminergic cell groups in <strong>the</strong> mesencephalon <strong>an</strong>d pons, <strong>the</strong><br />
mesencephalic <strong>an</strong>d principal nuclei of <strong>the</strong> trigeminal nerve, <strong>an</strong>d <strong>the</strong> dorsal raphe. We also<br />
observed a sparse population of strongly labeled cells throughout <strong>the</strong> medial <strong>an</strong>d lateral striatum,<br />
including song nucleus area X, whereas <strong>the</strong> number of labeled cells <strong>an</strong>d <strong>the</strong>ir labeling intensity<br />
were markedly lower throughout <strong>the</strong> rest of <strong>the</strong> brain. cck expression was conspicuously low in<br />
song nucleus HVC in comparison with <strong>the</strong> adjacent auditory shelf region, as well as absent in<br />
o<strong>the</strong>r telencephalic song control nuclei, including RA, LMAN <strong>an</strong>d NIf. We also show that both<br />
<strong>the</strong> overall cck mRNA expression levels <strong>an</strong>d <strong>the</strong> number of cck-labeled cells are increased in<br />
NCM after auditory stimulation with conspecific song relative to control unstimulated birds (n=4<br />
per group; P
Topic: F.04.c. Vocal/social communication<br />
Support: NIH Gr<strong>an</strong>t DC004724<br />
Title: Anatomical correlates of adult song plasticity in <strong>the</strong> zebra finch<br />
Authors: *K. S. MCDONALD 1 , J. R. KIRN 2 ;<br />
1 Dept Biol., 2 Wesley<strong>an</strong> Univ., Middletown, CT<br />
Abstract: Song learning <strong>an</strong>d related cellular plasticity in closed-ended vocal learners was<br />
thought to end following <strong>the</strong> critical period <strong>for</strong> song development. However, mounting evidence<br />
in one closed-ended learner, <strong>the</strong> zebra finch, suggests that m<strong>an</strong>y <strong>for</strong>ms of plasticity common<br />
during song learning continue into adulthood. Based on <strong>an</strong>atomical <strong>an</strong>d lesion studies (Bottjer &<br />
Arnold, 1997; Aronov et al., 2008) <strong>the</strong>re is a gradual shift in <strong>the</strong> relative influence of two cortical<br />
circuits on song structure during juvenile song learning. <strong>When</strong> song structure is highly variable<br />
early in song development, projections <strong>from</strong> <strong>the</strong> lateral magnocellular nucleus of <strong>the</strong> <strong>an</strong>terior<br />
nidopallium (LMAN) to <strong>the</strong> robust nucleus of <strong>the</strong> archopallium (RA) dominate over projections<br />
<strong>from</strong> HVC (used as a proper name) to RA. As song structure becomes more stereotyped, <strong>the</strong><br />
relative weighting of <strong>the</strong>se two inputs to RA is reversed. In previous work we showed that song<br />
stereotypy continues to be refined well into adulthood (Pytte et al., 2007). As a step toward<br />
identifying <strong>an</strong>atomical substrates <strong>for</strong> adult increases in stereotypy, we measured dendritic arbor<br />
of HVC-RA <strong>an</strong>d LMAN-RA neurons as a function of age <strong>an</strong>d experience by injecting<br />
Rhodamine Dextr<strong>an</strong> Amines into RA. We found that with increasing adult age <strong>the</strong>re were<br />
signific<strong>an</strong>t increases in <strong>the</strong> amount of dendritic arbor of RA-projecting neurons in HVC, but not<br />
LMAN. Increases in <strong>the</strong> ratio of HVC-RA:LMAN-RA dendritic arbor were also positively<br />
correlated with amount of prior singing in age-matched birds. These data suggest that <strong>the</strong> relative<br />
weighting of HVC-RA over LMAN-RA continues to increase in adulthood along with increases<br />
in song stereotypy, perhaps as a result of cumulative singing experience. We also found<br />
experience-dependent post-crystallization dendritic plasticity in both cell populations when birds<br />
of <strong>the</strong> same age were exposed to different social settings. Adult males housed individually with<br />
one adult female had signific<strong>an</strong>tly more dendritic arbor in both HVC-RA <strong>an</strong>d LMAN-RA<br />
populations compared to males in three o<strong>the</strong>r social settings, although <strong>the</strong> arbor ratio was<br />
preserved. Thus, both cell populations are capable of <strong>an</strong>atomical ch<strong>an</strong>ge under appropriate<br />
conditions. These data, toge<strong>the</strong>r with previous work, support a model in which <strong>the</strong> song system<br />
remains plastic well into adulthood in <strong>the</strong> zebra finch <strong>an</strong>d suggest that <strong>the</strong> ratio of HVC:LMAN<br />
arbor is causally linked to song stereotypy.<br />
Disclosures: K.S. McDonald, None; J.R. Kirn, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.6/GG107<br />
Topic: F.04.c. Vocal/social communication<br />
Support: 1 F31 MH082533<br />
1 R01 MH070712-01<br />
Title: Network <strong>an</strong>alysis of gene expression in area X during singing<br />
Authors: *A. T. HILLIARD 1 , J. E. MILLER 2 , S. A. WHITE 2,1 ;<br />
1 Interdepartmental Neurosci. PhD Program, 2 Physiological Sci., UCLA, Los Angeles, CA<br />
Abstract: The zebra finch is <strong>an</strong> established model <strong>for</strong> <strong>the</strong> study of neural systems supporting<br />
vocal learning, a prerequisite ability <strong>for</strong> song <strong>an</strong>d speech. Previous microarray studies in zebra<br />
finches have found genes regulated by singing in neural song control circuitry. However, genes<br />
were singled out based on differential expression, effectively identifying interesting genes only<br />
in isolation, <strong>an</strong>d were later mapped to known gene pathways to investigate <strong>the</strong>ir functional<br />
relationships. We used microarrays to measure mRNA expression of genes in area X <strong>an</strong>d <strong>the</strong><br />
outlying striatum of adult male zebra finches after 2 hours of singing or non-singing. To examine<br />
<strong>the</strong> rich body of co-expression in<strong>for</strong>mation present in our data, <strong>an</strong>d discover new song-activated<br />
gene networks, we per<strong>for</strong>med weighted gene co-expression network <strong>an</strong>alysis (WGCNA), <strong>an</strong><br />
unsupervised technique that uses a biologically me<strong>an</strong>ingful measure of gene similarity<br />
(topological overlap) to org<strong>an</strong>ize co-expressed genes into modules. High module membership<br />
(correlation to 1st principal component of <strong>the</strong> module) implies that a gene is well connected<br />
within <strong>the</strong> module, <strong>an</strong>d is critical <strong>for</strong> biological functions supported by <strong>the</strong> module. Such<br />
modules c<strong>an</strong> <strong>the</strong>n be correlated to biological trait data, <strong>an</strong>d past applications have shown <strong>the</strong>m to<br />
be functionally signific<strong>an</strong>t. To our knowledge, no studies have yet related modules to behavior.<br />
Here, we found 3 modules in area X that are strongly related to song measurements. For a given<br />
gene, <strong>the</strong> strength of song module membership is a good predictor of <strong>the</strong> correlation strength, or<br />
‘gene signific<strong>an</strong>ce’, between <strong>the</strong> gene <strong>an</strong>d song measurements to which <strong>the</strong> module is correlated.<br />
Of note, <strong>the</strong> gene signific<strong>an</strong>ce of area X song module genes is only weakly, or not at all,<br />
conserved in <strong>the</strong> outlying striatum, implying a unique functional specification of <strong>the</strong>se genes <strong>for</strong><br />
song in area X. Also, genes with <strong>the</strong> largest difference in gene signific<strong>an</strong>ce between tissues tend<br />
to be <strong>the</strong> strongest module members. The song modules contain genes such as egr-1 <strong>an</strong>d FoxP2<br />
that have previously been associated with song, as well as m<strong>an</strong>y that no prior studies have linked<br />
to song. Beyond producing a list of behaviorally regulated genes, our <strong>an</strong>alysis highlights <strong>the</strong><br />
most biologically signific<strong>an</strong>t ones, providing a roadmap <strong>for</strong> future gene validation <strong>an</strong>d<br />
m<strong>an</strong>ipulation studies. To illuminate <strong>the</strong> gene networks underlying song, <strong>an</strong>d by <strong>an</strong>alogy speech,<br />
<strong>the</strong> challenge now lies in characterizing <strong>the</strong> functional signific<strong>an</strong>ce of molecular networks<br />
populated by song genes found here, <strong>an</strong>d in future studies.<br />
Disclosures: A.T. Hilliard, None; J.E. Miller, None; S.A. White, None.
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.7/GG108<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NINDS<br />
NIMH<br />
Title: Unexpected relationships between <strong>for</strong>ebrain steroid <strong>an</strong>d neurotr<strong>an</strong>smitter levels in awake,<br />
behaving songbirds<br />
Authors: L. REMAGE-HEALEY 1 , N. T. MAIDMENT 1 , *B. A. SCHLINGER 2 ;<br />
1 UCLA, Los Angeles, CA; 2 UCLA, Top<strong>an</strong>ga, CA<br />
Abstract: Estrogens generally act to increase neuronal excitation, <strong>an</strong>d <strong>the</strong>y have been associated<br />
with behavioral arousal. A prediction <strong>from</strong> <strong>the</strong>se observations is that fluctuating levels of<br />
estradiol within <strong>the</strong> <strong>for</strong>ebrain should co-vary with levels of <strong>the</strong> excitatory amino acid<br />
neurotr<strong>an</strong>smitter glutamate, <strong>an</strong>d/or be inversely related to levels of <strong>the</strong> predomin<strong>an</strong>t inhibitory<br />
tr<strong>an</strong>smitter GABA. Using a recently-developed in vivo microdialysis system in zebra finches, we<br />
monitored levels of <strong>for</strong>ebrain estradiol (via ELISA) simult<strong>an</strong>eously with extracellular levels of<br />
amino acid neurotr<strong>an</strong>smitters (via HPLC) in males expressing a variety of natural behaviors. A<br />
pattern emerging <strong>from</strong> <strong>the</strong>se studies is that levels of estradiol are inversely related to extracellular<br />
glutamate within <strong>the</strong> auditory <strong>for</strong>ebrain (region NCM), but are not within Area X (a basalg<strong>an</strong>glia<br />
nucleus in <strong>the</strong> striatum). Second, when males are exposed to song playbacks, estradiol<br />
levels increase while extracellular glutamate levels decrease in <strong>the</strong> auditory <strong>for</strong>ebrain. Third,<br />
estradiol levels are unrelated to <strong>an</strong>y o<strong>the</strong>r amino acid tr<strong>an</strong>smitter we examined, including levels<br />
of GABA, aspartate, <strong>an</strong>d arginine. Fourth, acute infusion of glutamate into NCM suppresses<br />
local estradiol levels, while GABA infusion has no effect. Lastly, in vivo microdialysis has been<br />
adapted to a second songbird species, <strong>the</strong> Western scrub jay, <strong>for</strong> <strong>an</strong>alysis of steroidneurotr<strong>an</strong>smitter<br />
interactions in <strong>the</strong> hippocampus during spatial memory tasks. Preliminary<br />
results support a negative correlation between local estradiol <strong>an</strong>d glutamate levels in scrub jay<br />
hippocampus. Thus, estradiol <strong>an</strong>d glutamate appear to be inversely related within higher-order<br />
<strong>for</strong>ebrain regions in two species of songbirds. These observations are consistent with <strong>the</strong><br />
possibility of negative-feedback <strong>an</strong>d/or extra-synaptic interactions between glutamate <strong>an</strong>d<br />
estradiol in <strong>the</strong> regulation of complex circuits that support behavior. Support <strong>from</strong> NINDS <strong>an</strong>d<br />
NIMH.
Disclosures: L. Remage-Healey, None; N.T. Maidment, None; B.A. Schlinger, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.8/GG109<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NIH Gr<strong>an</strong>t R01DA020109<br />
Title: C<strong>an</strong>nabinoid-altered vocal learning is associated with elevated dendritic spine densities in<br />
Area X <strong>an</strong>d HVC<br />
Authors: *M. T. GILBERT, K. SODERSTROM;<br />
Pharmacol., Brody Sch. of Medicine|910001944|0, Greenville, NC<br />
Abstract: Postnatal CNS development includes normal decreases in dendritic spine densities.<br />
These ch<strong>an</strong>ges have been shown to occur within brain regions necessary <strong>for</strong> zebra finch song<br />
learning <strong>an</strong>d production. Our prior work indicates that developmental exposure to <strong>the</strong><br />
c<strong>an</strong>nabinoid agonist WIN55212-2 (WIN) alters vocal learning. We have begun to evaluate <strong>the</strong><br />
possibility that WIN-altered song may influence <strong>the</strong> normal process of spine density decreases.<br />
To test this, we measured spine densities following developmental WIN exposure. Zebra finches<br />
were tutored until 50 days of age <strong>an</strong>d assigned to treatment (1 mg/kg WIN) or vehicle control<br />
groups (n = 8). IM injections were given once daily <strong>from</strong> 50-75 days of age (during <strong>the</strong> sensory<br />
motor stage of song learning). After 100 days songs were recorded <strong>an</strong>d brains prepared <strong>for</strong><br />
Golgi-Cox staining. Dendritic spines were counted in Area X, HVC, RA, <strong>an</strong>d lMAN. WIN<br />
treatment resulted in elevated dendritic spine densities within Area X <strong>an</strong>d HVC. Spine density<br />
differences were not observed in lMAN or RA.<br />
Recent evidence suggests a non-apoptotic role <strong>for</strong> caspase-3 in resorption of dendritic spines.<br />
Caspase-3 activation has been shown to increase with novel song perception, indicating a role in<br />
zebra finch vocal memory. These findings led to <strong>the</strong> hypo<strong>the</strong>sis that WIN-induced ch<strong>an</strong>ges in<br />
spine densities may involve inhibition of caspase-3 activation. To test this, adults were assigned<br />
to WIN (3 mg/kg), vehicle or untreated control groups (n=4). Treatments were given 30 min<br />
be<strong>for</strong>e exposure to a recording of novel song. Following 10 min of novel song exposure, birds<br />
were <strong>an</strong>es<strong>the</strong>tized, perfused with para<strong>for</strong>m, <strong>an</strong>d brains prepared <strong>for</strong> immunohistochemistry with<br />
<strong>an</strong> <strong>an</strong>tibody against activated caspase-3. WIN treatment suppressed normally robust ch<strong>an</strong>ges in<br />
activated caspase-3 levels within auditory Field L2.<br />
Our results demonstrate that c<strong>an</strong>nabinoid-altered vocal development is associated with
inappropriately high dendritic spine densities within Area X of striatum, a brain region known to<br />
be critical <strong>for</strong> song learning, <strong>an</strong>d HVC, a region essential <strong>for</strong> learning <strong>an</strong>d producing song. In<br />
addition, we have shown that WIN alters normal activation of caspase-3 following auditory<br />
perception of song, raising <strong>the</strong> possibility that elevated Area X spine densities may due to similar<br />
caspase-3 inhibition during song learning. We are currently testing this possibility.<br />
Disclosures: M.T. Gilbert, 5R01DA020109-03, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); K.<br />
Soderstrom, 5R01DA020109-03, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or<br />
consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.9/GG110<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NSERC Discovery Gr<strong>an</strong>t Individual<br />
Title: Neural <strong>an</strong>d behavioral responses to playback of altered songs by male <strong>an</strong>d female blackcapped<br />
chickadees (Poecile atricapillus)<br />
Authors: *S. P. ROACH 1 , D. J. MENNILL 2 , L. S. PHILLMORE 1 ;<br />
1 2<br />
Psychology Dept, Dalhousie Univ., Halifax, NS, C<strong>an</strong>ada; Dept. of Biol. Sci., Univ. of Windsor,<br />
Windsor, ON, C<strong>an</strong>ada<br />
Abstract: The black-capped chickadee has a structurally simple song (containing two notes<br />
phonetically described as fee <strong>an</strong>d bee) that is produced mainly by males during <strong>the</strong> breeding<br />
season <strong>an</strong>d varies little across most of its North Americ<strong>an</strong> r<strong>an</strong>ge (Kroosdma et al., 1999).<br />
Although <strong>the</strong> song varies in absolute pitch, its structure is governed by specific rules regarding<br />
<strong>the</strong> relative pitch ratios between <strong>the</strong> beginning <strong>an</strong>d end of <strong>the</strong> first note (which contains a<br />
descending gliss<strong>an</strong>do) <strong>an</strong>d between <strong>the</strong> end of <strong>the</strong> first note <strong>an</strong>d <strong>the</strong> beginning of <strong>the</strong> second<br />
(Weism<strong>an</strong> et al., 1990). Previous behavioral field studies have shown that chickadees require one<br />
or both (depending on sex) of <strong>the</strong>se ratios to be intact in order to recognize <strong>the</strong> vocalization as a<br />
conspecific song <strong>an</strong>d thus respond appropriately (Shackleton et al., 1992; Ratcliffe <strong>an</strong>d Otter,<br />
1996). With <strong>the</strong> objective of establishing neural correlates to <strong>the</strong>se findings, we captured male<br />
(n=28) <strong>an</strong>d female (n=20) black-capped chickadees <strong>from</strong> <strong>the</strong> wild during <strong>the</strong> winter <strong>an</strong>d early<br />
spring, <strong>an</strong>d used photostimulation to bring <strong>the</strong> birds into full spring breeding condition. We
examined behavioral <strong>an</strong>d neural responses to “normal” chickadee song (following <strong>the</strong> pitch ratio<br />
rules) as well as songs altered in one of three ways: (1) Flattened fee gliss<strong>an</strong>do (i.e., pitch ratio<br />
across first note reduced to 1.0) (2) Eliminated inter-note ratio (i.e., pitch ratio between end of<br />
fee <strong>an</strong>d start of bee reduced to 1.0), <strong>an</strong>d (3) Both pitch ratios reduced to 1.0. Behaviorally, we<br />
measured <strong>the</strong> number of vocal responses, <strong>the</strong> average dist<strong>an</strong>ce <strong>from</strong> <strong>the</strong> sound source, <strong>an</strong>d<br />
general activity be<strong>for</strong>e, during, <strong>an</strong>d after playback. Neural response was qu<strong>an</strong>tified by measuring<br />
expression of <strong>the</strong> early immediate gene ZENK in <strong>the</strong> auditory perception-related caudal medial<br />
nidopallium (NCM) <strong>an</strong>d caudal medial mesopallium (CMM). Results are discussed in terms of<br />
<strong>the</strong> song components necessary <strong>for</strong> recognition of conspecific song by male <strong>an</strong>d female<br />
chickadees, as well as how <strong>the</strong>se results relate to those of previous field studies.<br />
Disclosures: S.P. Roach, None; D.J. Mennill, None; L.S. Phillmore, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.10/GG111<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NSERC<br />
CFI<br />
Alberta Ingenuity<br />
Title: Conspecific or heterospecific? Zenk activation in <strong>the</strong> nidopallium of black-capped<br />
chickadees<br />
Authors: *M. T. AVEY 1 , M. A. HOESCHELE 1 , M. K. MOSCICKI 1 , H. LEE 1 , L. L.<br />
BLOOMFIELD 3 , C. B. STURDY 2 ;<br />
1 Pscyhology, 2 Pscyhology, Ctr. <strong>for</strong> Neuroscience, Biocognition Unit, Univ. of Alberta,<br />
Edmonton, AB, C<strong>an</strong>ada; 3 Psychology, Algoma Univ., Sault Ste. Marie, ON, C<strong>an</strong>ada<br />
Abstract: Immediate early gene (ZENK) activity has been used as a marker <strong>for</strong> activity in <strong>the</strong><br />
songbird nidopallium <strong>for</strong> almost two decades. ZENK activity increases with <strong>the</strong> presentation of<br />
more ethologically relev<strong>an</strong>t stimuli classes, such that conspecific vocalizations induce more<br />
ZENK activity compared to heterospecific vocalizations or artificial stimuli, such as tones. We<br />
tested <strong>the</strong> relationship between increases in ZENK activity <strong>an</strong>d phylogenetic relatedness of vocal
stimuli, in o<strong>the</strong>r words, <strong>the</strong> degree of “heterospecifity”. We used vocalizations <strong>from</strong> <strong>the</strong> genus<br />
Poecile, all of which produce a “chick-a-dee” call that is used in a variety of contexts <strong>for</strong> a<br />
variety of putative purposes. The “chick-a-dee” calls used as stimuli were <strong>from</strong> black-capped<br />
chickadee (Poecile atricapillus), mountain chickadee (Poecile gambeli), <strong>an</strong>d boreal chickadee<br />
(Poecile hudsonica). Black-capped <strong>an</strong>d mountain chickadees are sister species whereas <strong>the</strong> boreal<br />
chickadee is closely related, but located on a separate br<strong>an</strong>ch of <strong>the</strong> Poecile clade. We played<br />
back one of <strong>the</strong> three types of chickadees to black-capped chickadees, which have previously<br />
been shown to have increased ZENK following conspecific calls <strong>an</strong>d, in oper<strong>an</strong>t conditioning<br />
experiments, to be able to discriminate between black-capped <strong>an</strong>d mountain chickadee calls.<br />
ZENK activity was qu<strong>an</strong>tified by <strong>the</strong> amount of labeled protein product in <strong>the</strong> caudal medial<br />
nidopallium (NCM) <strong>an</strong>d caudomedial mesopallium (CMM). We found no differences in <strong>the</strong><br />
amount of ZENK expressed between <strong>the</strong> three groups of Poecile vocalizations; however, all<br />
groups differed signific<strong>an</strong>tly <strong>from</strong> <strong>the</strong> silence control condition which had little ZENK induction.<br />
These results suggest that ZENK induction in <strong>the</strong> nidopallium is not a conspecific/heterospecific<br />
dichotomy but that a more complex relationship exists that may depend on vocal similarities or<br />
selective adv<strong>an</strong>tages to attending to vocalizations.<br />
Disclosures: M.T. Avey, None; M.A. Hoeschele, None; M.K. Moscicki, None; H. Lee,<br />
None; L.L. Bloomfield, None; C.B. Sturdy, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.11/GG112<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NSERC Discovery Gr<strong>an</strong>t Individual<br />
Title: Social context <strong>an</strong>d rates of proliferation in <strong>the</strong> brain of male <strong>an</strong>d female zebra finches,<br />
Taeniopygia guttata<br />
Authors: *L. S. PHILLMORE 1 , H. KINOSHITA 1 , S. P. ROACH 1 , H. A. ROBERTSON 2 ;<br />
1 Dept Psychol, 2 Dept Pharmacol., Dalhousie Univ., Halifax, NS, C<strong>an</strong>ada<br />
Abstract: Social context <strong>an</strong>d experience has been shown to alter neural activity in <strong>the</strong> brain of<br />
male zebra finches (Sasaki et al., 2006). For example, dopamine levels in Area X of <strong>the</strong> striatum<br />
in males singing directed song (i.e. to females) are higher th<strong>an</strong> when males sing undirected song<br />
(i.e. alone or to o<strong>the</strong>r males). Courtship behavior c<strong>an</strong> also increase dopaminergic ventral
tegmental area (VTA) cell activation similar to that seen in reward activation following <strong>the</strong><br />
administration of addictive drugs (Hu<strong>an</strong>g & Hessler, 2008). There<strong>for</strong>e social context <strong>an</strong>d<br />
resulting DA release has <strong>the</strong> potential to affect o<strong>the</strong>r neural processes such as neurogenesis (V<strong>an</strong><br />
Kampen & Robertson, 2005; V<strong>an</strong> Kampen & Eckm<strong>an</strong>, 2006). In this study we housed zebra<br />
finches in ei<strong>the</strong>r male-male or male-female pairs <strong>an</strong>d compared rates of proliferation in several<br />
regions of both sexes: <strong>the</strong> subventricular zone (SVZ), <strong>an</strong> area in which neurogenesis has been<br />
confirmed, <strong>an</strong>d <strong>the</strong> subst<strong>an</strong>tia nigra (SN), a region in which <strong>the</strong> existence of neurogenesis is<br />
currently debated in mammali<strong>an</strong> models (Sh<strong>an</strong> et al., 2006; Zhao et al., 2003; Frielingsdorf et al.,<br />
2004). Preliminary data indicates that proliferation was not different in SVZ of directed <strong>an</strong>d<br />
undirected males, however, proliferation in females varied with amount of directed singing<br />
heard. In addition, <strong>the</strong>re is some indication of new cells present in <strong>the</strong> SN. Underst<strong>an</strong>ding <strong>the</strong><br />
processes of neurogenesis <strong>an</strong>d cell proliferation is import<strong>an</strong>t in underst<strong>an</strong>ding mech<strong>an</strong>isms of<br />
plasticity <strong>an</strong>d repair in <strong>the</strong> adult brain. Specifically, evidence <strong>for</strong> neurogenesis or proliferation in<br />
<strong>the</strong> SN is of great interest <strong>for</strong> those searching <strong>for</strong> treatments <strong>for</strong> Parkinson’s disease, a<br />
neurodegenerative disorder involving neurons of <strong>the</strong> SN (Lie et al., 2002).<br />
Disclosures: L.S. Phillmore, None; H. Kinoshita, None; S.P. Roach, None; H.A. Robertson,<br />
None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.12/GG113<br />
Topic: F.04.c. Vocal/social communication<br />
Title: HVC-driven activity of <strong>an</strong>terior <strong>for</strong>ebrain song nuclei Area X <strong>an</strong>d LMAN c<strong>an</strong> be<br />
modulated by stimulation of VTA<br />
Authors: *N. AOKI, N. A. HESSLER;<br />
RIKEN BSI, Wako SAITAMA, Jap<strong>an</strong><br />
Abstract: For reproductive success, male songbirds need to sing attractively <strong>for</strong> females.<br />
Courtship song targeted to females (directed song, DS) is less variable th<strong>an</strong> song produced when<br />
<strong>the</strong> male is alone (undirected song, US). Neural activity in Area X, a striatal nucleus, is also less<br />
variable during DS th<strong>an</strong> US. The <strong>an</strong>terior <strong>for</strong>ebrain pathway (AFP), which includes Area X,<br />
appears to control <strong>the</strong> variability of song though LMAN activity which is <strong>the</strong> output of <strong>the</strong> AFP.<br />
We hypo<strong>the</strong>size that dopamine released <strong>from</strong> <strong>the</strong> ventral tegmental area (VTA) could modulate<br />
singing related activity of Area X <strong>an</strong>d LMAN, <strong>an</strong>d thus control singing variability.
To test this idea, we recorded neuronal activity of Area X <strong>an</strong>d LMAN <strong>from</strong> <strong>an</strong>es<strong>the</strong>tized birds, in<br />
response to stimulation of both <strong>the</strong> afferent song system motor nucleus HVC <strong>an</strong>d VTA. In Area<br />
X, putative medium spiny neurons whose firing rate was less th<strong>an</strong> 5 Hz responsed with shortlatency<br />
burst firing after HVC stimulation. Preceding stimulation of VTA did not affect <strong>the</strong> burst.<br />
For tonic firing neurons of Area X, we found two types of responses after HVC stimulation.<br />
Some Area X neurons bursted, <strong>an</strong>d latency of <strong>the</strong> bursts were longer <strong>an</strong>d more variable th<strong>an</strong><br />
those of medium spiny neurons. Elicited activity was slightly suppressed when preceded by VTA<br />
stimulation. Activity of o<strong>the</strong>r Area X neurons was inhibited following HVC stimulation, <strong>an</strong>d was<br />
not clearly altered by VTA stimulation. Activation of LMAN by HVC stimulation was<br />
conistently of loniger latency th<strong>an</strong> that of Area X neurons, <strong>an</strong>d was completely suppressed when<br />
preceded by VTA stimulation. With a longer delay between VTA <strong>an</strong>d HVC stimulation (300ms),<br />
such suppression was reduced, <strong>an</strong>d <strong>the</strong> variability of <strong>the</strong> burst latency was reduced. These results<br />
suggest that <strong>the</strong> higher precision of LMAN activity caused by dopamine release during courtship<br />
reduces <strong>the</strong> variability of song during DS.<br />
Disclosures: N. Aoki, None; N.A. Hessler, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.13/GG114<br />
Topic: F.04.c. Vocal/social communication<br />
Support: RIKEN<br />
KAKENHI(20700306)<br />
Title: Singing- <strong>an</strong>d sleep-related neuronal activity in songbird basal g<strong>an</strong>glia<br />
Authors: *S. YANAGIHARA, N. A. HESSLER;<br />
RIKEN, Wako-shi, Jap<strong>an</strong><br />
Abstract: Although <strong>the</strong> striatopallidal nucleus Area X is essential <strong>for</strong> normal song learning, its<br />
function in this process remains unclear. To underst<strong>an</strong>d <strong>the</strong> role of basal g<strong>an</strong>glia in this<br />
sensorimotor learning, we tried to characterize <strong>the</strong> activity patterns of neurons in Area X. We<br />
recorded single-unit activity <strong>from</strong> Area X of male juvenile zebra finches when <strong>the</strong> birds produce<br />
immature <strong>an</strong>d highly variable plastic songs. We found that <strong>the</strong> level of activity of Area X<br />
neurons was consistently increased during singing. This increase beg<strong>an</strong> several hundreds
millisecond be<strong>for</strong>e initiation of singing <strong>an</strong>d continued until termination of singing. These<br />
neurons did not respond to playback of <strong>the</strong> own song, or to hearing <strong>the</strong> tutor song. Moreover,<br />
most of <strong>the</strong> neurons that were active during singing also showed occasional phasic increase of<br />
activity during slow wave sleep. Similar singing- <strong>an</strong>d sleep-related activity was found in adult<br />
birds. These results suggest that activity of <strong>the</strong> Area X neurons during sleep as well as singing<br />
may have critical roles <strong>for</strong> song learning <strong>an</strong>d mainten<strong>an</strong>ce.<br />
Disclosures: S. Y<strong>an</strong>agihara, None; N.A. Hessler, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.14/GG115<br />
Topic: F.04.c. Vocal/social communication<br />
Title: Influence of RA neural spont<strong>an</strong>eous firing in zebra finch song production<br />
Authors: *A. AJIMA 1 , R. IMAI 2 , C. MORI 2 , K. WADA 2 , N. A. HESSLER 1 ;<br />
1 RIKEN, BSI, Saitama, Jap<strong>an</strong>; 2 Adv<strong>an</strong>ced Life Scicence, Hokkaido Univ., Sapporo, Jap<strong>an</strong><br />
Abstract: Birdsong, like hum<strong>an</strong> speech, requires precise control of vocal <strong>an</strong>d respiratory<br />
muscles. A key nucleus of <strong>the</strong> muscle-control is <strong>the</strong> robust nucleus of <strong>the</strong> arcopallium (RA),<br />
which projects to brain stem nuclei controlling vocal <strong>an</strong>d respiratory muscles <strong>an</strong>d directly<br />
controls fine structure of song. RA receives synaptic input <strong>from</strong> <strong>the</strong> premotor song-control<br />
nucleus HVC <strong>an</strong>d <strong>from</strong> LMAN, <strong>an</strong> area essential to song plasticity. In adults, RA neurons<br />
display a tonic, pacemaker-like activity at rest. During singing, activity becomes highly phasic,<br />
with prolonged silent periods. To underst<strong>an</strong>d <strong>the</strong> control mech<strong>an</strong>isms of RA neural activity, we<br />
examined <strong>the</strong> physiological characteristics in a slice preparation, <strong>an</strong>d in <strong>an</strong>es<strong>the</strong>tized birds. The<br />
rate of spont<strong>an</strong>eous activity was not influenced by synaptic inputs, but was regulated by I_h<br />
current. Pharmacological block of hyperpolarization -activated cyclic nucleotide-modulated<br />
(HCN) ch<strong>an</strong>nels reduced <strong>the</strong> spont<strong>an</strong>eous firing rate of RA neurons, <strong>an</strong>d HCN mRNA was<br />
expressed in RA neurons. An acute blockade of HCN ch<strong>an</strong>nels reduced <strong>the</strong> level of singing. This<br />
system may be a useful way to investigate <strong>the</strong> role of spont<strong>an</strong>eous activity in controlling song<br />
structure, <strong>an</strong>d may be <strong>an</strong> import<strong>an</strong>t target of neuromodulators such as norepinephrine.<br />
Disclosures: A. Ajima, None; R. Imai, None; C. Mori, None; K. Wada, None; N.A. Hessler,<br />
None.
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.15/GG116<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NIH Gr<strong>an</strong>t NS50436<br />
Title: A <strong>the</strong>oretical framework <strong>for</strong> <strong>the</strong> cultural evolution of birdsong<br />
Authors: *H. WANG, P. P. MITRA;<br />
Cold Spring Harbor Lab., Cold Spring Harbor, NY<br />
Abstract: Songbirds acquire <strong>the</strong>ir songs through social learning, <strong>an</strong>d members of a species show<br />
individual variation in song as well as geographically local song dialects. Moreover, different<br />
species exhibit distinct sets of wild type (WT) songs, suggesting constraints due to <strong>the</strong> species<br />
genome. Thus songbirds provide tractable models <strong>for</strong> studying <strong>the</strong> relationship between<br />
"culture", which consists of traits inherited epigenetically through social learning under genomic<br />
or genetic constraints. While this has been understood <strong>for</strong> some time, a ma<strong>the</strong>matical <strong>for</strong>mulation<br />
in <strong>the</strong> context of <strong>an</strong> articulated experimental research program has been lacking.<br />
This lacuna has been recently addressed in <strong>an</strong> experimental <strong>an</strong>d <strong>the</strong>oretical study of <strong>the</strong> evolution<br />
of song culture in zebra finch, where WT song culture was shown to emerge spont<strong>an</strong>eously over<br />
multiple generations, starting with isolate founders. Here we fur<strong>the</strong>r elaborate a set of models<br />
describing <strong>the</strong> recursive, multigenerational dynamics of song imitation. The models study <strong>the</strong><br />
song culture evolution in term of a qu<strong>an</strong>titative song phenotype P, which we partition into<br />
genotypic value G, <strong>an</strong> environmental component E0 independent of <strong>the</strong> tutor, <strong>an</strong>d <strong>an</strong><br />
environmental component corresponding to song imitation.<br />
In <strong>the</strong> Isolated Lineages model, individual tutors are imitated by individual pupils (which<br />
become tutors <strong>for</strong> <strong>the</strong> next generation). Assuming additive phenotypic variation <strong>an</strong>d a linear<br />
imitation function, we obtain P(n+1) = G(n+1) + c P(n) + E0(n+1). This recursive, stochastic<br />
difference equation models evolution of a population of birds. The emphasis on a population<br />
differentiates <strong>the</strong> present <strong>the</strong>oretical considerations <strong>from</strong> <strong>the</strong> modeling of song development or<br />
imitation in <strong>an</strong> individual bird.<br />
Starting <strong>from</strong> <strong>an</strong> initial population of isolate birds corresponds to <strong>the</strong> initial condition, P(1) =<br />
G(1) + E0(1). The recursion <strong>the</strong>n causes <strong>the</strong> initial distribution of phenotypic values to relax to<br />
<strong>an</strong> asymptotic distribution, corresponding to WT song culture, consistent with <strong>the</strong> experimental<br />
findings.<br />
The Isolated Lineages model c<strong>an</strong> be extended to simulate natural colony settings, by including
lateral song tr<strong>an</strong>smission <strong>from</strong> tutors to pupils, as well as between pupils, in <strong>an</strong> Interacting<br />
Lineages model. Genetic relatedness between tutor <strong>an</strong>d pupil c<strong>an</strong> be incorporated by introducing<br />
non-zero covari<strong>an</strong>ce between genotypic values across generations. The effects of non-linear<br />
imitation functions are also discussed. These models are <strong>an</strong>alogous to models of parental effects<br />
in qu<strong>an</strong>titative genetics, <strong>an</strong>d a number of <strong>an</strong>alogous models also exist in <strong>the</strong> <strong>the</strong>oretical literature<br />
on hum<strong>an</strong> cultural evolution.<br />
Disclosures: H. W<strong>an</strong>g, None; P.P. Mitra, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.16/GG117<br />
Topic: F.04.c. Vocal/social communication<br />
Support: R01 MH080225 to LVR<br />
CAC is a F.R.S.-FNRS Postdoctoral Researcher<br />
R01 NS 35467 to GFB<br />
Title: Alpha 2-noradrenergic receptor densities in brain regions involved in song control <strong>an</strong>d<br />
sexual motivation relate context-dependently to male song production in Europe<strong>an</strong> starlings<br />
(Sturnus vulgaris)<br />
Authors: *J. M. ELLIS 1 , S. A. HEIMOVICS 2 , C. CORNIL 3 , G. F. BALL 4 , L. RITERS 5 ;<br />
1 Zoology, Univ. of Wisconsin, Madison, Madison, WI; 2 Univ. of British Columbia, V<strong>an</strong>couver,<br />
BC, C<strong>an</strong>ada; 3 Univ. of Liege, Liege, Belgium; 4 John Hopkins Univ., Baltimore, MD; 5 Univ. of<br />
Wisconsin, Madison, WI<br />
Abstract: In seasonally breeding songbirds, seasonal alterations in testosterone (T) <strong>an</strong>d neuronal<br />
attributes parallel ch<strong>an</strong>ges in <strong>the</strong> function of song. The catecholamine norepinephrine (NE)<br />
modulates attention <strong>an</strong>d arousal, components of behavior likely import<strong>an</strong>t <strong>for</strong> a male to adjust his<br />
vocal response to match a seasonal context. Past work in male Europe<strong>an</strong> starlings identified<br />
relationships between seasonal ch<strong>an</strong>ges in α2-noradrenergic receptors (α2-R), circulating T, <strong>an</strong>d<br />
<strong>the</strong> function of song. In spring when T was high <strong>an</strong>d males were presumably singing to attract<br />
females <strong>the</strong> density of α2-R within <strong>the</strong> song control regions HVC <strong>an</strong>d <strong>the</strong> robust nucleus of <strong>the</strong><br />
arcopallium (RA) was low. In contrast, in fall when T was low <strong>an</strong>d male starlings were
presumably singing to maintain flock cohesion α2-R density in HVC <strong>an</strong>d RA was high. The<br />
present study explored <strong>the</strong> possibility that seasonal ch<strong>an</strong>ges in α2-R within song control regions<br />
<strong>an</strong>d regions implicated in male sexual motivation contribute to seasonal ch<strong>an</strong>ges in song<br />
behavior. We qu<strong>an</strong>tified song in aviary housed male starlings under spring breeding season<br />
conditions <strong>an</strong>d fall conditions both in <strong>the</strong> presence <strong>an</strong>d absence of a stimulus female. α2-R were<br />
identified with <strong>the</strong> selective lig<strong>an</strong>d [ 3 H]RX821002 using autoradiography. Under spring<br />
conditions males that occupied nest boxes s<strong>an</strong>g at high rates in response to a female. Those<br />
without nest boxes did not. Under fall conditions males did not occupy nest boxes <strong>an</strong>d s<strong>an</strong>g at <strong>the</strong><br />
highest rates when no female was present. The density of α2-R in both HVC <strong>an</strong>d RA was<br />
signific<strong>an</strong>tly higher in fall compared to spring, replicating past results. In area X, α2-R density<br />
correlated negatively with male song produced in <strong>the</strong> presence of a female in spring but not fall.<br />
In RA, α2-R density correlated positively with male song produced in <strong>the</strong> absence of a female<br />
only when data <strong>from</strong> spring <strong>an</strong>d fall were combined. In brain regions implicated in motivation,<br />
when data <strong>from</strong> spring <strong>an</strong>d fall were combined α2-R density in <strong>the</strong> ventral tegmental area related<br />
positively to male song in <strong>the</strong> presence of a female. In contrast, α2-R density in <strong>the</strong> medial<br />
preoptic nucleus related negatively to song in <strong>the</strong> absence of a female. Volumes of song nuclei<br />
<strong>an</strong>d <strong>the</strong> preoptic nucleus vary by season, thus density differences may reflect ch<strong>an</strong>ges in receptor<br />
number or <strong>the</strong> spread of receptors. Differences in <strong>the</strong> brain regions <strong>an</strong>d directional relationships<br />
between α2-R density <strong>an</strong>d song suggest that noradrenergic systems play a region <strong>an</strong>d contextspecific<br />
role in song. These data also suggest that individual variation in singing behavior may,<br />
in part, be explained by individual differences in α2-R density in brain regions implicated in song<br />
<strong>an</strong>d sexual motivation.<br />
Disclosures: J.M. Ellis, None; S.A. Heimovics, None; C. Cornil, None; G.F. Ball, None; L.<br />
Riters, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.17/GG118<br />
Topic: F.04.c. Vocal/social communication<br />
Support: NSF Gr<strong>an</strong>t 0717004<br />
Title: Effects of central injections of <strong>an</strong> alpha 1-noradrenergic receptor <strong>an</strong>tagonist on female<br />
responses to song depend on dose <strong>an</strong>d song stimulus in female Europe<strong>an</strong> starlings (Sturnus<br />
vulgaris)
Authors: *B. A. PAWLISCH 1,2 , S. A. STEVENSON 2 , L. V. RITERS 2 ;<br />
1 Zoology, Madison, WI; 2 Zoology, Univ. of Wisconsin, Madison, WI<br />
Abstract: Vocal communication plays <strong>an</strong> import<strong>an</strong>t role in social interactions in m<strong>an</strong>y <strong>an</strong>imal<br />
species. In seasonally-breeding songbirds, including Europe<strong>an</strong> starlings, females attend to<br />
differences in male song structure <strong>an</strong>d selectively approach particular conspecific male songs.<br />
The auditory processing <strong>an</strong>d perception of song have been relatively well-studied, but little is<br />
known about <strong>the</strong> neurochemicals or neural circuits underlying differential behavioral responses<br />
of females to male song. The catecholamine norepinephrine (NE) has been implicated in arousal,<br />
female sexual responses, <strong>an</strong>d <strong>the</strong> tr<strong>an</strong>slation of a choice into a behavioral response. The goal of<br />
<strong>the</strong> present study was to examine <strong>the</strong> role of alpha 1-noradrenergic receptors in female responses<br />
to male song. Six estradiol treated female starlings received c<strong>an</strong>nulae targeting <strong>the</strong> fourth<br />
ventricle. After surgery females were placed in observation cages with nest boxes <strong>for</strong> 10 days<br />
prior to <strong>the</strong> experiment. During this period females were presented with playbacks of male song<br />
<strong>an</strong>d live males to habituate <strong>the</strong>m to <strong>the</strong> test environment <strong>an</strong>d stimulate sexual interest. On each of<br />
4 test days females received in a counterbal<strong>an</strong>ced order central injections of vehicle or one of 3<br />
doses (0.2, 2, <strong>an</strong>d 20 nmol) of <strong>the</strong> alpha 1-noradrenergic receptor <strong>an</strong>tagonist prazosin. Behavioral<br />
discrimination responses to nest boxes sequentially playing male starling courtship song <strong>an</strong>d less<br />
biologically relev<strong>an</strong>t heterospecific (male purple martin, Progne subis) song were measured. A<br />
signific<strong>an</strong>t drug dose by song stimulus interaction was found <strong>for</strong> <strong>the</strong> number of times females<br />
l<strong>an</strong>ded on <strong>an</strong>d looked into (LAND+LOOK) nest boxes broadcasting song. The low dose of<br />
prazosin signific<strong>an</strong>tly increased female preferential responses to male starling song over<br />
heterospecific song, <strong>an</strong> effect that was absent at higher doses. Prazosin dose-dependently<br />
decreased LAND+LOOK in response to male starling song in a linear fashion. In contrast, in<br />
response to heterospecific song only <strong>the</strong> low <strong>an</strong>d intermediate doses of prazosin suppressed<br />
LAND+LOOK in response to song, suggesting <strong>an</strong> inverse U-shaped dose-response curve. No<br />
signific<strong>an</strong>t effects of drug or song were observed <strong>for</strong> general behaviors, including preening, beak<br />
wiping, or feeding. Overall, <strong>the</strong> results suggest that NE plays a complex modulatory role in<br />
female responses to male song stimuli that varies depending upon drug dose <strong>an</strong>d <strong>the</strong> biological<br />
relev<strong>an</strong>ce of <strong>the</strong> song stimulus.<br />
Disclosures: B.A. Pawlisch, None; S.A. Stevenson, None; L.V. Riters, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.18/GG119<br />
Topic: F.04.c. Vocal/social communication
Support: R01 MH080225 to LVR<br />
Title: High rates of male song production are associated with low densities of mu opioid<br />
receptors in <strong>the</strong> ventral tegmental area <strong>an</strong>d medial preoptic nucleus in male Europe<strong>an</strong> starlings<br />
(Sturnus vulgaris)<br />
Authors: *C. A. KELM 1 , R. M. FORBES-LORMAN 2 , S. A. STEVENSON 1 , C. J. AUGER 2 , L.<br />
V. RITERS 1 ;<br />
1 Zoology, 2 Psychology, Univ. Wisconsin-Madison, Madison, WI<br />
Abstract: Songbirds sing at high rates within multiple social contexts suggesting that <strong>the</strong>y are<br />
highly motivated to sing, <strong>an</strong>d <strong>the</strong> act of singing itself may be rewarding. Across species, opioid<br />
neuropeptides play a primary role in reward. In songbirds, opioid neuropeptides <strong>an</strong>d receptors<br />
are found in both <strong>the</strong> song control system <strong>an</strong>d within several brain regions implicated in both<br />
motivation <strong>an</strong>d reward, including <strong>the</strong> medial preoptic nucleus (POM) <strong>an</strong>d ventral tegmental area<br />
(VTA). Past work in male Europe<strong>an</strong> starlings demonstrated positive correlations between male<br />
song <strong>an</strong>d <strong>the</strong> density of <strong>the</strong> opioid met-enkephalin within <strong>the</strong> POM <strong>an</strong>d VTA. Treatment of male<br />
starlings with <strong>the</strong> opioid receptor <strong>an</strong>tagonist naloxone increased song production, whereas<br />
treatment with <strong>the</strong> opioid receptor agonist fent<strong>an</strong>yl decreased song production. This research<br />
suggests <strong>an</strong> inhibitory role <strong>for</strong> opioids in male song, which may reflect a role <strong>for</strong> opioids in<br />
reward leading to satiety <strong>an</strong>d <strong>an</strong> inhibition of song. Males display individual differences in song<br />
rate. Differential responses to opioid receptor pharmacological m<strong>an</strong>ipulations were identified <strong>for</strong><br />
birds naturally singing at low versus high rates, suggesting that differences might exist in opioid<br />
receptor densities or binding affinity in association with individual differences in <strong>the</strong> tendency to<br />
sing. We used immunocytochemistry to examine relationships between <strong>the</strong> density of mu opioid<br />
receptors <strong>an</strong>d song production. Nineteen spring condition male starlings were testosterone (T)<br />
impl<strong>an</strong>ted <strong>an</strong>d, over <strong>the</strong> course of one week, behaviorally observed in outdoor aviaries in <strong>the</strong><br />
presence of a female. Males that obtained <strong>an</strong>d defended nest boxes s<strong>an</strong>g at signific<strong>an</strong>tly higher<br />
rates th<strong>an</strong> males without nest boxes. Compared to males without nest boxes, males with nest<br />
boxes had lower densities of immunolabeled mu opioid receptors in both POM <strong>an</strong>d VTA. Mu<br />
opioid receptor densities also related positively to feeding behavior. No relationships were found<br />
between mu opioid receptor density <strong>an</strong>d T. The finding that males with higher receptor densities<br />
sing less is consistent with past studies in birds <strong>an</strong>d mammals suggesting <strong>an</strong> inhibitory role <strong>for</strong><br />
opioids in vocal communication. Overall, <strong>the</strong> results suggest that individual differences in <strong>the</strong><br />
densities of mu opioid receptors in VTA <strong>an</strong>d POM play a role in individual differences in <strong>the</strong><br />
motivation to communicate.<br />
Disclosures: C.A. Kelm, None; R.M. Forbes-Lorm<strong>an</strong>, None; S.A. Stevenson, None; C.J.<br />
Auger, None; L.V. Riters, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.19/GG120<br />
Topic: F.04.c. Vocal/social communication<br />
Support: RO1 NS35467<br />
Title: Comparison of song-dependent Arc mRNA expression within caudomedial mesopallium<br />
<strong>an</strong>d nidopallium using cellular compartmental <strong>an</strong>alysis of temporal activity in female zebra<br />
finches<br />
Authors: *K. S. LYNCH 1 , G. F. BALL 2 ;<br />
1 Psychol & Brain Sci., 2 Psychological <strong>an</strong>d Brain Sci., Johns Hopkins Univ., Baltimore, MD<br />
Abstract: Female songbirds selectively attend to conspecific male songs while simult<strong>an</strong>eously<br />
rejecting sounds <strong>from</strong> o<strong>the</strong>r sources. We explored how <strong>the</strong> female’s auditory system encodes<br />
various sound types by employing Arc, <strong>an</strong> immediate early gene (IEG). Arc mRNA c<strong>an</strong> be found<br />
within distinct cellular compartments, which allowed us to use this gene <strong>for</strong> cellular<br />
compartmental <strong>an</strong>alysis of temporal activity by florescent in situ hybridization (catFISH). Thus,<br />
we were able to examine neural responses of two song types within a subject. We presented<br />
female zebra finches with <strong>the</strong> following pairs of auditory stimuli: conspecific / reversed,<br />
conspecific / heterospecific, conspecific / noise. Control groups included: conspecific /<br />
conspecific <strong>an</strong>d silence. We compared <strong>the</strong> cellular compartment in which Arc appeared (nucleus,<br />
cytoplasm or both) within two regions of <strong>the</strong> auditory <strong>for</strong>ebrain; caudomedial mesopallium<br />
(CMM) <strong>an</strong>d caudomedial nidopallium (NCM). First, we labeled <strong>the</strong> cellular compartment in<br />
which Arc mRNA was located <strong>an</strong>d <strong>the</strong>n determined which auditory stimulus induced this<br />
expression. We <strong>the</strong>n compared <strong>the</strong> amount of Arc mRNA induction in response to a single song<br />
type. Arc expression was signific<strong>an</strong>tly different in response to <strong>the</strong> various auditory stimuli within<br />
CMM <strong>an</strong>d NCM. Post-hoc <strong>an</strong>alyses reveal that Arc induction in response to conspecific songs is<br />
signific<strong>an</strong>tly greater relative to all o<strong>the</strong>r sound types in CMM whereas in NCM conspecific songrelated<br />
Arc induction is only greater relative to noise <strong>an</strong>d silence. Secondly, we used pairwise<br />
comparisons to examine <strong>the</strong> difference in Arc expression in each compartment of <strong>the</strong> cell (i.e.<br />
nuclear or cytoplasmic) within subjects. Signific<strong>an</strong>tly greater Arc induction occurs in response to<br />
conspecific songs relative to reversed conspecific <strong>an</strong>d heterospecific songs in CMM whereas<br />
<strong>the</strong>re were no differences in Arc expression in nuclear or cytoplasmic compartments within<br />
NCM. Finally, we compared <strong>the</strong> number of cells expressing Arc in both cellular compartments<br />
relative to <strong>the</strong> control group (conspecific / conspecific). Again, only CMM contained a<br />
signific<strong>an</strong>tly greater number of cells that were labeled in both cellular compartments in response<br />
to <strong>the</strong> conspecific song control group relative to <strong>the</strong> groups that heard two distinct song types.<br />
Comparison of Arc mRNA patterns between CMM <strong>an</strong>d NCM reveals that more cells in CMM<br />
specialize in resolving differences in song type. Taken toge<strong>the</strong>r <strong>the</strong>se results are consistent with<br />
<strong>the</strong> hypo<strong>the</strong>sis that CMM <strong>an</strong>d NCM play distinct roles during song perception: cells in CMM<br />
function in song discrimination whereas cells in NCM function in song recognition.
Disclosures: K.S. Lynch, None; G.F. Ball, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.20/GG121<br />
Topic: F.04.d. Seasonal & sex differences<br />
Support: R01 NS35467<br />
Title: The roles of estrogenic <strong>an</strong>d <strong>an</strong>drogenic metabolites of testosterone in <strong>the</strong> regulation of<br />
doublecortin in <strong>the</strong> song system of adult female c<strong>an</strong>aries<br />
Authors: *T. YAMAMURA 1 , J. BALTHAZART 2 , G. F. BALL 1 ;<br />
1 2<br />
Psychological & Brain Sci., Johns Hopkins Univ., Baltimore, MD; GIGA Neurosciences, Univ.<br />
of Liège, Liège, Belgium<br />
Abstract: Vocal control nuclei in songbirds, such as HVC, <strong>the</strong> robust nucleus of <strong>the</strong> arcopallium<br />
(RA), <strong>an</strong>d Area X show seasonal ch<strong>an</strong>ges in brain nucleus volume that are regulated by <strong>the</strong><br />
action of gonadal testosterone (T) <strong>an</strong>d its metabolites. These ch<strong>an</strong>ges in nucleus volume are<br />
based on ch<strong>an</strong>ges in cell size, dendritic br<strong>an</strong>ching <strong>an</strong>d, in HVC, <strong>the</strong> incorporation of newborn<br />
neurons. Doublecortin (DCX) is a microtubule-associated protein expressed during development<br />
<strong>an</strong>d in adulthood in postmitotic migrating <strong>an</strong>d differentiating neurons in mammals. Our previous<br />
studies in male c<strong>an</strong>aries demonstrated that DCX is expressed in BrdU-positive neurons<br />
consistent with DCX being a marker of neurogenesis in adult c<strong>an</strong>aries. Within HVC, <strong>the</strong> number<br />
of DCX-immunoreacitive (ir) cells was increased by T treatment <strong>an</strong>d photostimulation. T c<strong>an</strong><br />
also induce marked increases in song nuclei volume in adult female c<strong>an</strong>aries so that <strong>the</strong>se nuclei<br />
are more male-like in volume. T c<strong>an</strong> be readily metabolized in <strong>the</strong> songbird brain to <strong>an</strong>drogenic<br />
metabolites such as 5alpha dihydrotestosterone (DHT) or estrogenic metabolites such as 17beta<br />
estradiol (E2). We found previously that both metabolites of T were required to induce increases<br />
in <strong>the</strong> volume of song nuclei in adult female c<strong>an</strong>aries. However, <strong>the</strong> cellular basis of <strong>the</strong> action of<br />
<strong>the</strong>se two metabolites on adult neuroplasticity is not well understood. We <strong>the</strong>re<strong>for</strong>e examined <strong>the</strong><br />
effect of <strong>the</strong>se two metabolites on DCX expression in <strong>the</strong> song nuclei of adult female c<strong>an</strong>aries.<br />
Intact female c<strong>an</strong>aries were impl<strong>an</strong>ted with Silastic tubing containing one of <strong>the</strong> following<br />
treatment regimens: crystalline T, DHT, E2, or a combination of DHT+E2. Control <strong>an</strong>imals<br />
received empty impl<strong>an</strong>ts. C<strong>an</strong>aries were kept under early spring-like photoperiodic conditions<br />
(11L:13D) <strong>for</strong> 3 weeks. In HVC, <strong>the</strong> number of fusi<strong>for</strong>m DCX-ir cells (presumptive migrating<br />
neurons) was signific<strong>an</strong>tly increased by all steroid treatments as compared to control birds. The
number of round DCX-ir cells (differentiating neurons) exhibited a similar but non-signific<strong>an</strong>t<br />
trend. In Area X, unlike our previous study of male c<strong>an</strong>aries, <strong>the</strong> treatment of T, DHT or<br />
E2+DHT was effective in eli<strong>citing</strong> signific<strong>an</strong>t increases in <strong>the</strong> number of fusi<strong>for</strong>m DCX-ir cells,<br />
while <strong>the</strong> number of round DCX-ir cells was increased by T treatments only. In o<strong>the</strong>r areas as<br />
LMAN (lateral magnocellular nucleus of <strong>the</strong> <strong>an</strong>terior nidopallium) <strong>an</strong>d <strong>the</strong> nidopallium, DCX<br />
expression was observed, but <strong>the</strong>re were no treatment effects. These results suggest that both<br />
metabolites of T, but particularly <strong>an</strong>drogenic metabolites, are involved in enh<strong>an</strong>cing <strong>the</strong><br />
migration of newborn neurons <strong>from</strong> <strong>the</strong> subventricular zone toward HVC <strong>an</strong>d Area X via effects<br />
on DCX expression.<br />
Disclosures: T. Yamamura, None; J. Balthazart, None; G.F. Ball, None.<br />
Poster<br />
584. Vocal Communication: Songbirds II<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 584.21/GG122<br />
Topic: F.04.c. Vocal/social communication<br />
Support: BOF-KP gr<strong>an</strong>t <strong>from</strong> Univ. Antwerp to CP<br />
NINDS gr<strong>an</strong>t (NS035467) to JB<br />
FWO project G.0420.02 to A.VdL<br />
GOA funding Univ. Antwerp to A.VdL<br />
EC-FP6 project DiMI LSHB-CT-2005-512146 to A.VdL<br />
EC-FP6 project EMIL LSHC-CT-2004-503569 to A.VdL<br />
PhD Fellowship <strong>from</strong> FWO to TB<br />
Title: Repetition-related neural plasticity in <strong>the</strong> songbird brain<br />
Authors: *C. C. POIRIER 1 , T. BOUMANS 1 , M. VERHOYE 1 , J. BALTHAZART 2 , A. VAN<br />
DER LINDEN 1 ;<br />
1 Bio-Imaging Lab., Antwerp, Belgium; 2 GIGA Neurosciences, Univ. Liege, Liege, Belgium
Abstract: Repetition priming is a <strong>for</strong>m of implicit memory <strong>an</strong>d refers to a behavioral ch<strong>an</strong>ge (in<br />
speed, accuracy,...) associated with <strong>the</strong> processing of a stimulus due to prior exposure to that<br />
same stimulus. At <strong>the</strong> neural level, repeated processing of a stimulus usually induces a reduction<br />
of <strong>the</strong> neural response, called repetition suppression <strong>an</strong>d in few cases, <strong>an</strong> increase of <strong>the</strong> neural<br />
response, called repetition enh<strong>an</strong>cement. Recognition memory is a <strong>for</strong>m of explicit memory <strong>an</strong>d<br />
refers to <strong>the</strong> ability to appreciate that a stimulus has been previously encountered (is it familiar or<br />
novel?). The relationship between repetition priming <strong>an</strong>d recognition memory remains unclear.<br />
Songbirds are a well-known model <strong>for</strong> learning, plasticity <strong>an</strong>d memory processes. Neural<br />
mech<strong>an</strong>isms supporting learning <strong>an</strong>d memory in songbirds are never<strong>the</strong>less poorly understood.<br />
Using BOLD fMRI, we investigated <strong>the</strong> neural effects of stimulus repetition using conspecific<br />
songs as stimuli <strong>an</strong>d assessed <strong>the</strong> interaction of <strong>the</strong>se repetition effects with <strong>the</strong> familiarity of <strong>the</strong><br />
experimental subjects (adult male zebra finches) with <strong>the</strong> song stimuli. Stimuli consisted of<br />
familiar (song <strong>from</strong> cage mate) <strong>an</strong>d novel (song <strong>from</strong> bird housed in <strong>an</strong>o<strong>the</strong>r room) conspecific<br />
songs which were presented 25 times each to <strong>the</strong> <strong>an</strong>es<strong>the</strong>tized subject. The 25 repetitions of each<br />
stimulus were spread over 5 periods of 5 repetitions. A repeated measure ANOVA was <strong>the</strong>n used<br />
to test <strong>the</strong> two main effects namely “repetition” (by testing linear trend effects over <strong>the</strong> 5 periods)<br />
<strong>an</strong>d "familiarity" (familiar vs. novel song), <strong>an</strong>d <strong>the</strong>ir interaction. Repetition suppression was<br />
found in a large set of regions, including <strong>the</strong> caudal mesopallium. Familiarity effects were found<br />
in <strong>the</strong> caudal nidopallium, caudal mesopallium <strong>an</strong>d lateral striatum. An interaction between<br />
“repetition” <strong>an</strong>d “familiarity” factors was found in two regions of <strong>the</strong> cerebellum. Post-hoc tests<br />
indicated that this interaction mainly resulted <strong>from</strong> a repetition enh<strong>an</strong>cement triggered by novel<br />
but not by familiar songs. As in hum<strong>an</strong>s, <strong>the</strong> neural effects of stimulus repetitions were thus<br />
observed in a large set of cerebral regions of zebra finches. In both species, most effects<br />
consisted in repetition suppressions of <strong>the</strong> neural activity while repetition enh<strong>an</strong>cements were<br />
only rarely observed. Additionally some effects were found to depend on <strong>the</strong> familiarity with <strong>the</strong><br />
stimulus whereas o<strong>the</strong>rs did not. A repetition enh<strong>an</strong>cement elicited only by novel songs was<br />
found, suggesting <strong>the</strong> <strong>for</strong>mation of new representations or new memory traces. This study thus<br />
reveals <strong>the</strong> presence of elaborate memory mech<strong>an</strong>isms in <strong>the</strong> songbird brain that are similar to<br />
those occurring in <strong>the</strong> hum<strong>an</strong> brain.<br />
Disclosures: C.C. Poirier , None; T. Boum<strong>an</strong>s, None; M. Verhoye, None; J. balthazart,<br />
None; A. V<strong>an</strong> Der Linden, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.1/GG123<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques
Title: LC/MS/MS <strong>an</strong>alysis of in vivo receptor occup<strong>an</strong>cy of <strong>the</strong> GABAA receptor in <strong>the</strong> rat brain<br />
Authors: *J. ZYSK, F. GHARAHDAGHI, A. ZACCO, M. DING, T. PENG, D. HEHMAN, D.<br />
SOTTUNG, V. CHHAJLANI, A. DUDLEY, D. MAIER;<br />
AstraZeneca, Wilmington, DE<br />
Abstract: In vivo receptor occup<strong>an</strong>cy (IVRO) is a methodology used to measure uptake,<br />
distribution <strong>an</strong>d specific binding of drugs or drug c<strong>an</strong>didates in specific target tissues or org<strong>an</strong>s.<br />
Much of <strong>the</strong> value of IVRO resides in its utility to assist <strong>the</strong> development of lig<strong>an</strong>ds <strong>for</strong> positron<br />
emission tomography (PET). St<strong>an</strong>dard IVRO procedures involve <strong>the</strong> administration of a<br />
radiolabeled tracer compound to a test <strong>an</strong>imal that has been treated with a blocking drug or<br />
control vehicle. A reduction in tracer counts, measured after sacrifice, solubilization <strong>an</strong>d<br />
radioactive counting of <strong>the</strong> selected target region of interest, is <strong>an</strong> indication of <strong>the</strong> binding of <strong>the</strong><br />
blocking compound or drug. Although sensitive, <strong>the</strong> radioactive method requires a bioavailable<br />
radiotracer, where <strong>the</strong> parent drug or drug metabolites are not easily measured <strong>an</strong>d where<br />
radioactive waste is generated. An alternative approach employs liquid chromatography/<br />
quadrapole mass spectroscopy (LC/MS/MS) <strong>for</strong> <strong>an</strong>alysis of tracer (nonradiolabeled) <strong>an</strong>d<br />
blocking compounds. The LC/MS/MS approach to IVRO has been previously demonstrated<br />
(Life Sciences 78 (2005) 340 -346). We have valided <strong>the</strong>se methods (with modification) <strong>for</strong> <strong>the</strong><br />
dopamine D2 receptor. In <strong>the</strong> present report, we have set up a plat<strong>for</strong>m <strong>for</strong> <strong>an</strong>alysis of <strong>the</strong><br />
GABAA receptor using flumazenil as <strong>the</strong> trace compound. This target provides us with <strong>the</strong><br />
opportunity to study a receptor of <strong>the</strong> major negative neurotr<strong>an</strong>smitter of <strong>the</strong> CNS. By testing<br />
diversely localized receptor systems, we were able to demonstrate <strong>the</strong> r<strong>an</strong>ge of this method <strong>for</strong><br />
IVRO. In our in vivo system (rat brain) we examined uptake <strong>an</strong>d binding of <strong>the</strong> unlabeled<br />
flumazenil tracer into <strong>the</strong> frontal cortex (FC), a region specific to this receptor subtype, <strong>an</strong>d<br />
compared <strong>the</strong> results with historic data which used radiolabeled [3H]-flumazenil tracer. Blocking<br />
of <strong>the</strong> nonradiolabeled tracer by receptor-selective compounds was compared with INVRO data<br />
using [ 3 H]-flumazenil. In <strong>the</strong> LC/MS/MS method, flumazenil at 0.062 nmole per <strong>an</strong>imal<br />
exhibited high uptake <strong>an</strong>d specific binding in <strong>the</strong> FC. The pons exhibited low (non-specific)<br />
binding, replicating our[ 3 H]-flumazenil findings. In <strong>the</strong> FC, flumazenil was displaced by<br />
pretreatment with diazepam ( 26-156 µmoles/kg, p.o.) or a potent GABAA selective compound<br />
(compound A) at ( 0.3-60 µmoles/kg, s.c.) with maximal receptor occup<strong>an</strong>cy values of<br />
approximately 84 <strong>an</strong>d 87%, respectively. The occup<strong>an</strong>cy values obtained using LC/MS/MS<br />
correlated with values obtained using <strong>the</strong> st<strong>an</strong>dard radio-labeled tracer method. These results<br />
demonstrate <strong>the</strong> utility of <strong>the</strong> LC/MS/MS plat<strong>for</strong>m as <strong>an</strong> alternative to <strong>the</strong> radio-labeled tracer<br />
approach to IVRO.<br />
Disclosures: J. Zysk, None; F. Gharahdaghi, None; A. Zacco, None; M. Ding, None; T.<br />
Peng, None; D. Hehm<strong>an</strong>, None; D. Sottung, None; V. Chhajl<strong>an</strong>i, None; A. Dudley, None; D.<br />
Maier, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.2/GG124<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: AASDAP<br />
CNPq<br />
FAPERN<br />
Title: Experience-dependent reactivation of <strong>the</strong> calcium signal tr<strong>an</strong>sduction pathway in <strong>the</strong> rat<br />
hippocampus during sleep<br />
Authors: *C. M. PEREIRA 1 , V. R. COTA 2 , S. SANTOS 1 , G. DIAS 1 , A. C. SOUZA 1 , S.<br />
RIBEIRO 1,3 , M. A. L. NICOLELIS 1,4 ;<br />
1 Edmond <strong>an</strong>d Lily Safra Intl. Inst. of Neurosci. of Natal, Natal, Brazil; 2 Federal Univ. of São<br />
João Del-Rei, São João Del-Rei, Brazil; 3 Federal Univ. of Rio Gr<strong>an</strong>de do Norte, Natal, Brazil;<br />
4 Duke Univ., Durham, NC<br />
Abstract: Sleep-dependent plastic ch<strong>an</strong>ges play a key role in <strong>the</strong> consolidation of newly<br />
acquired memories. Two distinct <strong>an</strong>d successive phases of sleep, slow wave sleep (SWS), <strong>an</strong>d<br />
rapid eye movement (REM) sleep c<strong>an</strong> be recognized in mammals. Both phases have been<br />
implicated in <strong>the</strong> sensorimotor processing of daytime events, but <strong>the</strong> molecular mech<strong>an</strong>isms<br />
involved remain poorly understood. Brain expression of <strong>the</strong> plasticity-associated immediateearly<br />
gene (IEG) zif-268 is upregulated during REM sleep in <strong>the</strong> cerebral cortex <strong>an</strong>d<br />
hippocampus of <strong>an</strong>imals exposed to rich sensorimotor experience in <strong>the</strong> preceding waking period<br />
(Learn Mem. 6; 500, 1999). Zif-268 integrates a major calcium signal tr<strong>an</strong>sduction pathway<br />
which includes Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) <strong>an</strong>d mitogen activated<br />
protein kinase (MAPK). CaMKII autophosphorylation of T286 is of special import<strong>an</strong>ce because<br />
it makes <strong>the</strong> enzyme active in <strong>the</strong> absence of Ca(2+), providing a biochemical memory that is<br />
critical <strong>for</strong> plasticity. MAPK, <strong>an</strong> integral component of cellular signaling during mitotic cell<br />
differentiation, has been implicated in hippocampal long-term potentiation (LTP) <strong>an</strong>d learning<br />
<strong>an</strong>d memory in behaving <strong>an</strong>imals.<br />
Our goal here is to investigate <strong>the</strong> phosphorylation levels of CaMKII <strong>an</strong>d MAPK during sleep in<br />
rats exposed to a new rich environment in <strong>the</strong> preceding waking period. Intracr<strong>an</strong>ial local field<br />
potentials (LFPs) recorded in <strong>the</strong> cortex <strong>an</strong>d hippocampus were used to characterize <strong>the</strong> wakesleep<br />
cycle (J. Neurosci. 24; 11137, 2004). The phosphorylation levels of CaMKII <strong>an</strong>d MAPK<br />
were assessed using specific <strong>an</strong>tibodies <strong>for</strong> western blots <strong>an</strong>d immunohistochemistry. Our<br />
preliminary data (WK n=3, SWS n=5 <strong>an</strong>d REM n=3)) indicate that <strong>the</strong> MAPK pathway was<br />
reactivated in <strong>the</strong> hippocampus after a few minutes of SWS. Interestingly, <strong>for</strong> reasons still<br />
unknown, MAPK phosphorylation decreased to WK level after a single episode of REM sleep.<br />
Our results also showed CaMKII reactivation during REM sleep in <strong>the</strong> hippocampus of rats
previously exposed to novel objects in <strong>the</strong> preceding WK period. Controls unexposed to novel<br />
experience did not show kinase reactivation during sleep. Our results support <strong>the</strong> notion that<br />
sleep harbors experience-dependent mech<strong>an</strong>isms of synaptic upscaling (Learn Mem. 6; 500,<br />
1999, J. Neurosci. 22; 10914, 2002, J. Neurochem. 95; 418, 2005, Science, 313; 1775, 2006,<br />
FINS 1; 43, 2007, Neuron 61; 454, <strong>2009</strong>).<br />
Disclosures: C.M. Pereira, None; V.R. Cota, None; S. S<strong>an</strong>tos, None; G. Dias, None; A.C.<br />
Souza, None; S. Ribeiro, None; M.A.L. Nicolelis, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.3/GG125<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: Aid <strong>for</strong> Scientific Research <strong>from</strong> <strong>the</strong> Ministry of Education, Culture, Sport, <strong>an</strong>d<br />
Technology of Jap<strong>an</strong> (Tokyo, Jap<strong>an</strong>) 20791065<br />
Title: A long-term effect of sciatic nerve block with slow releasing lidocaine in a rat model of<br />
postoperative pain<br />
Authors: *M. TOBE 1 , H. OBATA 2 , S. SAITO 2 ;<br />
1 Maebashi Gunma, Jap<strong>an</strong>; 2 Univ. Gunma, Maebashi Gunma, Jap<strong>an</strong><br />
Abstract: Background: Postoperative pain m<strong>an</strong>agement is import<strong>an</strong>t <strong>for</strong> preventing<br />
perioperative complications. In a recent meta-<strong>an</strong>alysis, however, about 40% of all surgical<br />
patients still experience moderate to severe acute postoperative pain. We made control released<br />
lidocaine <strong>an</strong>d use it <strong>for</strong> a sciatic nerve block in <strong>the</strong> rat model of postoperative pain.<br />
Methods: We made a novel slow releasing lidocaine sheet (SRLS) with polylactic-co-glycolic<br />
acid (PLGA). were used <strong>for</strong> all experiments. We made a hind paw incision using male Sprague-<br />
Dawley rats (postoperative pain model), <strong>an</strong>d SRLS, lidocaine itself, or PLGA only (control) was<br />
applied near <strong>the</strong> ipsilateral sciatic nerve just be<strong>for</strong>e <strong>the</strong> paw incision. The development of<br />
mech<strong>an</strong>ical hypersensitivity was assessed using von Frey filaments. We also examined c-fos<br />
expression in <strong>the</strong> spinal dorsal horn of segments L4-5 in each group.<br />
Results: We could prepare a SRLS (30%, w/w), which continuously release lidocaine <strong>for</strong> a week.<br />
In <strong>the</strong> behavioral studies, withdrawal threshold in <strong>the</strong> SRLS-treated group was higher th<strong>an</strong> that in<br />
<strong>the</strong> control group at all time point measured (2 h-7 days). In contrast, withdrawal threshold in <strong>the</strong><br />
lidocaine-treated group was higher th<strong>an</strong> that in <strong>the</strong> control group only at 2 h after paw incision.
The numbers of c-fos positive neurons in <strong>the</strong> SRLS-treated group were smaller th<strong>an</strong> that of<br />
control group at 2 h, 5 h, 48 h after paw incision. The numbers of c-fos positive neurons in <strong>the</strong><br />
SRLS-treated group was also smaller th<strong>an</strong> that of lidocaine-treated group at 5 h, 48 h after paw<br />
incision.<br />
Conclusions: Single treatment with this SRLS inhibited nociceptive behavior <strong>an</strong>d c-fos<br />
expression in <strong>the</strong> spinal cord dorsal horn <strong>for</strong> a week. slow releasing technique of local <strong>an</strong>es<strong>the</strong>tics<br />
might be a promising method <strong>for</strong> m<strong>an</strong>agement of postoperative pain.<br />
Disclosures: M. Tobe, None; H. Obata, None; S. Saito, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.4/GG126<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: Start-Up funds <strong>from</strong> <strong>the</strong> Department of Anatomy (E.W.D)<br />
Institute <strong>for</strong> Complex Matter postdoctoral fellowship (C.S)<br />
NSF gr<strong>an</strong>t NSEC DMR-0425880 (S.N.C, M.A.E, C.S.)<br />
Wisconsin Alumni Research Foundation (C.S., S.N.C.)<br />
Department of Energy gr<strong>an</strong>t (M.A.E., C.S.)<br />
Title: Controlling neuronal growth on gold surfaces by directed assembly of extracellular matrix<br />
proteins<br />
Authors: *E. W. DENT 1 , C. STAII 2 , C. VIESSELMANN 1 , J. BALLWEG 1 , Y. HUANG 3 , J. C.<br />
WILLIAMS 3 , S. N. COOPERSMITH 2 , M. A. ERIKSSON 2 ;<br />
1 Anat., 2 Physics, 3 Biomed. Engin., Univ. Wisconsin, Madison, WI<br />
Abstract: Studying how individual neuronal cells grow <strong>an</strong>d interact with each o<strong>the</strong>r is of<br />
fundamental import<strong>an</strong>ce <strong>for</strong> underst<strong>an</strong>ding <strong>the</strong> functions of <strong>the</strong> nervous system. However, <strong>the</strong><br />
mech<strong>an</strong>ism of axonal navigation to <strong>the</strong>ir target region <strong>an</strong>d <strong>the</strong>ir specific interactions with<br />
guid<strong>an</strong>ce factors such as membr<strong>an</strong>e-bound proteins, chemical <strong>an</strong>d temperature gradients,<br />
mech<strong>an</strong>ical guid<strong>an</strong>ce cues, etc. are not well understood. Here we describe a new approach <strong>for</strong>
controlling <strong>the</strong> adhesion, growth <strong>an</strong>d interconnectivity of cortical neurons on gold surfaces.<br />
Specifically, we use Atomic Force Microscopy (AFM) n<strong>an</strong>olithography to immobilize<br />
extracellular matrix proteins at well-defined locations on Au surfaces. These surfaceimmobilized<br />
proteins act as a) adhesion proteins <strong>for</strong> neuronal cells (i.e. well-defined locations<br />
where <strong>the</strong> cells “stick” to <strong>the</strong> surface), <strong>an</strong>d b) promoters/inhibitors <strong>for</strong> <strong>the</strong> growth of neurites. Our<br />
results show that protein patterns c<strong>an</strong> be used to confine neuronal cells <strong>an</strong>d to control <strong>the</strong>ir<br />
growth <strong>an</strong>d interconnectivity on gold surfaces. We also show that AFM n<strong>an</strong>olithography presents<br />
unique adv<strong>an</strong>tages <strong>for</strong> this type of work: 1) high degree of control over location <strong>an</strong>d shape of <strong>the</strong><br />
protein patterns, 2) <strong>the</strong> procedure is carried out in aqueous solutions (protein buffers), such that<br />
<strong>the</strong> proteins are very likely to retain <strong>the</strong>ir folding con<strong>for</strong>mation/bioactivity, <strong>an</strong>d 3) minimum<br />
protein feature size c<strong>an</strong> be reduced down to several tens of nm (typically 50nm).<br />
Disclosures: E.W. Dent, None; C. Staii, None; C. Viesselm<strong>an</strong>n, None; J. Ballweg, None; Y.<br />
Hu<strong>an</strong>g, None; J.C. Williams, None; S.N. Coopersmith, None; M.A. Eriksson, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.5/HH1<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: Volgenau School of In<strong>for</strong>mation Technology <strong>an</strong>d Engineering<br />
Title: Inflammatory reactions to carbon n<strong>an</strong>otube stimulating electrodes in acute impl<strong>an</strong>ts<br />
Authors: *N. PEIXOTO 1 , S. MINNIKANTI 1 , M. G. PEREIRA 2 , C. COSTA-NETO 2 ;<br />
1 2<br />
Electrical & Computer Eng, George Mason Univ., Fairfax, VA; Med. Sch., Univ. Sao Paulo,<br />
Ribeirao Preto, Brazil<br />
Abstract: We have designed multi walled carbon n<strong>an</strong>otube (MWCNT) mesh coatings <strong>for</strong><br />
stimulating <strong>an</strong>d recording electrodes. Electrodes were acutely impl<strong>an</strong>ted in deep brain structures.<br />
They were <strong>the</strong>n characterized when used <strong>for</strong> low frequency stimulation in deep brain structures.<br />
Questions focusing on <strong>the</strong> survival of electrode coatings after <strong>the</strong> impl<strong>an</strong>t were addressed<br />
electrochemically through galv<strong>an</strong>ostatic <strong>an</strong>alyses prior to <strong>an</strong>d after impl<strong>an</strong>tation. Questions<br />
referring to <strong>the</strong> tissue response to <strong>the</strong> damage caused by <strong>the</strong> mech<strong>an</strong>ical insertion of <strong>the</strong> electrode<br />
<strong>an</strong>d by <strong>the</strong> electric field application were tackled using molecular biology techniques.<br />
Cyclic voltammetry spectra showed increased charge delivery capacity of MWCNT coatings in<br />
vivo when compared to in vitro results. However, differently <strong>from</strong> previous results reported in
<strong>the</strong> literature, <strong>the</strong> decrease in imped<strong>an</strong>ce across <strong>the</strong> r<strong>an</strong>ge tested (0.1Hz to 100kHz) is not directly<br />
proportional to <strong>the</strong> increase in charge delivered. These results point to a non-st<strong>an</strong>dard interface<br />
between <strong>the</strong> n<strong>an</strong>ofeatures coating <strong>the</strong> electrode <strong>an</strong>d <strong>the</strong> biological tissue being stimulated.<br />
Qu<strong>an</strong>titative <strong>an</strong>alysis of mRNA levels showed upregulation expression of IL-1beta, <strong>an</strong><br />
inflammatory cytokine. O<strong>the</strong>r indicators of central nervous system innate <strong>an</strong>d adaptive immune<br />
reactions were also tested (e.g. TLR-2) but found not to be signific<strong>an</strong>tly altered. We hypo<strong>the</strong>size<br />
that <strong>the</strong> short term damage to <strong>the</strong> CNS <strong>an</strong>d early responses measurable <strong>from</strong> <strong>the</strong> tissue being<br />
stimulated will yield insights into <strong>the</strong> mech<strong>an</strong>isms of biofouling <strong>an</strong>d reaction to electrode<br />
damage, both in recording <strong>an</strong>d stimulating scenarios.<br />
Disclosures: N. Peixoto, None; S. Minnik<strong>an</strong>ti, None; M.G. Pereira, None; C. Costa-Neto,<br />
None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.6/HH2<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: NIH Gr<strong>an</strong>t AI15429<br />
Title: Mapping neuropeptide expression by mass spectrometry in single dissected identified<br />
neurons <strong>from</strong> <strong>the</strong> dorsal g<strong>an</strong>glion of <strong>the</strong> nematode Ascaris suum<br />
Authors: J. L. JARECKI 1 , K. M. ANDERSON 2 , M. M. VESTLING 3 , *A. O. STRETTON 4 ;<br />
1 Neurosci. Training Program, 2 Dept of Zoology, 3 Dept of Chem., 4 Univ. Wisconsin, Madison,<br />
WI<br />
Abstract: Detailed knowledge of cellular peptide expression is vital to <strong>the</strong> underst<strong>an</strong>ding of <strong>the</strong><br />
neural control of behavior. The nematode Ascaris suum is <strong>an</strong> excellent model system to achieve<br />
such knowledge because of <strong>the</strong> small number of neurons in its nervous system. In addition, this<br />
nervous system is remarkably similar to that of <strong>the</strong> well-studied nematode Caenorhabditis<br />
eleg<strong>an</strong>s: <strong>the</strong> morphology of each class of neuron is highly conserved, despite dramatic<br />
differences in size. This allows interesting cross-species comparisons. In A. suum, isolation of<br />
single neuronal cell bodies has been difficult because of <strong>the</strong> close association between<br />
hypodermis <strong>an</strong>d neurons. However, recent adv<strong>an</strong>ces in our dissection technique have enabled<br />
dissections of single identified neurons, <strong>an</strong>d subsequent direct <strong>an</strong>alysis of <strong>the</strong>ir peptide content<br />
by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS). Previously this approach had been limited to direct tissue <strong>an</strong>alysis of whole g<strong>an</strong>glia in A.<br />
suum (Yew et al., 2005). Direct MS <strong>an</strong>alysis of individual cells has several adv<strong>an</strong>tages: spatial<br />
localization is maintained, <strong>the</strong>re is minimal sample loss, <strong>the</strong> possibility of chemical degradation<br />
is lessened, <strong>an</strong>d <strong>the</strong> reduction in <strong>the</strong> complexity of <strong>the</strong> peptides present decreases <strong>the</strong> effects of<br />
ion suppression. Here we present a peptide expression map of <strong>the</strong> simplest g<strong>an</strong>glion, <strong>the</strong> dorsal<br />
g<strong>an</strong>glion (DG). The DG contains two morphologically distinct cells, named ALA <strong>an</strong>d RID.<br />
These identifiable cells have yielded different, <strong>an</strong>d highly reproducible, peptide profiles. The<br />
profiles match known results <strong>from</strong> in situ hybridization with probes complementary to known<br />
tr<strong>an</strong>scripts that encode <strong>the</strong> endogenous peptides (J. N<strong>an</strong>da, PhD <strong>the</strong>sis, University of Wisconsin-<br />
Madison). De novo sequencing has also been per<strong>for</strong>med on peptides <strong>from</strong> <strong>the</strong>se cells through <strong>the</strong><br />
use of TOF/TOF. Confirmation of de novo sequencing by on-target chemical modification of<br />
peptides is also possible at <strong>the</strong> single-cell level, as it was in whole g<strong>an</strong>glia. Several novel<br />
peptides have been sequenced. The sequences have been confirmed by BLAST searches of a 1x<br />
coverage, non-assembled genome of A. suum (courtesy M. Mitreva, Washington University, St<br />
Louis MO), <strong>an</strong>d by gene cloning. These techniques have been extended to identified cells in<br />
o<strong>the</strong>r g<strong>an</strong>glia. The limited number of neurons in this org<strong>an</strong>ism makes it feasible to map peptide<br />
expression at <strong>the</strong> cellular level throughout <strong>the</strong> worm.<br />
Disclosures: J.L. Jarecki, None; K.M. Anderson, None; M.M. Vestling, None; A.O.<br />
Stretton, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.8/HH3<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: NIDA DA022506<br />
NIDA DA06511<br />
NIDA DA020087-03<br />
NIDA DA07287-11<br />
Title: Syn<strong>the</strong>sis <strong>an</strong>d functional characterization of novel serotonin (5-HT) 2 receptor (5-HT2R)<br />
<strong>an</strong>tagonists <strong>an</strong>d agonists: Towards development of a bivalent lig<strong>an</strong>d
Authors: *P. K. SEITZ 1 , M. J. SHASHACK 1 , L. SUN 1 , C. S. WATSON 2 , S. R.<br />
GILBERTSON 3 , K. A. CUNNINGHAM 1 ;<br />
1 Ctr. <strong>for</strong> Addiction Research, Dept. of Pharmacol., 2 Ctr. <strong>for</strong> Addiction Research, Biochem. &<br />
Mol. Biol., Univ. Texas Med. Brch, Galveston, TX; 3 Chem., Univ. of Houston, Houston, TX<br />
Abstract: The 5-HT2AR <strong>an</strong>d 5-HT2CR play import<strong>an</strong>t oppositional roles in modulating cocaineinduced<br />
behaviors: at doses that do not modify basal behavior, 5-HT2AR <strong>an</strong>tagonists <strong>an</strong>d 5-<br />
HT2CR agonists block while 5-HT2AR agonists <strong>an</strong>d 5-HT2CR <strong>an</strong>tagonists enh<strong>an</strong>ce <strong>the</strong><br />
hypermotive <strong>an</strong>d discriminative stimulus effects of cocaine. Preclinical findings strongly suggest<br />
that a 5-HT2AR <strong>an</strong>tagonist, a 5-HT2CR agonist or a combination of <strong>the</strong>se actions might be useful<br />
to reduce craving <strong>an</strong>d enh<strong>an</strong>ce abstinence in cocaine addicts. Syn<strong>the</strong>tic bivalent lig<strong>an</strong>ds have<br />
been used in several o<strong>the</strong>r receptor systems, with increased potency <strong>an</strong>d fewer side effects<br />
relative to application of single lig<strong>an</strong>ds in combination. Thus, a bivalent 5-HT2AR <strong>an</strong>tagonist/5-<br />
HT2CR agonist may have utility to suppress cocaine-seeking <strong>an</strong>d/or -taking, with <strong>the</strong> potential to<br />
serve as <strong>an</strong> abstinence enh<strong>an</strong>cer in hum<strong>an</strong>s. We hypo<strong>the</strong>size that covalent linkage of M100907<br />
(selective 5-HT2AR <strong>an</strong>tagonist) <strong>an</strong>d WAY 470 (selective 5-HT2CR agonist) will result in a series<br />
of more selective <strong>an</strong>d efficacious bifunctional compounds. Since <strong>the</strong>re is limited structure<br />
activity data <strong>for</strong> M100907 <strong>an</strong>d WAY 470, <strong>the</strong> possiblitly exists that attachment to a te<strong>the</strong>r may<br />
affect binding of <strong>the</strong> lig<strong>an</strong>ds to <strong>the</strong>ir receptors. There<strong>for</strong>e, we have syn<strong>the</strong>sized monomers<br />
possessing simple modifications at various potential te<strong>the</strong>r sites to test <strong>for</strong> functional activity:<br />
four derivatives of M100907 <strong>an</strong>d three derivatives of WAY 470. We have tested <strong>the</strong> activity of<br />
M100907 <strong>an</strong>d its derivatives to inhibit <strong>the</strong> release of Cai ++ induced by 1µM 1-[2,5-dimethoxy-4iodophenyl]-2-aminoprop<strong>an</strong>e<br />
(DOI) in CHO cells tr<strong>an</strong>sfected with 5-HT2AR. Cells were loaded<br />
with <strong>the</strong> Cai ++ -sensitive dye Calcium4, agonist was added <strong>an</strong>d peak fluorescence was measured<br />
(485 nm excitation, 525 nm emission) using a FlexStation 3 (Molecular Devices). Stimulation of<br />
DOI-induced Cai ++ release was inhibited by M100907 (ED50=30 nM) <strong>an</strong>d three of its derivatives<br />
with ED50 values between 90 nM <strong>an</strong>d 9 µM; one compound did not reduce <strong>the</strong> Cai ++ signal to<br />
baseline levels, even at 10 -4 M. None of <strong>the</strong> putative 5-HT2AR <strong>an</strong>tagonists (up to 10 -5 M)<br />
exhibited agonist activity at <strong>the</strong> 5-HT2AR. We are in <strong>the</strong> process of testing activity of <strong>the</strong> WAY<br />
470 derivatives to stimulate Cai ++ in CHO cells tr<strong>an</strong>sfected with 5-HT2CR.<br />
Disclosures: P.K. Seitz, None; M.J. Shashack, None; L. Sun, None; C.S. Watson, None; S.R.<br />
Gilbertson, None; K.A. Cunningham, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.9/HH4
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: NIH<br />
Title: Chromosome con<strong>for</strong>mation capture reveals higher order chromatin at <strong>the</strong> n-methyl daspartate<br />
receptor gene Grin2b<br />
Authors: *R. BHARADWAJ 1,2 , Y. JIANG 1,2 , S. AKBARIAN 1,2 ;<br />
1 UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL, Worcester, MA; 2 Psychiatry,<br />
BRUDNICK NEUROPSYCHIATRIC RESEARCH INSTITUTE, Worcester, MA<br />
Abstract: Chromosome con<strong>for</strong>mation capture (3C) is <strong>an</strong> effective tool to identify physical<br />
interactions between pairs of genomic loci both in cis (within <strong>the</strong> same chromosome) <strong>an</strong>d in tr<strong>an</strong>s<br />
(between different chromosomes). 3C entails <strong>for</strong>maldehyde cross-linking of DNA fragments in<br />
close proximity followed by restriction enzyme digestion <strong>an</strong>d intra-molecular ligation resulting<br />
in a library of ligated products that c<strong>an</strong> be <strong>an</strong>alyzed <strong>for</strong> <strong>the</strong> identification of specific genetic<br />
interactions. We identified a specific binding site <strong>for</strong> Setdb1 (SET domain, bifurcated 1), a<br />
histone 3 lysine 9 specific methyltr<strong>an</strong>sferase, within <strong>the</strong> 127 KB intron 3 of <strong>the</strong> ionotropic<br />
glutamate receptor gene Grin2b, which encodes <strong>the</strong> NMDA receptor 2B subunit. To fur<strong>the</strong>r<br />
investigate this, we per<strong>for</strong>med 3C <strong>an</strong>alysis to capture <strong>the</strong> potential genetic interactions within a<br />
70 KB fragment surrounding <strong>the</strong> Grin2b tr<strong>an</strong>scription start site using a rat neuronal progenitor<br />
cell culture system. A single loop was identified, te<strong>the</strong>ring <strong>the</strong> 760 bp Setdb1-target sequence to<br />
a portion of <strong>the</strong> same intronic region around 12 KB downstream of <strong>the</strong> tr<strong>an</strong>scription start site,<br />
which was also highly enriched with KAP1 (KRAB-associated protein 1), a known<br />
tr<strong>an</strong>scriptional co-repressor that physically interacts with Setdb1. Moreover, both ends of <strong>the</strong><br />
loop harbored putative binding elements <strong>for</strong> KRAB zinc-finger tr<strong>an</strong>scription factors. Similar loop<br />
<strong>for</strong>mations were also observed <strong>for</strong> <strong>the</strong> GRIN2B locus in a hum<strong>an</strong> glioblastoma cell line U87-<br />
MG, as well as in nuclei isolated <strong>from</strong> <strong>the</strong> hum<strong>an</strong> cerebral cortex. Taken toge<strong>the</strong>r, by using 3C,<br />
we revealed <strong>the</strong> presence of a highly conserved chromatin loop <strong>for</strong>mation at Grin2b locus across<br />
different species, which could be involved in tr<strong>an</strong>scriptional regulation. GRIN2B is associated<br />
with genetic risk <strong>for</strong> bipolar disorder in selected populations, <strong>an</strong>d NMDA receptor <strong>an</strong>tagonists<br />
including Grin2b selective drugs were recently used with success in subjects with treatment<br />
resist<strong>an</strong>t depression. There<strong>for</strong>e, our findings could be import<strong>an</strong>t <strong>for</strong> <strong>the</strong> neurobiology <strong>an</strong>d<br />
pharmacology of depression.<br />
Acknowledgements: This work was supported by gr<strong>an</strong>ts <strong>from</strong> <strong>the</strong> NIH.<br />
We would like to th<strong>an</strong>k Dr.Job Dekker <strong>an</strong>d Dr.V<strong>an</strong>Berkum Nynke <strong>for</strong> <strong>the</strong>ir technical expertise<br />
with chromosome con<strong>for</strong>mation capture.<br />
We would like to th<strong>an</strong>k Ca<strong>the</strong>rine Whittle <strong>for</strong> technical assist<strong>an</strong>ce with cell culture work.<br />
Disclosures: R. Bharadwaj, None; Y. Ji<strong>an</strong>g, None; S. Akbari<strong>an</strong>, None.<br />
Poster<br />
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.10/HH5<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: Brains On-Line US LLC<br />
Title: Relev<strong>an</strong>ce of in vivo recovery when studying re-uptake inhibitors using intracerebral<br />
microdialysis<br />
Authors: *M. G. VAN DER HART 1,2 , M. E. JONGSMA 1,2 , L. TECOTT 1 , T. CREMERS 3 , B.<br />
H. C. WESTERINK 4 ;<br />
1 UCSF, S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 2 Brains On-Line US LLC, South S<strong>an</strong> Fr<strong>an</strong>cisco, CA; 3 Brains On-<br />
Line B.V., Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds; 4 Pharm., Univ. of Groningen, Groningen, Ne<strong>the</strong>rl<strong>an</strong>ds<br />
Abstract: Pharmacological studies typically rely on <strong>the</strong> assumption that <strong>the</strong> expression of data as<br />
percentages of basal levels obviates <strong>the</strong> need to account <strong>for</strong> recovery. However, it is possible that<br />
pharmacological interventions could alter <strong>the</strong> in vivo recovery of neurotr<strong>an</strong>smitters, confounding<br />
data interpretation.<br />
Using st<strong>an</strong>dard non-qu<strong>an</strong>titative microdialysis methods, most studies report reuptake inhibitors<br />
to increase levels of monoamines, leading to <strong>the</strong> conclusion that <strong>the</strong>se compounds actually<br />
increase extracellular levels of monoamines. However, <strong>the</strong>se studies have not accounted <strong>for</strong><br />
possible influences of reuptake inhibitors on in vivo recovery (Cos<strong>for</strong>d et al 1996).<br />
Recently, we developed a new microdialysis technique that facilitates qu<strong>an</strong>titative microdialysis<br />
by employing a modified ultraslow method using <strong>the</strong> ‘metaqu<strong>an</strong>t’ technique (Cremers et al,<br />
<strong>2009</strong>).<br />
To investigate <strong>the</strong> extent to which monoamine uptake inhibitors alter in vivo recovery, we<br />
compared qu<strong>an</strong>titative microdialysis with conventional microdialysis <strong>an</strong>alyses of <strong>the</strong><br />
pharmacological effects of monoamine uptake inhibitors.<br />
It appears that serotonin recovery is dependent on pharmacological intervention, which c<strong>an</strong> result<br />
in <strong>an</strong> overestimation of <strong>the</strong> pharmacological effects of <strong>the</strong> compounds tested. Norepinephrine<br />
recovery does not seem to alter by pharmacological intervention. Dopamine recovery appears to<br />
be dependent of <strong>the</strong> pharmacological agent used. These data suggest that data obtained <strong>from</strong><br />
conventional microdialysis studies should be interpreted, with great care.<br />
Disclosures: M.G. v<strong>an</strong> der Hart, Brains On-Line, A. Employment (full or part-time); M.E.<br />
Jongsma, Brains On-Line, A. Employment (full or part-time); L. Tecott, None; T. Cremers,<br />
Brains On-Line, A. Employment (full or part-time); B.H.C. Westerink, Brains On-Line, F.<br />
Consult<strong>an</strong>t/Advisory Board.<br />
Poster
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.11/HH6<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Title: Micropatterning of neuroactive molecules: Strategies <strong>for</strong> guiding neuronal growth<br />
Authors: *E. MARCONI 1,3 , M. MESSA 3 , A. MACCIONE 3 , S. DANTE 3 , S. CASAGRANDE 2 ,<br />
P. BALDELLI 2,3 , L. BERDONDINI 3 , F. BENFENATI 3,2 ;<br />
1 2 3<br />
DIST, Dimes, Univ. of Genova, Genova, Italy; Neurosci. <strong>an</strong>d Brain Technologies, Itali<strong>an</strong> Inst.<br />
of Technol., Genova, Italy<br />
Abstract: Microcontact printing (µCP) recently became a diffuse technique to create<br />
geometrically-defined patterns of molecules on target substrates <strong>an</strong>d was previously used <strong>for</strong><br />
growing patterned neuronal networks. In this work we generated architecture-controlled neuronal<br />
networks on microelectrode arrays in order to i) locate neurons on electrode sites <strong>for</strong> improving<br />
neuroelectrode couplings <strong>an</strong>d to ii) define network connections <strong>for</strong> electrophysiological studies<br />
in combination with patch-clamp. Results were achieved using two methodologies to print<br />
patterns of neuroactive molecules aligned to arrays of microelectrodes. The first consisted in<br />
using poly-dimethylsilox<strong>an</strong>e (PDMS) to create negative patterns to be printed on MEAs by using<br />
a custom alignment setup. The second consisted in using a n<strong>an</strong>odrop printing system (Henderson<br />
E., N<strong>an</strong>omed 2007) <strong>for</strong> deposition of <strong>the</strong> aligned pattern. Since a variety of extracellular <strong>an</strong>d celladhesion<br />
molecules have repulsive or adhesive effects, various combinations of native or<br />
recombin<strong>an</strong>t molecules were evaluated. In particular, we realized adhesive patterns by<br />
depositions of Poly-D-Lysine (PDL) <strong>an</strong>d extracellular matrix molecules (ECM) <strong>an</strong>d repulsive<br />
layers based on agarose. After optimization of <strong>the</strong> culturing conditions, neurons were able to<br />
grow on patterned substrates. Interestingly, <strong>the</strong> accurate control of very low deposition volumes<br />
enabled by <strong>the</strong> n<strong>an</strong>odropping method highlighted minimal requirements of PDL to sustain<br />
neuronal growth. Moreover, we observed a well-<strong>for</strong>med network with a better morphology by<br />
using ECM as adhesive molecule as compared to PDL layers. Neurite outgrowth, <strong>for</strong>mation <strong>an</strong>d<br />
function of synaptic contacts of hippocampal neurons grown on <strong>the</strong>se patterns were evaluated by<br />
using immunofluorescence <strong>an</strong>d patch-clamp techniques. Although neurons were restrained <strong>an</strong>d<br />
plated at very low densities, synapses <strong>for</strong>med normally <strong>an</strong>d <strong>the</strong>ir physiological properties were<br />
comparable with those observed in r<strong>an</strong>dom neuronal cultures.<br />
Disclosures: E. Marconi, None; M. Messa, None; A. Maccione, None; S. D<strong>an</strong>te, None; S.<br />
Casagr<strong>an</strong>de, None; P. Baldelli, None; L. Berdondini, None; F. Benfenati, None.<br />
Poster
585. Molecular Methods to Monitor <strong>an</strong>d M<strong>an</strong>ipulate Neuronal Properties<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 585.12/HH7<br />
Topic: G.01. Molecular, Biochemical, <strong>an</strong>d Genetic Techniques<br />
Support: CJD Foundation<br />
NIHR01NS062787<br />
NIA AG-14359<br />
CDC UR8/CCU515004<br />
Charles S. Britton Fund<br />
Title: Characterization of caveolin-1 <strong>from</strong> hum<strong>an</strong> brains<br />
Authors: *X. XIAO, J. YUAN, H. BI, M. SHIMOJI, W.-Q. ZOU;<br />
Dept. of Pathology & Natl. Prion Dis. Pathology Surveill<strong>an</strong>ce Ctr., Case Western Reserve Univ.,<br />
Clevel<strong>an</strong>d, OH<br />
Abstract: Caveolin-1 is a major component protein of <strong>the</strong> caveolae that are 50-100 nm<br />
nonclathrin-coated, flask-shaped plasma membr<strong>an</strong>e microdomains in most mammali<strong>an</strong> cells. It<br />
functions as a scaffolding protein to org<strong>an</strong>ize <strong>an</strong>d concentrate signaling molecules within<br />
caveolae, which may be associated with its unique physicochemical properties including<br />
oligomerization, acquisition of detergent insolubility, <strong>an</strong>d association with cholesterol. Evidence<br />
indicates that caveolin-1 is <strong>an</strong> unusual protein that c<strong>an</strong> be both <strong>an</strong> integral membr<strong>an</strong>e protein <strong>an</strong>d<br />
soluble in multiple cellular compartments. Here we demonstrate that caveolin-1 is detected in all<br />
brain areas examined <strong>an</strong>d recovered in both detergent-soluble <strong>an</strong>d -insoluble fractions.<br />
Surprisingly, <strong>the</strong> molecules in <strong>the</strong> two different fractions share a similar molecular size r<strong>an</strong>ging<br />
<strong>from</strong> 200 to 2,000 kDa, indicated by gel filtration. Fur<strong>the</strong>rmore, both soluble <strong>an</strong>d insoluble<br />
caveolin-1 molecules generate a C-terminal core fragment over protease digestion, by removing<br />
~36 amino acids <strong>from</strong> <strong>the</strong> N-terminus of <strong>the</strong> protein. Although <strong>the</strong>y recognize caveolin-1 <strong>from</strong><br />
A431 cell lysate, two <strong>an</strong>tibodies against <strong>the</strong> C-terminus of caveolin-1 failed to detect <strong>the</strong> protein<br />
by Western blotting, suggesting that <strong>the</strong> epitopes in <strong>the</strong> brain caveolin-1 are concealed.<br />
Disclosures: X. Xiao, None; J. Yu<strong>an</strong>, None; H. Bi, None; M. Shimoji, None; W. Zou, None.<br />
Poster
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.1/HH8<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NIH P01 NS032636<br />
NIH R01 NS036265<br />
AHA 0615509Z<br />
Title: An AAV2 vector with axon-targeted reporter expression after injection in mouse motor<br />
cortex<br />
Authors: *R. E. SMITH 1 , C. M. LAPASH DANIELS 2 , J. R. FISHER 1 , M. P. GOLDBERG 2 ;<br />
1 2<br />
Div. of Biol. <strong>an</strong>d Biomed. Sci., Washington Univ. In St. Louis, Saint Louis, MO; Hope Ctr. <strong>for</strong><br />
Neurolog. Disorders, Saint Louis, MO<br />
Abstract: Viral vectors expressing fluorescent proteins are useful tools <strong>for</strong> labeling CNS<br />
projections in vivo. However, reporter proteins distributed in <strong>the</strong> cytosol may take weeks to<br />
accumulate at distal ends of axons. Previous studies have shown that insertion of <strong>the</strong> 20-amino<br />
acid N-terminus sequence of <strong>the</strong> neuronal growth associated protein GAP-43 targets reporter<br />
proteins to neuronal membr<strong>an</strong>es. We compared axon labeling after viral expression of cytosolic<br />
fluorescent proteins, GAP-43 targeted fluorescent proteins, <strong>an</strong>d full-length GAP-43. AAV2 was<br />
created carrying ei<strong>the</strong>r a green fluorescent protein after <strong>an</strong> IRES (10 8 TU/ml), a cy<strong>an</strong> fluorescent<br />
protein with <strong>the</strong> 20-amino acid sequence <strong>from</strong> GAP-43 after <strong>an</strong> IRES (10 12 TU/ml), or a fulllength<br />
GAP-43 with a C-terminal FLAG-tag followed by <strong>an</strong> IRES <strong>an</strong>d a green fluorescent<br />
protein (10 8 TU/ml). Two or four microliter injections of virus into mouse motor cortex were<br />
per<strong>for</strong>med following stereotaxic coordinates. Two weeks after injection of <strong>the</strong> GAP-43 targeted<br />
CFP virus, we observed labeling of axons in <strong>the</strong> corpus callosum <strong>an</strong>d ipsilateral striatum, with<br />
minimal expression seen in cell bodies. By four weeks, we observed fluorescently labeled<br />
corticospinal axons to <strong>the</strong> level of cervical spinal cord. The distribution of GAP-43-targeted CFP<br />
was similar to a full-length GAP-43. FLAG-positive (GAP-43 over-expressing) processes were<br />
also targeted primarily to axons <strong>an</strong>d were found in ipsilateral cortex <strong>an</strong>d striatum by 1 week after<br />
injection <strong>an</strong>d numerous axons were found throughout <strong>the</strong> brain <strong>an</strong>d cervical spinal cord by 5<br />
weeks post-injection. By comparison, cytosolic GFP expression without <strong>the</strong> GAP-43 targeting<br />
sequence resulted in labeling of cell bodies throughout motor cortex layers, with prominent<br />
apical dendrites by 2 weeks post-injection <strong>an</strong>d limited labeling of axons descending into <strong>the</strong><br />
ipsilateral striatum by 4 weeks. Altoge<strong>the</strong>r, GAP-43-targeted CFP is a valuable tool <strong>for</strong> future<br />
experiments to identify <strong>the</strong> distal axons of cortical neurons tr<strong>an</strong>sduced by virus.
Disclosures: R.E. Smith, None; C.M. Lapash D<strong>an</strong>iels, None; J.R. Fisher, None; M.P.<br />
Goldberg, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.2/HH9<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: Falk Medical Research Trust<br />
Title: Ex vivo diffusion tensor imaging indices in rat cervical spinal cord gray matter following<br />
distal contusion spinal cord injury<br />
Authors: *R. E. MOTTACKEL 1 , B. M. ELLINGSON 2,3 , S. N. KURPAD 3 , B. D. SCHMIT 1 ;<br />
1 2 3<br />
Biomed. Engin., Marquette Univ., Milwaukee, WI; Dept. of Radiology, Dept. of Neurosurg.,<br />
Med. Col. of Wisconsin, Milwaukee, WI<br />
Abstract: The purpose of this study was to delineate <strong>the</strong> diffusion tensor imaging (DTI)<br />
parameters across <strong>the</strong> cervical spinal cord gray matter (GM) in a distal rat contusion spinal cord<br />
injury (SCI) model at T8. DTI data were obtained <strong>from</strong> ex vivo rat spinal cords <strong>an</strong>d registered to<br />
corresponding histological slices in samples <strong>from</strong> <strong>the</strong> acute through chronic stages of SCI<br />
including uninjured control, 2 weeks post injury, 15 weeks post injury <strong>an</strong>d 25 weeks post injury<br />
groups. At <strong>the</strong> predesignated time post-injury, <strong>an</strong>imals were perfused with 10% <strong>for</strong>malin <strong>an</strong>d<br />
spinal cords were excised, post-fixed <strong>for</strong> 7 days, placed in NMR tubes <strong>an</strong>d imaged using a<br />
Bruker 9.4T small <strong>an</strong>imal MR system. A single T2-weighted image (b=0) <strong>an</strong>d 6 ex vivo<br />
diffusion weighted images (DWIs) at b=500 were acquired at TE/TR = 31.6 ms /14 s, slice<br />
thickness of 2 mm with 1 mm interslice gap, FOV = 5.12 cm, NEX = 1, <strong>an</strong>d <strong>an</strong> acquisition<br />
matrix of 512 x 512 using a st<strong>an</strong>dard PG-SE sequence. After imaging, samples were dehydrated,<br />
blocked in paraffin, sliced axially <strong>an</strong>d stained with eriochrome cy<strong>an</strong>ine R stain <strong>an</strong>d H&E<br />
counter-stain. Histology images were captured at 4x using a Nikon Eclipse E600 research<br />
microscope. The histology images were <strong>the</strong>n segmented into white matter (WM) <strong>an</strong>d GM using a<br />
custom cluster <strong>an</strong>alysis. Using whole cord templates, <strong>the</strong> DTI indices <strong>for</strong> each <strong>an</strong>imal were <strong>the</strong>n<br />
registered to <strong>the</strong> corresponding histology images. The WM <strong>an</strong>d <strong>the</strong> GM histological templates<br />
were <strong>the</strong>n used to map DTI indices, including fractional <strong>an</strong>isotropy (FA), longitudinal apparent<br />
diffusion coefficient (lADC), tr<strong>an</strong>sverse apparent diffusion coefficient (tADC), volume ratio<br />
(VR) <strong>an</strong>d Me<strong>an</strong> Diffusivity (MD) across <strong>the</strong> GM. The average values <strong>for</strong> each index was also<br />
calculated in <strong>the</strong> gray matter. It was observed that <strong>the</strong> average FA value decreased at 2 weeks
post injury as compared to that of <strong>the</strong> control group. This decrease in FA was followed by <strong>an</strong><br />
increase at 15 weeks <strong>an</strong>d 25 weeks post injury as compared to control FA value. The average<br />
tADC value increased at 2 weeks post injury as compared to that of <strong>the</strong> control group. This<br />
increase in tADC was followed by a drop to values similar to control values at 15 <strong>an</strong>d 25 weeks<br />
post injury. Fur<strong>the</strong>rmore, it appeared that <strong>the</strong> increase in tADC values at 2 weeks post injury<br />
corresponded to <strong>an</strong> overall increase in <strong>the</strong> tADC intensity localized to <strong>the</strong> ventral horn in <strong>the</strong><br />
diffusion map. These results suggest that DTI c<strong>an</strong> be used as <strong>an</strong> indicator of spatial as well as<br />
temporal ch<strong>an</strong>ges in cervical GM with distal SCI.<br />
Disclosures: R.E. Mottackel, None; B.M. Ellingson, None; S.N. Kurpad, None; B.D. Schmit,<br />
None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.3/HH10<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: US Air<strong>for</strong>ce FA4869-07-1-4072<br />
NBS-ERC R11-2000-075-01001-0<br />
Brain Research Center M103KV010024-08K2201-02410<br />
Title: Optical spectrum measurement of neural activity in brain tissues <strong>an</strong>d its <strong>the</strong>oretical origin<br />
Authors: *J. LEE, S. KIM;<br />
Gw<strong>an</strong>ak Gu, Seoul Natl. Univ., Seoul, Republic of Korea<br />
Abstract: This study is aimed at measuring a tr<strong>an</strong>sient ch<strong>an</strong>ge in optical properties of brain bulk<br />
tissue during neural activation <strong>an</strong>d at providing a qu<strong>an</strong>titative elucidation on its origin. We<br />
monitored <strong>the</strong> tr<strong>an</strong>smission <strong>an</strong>d reflection spectra (<strong>for</strong> wavelengths <strong>from</strong> 800 to 1300 nm) of rat<br />
cortical slices while electrically evoking <strong>the</strong> neurons <strong>an</strong>d simult<strong>an</strong>eously recording <strong>the</strong> neural<br />
activity. We developed a high-speed confocal near-infrared (NIR) spectrometer. Results show<br />
that, both <strong>the</strong> tr<strong>an</strong>smitt<strong>an</strong>ce <strong>an</strong>d reflect<strong>an</strong>ce of <strong>the</strong> tissue increased at <strong>the</strong> onset of <strong>the</strong> local field<br />
potential (LFP), <strong>an</strong>d relaxed slowly relative to <strong>the</strong> LFP. The LFP <strong>an</strong>d concurrent optical ch<strong>an</strong>ge<br />
disappeared when <strong>the</strong> tetrodotoxin was applied. Although <strong>the</strong> optical signal was verified to<br />
originate <strong>from</strong> neural activity, <strong>the</strong> time course was different <strong>from</strong> <strong>the</strong> LFP. The optical signal
showed a monophasic ch<strong>an</strong>ge <strong>an</strong>d slow relaxation, which implies that <strong>the</strong> optical ch<strong>an</strong>ge is not<br />
directly attributed to <strong>the</strong> membr<strong>an</strong>e potential variation caused by neural excitation. To elucidate<br />
this difference qu<strong>an</strong>titatively, we developed a new neuron model with variable cellular volume<br />
<strong>an</strong>d intracellular ion concentration, which were originally const<strong>an</strong>t qu<strong>an</strong>tities in <strong>the</strong> conventional<br />
Hodgkin-Huxley model. Using physical <strong>the</strong>ories such as <strong>the</strong> continuity equation of mass<br />
conservation <strong>an</strong>d <strong>the</strong> Fick's 1st law of diffusion, we built a set of coupled nonlinear differential<br />
equations. Results show that, a cellular volume tr<strong>an</strong>siently increases during excitation, <strong>an</strong>d its<br />
time course is similar to that of <strong>the</strong> optical signal. Also, <strong>the</strong> magnitude of <strong>the</strong> tr<strong>an</strong>sient cellular<br />
volume ch<strong>an</strong>ge (tCVC) agrees with that of <strong>the</strong> optical signal in <strong>the</strong> order of magnitude (10 -4 -10 -<br />
5 ). In addition to this modeling, we per<strong>for</strong>med a finite-difference time-domain (FDTD)<br />
simulation to investigate how <strong>the</strong> tCVC produces a ch<strong>an</strong>ge in <strong>the</strong> optical properties of <strong>the</strong> cell.<br />
According to our FDTD results, both <strong>the</strong> tr<strong>an</strong>smitt<strong>an</strong>ce <strong>an</strong>d reflect<strong>an</strong>ce increase as <strong>the</strong> cell<br />
diameter increases. These agreements support that <strong>the</strong> observed optical signal might domin<strong>an</strong>tly<br />
originate <strong>from</strong> <strong>the</strong> tCVC.<br />
Disclosures: J. Lee, None; S. Kim, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.4/HH11<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: "Research fund of <strong>the</strong> University of Pisa 2007"<br />
"Istituto Tosc<strong>an</strong>o Tumori - Gr<strong>an</strong>t 2007"<br />
Title: Identification of new molecular diagnosis tools targeting <strong>the</strong> tr<strong>an</strong>slocator protein (18kDa)<br />
Authors: E. DA POZZO 1 , S. TALIANI 2 , S. BENDINELLI 1 , M. BELLANDI 2 , L. ROSSI 3 , V.<br />
GREMIGNI 3 , F. DA SETTIMO 2 , *C. MARTINI 1 ;<br />
1 2 3<br />
Psychiatry, Neurobiology, Pharmacol. & Biotech., Medicinal Chem., Hum<strong>an</strong> Morphology <strong>an</strong>d<br />
Applied Biol., Univ. of Pisa, Pisa, Italy<br />
Abstract: The 18 kDa Tr<strong>an</strong>slocator Protein (TSPO), previously known as Peripheral<br />
Benzodiazepine Receptor, is a mitochondrial protein, which is widely <strong>an</strong>d constitutively<br />
expressed in glial cells <strong>an</strong>d in <strong>the</strong> peripheral tissues. TSPO is associated with a plethora of<br />
biological functions such as cell proliferation, cholesterol tr<strong>an</strong>sport, steroidogenesis <strong>an</strong>d
apoptosis (1). In <strong>the</strong> last decade m<strong>an</strong>y studies have reported that both TSPO basal density <strong>an</strong>d its<br />
cellular localization are altered in several diseases as c<strong>an</strong>cer, psychiatric <strong>an</strong>d neurodegenerative<br />
disorders. In addition, a tight correlation between TSPO expression level <strong>an</strong>d <strong>the</strong> grade of c<strong>an</strong>cer<br />
malign<strong>an</strong>cy has been reported in glioma cells (2). For such reasons, <strong>the</strong> evaluation of TSPO<br />
expression level <strong>an</strong>d subcellular distribution may represent a promising diagnostic marker to<br />
investigate <strong>the</strong> state <strong>an</strong>d progression of certain diseases. The TSPO has become <strong>an</strong> interesting<br />
target in Molecular Imaging Techniques, prompting <strong>the</strong> development of specific labelled lig<strong>an</strong>ds<br />
as powerful tools to image <strong>an</strong>d measure <strong>the</strong> protein expression level in both living cells <strong>an</strong>d<br />
isolated tissues.<br />
We have previously described a series of N,N-dialkyl-(2-phenylindol-3-yl)glyoxylamides as<br />
potent <strong>an</strong>d selective TSPO lig<strong>an</strong>ds (Ki values in <strong>the</strong> n<strong>an</strong>omolar-subn<strong>an</strong>omolar r<strong>an</strong>ge) (3). These<br />
derivatives have represented <strong>the</strong> basis <strong>for</strong> <strong>the</strong> design of novel TSPO fluorescent probes, which<br />
display n<strong>an</strong>omolar affinity, <strong>an</strong>d <strong>for</strong> new TSPO irreversible fluorescent lig<strong>an</strong>ds, bearing a<br />
chemoreactive group able to irreversibly <strong>an</strong>d covalently bind <strong>the</strong> receptor protein.<br />
The structure-activity relationship data indicated that <strong>the</strong> position 5 of <strong>the</strong> indole nucleus was<br />
suitable to support <strong>the</strong> chemo-reactive group without affecting <strong>the</strong> affinity <strong>an</strong>d <strong>the</strong> selectivity of<br />
<strong>the</strong> parent compound <strong>for</strong> TSPO. Among <strong>the</strong> possible chemo-reactive moieties, <strong>the</strong> electrophilic<br />
isothiocy<strong>an</strong>ato group (CNS) has been introduced. The affinity <strong>for</strong> TSPO of <strong>the</strong> newly<br />
syn<strong>the</strong>sized irreversible lig<strong>an</strong>ds were tested by proper 3H-Ro5-4864 radiobinding assays,<br />
conducted on rat tissue homogenates: <strong>the</strong> new compounds have shown high affinities <strong>for</strong> <strong>the</strong><br />
TSPO. Moreover, <strong>the</strong> fluorescent probes were also examined by using <strong>the</strong> fluorescent<br />
microscopy to explore <strong>the</strong> TSPO subcellular distribution in rat <strong>an</strong>d hum<strong>an</strong> glioma cells. Such<br />
results shed light on new fluorescent molecules targeting TSPO as promising imaging diagnostic<br />
tools <strong>for</strong> c<strong>an</strong>cer <strong>an</strong>d neurodegenerative diseases.<br />
References: (1) V. Papadopoulos et al, Trends Pharmacol. Sci. 2006, 27, 402. (2) M. Kassiou et<br />
al, Brain Res. Rev. 2005, 48, 207. (3) Da Settimo et al, J. Med. Chem. 2008, 51, 5798.<br />
Disclosures: E. Da Pozzo, None; S. Tali<strong>an</strong>i, None; S. Bendinelli, None; M. Bell<strong>an</strong>di, None; L.<br />
Rossi, None; V. Gremigni, None; F. Da Settimo, None; C. Martini, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.5/HH12<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: Max Pl<strong>an</strong>ck <strong>Society</strong>
SFB636/A4<br />
Title: Genetic activity-induced tagging of cells<br />
Authors: *W. TANG 1 , J.-M. WEISLOGEL 2 , R. SPRENGEL 3 , P. H. SEEBURG 3 , H.<br />
BADING 2 , M. T. HASAN 3 ;<br />
1 MPIMF, Heidelberg, Germ<strong>an</strong>y; 2 Interdisciplinary Ctr. <strong>for</strong> Neurosciences, Univ. of Heidelberg,<br />
Heidelberg, Germ<strong>an</strong>y; 3 MPIMF Heidelberg, Heidelberg, Germ<strong>an</strong>y<br />
Abstract: For in<strong>for</strong>mation processing in <strong>the</strong> brain, different cellular <strong>an</strong>d molecular signaling<br />
pathways lead to rise in intracellular [Ca2+]. For example, backpropagating action-potentials<br />
when coinciding with EPSPs cause supralinear Ca2+ influx. This results in <strong>the</strong> generation of<br />
long-term potentiation, a c<strong>an</strong>didate cellular model of synaptic plasticity <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e also<br />
import<strong>an</strong>t <strong>for</strong> learning <strong>an</strong>d memory. Increases in somatic [Ca2+] activate tr<strong>an</strong>scription of<br />
immediate-early genes such as c-fos which in turn activate late-response gene-products.<br />
There<strong>for</strong>e, activity-induced ch<strong>an</strong>ges in c-fos expression in neurons are used <strong>for</strong> mapping active<br />
brain regions at a cellular resolution.<br />
We have engineered a novel genetic latch which is based on a c-fos promoter in a recombin<strong>an</strong>t<br />
adeno-associated virus (AAV) <strong>for</strong> Genetic Activity-Induced Tagging of cells (or GAIT method)<br />
potentially in <strong>an</strong>y mammali<strong>an</strong> system. In <strong>the</strong> GAIT method, AAV-delivered c-fos promoter<br />
drives <strong>the</strong> reverse tetracycline tr<strong>an</strong>sactivator (rtTA) in cells allowing <strong>the</strong> beginning of <strong>the</strong><br />
‘recording period’ to be exogenously controlled, rapidly, by <strong>an</strong> inducer, doxycycline (Dox) <strong>an</strong>d<br />
c<strong>an</strong> be returned back to pre-induced base-line state by removing Dox. In <strong>the</strong> presence of Dox,<br />
only a tr<strong>an</strong>sient activation of c-fos leads to perm<strong>an</strong>ent rtTA expression via autoregulated rtTA<br />
expression. The “GAIT” method should allows perm<strong>an</strong>ent labeling of cells that have participated<br />
in activity during a specific task. Different reporter genes c<strong>an</strong> be put under <strong>the</strong> control of <strong>the</strong><br />
rtTA gene <strong>an</strong>d c<strong>an</strong> be read out, <strong>for</strong> example, ei<strong>the</strong>r by two-photon fluorescent imaging of living<br />
brain slices or non-invasively, in vivo. It is possible to use fluorescent indicator proteins as<br />
reporters <strong>an</strong>d thus measure, <strong>for</strong> example, Ca2+ activity in c-fos activated cells. It should also be<br />
possible to introduce in c-fos activated cells ch<strong>an</strong>nelrhodopsin <strong>an</strong>d halorhodopsin <strong>for</strong> lightinduced<br />
rapid m<strong>an</strong>ipulation of neuronal activity.<br />
We have functionally characterized <strong>the</strong> GAIT method in cultured neurons <strong>an</strong>d rat hippocampal<br />
org<strong>an</strong>otypic slices <strong>an</strong>d have found that it c<strong>an</strong> label activated neurons. Preliminary results with<br />
single-whisker experience showed that, at large, neurons within a single cortical column were<br />
labeled. We will next investigate whe<strong>the</strong>r single-whisker experience may result in <strong>an</strong> increase in<br />
synaptic strength or release probability at unitary neuronal connections within or between<br />
cortical layers.<br />
The GAIT method <strong>for</strong> perm<strong>an</strong>ently labeling of experience-activated neurons should be widely<br />
applicable in different biological systems <strong>an</strong>d <strong>an</strong>imal species.<br />
Disclosures: W. T<strong>an</strong>g, None; J. Weislogel, None; R. Sprengel, None; P.H. Seeburg, None; H.<br />
Bading, None; M.T. Has<strong>an</strong>, None.<br />
Poster
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.6/HH13<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: DBT<br />
DAE/SRC<br />
NCBS<br />
Title: Large scale functional mapping of hippocampal CA3-CA1 connections<br />
Authors: *R. MADHAVAN, M. N. MODI, U. S. BHALLA;<br />
Natl. Ctr. Biol Sci., B<strong>an</strong>galore, India<br />
Abstract: Brain mapping techniques have come a long way since <strong>the</strong> work of Ramón y Cajal.<br />
Never<strong>the</strong>less, we are far <strong>from</strong> reading out network-wide synaptic weights <strong>an</strong>d large-scale<br />
connection maps. Ch<strong>an</strong>nelrhodpsin-2 <strong>an</strong>d glutamate uncaging methods provide a functional<br />
readout, but currently only <strong>for</strong> single target neurons. In this study, we experimentally evaluate a<br />
new hybrid method to build large-scale synapse-resolution functional connection maps by<br />
combining electrical stimulation <strong>an</strong>d optical readouts. This method constructs <strong>the</strong> connectivity<br />
matrix <strong>for</strong> <strong>the</strong> CA3-CA1 network in <strong>the</strong> slice by detecting potential synaptic contacts <strong>an</strong>d<br />
estimating <strong>the</strong>ir synaptic weights. Input electrical stimulation was provided to <strong>the</strong> Schaffer<br />
collaterals through <strong>an</strong> electrode array, <strong>an</strong>d responses of individual CA1 neurons loaded with<br />
calcium indicator were monitored optically in a rat hippocampal slice preparation. The basic<br />
experimental protocol was to deliver a strong baseline stimulus to a block of Schaffer collateral<br />
axons to bring <strong>the</strong> CA1 cells above firing threshold. Riding on <strong>the</strong> baseline stimulus, minimal<br />
stimulation was delivered to <strong>an</strong>o<strong>the</strong>r probe electrode. We reduced stimulus current <strong>an</strong>d<br />
monitored EPSCs <strong>an</strong>d response statistics to ensure that this minimal stimulus activated a single<br />
axon projecting onto <strong>the</strong> patched neuron. By comparing <strong>the</strong> statistics of responses to <strong>the</strong><br />
‘baseline’ <strong>an</strong>d ‘baseline+probe’ stimuli, <strong>the</strong> presence <strong>an</strong>d synaptic weight of potential synapses<br />
were estimated <strong>from</strong> optical signals (Bhalla, PLOS Comp. Biol, 2008). Whole-cell patch<br />
recordings <strong>from</strong> loaded neurons were used to validate <strong>the</strong>se optical predictions, <strong>an</strong>d to directly<br />
measure synaptic weights. In order to scale up <strong>the</strong> number of potential synapses detected with<br />
this approach, a large array of custom-built stimulation electrodes <strong>an</strong>d 2-photon imaging were<br />
used <strong>for</strong> large-scale network characterization. We <strong>an</strong>ticipate that such detailed wiring diagrams<br />
that specify individual synaptic connection weights between distinct input axons <strong>an</strong>d output<br />
neurons will provide valuable insights into hippocampal connectivity <strong>an</strong>d ch<strong>an</strong>ges induced by<br />
learning.
Disclosures: R. Madhav<strong>an</strong>, None; M.N. Modi, None; U.S. Bhalla, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.7/HH14<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NIH Gr<strong>an</strong>t DK20572<br />
NIH Gr<strong>an</strong>t NS38849<br />
NIH Gr<strong>an</strong>t DK 076160<br />
Title: Enh<strong>an</strong>ced visualization of BrdU <strong>an</strong>d EdU incorporation into newly syn<strong>the</strong>sized<br />
mitochondrial DNA of neuroblastoma cells <strong>an</strong>d primary dorsal root g<strong>an</strong>glion neurons<br />
Authors: *S. I. LENTZ 1 , J. L. EDWARDS 4 , C. BACKUS 2 , L. L. MCLEAN 2 , K. M. HAINES 3 ,<br />
E. L. FELDMAN 2 ;<br />
1 Intrnl. Med., 2 Neurol., 3 Michig<strong>an</strong> Res. Community, Univ. of Michig<strong>an</strong>, Ann Arbor, MI; 4 Ctr. <strong>for</strong><br />
Adv<strong>an</strong>ced Res. in Biotech., Univ. of Maryl<strong>an</strong>d Biotech. Inst., Rockville, MD<br />
Abstract: Mitochondria (Mt) are key regulators of cellular energy <strong>an</strong>d a large number of studies<br />
are focused on <strong>the</strong> regulation of mitochondrial dynamics <strong>an</strong>d biogenesis in healthy <strong>an</strong>d diseased<br />
conditions. We hypo<strong>the</strong>size that mitochondrial biogenesis is altered by hyperglycemia <strong>an</strong>d could<br />
contribute to <strong>the</strong> pathogenesis of diabetic neuropathy. Mt continually undergo fission <strong>an</strong>d fusion<br />
processes regulated by specialized proteins. Mutations in Mt fusion proteins are implicated in<br />
hereditary Charcot-Marie-Tooth sensory type 2A <strong>an</strong>d domin<strong>an</strong>t optic neuropathies suggesting<br />
that <strong>an</strong> imbal<strong>an</strong>ce in Mt networks favoring Mt fission underlies <strong>the</strong> development of neuropathy.<br />
We previously developed a sensitive assay to visualize newly syn<strong>the</strong>sized mtDNA with BrdU.<br />
We demonstrated that cultured embryonic dorsal root g<strong>an</strong>glion (DRG) neurons treated with high<br />
glucose had increased numbers of newly syn<strong>the</strong>sized mtDNA that were primarily in <strong>the</strong> soma<br />
<strong>an</strong>d present along <strong>the</strong> neurites. We exp<strong>an</strong>ded <strong>the</strong>se studies by developing a technique to amplify<br />
<strong>the</strong> incorporation of <strong>the</strong> recently introduced thymidine <strong>an</strong>alog, EdU, into newly syn<strong>the</strong>sized<br />
mtDNA. The milder labeling procedure <strong>for</strong> EdU allows <strong>for</strong> more comprehensive results by<br />
gaining <strong>the</strong> ability to compare its incorporation with o<strong>the</strong>r intracellular markers. Employing this<br />
technique, we visualized <strong>an</strong>d qu<strong>an</strong>tified <strong>the</strong> incorporation of EdU into newly syn<strong>the</strong>sized<br />
mtDNA in individual cells, <strong>an</strong>d import<strong>an</strong>tly, in specific subcellular compartments of neurons,
such as axons. EdU signal was present in mtDNA (arrows below) of embryonic (A) <strong>an</strong>d adult<br />
DRG neurons identified by a neuronal marker (B, TuJ1) or a subtype specific marker (C, trkA)<br />
after 24h incubation with EdU. In addition, utilization of both BrdU <strong>an</strong>d EdU permitted <strong>the</strong><br />
opportunity to per<strong>for</strong>m sequential pulse-chase paradigms to follow <strong>the</strong> intracellular localization<br />
of mtDNA replication. The ability to qu<strong>an</strong>tify mitochondrial biogenesis at <strong>the</strong> subcellular level<br />
will provide investigators with tools <strong>for</strong> elucidating mech<strong>an</strong>isms that underly <strong>the</strong> pathogenesis of<br />
a variety of neurological disorders.<br />
Disclosures: S.I. Lentz, None; J.L. Edwards, None; C. Backus, None; L.L. McLe<strong>an</strong>,<br />
None; K.M. Haines, None; E.L. Feldm<strong>an</strong>, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.8/HH15<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: National Parkinson's Foundation<br />
Title: Reproducibility <strong>an</strong>d validity of dopamine tr<strong>an</strong>sporter PET imaging lig<strong>an</strong>d 18 F-FECNT in<br />
MPTP-treated monkeys<br />
Authors: Y. SMITH 1 , *G. M. JEYARAJ 2 , J. VOTAW 3 , L. HOWELL 2 , T. WICHMANN 2 ;<br />
1 Neurol., 3 Radiology, 2 Emory Univ., Atl<strong>an</strong>ta, GA<br />
Abstract: The positron emission tomography (PET) tracer 2β-carbomethoxy-3β-(4chlorophenyl)-8-(2-[18F]-fluoroethyl)-nortrop<strong>an</strong>e<br />
(18F-FECNT) has a much higher affinity <strong>for</strong><br />
<strong>the</strong> dopamine tr<strong>an</strong>sporter (DAT) th<strong>an</strong> <strong>the</strong> serotonin tr<strong>an</strong>sporter, yields <strong>the</strong> highest peak striatumto-cerebellum<br />
ratios <strong>an</strong>d has among <strong>the</strong> most favorable kinetics of 18F-radiolabeled DAT lig<strong>an</strong>ds<br />
(Goodm<strong>an</strong> et al., 2000, Nucl. Med. Biol. 27:1; Davis et al., 2003, J. Nucl. Med. 44: 855). The use<br />
of this lig<strong>an</strong>d as a surrogate biomarker to assess <strong>the</strong> integrity of <strong>the</strong> dopamine system in<br />
parkinsonism is highly dependent on its longitudinal reproducibility <strong>an</strong>d pathological validation,<br />
which have not yet been adequately characterized.<br />
In <strong>the</strong> present study, we tested <strong>the</strong> reproducibility <strong>an</strong>d validity of <strong>the</strong> 18 F-FECNT PET radiotracer<br />
in MPTP-treated parkinsoni<strong>an</strong> monkeys <strong>an</strong>d controls. Three Rhesus monkeys received weekly<br />
injections of MPTP (0.2-0.5 mg/kg) <strong>for</strong> 21 weeks. This protocol induces a slowly progressive<br />
<strong>for</strong>m of parkinsonism. We carried out 18 F-FECNT PET at baseline (twice; ten weeks apart) <strong>an</strong>d<br />
at week 21. Postmortem stereological cell counts of dopaminergic neurons in <strong>the</strong> ventral<br />
midbrain, <strong>an</strong>d intensity measurements of DAT <strong>an</strong>d tyrosine hydroxylase (TH) immunoreactivity<br />
in <strong>the</strong> striatum were per<strong>for</strong>med <strong>an</strong>d correlated with PET data. Three additional (untreated)<br />
monkeys were used as controls to generate cell counts, <strong>an</strong>d DAT <strong>an</strong>d TH immunoreactivity<br />
measurements. For fur<strong>the</strong>r <strong>an</strong>alysis, <strong>the</strong> nigral cell counts, <strong>an</strong>d DAT <strong>an</strong>d TH measurements in <strong>the</strong><br />
MPTP-treated <strong>an</strong>imals were expressed as a percentage of <strong>the</strong> respective values generated in <strong>the</strong><br />
control <strong>an</strong>imals. The correlation <strong>an</strong>d coefficient of vari<strong>an</strong>ce between 18 F-FECNT test-retest<br />
binding potentials were 0.9926 (R 2 ) <strong>an</strong>d 2.65%, respectively. The 18 F-FECNT binding potential<br />
of <strong>the</strong> subst<strong>an</strong>tia nigra, putamen, caudate nucleus <strong>an</strong>d accumbens tightly correlated with<br />
postmortem stereological cell counts of nigral dopaminergic neurons (R 2 = 0.9092), <strong>an</strong>d striatal<br />
DAT (R 2 = 0.8505) or TH (R 2 = 0.8938) immunoreactivity intensity measurements. We conclude<br />
that 18 F-FECNT is a reliable PET imaging lig<strong>an</strong>d to visualize <strong>an</strong>d qu<strong>an</strong>tify ch<strong>an</strong>ges in dopamine<br />
cell counts, <strong>an</strong>d striatal DAT <strong>an</strong>d TH levels in <strong>the</strong> nigrostriatal system in Parkinson’s disease.<br />
Disclosures: Y. Smith, None; G.M. Jeyaraj, None; J. Votaw, None; L. Howell, None; T.<br />
Wichm<strong>an</strong>n, None.<br />
Poster
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.9/HH16<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NSH<br />
PDS<br />
Title: Antigen retrieval improves <strong>the</strong> immunoreactivity of cells <strong>an</strong>d receptor sites in <strong>the</strong> basal<br />
g<strong>an</strong>glia: Studies with paraffin embedded rat <strong>an</strong>d hum<strong>an</strong> brain sections<br />
Authors: *R. T. VONTELL 1 , K. SEGOVIA 2 , D. DEXTER 1 , K. GOLDRING 1 , J.<br />
SALAMONE 2 ;<br />
1 UK Parkinson's Dis. Tissue B<strong>an</strong>k, Imperial Col. London, London, United Kingdom;<br />
2 Psychology <strong>an</strong>d Behavorial Neurosci., Univ. of Connecticut, Storrs, CT<br />
Abstract: M<strong>an</strong>y features of <strong>the</strong> neural circuitry of <strong>the</strong> basal g<strong>an</strong>glia in <strong>the</strong> rat are thought to be<br />
very similar to those of hum<strong>an</strong>s. One particular component of <strong>the</strong> basal g<strong>an</strong>glia, <strong>the</strong><br />
caudate/putamen (also known as neostriatum or “striatum”), contains a very large number of<br />
GABAergic medium-sized spiny neurons. Characterization of <strong>the</strong> subpopulations of <strong>the</strong>se<br />
medium spiny neurons is usually done by <strong>the</strong> use of immunohistochemistry,<br />
immunofluorescence <strong>an</strong>d in situ hybridization techniques. The established immunohistochemical<br />
techniques c<strong>an</strong> be more effective if additional steps are taken to amplify <strong>an</strong>tigen sensitivity <strong>an</strong>d<br />
decrease background staining. Antigen retrieval methods are used in various areas of histology to<br />
increase <strong>an</strong>tigen sensitivity, so that immunohistochemical methods may prove to be more<br />
effective <strong>for</strong> visualizing cells <strong>an</strong>d receptor sites. However, most of <strong>the</strong> published<br />
immunocytochemical studies on striatal neurons do not incorporate <strong>an</strong>tigen retrieval methods.<br />
The aim of <strong>the</strong> present studies was to determine if <strong>the</strong> use of <strong>an</strong>tigen retrieval techniques could<br />
improve <strong>the</strong> immunocytochemical characterization of <strong>the</strong> rat <strong>an</strong>d hum<strong>an</strong> striatum. Our laboratory<br />
uses paraffin-embedded whole sections of basal g<strong>an</strong>glia <strong>from</strong> hum<strong>an</strong>s <strong>an</strong>d rat brains. Utilizing<br />
st<strong>an</strong>dard immunoperoxidise techniques, we compared <strong>the</strong> immunoreactivity of cells in tissue<br />
sections with <strong>an</strong>d with out <strong>the</strong> application of steamed <strong>an</strong>tigen retrieval (pH 6.0) using <strong>an</strong>tityrosine<br />
hydroxylase, <strong>an</strong>ti-enkephalin, <strong>an</strong>ti-subst<strong>an</strong>ce P, <strong>an</strong>ti-adenosine A2A receptor, <strong>an</strong>ti-D1<br />
receptor, <strong>an</strong>ti-D2 receptor <strong>an</strong>d <strong>an</strong>ti-calbindin. Steam <strong>an</strong>tigen retrieval did not have <strong>an</strong>y<br />
detrimental affects on <strong>the</strong> general cellular morphology, <strong>an</strong>d appeared to produce <strong>an</strong> amplification<br />
of <strong>an</strong>tigen sensitivity with a reduction of background staining, thus enh<strong>an</strong>cing <strong>the</strong> detection <strong>an</strong>d<br />
visualization of different cell types <strong>an</strong>d receptors. These studies indicate that steam <strong>an</strong>tigen<br />
retrieval methods could be useful <strong>for</strong> enh<strong>an</strong>cing immunocytochemical detection of<br />
subpopulations of neurons <strong>an</strong>d <strong>the</strong>ir receptors in <strong>the</strong> hum<strong>an</strong> <strong>an</strong>d rat striatum.
Disclosures: R.T. Vontell, None; K. Segovia, None; D. Dexter, None; K. Goldring, None; J.<br />
Salamone, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.10/HH17<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NIH gr<strong>an</strong>t 5K25AG029415-03<br />
NIH gr<strong>an</strong>t 5R01AG008487-18<br />
E.P. Richardson Fellowship, MGH<br />
Title: Characterization of a novel RNA aptamer probe <strong>for</strong> imaging amyloid plaques <strong>an</strong>d<br />
oligomeric Aβ<br />
Authors: *C. T. FARRAR 1 , C. M. WILLIAM 2 , B. T. HYMAN 3 ;<br />
1 2 3<br />
Martinos Ctr. <strong>for</strong> Biomed. Imaging, Pathology, Neurol., Massachusetts Gen. Hopsital,<br />
Charlestown, MA<br />
Abstract: Introduction: Given <strong>the</strong> increasing evidence <strong>for</strong> a direct role of Aβ oligomers in<br />
Alzheimer’s Disease (AD) neuro-degeneration, <strong>the</strong>re is a great need <strong>for</strong> new in vivo imaging<br />
agents capable of detecting not only mature amyloid plaques but also Aβ oligomers. A novel<br />
RNA aptamer, β55, has recently been shown to bind syn<strong>the</strong>tic Aβ fibrils in vitro. 1 The β55<br />
aptamer was screened <strong>for</strong> binding to immobilized syn<strong>the</strong>tic Aβ1-40 monomer <strong>an</strong>d <strong>the</strong>re<strong>for</strong>e may<br />
also be a good probe <strong>for</strong> oligomeric Aβ. RNA aptamers are ideally suited as Aβ imaging probes<br />
due to <strong>the</strong>ir [1] high affinities (comparable to or better th<strong>an</strong> those of <strong>an</strong>tibodies), [2] small size,<br />
which improves <strong>the</strong>ir biodistribution <strong>an</strong>d may allow <strong>the</strong>m to cross <strong>the</strong> blood-brain barrier (BBB),<br />
[3] ease of chemical modification, <strong>an</strong>d [4] non-immunogenic nature.<br />
Results:<br />
Histology of hum<strong>an</strong> AD brain tissue: Frozen-section brain tissue <strong>from</strong> AD subjects was stained<br />
with ei<strong>the</strong>r biotinylated β55 aptamer or its reverse complement, β55rc, which is used as a control<br />
probe. Probes were visualized by reacting with avidin binding complex followed by tyramide<br />
signal amplification. The β55 RNA probe was shown to bind senile plaques in <strong>the</strong> cortex. In<br />
contrast, no specific binding was observed <strong>for</strong> <strong>the</strong> β55rc control probe. M<strong>an</strong>y of <strong>the</strong> stained<br />
plaques had a halo like appear<strong>an</strong>ce, similar to that observed with <strong>an</strong>tibody probes of oligomeric
Aβ. Tissue sections co-stained with Thioflavin-S indicate that β55 co-localizes with Thioflavin-<br />
S.<br />
Western blot <strong>an</strong>alysis of Aβ fractions: Frozen-section brain tissue <strong>from</strong> <strong>an</strong> AD subject was<br />
homogenized <strong>an</strong>d protein fractions were extracted following successive treatment with 10 mM<br />
Tris-HCl, 2% Triton-X100, <strong>an</strong>d 0.5% SDS. A western blot of <strong>the</strong> different protein fractions was<br />
stained with both 6E10 <strong>an</strong>tibody <strong>an</strong>d biotinylated β55. Probes were visualized by secondary<br />
staining with AlexaFluor-700 streptavidin <strong>an</strong>d AlexaFluor-800 secondary <strong>an</strong>tibody <strong>an</strong>d imaged<br />
on a LI-COR infrared imaging system. β55 co-localizes with m<strong>an</strong>y of <strong>the</strong> same b<strong>an</strong>ds as<br />
observed with 6E10, with molecular weights r<strong>an</strong>ging <strong>from</strong> 8-100 kDa. The observation of low<br />
molecular weight b<strong>an</strong>ds suggests that β55 does bind Aβ oligomers.<br />
Conclusions: β55 binds senile plaques in frozen-section AD brain tissue. The halo-like plaque<br />
staining <strong>an</strong>d staining of low-molecular weight b<strong>an</strong>ds in <strong>the</strong> western blot suggests that β55 binds<br />
oligomeric <strong>for</strong>ms of Aβ. In vivo studies are underway to examine <strong>the</strong> ability of β55 to cross <strong>the</strong><br />
BBB in mouse AD models <strong>an</strong>d bind Aβ in vivo.<br />
References:<br />
1. Ylera F, Lurz R, Erdm<strong>an</strong>n VA, Furste JP. Selection of RNA aptamers to <strong>the</strong> Alzheimer's<br />
disease amyloid peptide. Biochem Biophys Res Commun. 2002;290(5):1583-1588.<br />
Disclosures: C.T. Farrar, None; C.M. William, None; B.T. Hym<strong>an</strong>, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.11/HH18<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NIH Gr<strong>an</strong>t AA016748<br />
NIH Gr<strong>an</strong>t AA014106<br />
Title: Magnetic reson<strong>an</strong>ce spectroscopic assessment of eth<strong>an</strong>ol’s metabolic effects in vervet<br />
monkey brain<br />
Authors: *J. B. DAUNAIS 1 , N. C. BUCHHEIMER 2 , E. J. BURNETT 3 , A. T. DAVENPORT 3 ,<br />
V. M. MAXEY 3 , Q. K. TELESFORD 4 , R. A. KRAFT 4 , D. P. FRIEDMAN 3 ;<br />
1 Dept Physiol & Pharmacol, 2 Dept Neurol., 3 Dept Physiol. <strong>an</strong>d Pharmacol., 4 Dept Biomed.<br />
Engin., Wake Forest Univ. Sch. of Med., Winston-Salem, NC
Abstract: Alcohol consumption is known to influence magnetic reson<strong>an</strong>ce (MR) signal of brain<br />
metabolites but it is difficult to get a clear picture of alcohol’s metabolic effects due to various<br />
conditions under which studies are conducted in hum<strong>an</strong>s. Much of what is known derives <strong>from</strong><br />
older clinical populations that have been abusing <strong>for</strong> m<strong>an</strong>y decades. Age is also a factor because<br />
<strong>the</strong> aging process alters N-acetylaspartate (NAA), creatine (Cr) <strong>an</strong>d choline. Fur<strong>the</strong>r<br />
complicating <strong>the</strong> picture is <strong>the</strong> impact of <strong>the</strong> current alcohol state since metabolite levels are<br />
reported to ch<strong>an</strong>ge differentially in response to acute or chronic exposure as well as during<br />
abstinence/detoxification. High levels of cortisol also impact metabolite levels. Hypercortisolism<br />
results in decreased Cho/Cr ratios without impacting NAA levels (Khiat et al., 1999). Oral<br />
cortisol decreases metabolite ratios (Michaelis et al., 2000, 2001) while psychosocial stress<br />
increases Ins/Cr <strong>an</strong>d NAA/Cr (Michaelis et al., 2000). Nonhum<strong>an</strong> primates provide unique<br />
research models to study alcohol use disorders in part because <strong>the</strong>y c<strong>an</strong> be studied in <strong>the</strong> eth<strong>an</strong>olnaïve<br />
state <strong>an</strong>d <strong>the</strong>n longitudinally after various lengths of eth<strong>an</strong>ol exposure. We conducted<br />
magnetic reson<strong>an</strong>ce spectroscopy (MRS) in a group of ten young (3.5-4.0 yr) eth<strong>an</strong>ol naïve<br />
vervet monkeys selected based on <strong>the</strong>ir cortisol response to novelty [high cortisol/high reactivity<br />
(n=5); low cortisol/low reactivity (n=5) to determine if metabolite levels ch<strong>an</strong>ged as a result of<br />
cortisol classification. Single voxel MRS per<strong>for</strong>med on a 3 Tesla GE Signa sc<strong>an</strong>ner using a<br />
dedicated RF coil (Litzcage, Doty Scientific, Columbia, SC) did not detect <strong>an</strong>y signific<strong>an</strong>t<br />
differences in <strong>the</strong> major metabolites in 1 x 1 x 1 cm voxels placed in <strong>the</strong> frontal cortex NAA<br />
(p
Support: AEG: Natl. Defense Science & Engineering Graduate Fellowship<br />
MLS: NJ Commission on Spinal Cord Research<br />
SS-HW: W.M. Keck Foundation, NIH R01 NS045193, NJ Gov. Council on Autism<br />
LWE: NIH gr<strong>an</strong>t R01 NS60699<br />
Title: Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing<br />
pseudorabies virus<br />
Authors: A. E. GRANSTEDT, *M. L. SZPARA, B. KUHN, S. S.-H. WANG, L. W.<br />
ENQUIST;<br />
Mol. Biology, Neurosci. Inst., Princeton Univ., Princeton, NJ<br />
Abstract: The study of coordinated activity in neuronal circuits would be facilitated by a method<br />
to report activity <strong>an</strong>d connectivity in <strong>the</strong> same preparation. Here we present <strong>the</strong> first use of<br />
pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a<br />
fluorescent calcium indicator protein <strong>an</strong>d monitor neuronal activity in vivo. Using <strong>the</strong> strong<br />
CMV promoter, we expressed G-CaMP2, a fluorescent calcium indicator protein that is stable at<br />
physiological pH <strong>an</strong>d mammali<strong>an</strong> temperature (Tallini et al. PNAS 2006), in <strong>the</strong> genome of <strong>the</strong><br />
classical PRV-Bartha tracing strain (Ekstr<strong>an</strong>d et al. Trends Mol Med 2008). In dissociated mouse<br />
superior cervical g<strong>an</strong>glia neurons, fluorescence signals were proportional to <strong>the</strong> number of action<br />
potentials <strong>an</strong>d could reliably detect single action potentials. In vivo, we injected <strong>the</strong> virus into <strong>the</strong><br />
salivary gl<strong>an</strong>ds of mice to infect <strong>the</strong> subm<strong>an</strong>dibular g<strong>an</strong>glia (SMG), peripheral parasympa<strong>the</strong>tic<br />
g<strong>an</strong>glia that innervate <strong>the</strong> salivary gl<strong>an</strong>ds <strong>an</strong>d receive input <strong>from</strong> <strong>the</strong> superior salivatory nucleus.<br />
Using in vivo two-photon imaging, we observed both spont<strong>an</strong>eous <strong>an</strong>d elicited activity in SMG<br />
neurons. Single-neuron responses were observed after direct electrical stimulation of <strong>the</strong><br />
presynaptic axons, <strong>an</strong>d after stimulation of <strong>the</strong> oral cavity with hot water. Spont<strong>an</strong>eous tr<strong>an</strong>sients<br />
were relatively brief <strong>an</strong>d infrequent at 24 <strong>an</strong>d 48 hours post inoculation, with cells being active,<br />
on average, 1-2% of recorded time. By 72 hours, calcium bursts were twice as long <strong>an</strong>d occurred<br />
six times more often. It is unclear if <strong>the</strong>se responses represent intrinsic ch<strong>an</strong>ges in neurons, or if<br />
<strong>the</strong>y reflect a more global tissue response to viral infection. Compared to o<strong>the</strong>r recently<br />
published work (Boldogkoi et al. Nat Methods <strong>2009</strong>), <strong>the</strong> reduced virulence of PRV-Bartha <strong>an</strong>d<br />
<strong>the</strong> increased sensitivity of G-CaMP2 should allow <strong>the</strong> monitoring of relatively unperturbed<br />
circuits within 48 hours after inoculation, with single cell resolution in vivo.<br />
Disclosures: A.E. Gr<strong>an</strong>stedt, None; M.L. Szpara, None; B. Kuhn, None; S.S. W<strong>an</strong>g,<br />
None; L.W. Enquist, None.<br />
Poster<br />
586. Imaging Techniques VI
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.13/HH20<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: IBRO-SfN Travel Gr<strong>an</strong>ts <strong>2009</strong><br />
Title: Putative cholinergic elements in <strong>the</strong> ray-finned fish (Leporinus macrocephalus)<br />
telencephalon: Histochemical <strong>an</strong>alyses<br />
Authors: A. BARBOSA-JÚNIOR 1 , L. IDE 2 , *A. HOFFMANN 1 ;<br />
1 Physiol. Dept., Sch. of Med. of Ribeirao Preto - Univ. of Sao Paulo, Ribeirao Preto / SP, Brazil;<br />
2 Dept. of Natural Sci., Federal Univ. of São João del-Rei, São João del-Rei / MG, Brazil<br />
Abstract: In fish, <strong>the</strong> cholinergic systems of <strong>the</strong> central nervous system takes part in <strong>the</strong><br />
alteration of <strong>the</strong> visual responsiveness of optic circuitries, in <strong>the</strong> processing of gustatory<br />
in<strong>for</strong>mation during feeding, in <strong>the</strong> modulation of <strong>the</strong> telencephalic circuitry <strong>an</strong>d in motor<br />
in<strong>for</strong>mation processing. The aim of this study is to compare our data with previous reports on<br />
o<strong>the</strong>r fish <strong>an</strong>d o<strong>the</strong>r vertebrates to provide additional in<strong>for</strong>mation on <strong>the</strong> cholinergic system <strong>from</strong><br />
a phylogenetic point of view. The distribution of telencephalic putative cholinergic neurons was<br />
<strong>an</strong>alyzed by using acetylcholinesterase (AChE) histochemistry in telencephalic frozen tissue of a<br />
ray-finned fish piauçu (Leporinus macrocephalus). AChE-positive small neurons, usually<br />
aggregated in groups of two or three cells, appeared within <strong>the</strong> mitral <strong>an</strong>d internal cell layer of<br />
<strong>the</strong> olfactory bulb. AChE-positive small neurons were observed in both <strong>the</strong> telencephalic area<br />
dorsalis (pallium) <strong>an</strong>d ventralis (subpallium). The density of cells in <strong>the</strong> area dorsalis was higher<br />
th<strong>an</strong> in <strong>the</strong> area ventralis. Small AChE-positive cells were present in <strong>the</strong> dorsal, medial, central,<br />
posterior <strong>an</strong>d lateral divisions of area dorsalis. The highest density of positive neurons in <strong>the</strong> area<br />
dorsalis was observed in <strong>the</strong> dorsal <strong>an</strong>d lateral divisions. In addition, <strong>the</strong> highest density <strong>an</strong>d <strong>the</strong><br />
largest AChE-positive cells of <strong>the</strong> area ventralis were observed in <strong>the</strong> intermediate nucleus. The<br />
distribution of putative cholinergic cells in <strong>the</strong> telencephalon of piauçu shows some similarities<br />
<strong>an</strong>d differences in comparison to o<strong>the</strong>r teleosts <strong>an</strong>d o<strong>the</strong>r vertebrates. Thought putative<br />
cholinergic cells have been observed in <strong>the</strong> pallium, <strong>the</strong>re is a consensus that telencephalic<br />
cholinergic cells are restricted to <strong>the</strong> teleoste<strong>an</strong> area ventralis. Never<strong>the</strong>less, <strong>the</strong>se subpallial<br />
populations may all be homologous to one or more septal cholinergic basal <strong>for</strong>ebrain populations<br />
in mammals (e.g., <strong>the</strong> nucleus basalis of Meynert). In contrast to <strong>the</strong>se cholinergic septal cells<br />
found in <strong>the</strong> subpallial ventral tier, no such putative cholinergic cells are seen in <strong>the</strong> dorsal tier<br />
nuclei of <strong>the</strong> subpallium (i.e., dorsal <strong>an</strong>d pré <strong>an</strong>d postcommissural nuclei, representing <strong>the</strong> striatal<br />
<strong>for</strong>mation), as similarly reported in o<strong>the</strong>r teleosts be<strong>for</strong>e. This situation in teleosts is similar to<br />
that in tetrapod <strong>an</strong>amniotes where extensive septal, but very few striatal cholinergic or putative<br />
cholinergic neurons are present, unlike in amniotes which display m<strong>an</strong>y cholinergic striatal<br />
interneurons.<br />
Disclosures: A. Barbosa-Júnior, None; L. Ide, None; A. Hoffm<strong>an</strong>n, None.
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.14/HH21<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NIH R21 MH076289<br />
DOD W81XWH-07-2-0092<br />
Howard Hughes Medical Institute<br />
Title: Gene-targeted viral-based tracers reveal monosynaptic connections between specific cell<br />
types<br />
Authors: *A. P. WEIBLE 1 , L. SCHWARCZ 2 , I. R. WICKERSHAM 3 , L. DEBLANDER 2 , H.<br />
WU 2 , E. M. CALLAWAY 4 , H. S. SEUNG 3 , C. KENTROS 2 ;<br />
1 Psychol, Univ. Oregon, Eugene, OR; 2 Univ. of Oregon, Eugene, OR; 3 Howard Hughes Med.<br />
Inst. <strong>an</strong>d Massachusetts Inst. of Technol., Cambridge, MA; 4 Salk Inst. <strong>for</strong> Biol. Studies, La Jolla,<br />
CA<br />
Abstract: Knowing <strong>the</strong> connections of specific cell types within a neural network is crucial to<br />
underst<strong>an</strong>ding how those cells function within that network. Traditional tract tracing techniques<br />
(e.g., WGA-HRP, FluroGold) do not differentiate between different cell types, <strong>an</strong>d instead<br />
reflect <strong>the</strong> aggregate of connections between regions. Genetically-targeted tr<strong>an</strong>ssynaptic tracers<br />
provide a potential solution <strong>for</strong> this problem, as tr<strong>an</strong>sgenes c<strong>an</strong> be targeted to specific neuronal<br />
cell types. Wickersham <strong>an</strong>d colleagues (Wickersham et al 2007) recently developed a modified<br />
rabies virus-based system that selectively labels neurons directly presynaptic to a target<br />
population. This system requires factors to be supplied in tr<strong>an</strong>s <strong>for</strong> both infection <strong>an</strong>d replication.<br />
To take adv<strong>an</strong>tage of <strong>the</strong> cellular specificity af<strong>for</strong>ded by <strong>the</strong> tTA/tetO system (in which mice<br />
expressing <strong>the</strong> tTA fusion protein are crossed with mice that c<strong>an</strong> express tr<strong>an</strong>sgenes under <strong>the</strong><br />
control of <strong>the</strong> tetO element), we created lines of tr<strong>an</strong>sgenic mice that c<strong>an</strong> bicistronically express<br />
<strong>the</strong> two tr<strong>an</strong>s factors, <strong>an</strong> avi<strong>an</strong> viral receptor (TVA) to supply infectivity <strong>an</strong>d <strong>the</strong> viral<br />
glycoprotein (G, TVAG toge<strong>the</strong>r) to enable replication, under <strong>the</strong> control of <strong>the</strong> tetO element<br />
(tetO-TVAG mice). Thus, <strong>the</strong> virus should only infect <strong>an</strong>d replicate in those neurons that express<br />
<strong>the</strong> TVAG tr<strong>an</strong>sgenes. In <strong>the</strong> present study, a recombin<strong>an</strong>t rabies virus expressing <strong>the</strong><br />
fluorophore mCherry <strong>an</strong>d pseudotyped with <strong>the</strong> appropriate avi<strong>an</strong> viral coat protein (EnvA) was<br />
injected into <strong>the</strong> subiculum of tetO/TVAG mice crossed to a tTA line to drive <strong>the</strong> expression of
<strong>the</strong> TVAG tr<strong>an</strong>sgenes in individual subicular <strong>an</strong>d presubicular neurons. The results confirm <strong>the</strong><br />
efficacy of <strong>the</strong> technique: fluorescence was observed at <strong>the</strong> injection site which clearly spread to<br />
monosynaptically connected regions such as CA1 hippocampus <strong>an</strong>d <strong>an</strong>terior dorsal (AD)<br />
thalamus. No evidence of uptake or tr<strong>an</strong>sport was observed in single positive TVAG+ or tTA+ or<br />
-/- control mice, confirming <strong>the</strong> requirement of tr<strong>an</strong>sgene expression <strong>for</strong> viral infection <strong>an</strong>d<br />
tr<strong>an</strong>sport. <strong>When</strong> mated to specific tTA lines, <strong>the</strong>se tetO/TVAG mice should prove invaluable in<br />
<strong>the</strong> study of <strong>the</strong> circuitry of <strong>the</strong> mammali<strong>an</strong> CNS by determining <strong>the</strong> connectivity of specific cell<br />
neuronal cell types.<br />
Disclosures: A.P. Weible, None; L. Schwarcz, None; I.R. Wickersham, None; L. DeBl<strong>an</strong>der,<br />
None; H. Wu, None; E.M. Callaway, None; H.S. Seung, None; C. Kentros, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.15/HH22<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Title: Milnacipr<strong>an</strong>, one of <strong>an</strong>tidepress<strong>an</strong>t, bound with fluorescence-NBD-F passed <strong>an</strong>d<br />
accumulated into HT22 cell<br />
Authors: *Y. OGAWA 1 , K. OSADA 2 , T. HAGA 2 , H. MATSUI 3 , M. NAKANO 2 , S. KANAI 2 ,<br />
D. TANAKA 2 , T. YANAGIDA 2 , K. FUJIWARA 2 , Y. SASUGA 2 , K. TAKAHASH 2 , M.<br />
ASAKURA 2 ;<br />
1 Kawasaki, Jap<strong>an</strong>; 2 St.mari<strong>an</strong>na Univ.Sch.Med, Kawasaki,K<strong>an</strong>agawa, Jap<strong>an</strong>; 3 Inst.Radioisotope<br />
Res.St.mari<strong>an</strong>na Univ.Sch.Med, Kawasaki,K<strong>an</strong>agawa, Jap<strong>an</strong><br />
Abstract: Derivatization of amino acids with fluorescent dyes, 4-Fluoro-7-nitrobenzofuraz<strong>an</strong><br />
(NBD-F) has <strong>the</strong> potential to signific<strong>an</strong>tly improve <strong>the</strong> sensitivity <strong>an</strong>d specificity of <strong>an</strong>alyze<br />
detection. NBD-F is could achieve up to 10-fold higher sensitivity th<strong>an</strong> ortho-phthaldialdehyde<br />
(OPA) <strong>for</strong> most amino acids. NBD-F reacts with amino acid at n<strong>an</strong>omolar level.<br />
We developmented <strong>the</strong> new method that <strong>the</strong> drug with amino group (-NH2) bound with NBD-F<br />
<strong>an</strong>d observe <strong>the</strong> NBD-F labeled drug in vivo.<br />
It was thought that <strong>an</strong>tidepress<strong>an</strong>t agents react through receptor <strong>an</strong>d tr<strong>an</strong>sporter on neuronal cell<br />
surface. But clinical <strong>an</strong>tidepress<strong>an</strong>ts effects need more th<strong>an</strong> 3-4 weeks. We have hypo<strong>the</strong>sis that<br />
<strong>an</strong>tidepress<strong>an</strong>ts pass within <strong>the</strong> cell, <strong>an</strong>d react directly to <strong>the</strong> protein in <strong>the</strong> cell.<br />
We examined whe<strong>the</strong>r Milnacipr<strong>an</strong>, one of <strong>the</strong> SNRIs, labeled with NBD-F passed into <strong>the</strong> cell<br />
<strong>an</strong>d which org<strong>an</strong> Milnacipr<strong>an</strong> was accumulated into cell.
Disclosures: Y. Ogawa, None; K. Osada, None; T. Haga, None; H. Matsui, None; M.<br />
Nak<strong>an</strong>o, None; S. K<strong>an</strong>ai, None; D. T<strong>an</strong>aka, None; T. Y<strong>an</strong>agida, None; K. Fujiwara, None; Y.<br />
sasuga, None; K. Takahash, None; M. Asakura, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.16/HH23<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: Invitrogen/Life Technologies Collaborative Research Compact Funding, 2008-09<br />
Title: BacMam gene delivery <strong>for</strong> functional <strong>an</strong>d imaging applications<br />
Authors: *D. W. BEACHAM 1 , G. T. HANSON 1 , U. LAKSHMIPATHY 2 , T. BLACKMER 1 ,<br />
M. O'GRADY 1 , F. BOYCE 3 ;<br />
1 2 3<br />
Life Technologies, Eugene, OR; Life Technologies, Carlsbad, CA; Dept. of Neurol.,<br />
Massachusetts Gen. Hosp., Boston, MA<br />
Abstract: Gene expression in primary neurons has long been a stumbling block <strong>for</strong> basic science<br />
<strong>an</strong>d drug discovery ef<strong>for</strong>ts that rely on functional measurements of activity in living cells. Like<br />
m<strong>an</strong>y postmitotic, terminally differentiated cell types, neurons <strong>an</strong>d <strong>the</strong>ir highly specialized roles<br />
are difficult to recapitulate in vitro, with particular challenges around expression of recombin<strong>an</strong>t<br />
proteins <strong>an</strong>d knockdown of endogenous genes. Currently, <strong>the</strong>se m<strong>an</strong>ipulations are made with<br />
physical methods such as <strong>the</strong> biolistic "gene gun" <strong>an</strong>d electroporation technologies, or with<br />
biological <strong>an</strong>d chemical methods such as infective virions <strong>an</strong>d lipid tr<strong>an</strong>sfection reagents.<br />
Despite low efficiencies of delivery, risk of viral propagation <strong>an</strong>d high cell mortality rates, <strong>the</strong>se<br />
techniques have been applied with varying degrees of success in a variety of neuronal studies.<br />
Here, we describe recent adv<strong>an</strong>ces in <strong>the</strong> engineering of mammali<strong>an</strong>-targeted baculovirus<br />
(BacMam) expression vectors <strong>for</strong> neuronal gene expression. BacMam expresses large gene<br />
products (up to 38 kB) in mammali<strong>an</strong> cells by virtue of mammali<strong>an</strong> promoter elements placed<br />
upstream of <strong>the</strong> gene or genes of interest. In contrast to replicating mammali<strong>an</strong> cell virions,<br />
native baculoviral genes under insect promoters are ignored once <strong>the</strong> virus is taken up by <strong>the</strong> host<br />
cell. BacMam is tolerated by cells across a broad r<strong>an</strong>ge of infectivity, allowing titration of<br />
expression as well as convenient co-delivery of multiple gene products including biosensors, ion
ch<strong>an</strong>nel <strong>an</strong>d GPCR subunits, or photoproteins engineered to measure or control neuronal<br />
function.<br />
Disclosures: D.W. Beacham, Life Technologies, A. Employment (full or part-time); Life<br />
Technologies, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
G.T. H<strong>an</strong>son, Life Technologies, A. Employment (full or part-time); Life Technologies, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); U.<br />
Lakshmipathy, Life Technologies, A. Employment (full or part-time); Life Technologies, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); T. Blackmer,<br />
Life Technologies, A. Employment (full or part-time); Life Technologies, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); M. O'Grady, Life Technologies, A.<br />
Employment (full or part-time); Life Technologies, E. Ownership Interest (stock, stock options,<br />
patent or o<strong>the</strong>r intellectual property); F. Boyce, Life Technologies, C. O<strong>the</strong>r Research Support<br />
(receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support).<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.17/HH24<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Title: A novel radiolig<strong>an</strong>d <strong>for</strong> calcitonin gene-related peptide (CGRP) receptors: In vitro<br />
autoradiography <strong>an</strong>d tissue homogenate binding studies
Authors: *Z. ZENG 1 , S. O'MALLEY 2 , P. PATRICIA MILLER 2 , E. HOSTETLER 2 , H. FAN 2 ,<br />
J. KIM 2 , H. SELNICK 2 , M. WOOD 2 , S. GRAHAM 2 , S. KANE 2 , C. CHRISTOPHER<br />
SALVATORE 2 , R. HARGREAVES 2 , C. SUR 2 , D. WILLIAMS 2 ;<br />
1 2<br />
Imaging Dept, Merck Res. Lab. Merck & Co. Inc, West Point, PA; Merck Res. Labs., West<br />
Point, PA<br />
Abstract: Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide (1). CGRP is<br />
a very potent vasodilator, <strong>an</strong>d its levels in <strong>the</strong> venous flow <strong>from</strong> <strong>the</strong> head show dramatic increase<br />
during <strong>the</strong> onset of migraine, supporting its role in mediation of primary headaches, including<br />
migraine (2). The receptors mediating CGRP effects are comprised of calcitonin receptor-like<br />
receptor (CLR), <strong>an</strong>d receptor activity-modifying protein 1 (RAMP1) (3). A CGRP receptor<br />
<strong>an</strong>tagonist was able to attenuate neurogenic increases in dural blood flow in <strong>an</strong> <strong>an</strong>imal study (4),<br />
<strong>an</strong>d clinical studies with MK-0974 (telcagep<strong>an</strong>t), have demonstrated <strong>an</strong>timigraine effects (5),<br />
supporting its <strong>the</strong>rapeutic application as a potential new class of <strong>an</strong>timigraine <strong>the</strong>rapy.<br />
Fur<strong>the</strong>rmore, recent preclinical studies indicate <strong>the</strong> <strong>an</strong>timigraine effects of MK-0974 may rely on<br />
its interaction with CGRP receptors located not only in <strong>the</strong> peripheral but also in <strong>the</strong> central<br />
nervous system (CNS) (6).<br />
Development of a CNS PET tracer <strong>for</strong> CGRP receptors would enable us to study target<br />
distribution in tissues, measure target occup<strong>an</strong>cy, <strong>an</strong>d define doses <strong>for</strong> clinical studies. As part of<br />
a CGRP receptor PET tracer development program, [ 3 H]-CGRP-A2 was syn<strong>the</strong>sized <strong>an</strong>d<br />
evaluated by in vitro autoradiography <strong>an</strong>d tissue homogenate binding. [ 3 H]-CGRP-A2 binds to<br />
CGRP receptors with high affinity, Kd = 0.14 nM (rhesus), <strong>an</strong>d 0.15 nM (hum<strong>an</strong>) in brain<br />
homogenates. The total number of binding sites (Bmax) of [ 3 H]-CGRP-A2 in <strong>the</strong> cerebellum of<br />
rhesus monkey <strong>an</strong>d hum<strong>an</strong> is 21 nM <strong>an</strong>d 22 nM (wet tissue weight), respectively. Binding<br />
potentials as defined using Bmax / Kd ratio are 139 in rhesus cerebellum, <strong>an</strong>d 129 in hum<strong>an</strong><br />
cerebellum. Binding of [ 3 H]-CGRP-A2 to brain slices was uni<strong>for</strong>mly inhibited by 10 µM<br />
unlabeled CGRP-A2 as a self blocker. Displaceable binding sites of [ 3 H]-CGRP-A2 in brain<br />
slices of rhesus monkey were unevenly distributed throughout <strong>the</strong> whole brain with <strong>the</strong> highest<br />
levels found in <strong>the</strong> cerebellum, brainstem, subst<strong>an</strong>tia nigra, <strong>an</strong>d meninges. Relatively high levels<br />
of binding were also observed in <strong>the</strong> hippocampus <strong>an</strong>d <strong>the</strong> gray matter of spinal cord. Low levels<br />
of binding were shown in <strong>the</strong> caudate-putamen, cerebral cortex <strong>an</strong>d thalamus. Similar patterns of<br />
[ 3 H]-CGRP-A2 binding were shown in hum<strong>an</strong> brain slices examined. The distribution patterns of<br />
[ 3 H]-CGRP-A2 binding sites observed in rhesus monkey <strong>an</strong>d hum<strong>an</strong> brain slices are similar to<br />
those of CGRP binding sites reported by Inagaki <strong>an</strong>d colleagues (7). The results of <strong>the</strong>se studies<br />
demonstrate [ 3 H]-CGRP-A2 is a novel radiolig<strong>an</strong>d <strong>for</strong> <strong>the</strong> CGRP receptor, <strong>an</strong>d highlight <strong>the</strong><br />
feasibility of developing a CNS PET tracer <strong>for</strong> <strong>the</strong> in vivo study of receptor occup<strong>an</strong>cy in <strong>the</strong><br />
brain.<br />
Disclosures: Z. Zeng, Merck & Co. Inc., A. Employment (full or part-time); S. O'Malley,<br />
Merck & Co. Inc., A. Employment (full or part-time); P. Patricia Miller, Merck & Co. Inc., A.<br />
Employment (full or part-time); E. Hostetler, Merck & Co. Inc., A. Employment (full or parttime);<br />
H. F<strong>an</strong>, Merck & Co. Inc., A. Employment (full or part-time); J. Kim, Merck & Co. Inc.,<br />
A. Employment (full or part-time); H. Selnick, Merck & Co. Inc., A. Employment (full or parttime);<br />
M. Wood, Merck & Co. Inc., A. Employment (full or part-time); S. Graham, Merck &<br />
Co. Inc., A. Employment (full or part-time); S. K<strong>an</strong>e, Merck & Co. Inc., A. Employment (full or<br />
part-time); C. Christopher Salvatore, Merck & Co. Inc., A. Employment (full or part-time); R.
Hargreaves, Merck & Co. Inc., A. Employment (full or part-time); C. Sur, Merck & Co. Inc.,<br />
A. Employment (full or part-time); D. Williams, Merck & Co. Inc., A. Employment (full or parttime).<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.18/HH25<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NSF ERC on BMES<br />
Whittier Foundation<br />
AFOSR funded DURINT Program<br />
Title: Seeing <strong>the</strong> death of cell on <strong>the</strong> n<strong>an</strong>oscale: Real-time molecular imaging of retinal g<strong>an</strong>glion<br />
cell apoptosis under elevated pressure<br />
Authors: *J. LEE 1 , S. LU 2 , M. HUMAYUN 3 , A. MADHUKAR 4 ;<br />
1 Biomed. Engin., 2 Physics <strong>an</strong>d Ophthalmology, 3 Ophthalmology <strong>an</strong>d Biomed. Engin., 4 Chem.<br />
Engin. <strong>an</strong>d Materials Science, Physics, <strong>an</strong>d Biomed. Engin., USC, Los Angeles, CA<br />
Abstract: The infusion of n<strong>an</strong>otechnologies into neuroscience provides new insights into<br />
molecular-level underst<strong>an</strong>ding of <strong>the</strong> mech<strong>an</strong>isms of neuropathogenesis <strong>an</strong>d thus early diagnosis<br />
of neuropathy. In <strong>the</strong> present research, n<strong>an</strong>oscale molecular imaging is employed to gain better<br />
underst<strong>an</strong>ding of <strong>the</strong> molecular mech<strong>an</strong>isms of retinal g<strong>an</strong>glion cell (RGC) apoptosis under<br />
elevated hydrostatic pressure, a key contributing factor to <strong>the</strong> disease of glaucoma Tr<strong>an</strong>slocation<br />
of phosphatidyl serine (PS) <strong>from</strong> inner to outer leaflet of plasma membr<strong>an</strong>e is a molecular<br />
process known to occur in <strong>the</strong> progress of apoptosis. Thus PS is particularly suited <strong>for</strong><br />
examination utilizing combined surface sensitive techniques of atomic <strong>for</strong>ce microscopy (AFM)<br />
<strong>an</strong>d near-field sc<strong>an</strong>ning optical microscopy (NSOM) <strong>for</strong> <strong>the</strong> simult<strong>an</strong>eous imaging of <strong>the</strong><br />
morphology of <strong>the</strong> cells reflecting <strong>the</strong> state of <strong>the</strong> cytoskeleton disintegration <strong>an</strong>d <strong>the</strong> surface<br />
density of <strong>the</strong> PS on <strong>the</strong> n<strong>an</strong>oscale beyond <strong>the</strong> diffraction-limited optical resolution. Our<br />
NSOM/AFM imaging of PS in differentiated RGC-5 cells under elevated pressure reveal a<br />
positive correlation between <strong>the</strong> geometrical cell height <strong>an</strong>d PS density probed by <strong>an</strong>nexin-Vconjugated<br />
qu<strong>an</strong>tum dot (QD) labeling. To underst<strong>an</strong>d <strong>the</strong> dynamics of molecular processes in<br />
apoptosis we have carried out real-time simult<strong>an</strong>eous imaging of Ca2+ elevation <strong>an</strong>d caspase-3/7
activation, along with <strong>the</strong> morphological ch<strong>an</strong>ges in differentiated RGC-5 cells under elevated<br />
pressure <strong>for</strong> prolonged times (~24 hours) utilizing a pressurized incubation chamber. The<br />
different temporal profiles observed - tr<strong>an</strong>sient Ca2+ at <strong>an</strong> early time versus gradual increase of<br />
caspase-3/7 activation - yet with <strong>the</strong> strong correlation of occurrence between <strong>the</strong> two reveals<br />
<strong>the</strong>ir distinct roles in apoptosis as initiator/amplifier <strong>an</strong>d final effector of <strong>the</strong> apoptotic process.<br />
The combination of n<strong>an</strong>oscale imaging of certain biomarkers at fixed stages <strong>an</strong>d real-time<br />
imaging of o<strong>the</strong>r biomarkers in live cell will enable a better underst<strong>an</strong>ding of stress induced cell<br />
apoptosis <strong>an</strong>d thus may allow <strong>the</strong> development of underst<strong>an</strong>ding-based remedy or prevention of<br />
relev<strong>an</strong>t diseases.<br />
Disclosures: J. Lee, None; S. Lu, None; M. Humayun, None; A. Madhukar, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.19/HH26<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: ANR Programme Bl<strong>an</strong>c No ANR-06-BLAN-0356<br />
Title: Design of new microelectrode arrays <strong>for</strong> a 3D sampling of <strong>the</strong> neural tissue<br />
Authors: *L. ROUSSEAU, Mr 1 , F. VERJUS 3 , J. ROUESSARD 3 , O. ABDOUN 4 , C.<br />
MAZZOCCO 4 , G. LISSORGUES 2 , B. YVERT 4 ;<br />
1 Groupe ESIEE, Noisy le gr<strong>an</strong>d, Fr<strong>an</strong>ce; 2 Groupe ESIEE, Noisy le Gr<strong>an</strong>d, Fr<strong>an</strong>ce; 3 Cap-VRF,<br />
Hérouville, Fr<strong>an</strong>ce; 4 CNRS & Univ. of Bordeaux, Talence, Fr<strong>an</strong>ce<br />
Abstract: Microelectrode Arrays (MEAs) offer a nice way to probe <strong>the</strong> electrical activity<br />
distributed over large populations of neurons ei<strong>the</strong>r in vitro or in vivo <strong>an</strong>d give <strong>the</strong> possibility to<br />
deliver appropriate electrical stimulations to neuronal networks. However, to date, MEAs offer<br />
only a two-dimensional sampling of <strong>the</strong> neural tissue, while in<strong>for</strong>mation is distributed in all<br />
dimensions of <strong>the</strong> Central Nervous System. Here we report on preliminary realization of true 3D<br />
MEA probes. These arrays are built by assembling comb-like 2D arrays. Each comb, composed<br />
of several multisite sh<strong>an</strong>ks, is microfabricated by Deep Reactive Ion Etching (DRIE) of a silicon<br />
substrate. First, <strong>an</strong> oxidation step of a silicon wafer (500 nm) is per<strong>for</strong>med. Using<br />
photolithography, a metallic layer (Gold or Platinum) is <strong>the</strong>n sputtered on <strong>the</strong> silicon substrate to<br />
define microelectrodes, leads, <strong>an</strong>d contacting pads. A PECVD (Plasma Enh<strong>an</strong>ced Chemical<br />
Vapor Deposition) silicon nitride layer is next deposited to insulate <strong>the</strong> leads on <strong>the</strong> comb shaft
<strong>from</strong> <strong>the</strong> liquid environment. Finally, top side DRIE is done to define <strong>the</strong> comb shafts <strong>an</strong>d a<br />
second bottom side DRIE to release <strong>the</strong> structures. Each comb is composed of ei<strong>the</strong>r 1, 2, or 4<br />
sh<strong>an</strong>ks, <strong>the</strong> overall number of sites being ei<strong>the</strong>r 12 or 16. In order to test <strong>for</strong> optimal electrode<br />
size, we also designed electrode arrays with variable electrode sizes. The recording noise level<br />
was found to be inversely correlated to <strong>the</strong> electrode diameter: while 64-µm diameter gold<br />
electrodes lead to noise level as low as 2 µV RMS, decreasing electrode size down to 5-10 µm in<br />
diameter only moderately increased <strong>the</strong> noise level, which remained around 3-4 µV RMS. We<br />
finally designed a specific micro-connecting device to assemble several combs into 3D arrays on<br />
a PCB. Sixty-four-electrode prototypes of such 3D arrays have been designed. In conclusion, we<br />
present a process to fabricate new MEA devices dedicated to 3D sampling of neural tissue.<br />
Disclosures: L. rousseau, None; F. Verjus, None; J. Rouessard, None; O. Abdoun, None; C.<br />
Mazzocco, None; G. Lissorgues, None; B. Yvert, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.20/HH27<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: Max Pl<strong>an</strong>ck <strong>Society</strong><br />
Volkswagenstiftung<br />
SFB636/A4<br />
Title: Recombin<strong>an</strong>t adeno-associated viruses equipped with tetracycline-controlled genetic<br />
switches <strong>for</strong> rapid <strong>an</strong>d efficient gene activation in <strong>the</strong> mammali<strong>an</strong> brain<br />
Authors: *G. K. DOGBEVIA, W. TANG, P. H. SEEBURG, R. SPRENGEL, M. T. HASAN;<br />
Max-Pl<strong>an</strong>ck Inst. For Med. Res., Heidelberg, Germ<strong>an</strong>y<br />
Abstract: Regulatable control of gene expression in <strong>the</strong> brain is of great value <strong>for</strong> investigating<br />
<strong>the</strong> role of neuronal circuits in sensory in<strong>for</strong>mation processing <strong>an</strong>d behavior. Previously, we<br />
discovered that stably-integrated tetracycline (Tet) promoter (Ptet) becomes functionally silent in<br />
a majority of neurons when it is inactive during development (Zhu et al., 2007 PLoS ONE. 2007<br />
2(6):e533). To avoid integration-induced gene silencing, we developed tetracycline-regulatable<br />
adeno-associated viruses (AAVs) <strong>for</strong> efficient <strong>an</strong>d long-term expression of genes in neurons in
vivo. Because co-infectivity of AAVs in neurons is very high, we used two viruses <strong>for</strong><br />
doxycycline (Dox) controlled gene expression in vivo: one virus delivers, under control of a celltype-specific<br />
promoter, <strong>the</strong> rtTA gene while <strong>the</strong> o<strong>the</strong>r virus delivers two different genes under<br />
<strong>the</strong> control of a bidirectional Tet promoter (Ptetbi). Our data clearly demonstrate that gene<br />
expression, visualized by fluorescence imaging, c<strong>an</strong> be observed within a few hours after Dox<br />
administration. By non-invasive bioluminescence imaging of firefly luciferase activity in <strong>the</strong><br />
cortex, we will monitor in same individual mice repeated cycles of gene activation <strong>an</strong>d<br />
inactivation by administering or removing Dox <strong>from</strong> <strong>the</strong> <strong>an</strong>imals' drinking water, respectively<br />
(Genesis. 2001 29:116-22). By in vivo two-photon imaging, we will monitor Dox-induced realtime<br />
gene activation in a large number of cortical neurons in vivo. With <strong>the</strong> AAV-based rtTA<br />
system, it should be feasible to per<strong>for</strong>m genetic m<strong>an</strong>ipulation of hippocampal <strong>an</strong>d cortical<br />
circuits, <strong>for</strong> example, by expressing tet<strong>an</strong>us toxin light chain to inhibit synaptic tr<strong>an</strong>smission or<br />
by siRNA-mediated expression knockdown to ch<strong>an</strong>ge cellular signaling. Such studies should<br />
help to dissect <strong>the</strong> role of neural circuits <strong>an</strong>d gene activity in a variety of brain functions<br />
including learning <strong>an</strong>d memory consolidation.<br />
Disclosures: G.K. Dogbevia, None; W. T<strong>an</strong>g, None; P.H. Seeburg, None; R. Sprengel,<br />
None; M.T. Has<strong>an</strong>, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.21/HH28<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: FDA protocol 731201<br />
Title: Development <strong>an</strong>d characterization of novel histochemical markers of brain vascular<br />
elements in normal <strong>an</strong>d neurotoxic<strong>an</strong>t exposed rats<br />
Authors: *L. C. SCHMUED, S. SARKAR, D. HEARD;<br />
Div. Of Neurotox, Natl. Ctr. For Tox Res., Jefferson, AR<br />
Abstract: Although routine histological stains are useful <strong>for</strong> staining all cell bodies within <strong>the</strong><br />
brain, <strong>the</strong>ir value is limited by <strong>the</strong>ir lack of specificity <strong>for</strong> <strong>an</strong>y one cell type. There<strong>for</strong>e, specific<br />
tracers have been developed to allow <strong>for</strong> <strong>the</strong> localization of specific cell types within <strong>the</strong> brain<br />
such as neurons, degenerating neurons, astrocytes, microglia, <strong>an</strong>d oligodendorcytes <strong>an</strong>d <strong>the</strong>ir<br />
associated myelin sheaths. One class of cells that has not received much attention comprises <strong>the</strong>
vascular elements of <strong>the</strong> brain, such as endo<strong>the</strong>lial, smooth muscle <strong>an</strong>d pericyte cells.<br />
There<strong>for</strong>e, <strong>the</strong> goal of this study was to develop novel histochemical markers <strong>for</strong> <strong>the</strong> labeling of<br />
brain pericytes <strong>an</strong>d endo<strong>the</strong>lial cells. Pericyte labeling was accomplished by <strong>the</strong><br />
intracerebroventricular injection of certain fluorescent dextr<strong>an</strong> conjugates such as Fluoro-<br />
Gold/dextr<strong>an</strong> [FG/Dex] or TRITC/dextr<strong>an</strong> [Fluoro-Ruby; FR]. Following post operative survival<br />
intervals r<strong>an</strong>ging <strong>from</strong> 1-7 days, extensive labeling of vascular pericytes was seen throughout <strong>the</strong><br />
entire brain. These cells were found distal to <strong>the</strong> endo<strong>the</strong>lial cells <strong>an</strong>d exhibited large dye<br />
containing vacuoles. The morphology of <strong>the</strong> pericytes was somewhat variable, exhibiting a more<br />
round or amoeboid shape in larger vesicles <strong>an</strong>d a more elongated appear<strong>an</strong>ce in small capillaries.<br />
The use of FG/DEX resulted in yellow labeled pericytes, while FR resulted in red labeling.<br />
Two different strategies were used to label brain endo<strong>the</strong>lial cells. One approach involved <strong>the</strong><br />
intravascular perfusion with ei<strong>the</strong>r silver salts or certain fluorescent dyes such as CBR-3B or FB-<br />
28 at <strong>the</strong> time of sacrifice. This procedure results in <strong>the</strong> respective bright-field or fluorescent<br />
labeling of all vascular endo<strong>the</strong>lial cells within <strong>the</strong> brain. Alternatively, <strong>the</strong> a<strong>for</strong>ementioned<br />
fluorescent tracers c<strong>an</strong> be used as histological stains to label <strong>the</strong> vascular endo<strong>the</strong>lial cells, thus<br />
revealing <strong>the</strong>ir respective distribution, density <strong>an</strong>d lumen size. Preliminary findings in <strong>the</strong> kainic<br />
acid treated <strong>an</strong>imal suggest that labeled pericytes in brain regions containing degenerating<br />
neurons, as revealed by Fluoro-Jade C, exhibit filamentous dysplastic morphology. In contrast,<br />
3-nitropropionic acid exposure results in a decrease in <strong>the</strong> number <strong>an</strong>d intensity of labeled<br />
pericytes in regions exhibiting neuronal degeneration such as <strong>the</strong> striatum. This suggests that<br />
vascular pericytes respond differently to excitotoxic vs. necrotic lesions. Supported by FDA<br />
protocol E731201<br />
Disclosures: L.C. Schmued, None; S. Sarkar, None; D. Heard, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.22/HH29<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Support: NRSA Gr<strong>an</strong>t F31 MH081508-01A2<br />
NIMH Gr<strong>an</strong>t R01 MH47340<br />
Title: Projections of rabbit whisker barrel cortex: Possible targets <strong>for</strong> trace eyeblink conditioning
Authors: *L. C. FLORES, R. GALVEZ, J. F. DISTERHOFT;<br />
Northwestern Univ., Chicago, IL<br />
Abstract: Our laboratory has established that S1 barrel cortex is essential <strong>for</strong> trace eyeblink<br />
conditioning when whisker stimulation is used as a conditioned stimulus. In order to determine<br />
future targets of study, we injected horse radish peroxidase conjugated with wheat germ agglutin<br />
(HRP-WGA) into S1 barrel cortex of two New Zeal<strong>an</strong>d albino rabbits. All surgical procedures<br />
were approved by <strong>the</strong> Northwestern University ACUC. Under sterile conditions, <strong>the</strong> cortex was<br />
exposed over <strong>the</strong> left S1 barrel cortex. An electrode was lowered into <strong>the</strong> cortex <strong>an</strong>d <strong>the</strong> whiskers<br />
on <strong>the</strong> right side of <strong>the</strong> face were stimulated to determine correct placement. After removing <strong>the</strong><br />
electrode, a Hamilton syringe was slowly lowered 400µm into <strong>the</strong> cortex over a course of 5<br />
minutes. The syringe was left in place <strong>for</strong> 5 minutes, <strong>the</strong>n 10nL of 2% HRP/WGA (Sigma,<br />
dissolved in 0.5M NaCl) was pressure injected over a course of 10 minutes. The syringe was left<br />
in place <strong>for</strong> 5 minutes after <strong>the</strong> injection be<strong>for</strong>e being removed, <strong>an</strong>d <strong>the</strong> skin above <strong>the</strong> skull was<br />
sutured closed. 48 hours after injection, <strong>the</strong> rabbit was sacrificed <strong>an</strong>d perfused through <strong>the</strong> heart<br />
with saline followed by 3% para<strong>for</strong>maldeyde <strong>an</strong>d glycerol. The brain was cryoprotected <strong>for</strong> 1<br />
week in glycerol, frozen in isopent<strong>an</strong>e, <strong>an</strong>d cut into 50µm sections. Sections were reacted in<br />
tetramethylbenzidine <strong>an</strong>d sodium nitroferricy<strong>an</strong>ide baths using glucose oxidase <strong>an</strong>d β-d-glucose<br />
to promote <strong>the</strong> reaction. After counterstaining with Neutral Red, a light microscope was used to<br />
determine reaction product. Dense <strong>an</strong>terograde projections were found ipsilaterally in <strong>the</strong><br />
claustrum <strong>an</strong>d <strong>the</strong> VPM of <strong>the</strong> thalamus. Moderate projections were found in <strong>the</strong> caudate<br />
nucleus, lateral pons, <strong>an</strong>d SII. Light projections were found in <strong>the</strong> putamen <strong>an</strong>d <strong>the</strong> <strong>an</strong>terior<br />
cingulate/premotor region of cortex. Retrograde projections were only found in <strong>the</strong> VPM nucleus<br />
of <strong>the</strong> thalamus. Fibers were seen projecting to <strong>the</strong> contralateral hemisphere via <strong>the</strong> corpus<br />
callosum, but terminations could not be visualized. Our lab has chosen to look at <strong>the</strong> caudate<br />
nucleus, VPM nucleus of <strong>the</strong> thalamus, <strong>an</strong>d pons <strong>for</strong> contributions to trace eyeblink conditioning.<br />
Disclosures: L.C. Flores, None; R. Galvez, None; J.F. Disterhoft, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.23/HH30<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Title: Assessment of histamine H3 receptor occup<strong>an</strong>cy by ex vivo radiolig<strong>an</strong>d binding utilizing<br />
<strong>the</strong> H3 receptor <strong>an</strong>tagonist radiolig<strong>an</strong>d [ 3 H]A-349821 <strong>an</strong>d autoradiography
Authors: *T. R. MILLER, R. S. BITNER, T. A. ESBENSHADE, J. D. BRIONI;<br />
Global Pharmaceut. R&D, Abbott Labs., Abbott Park, IL<br />
Abstract: The histamine H3 receptor <strong>an</strong>tagonist radiolig<strong>an</strong>d [ 3 H]A-349821 was evaluated as a<br />
radiolig<strong>an</strong>d <strong>for</strong> conducting preclinical H3 receptor occup<strong>an</strong>cy studies using <strong>the</strong> ex vivo binding<br />
approach. In this study, various doses of <strong>the</strong> H3 receptor <strong>an</strong>tagonist ABT-239 were administered<br />
intraperitoneally to adult Sprague-Dawley rats; following <strong>the</strong> in vivo treatments, [ 3 H]A-349821<br />
binding assays were conducted with coronal sections of excised brain tissue. [ 3 H]A-349821<br />
binding was <strong>an</strong>alyzed by autoradiography using <strong>the</strong> BetaImager (Biospace Lab, Paris, Fr<strong>an</strong>ce),<br />
<strong>an</strong>d H3 receptor occup<strong>an</strong>cy by ABT-239 was <strong>the</strong>n qu<strong>an</strong>tified as <strong>the</strong> reduction in [ 3 H]A-349821 ex<br />
vivo binding in comparison with vehicle-treated controls. Results <strong>from</strong> this ex vivo binding<br />
method were compared to those <strong>from</strong> <strong>an</strong> in vivo approach, where both ABT-239 <strong>an</strong>d [ 3 H]A-<br />
349821 were administered systemically <strong>an</strong>d competed <strong>for</strong> H3 receptor occup<strong>an</strong>cy in vivo. As<br />
assessed by autoradiography with coronal rat brain sections, <strong>the</strong> regional distribution of specific<br />
[ 3 H]A-349821 binding was consistent with H3 receptor localization in rat brain, i.e. [ 3 H]A-<br />
349821 binding levels were greatest in cerebral cortex, caudate putamen <strong>an</strong>d hypothalamus. With<br />
ABT-239 pretreatment, [ 3 H]A-349821 binding was reduced in <strong>an</strong> ABT-239 dose-dependent<br />
m<strong>an</strong>ner; <strong>the</strong> ED50 <strong>for</strong> ABT-239 <strong>for</strong> H3 receptor occup<strong>an</strong>cy was 0.56 mg/kg. The potency of<br />
ABT-239 <strong>for</strong> H3 receptor occup<strong>an</strong>cy as obtained <strong>from</strong> this <strong>an</strong>alysis was very similar to that<br />
obtained using <strong>the</strong> in vivo approach (ED50 = 0.44 mg/kg). Thus, this preclinical model of H3<br />
receptor occup<strong>an</strong>cy utilizing [ 3 H]A-349821 ex vivo binding <strong>an</strong>d autoradiography provides a valid<br />
measure of in vivo H3 receptor occup<strong>an</strong>cy, which c<strong>an</strong> <strong>the</strong>n be related to <strong>an</strong>tagonist dose, blood<br />
exposure levels <strong>an</strong>d efficacy in preclinical models to help guide <strong>an</strong>d interpret clinical studies of<br />
adv<strong>an</strong>cing H3 receptor <strong>an</strong>tagonists.<br />
Disclosures: T.R. Miller, Abbott Laboratories, A. Employment (full or part-time); R.S.<br />
Bitner, Abbott Laboratories, A. Employment (full or part-time); T.A. Esbenshade, Abbott<br />
Laboratories, A. Employment (full or part-time); J.D. Brioni, Abbott Laboratories, A.<br />
Employment (full or part-time).<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.24/HH31<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Title: Laser capture microdissection <strong>an</strong>d microarray <strong>an</strong>alysis of ascending spinal projection<br />
neurones
Authors: J. A. CURRY, A. MOSS, S. TRIM, M. FIDOCK, H. WEST, K. PATEL, S.<br />
PHILLIPS, P. COX, D. WILLIAMS, *K. H. HOLMBERG;<br />
Pfizer Ltd, S<strong>an</strong>dwich, United Kingdom<br />
Abstract: The functional role of <strong>the</strong> dorsal horn within pain has long been recognised[1].<br />
Mediation of noxious stimuli <strong>from</strong> peripheral nociceptors occurs via 2 nd order projection neurons<br />
within lamina I/V of <strong>the</strong> spinal grey matter. Although <strong>the</strong> functional signific<strong>an</strong>ce of <strong>the</strong>se<br />
neurons has been established, little is known about <strong>the</strong>ir genetic profile. To investigate this, we<br />
have combined stereotaxic injections of dextr<strong>an</strong> fluororuby into <strong>the</strong> brainstem with LMPC (Laser<br />
Microdissection with Pressure Catapulting), <strong>an</strong>d whole genome microarray <strong>an</strong>alysis.<br />
With local ethical approval, injections were made into both <strong>the</strong> spinal parabrachial nucleus <strong>an</strong>d<br />
<strong>the</strong> lateral reticular nucleus of 6 male SD rats (weight 250-300g). 7 days subsequent to injection<br />
<strong>an</strong>imals were euth<strong>an</strong>ized, spinal cords removed <strong>an</strong>d snap frozen on dry-ice chilled isopent<strong>an</strong>e.<br />
20µm sections <strong>from</strong> <strong>the</strong> lumbar enlargement were cryosectioned <strong>an</strong>d prepared <strong>for</strong> LMPC.<br />
Approximately 50 traced cells within Lamina I were collected, as well as both traced <strong>an</strong>d<br />
untraced cells <strong>from</strong> Lamina V as control cell populations. RNA was <strong>the</strong>n extracted <strong>an</strong>d<br />
qu<strong>an</strong>tified. The RNA qu<strong>an</strong>tity (A260) <strong>an</strong>d purity (260/280 ratio) was assessed using<br />
spectrophotometery, <strong>an</strong>d integrity via 2100 Bio<strong>an</strong>alyzer (Agilent technologies). Samples <strong>the</strong>n<br />
underwent two-step amplification <strong>an</strong>d microarray <strong>an</strong>alysis.<br />
These methods have demonstrated approximately 300 genes which are preferentially expressed<br />
in <strong>the</strong> lamina I population vs control populations. Of <strong>the</strong>se, genes that have been associated with<br />
pain such as NPY, neurokinin <strong>an</strong>d prodynorphin were found to be expressed at levels up to10fold<br />
higher th<strong>an</strong> both control groups.<br />
Future experiments will use qPCR to confirm <strong>the</strong> ch<strong>an</strong>ges observed <strong>from</strong> <strong>the</strong> array <strong>an</strong>alysis, as<br />
well as pathway mining of novel hits in <strong>an</strong> attempt to elucidate novel drug targets.<br />
[1] D'Mello R, Dickenson AH. Spinal cord mech<strong>an</strong>isms of pain. British Journal of Anaes<strong>the</strong>sia.<br />
101(1):8-16; 2008<br />
Disclosures: J.A. Curry, None; A. Moss, None; S. Trim, None; M. Fidock, None; H. West,<br />
None; K. Patel, None; S. Phillips, None; P. Cox, None; D. Williams, None; K.H. Holmberg,<br />
None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.25/HH32<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques
Title: Essential role of AKAP79/150 <strong>an</strong>choring protein in regulation of CREB signaling<br />
Authors: *G. ARAMUNI, M. FRIEDRICH, M. MANK, O. GRIESBECK;<br />
Lab. of Cell. Dynamics, MPI of Neurobio., Munich, Germ<strong>an</strong>y<br />
Abstract: The cAMP response element binding protein CREB <strong>an</strong>d its close relatives activating<br />
tr<strong>an</strong>scription factor 1 (ATF1) <strong>an</strong>d <strong>the</strong> cAMP response element modulatory protein (CREM) are<br />
tr<strong>an</strong>scription factors that mediate tr<strong>an</strong>scriptional activation of genes in response to various<br />
extracellular signals <strong>an</strong>d are involved in diverse processes r<strong>an</strong>ging <strong>from</strong> development to<br />
plasticity to disease. To investigate activation of CREB due to phosphorylation of <strong>the</strong> critical<br />
serine 133, we present a genetically encoded fluorescent reporter <strong>for</strong> CREB consisting of fusions<br />
of cy<strong>an</strong> fluorescent protein (CFP), a kinase inducible domain (KID) of CREB, a KIX of CREB<br />
binding protein (CBP), <strong>an</strong>d yellow fluorescent protein (YFP). The Indicator of CREB Activation<br />
due to Phosphorylation (ICAP) shows 20-40 % emission ratio ch<strong>an</strong>ge in response to applications<br />
of PKA or CaMK IV in vitro <strong>an</strong>d in vivo. ICAP allows studying <strong>the</strong> role of <strong>the</strong> scaffold <strong>an</strong>d<br />
<strong>an</strong>choring protein AKAP79/150 (79 hum<strong>an</strong>/150 rodent) in CREB regulation in response to<br />
different chemical <strong>an</strong>d electrical stimulations in living hippocampal neurons using FRET <strong>an</strong>d<br />
electrophysiological measurements. It has been previously demonstrated that AKAP79/150 binds<br />
protein kinase A (PKA), protein kinase C (PKC), Calcineurin <strong>an</strong>d modulates L-type voltage<br />
dependent calcium ch<strong>an</strong>nels. <strong>When</strong> acutely disrupting AKAP79/150 <strong>an</strong>choring to PKA, we<br />
observed dramatical reduction in PKA mediated CREB activation. In contrast, disrupting <strong>the</strong><br />
binding of Calcineurin to AKAP79/150 <strong>an</strong>d <strong>the</strong> L-type calcium ch<strong>an</strong>nel, had only a subtle effect<br />
on depolarization induced CREB activation by enh<strong>an</strong>cing CREB activation in depolarized<br />
neurons in <strong>the</strong> presence of <strong>for</strong>skolin. Thus, PKA <strong>an</strong>d Calcineurin <strong>an</strong>choring to AKAP79/150 is<br />
import<strong>an</strong>t <strong>for</strong> CREB regulation. These observations demonstrate <strong>the</strong> high potential of our new<br />
fluorescent reporter ICAP <strong>for</strong> studying <strong>the</strong> dynamics of various signaling complexes in specific<br />
cell types <strong>an</strong>d tissues.<br />
Disclosures: G. Aramuni, None; M. Friedrich, None; M. M<strong>an</strong>k, None; O. Griesbeck, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.26/HH33<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques<br />
Title: Novel multi-color immunofluorescence technique using primary <strong>an</strong>tibodies raised in <strong>the</strong><br />
same host species
Authors: *A. E. KALYUZHNY 1 , J. P. HOUCHINS 1 , M. GRAHEK 1 , J. FRISCH 1 , J.<br />
SCHOEPHOERSTER 1 , J. HAGEN 1 , J. SWEET 1 , L. G. MENDOZA 2 , D. SCHWARTZ 2 ;<br />
1 R&D Systems, Inc., Minneapolis, MN; 2 SoluLink, Inc., S<strong>an</strong> Diego, CA<br />
Abstract: Simult<strong>an</strong>eous detection of multiple tissue <strong>an</strong>tigens is one of <strong>the</strong> most frequently used<br />
immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of secondary<br />
<strong>an</strong>tibodies with unw<strong>an</strong>ted primary <strong>an</strong>tibodies when doing ei<strong>the</strong>r dual- or triple-labeling<br />
immunofluorescence it is necessary to use primary <strong>an</strong>tibodies raised in different host species<br />
such as mouse, rabbit <strong>an</strong>d goat <strong>an</strong>tibodies. However, in m<strong>an</strong>y cases suitable primary <strong>an</strong>tibodies<br />
raised in different species are unavailable. We have developed a novel technique <strong>for</strong> triplelabeling<br />
immunofluorescence IHC that c<strong>an</strong> be used with all primary <strong>an</strong>tibodies derived <strong>from</strong> <strong>an</strong>y<br />
single host source. This technique includes modification of one primary <strong>an</strong>tibody with biotin<br />
(ChromaLink TM Biotin) <strong>an</strong>d a second primary <strong>an</strong>tibody with DIG (ChromaLink TM Digoxigenin).<br />
For IHC staining cells or tissue are incubated first with unconjugated primary <strong>an</strong>tibody against<br />
1 st target protein followed by detection with <strong>an</strong>ti-primary secondary <strong>an</strong>tibody conjugated to<br />
Nor<strong>the</strong>rnLights TM NL-493 tag (green fluorescence). Subsequently, <strong>the</strong> same tissue sections were<br />
incubated with a mixture of same species biotin-labeled primary <strong>an</strong>tibody (against <strong>the</strong> 2 nd target<br />
protein) <strong>an</strong>d DIG-labeled primary <strong>an</strong>tibody (against <strong>the</strong> 3 rd target protein) followed by detection<br />
using a mixture of Streptavidin Nor<strong>the</strong>rnLights TM NL-557 tag (red fluorescence) <strong>an</strong>d <strong>an</strong>ti-DIG<br />
secondary <strong>an</strong>tibody conjugated to a Cy5 TM tag (fluorescence in far red spectral region).<br />
This novel technique provides good spectral separation of colors depicting different <strong>an</strong>tigens of<br />
interest while avoiding cross-reactivity between irrelev<strong>an</strong>t primary <strong>an</strong>d secondary <strong>an</strong>tibodies. In<br />
addition, this multiplexed IHC technique provides signific<strong>an</strong>t convenience to researchers who<br />
have at <strong>the</strong>ir disposal only primary <strong>an</strong>tibodies raised in <strong>the</strong> same host species.<br />
ChromaLink is a trademark of SoluLink, Inc.<br />
Nor<strong>the</strong>rnLights is a trademark of R&D Systems, Inc.<br />
Cy5 is a trademark of GE Healthcare<br />
Disclosures: A.E. Kalyuzhny, University of Minnesota, A. Employment (full or parttime);<br />
J.P. Houchins, None; M. Grahek, None; J. Frisch, University of Minnesota, A.<br />
Employment (full or part-time); J. Schoephoerster, None; J. Hagen, None; J. Sweet,<br />
None; L.G. Mendoza, None; D. Schwartz, None.<br />
Poster<br />
586. Imaging Techniques VI<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 586.27/HH34<br />
Topic: G.03. Staining, Tracing, <strong>an</strong>d Imaging Techniques
Support: Tore Nilsson foundation<br />
Title: Cell proliferation in <strong>the</strong> adult hum<strong>an</strong> dentate gyrus<br />
Authors: O. BENDEL 1 , K. ALKASS 2 , *H. DRUID 3 ;<br />
1 Dept of Forensic Med., Karolinska Institutet, Stockholm, Sweden; 2 Dept of Oncology-<br />
Pathology, Forensic Med., Stockholm, Sweden; 3 Dept of Forensic Med., Fac. of Med., SE-171<br />
77 Stockholm, Sweden<br />
Abstract: Formation of new neurons <strong>from</strong> neuronal stem or progenitor cells, neurogenesis,<br />
continues throughout life in discrete brain regions of vertebrate species including hum<strong>an</strong>s<br />
(Altm<strong>an</strong> <strong>an</strong>d Das, 1965, Eriksson, et al., 1998, Kapl<strong>an</strong> <strong>an</strong>d Hinds, 1977). The adult rat brain<br />
contains two neurogenic regions: <strong>the</strong> subventrical zone (SVZ) of <strong>the</strong> lateral ventricles <strong>an</strong>d <strong>the</strong><br />
subgr<strong>an</strong>ular zone (SGZ) at <strong>the</strong> border of <strong>the</strong> hilar region <strong>an</strong>d gr<strong>an</strong>ule cell layer of <strong>the</strong> dentate<br />
gyrus (DG). The aim of <strong>the</strong> present study was to investigate cell proliferation in <strong>the</strong> adult hum<strong>an</strong><br />
subgr<strong>an</strong>ular zone (SGZ). Brain samples were collected <strong>from</strong> eight deceased donors subjected to a<br />
<strong>for</strong>ensic autopsy at <strong>the</strong> Department of Forensic Medicine in Stockholm. All donors had earlier<br />
history of drug abuse or depression, or both (26-67 years of age). Samples were frozen in icecold<br />
isopent<strong>an</strong>e <strong>an</strong>d later cut in 20 µm sections. For detection of newborn cells, sections were<br />
immunohistochemically labeled with <strong>an</strong>tibody against KI-67. For detection of progenitor cells,<br />
we also used <strong>an</strong> <strong>an</strong>tibody against hum<strong>an</strong> Nestin. In <strong>the</strong> DG, we occasionally observed a small<br />
number of KI-67 positive cells, suggesting that <strong>the</strong> generation of new gr<strong>an</strong>ule cells in <strong>the</strong> hum<strong>an</strong><br />
adult hippocampus occurs at a slow rate. However, several donors had <strong>an</strong> accumulation of<br />
Nestin-positive cells near <strong>the</strong> outer blade of <strong>the</strong> DG. Because of <strong>the</strong>ir proximity to <strong>the</strong> lateral<br />
ventricle, such Nestin-positive cells may originate <strong>from</strong> <strong>the</strong> SVZ <strong>an</strong>d later migrate to <strong>the</strong> DG.<br />
Our results show that fur<strong>the</strong>r work is needed be<strong>for</strong>e we c<strong>an</strong> underst<strong>an</strong>d <strong>the</strong> complexity of adult<br />
neurogenesis, <strong>an</strong>d that hum<strong>an</strong> post-mortem studies are necessary in this respect.<br />
Disclosures: O. Bendel, None; K. Alkass, None; H. Druid, None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.1/HH35<br />
Topic: G.04. Physiological Methods<br />
Support: HFSP gr<strong>an</strong>t (RGP0049/2002) to LJG
Agence Nationale de Recherche gr<strong>an</strong>t (FUNVISYNIN) to LJG<br />
Title: Estimation of access resist<strong>an</strong>ce <strong>for</strong> whole-cell patch recordings in vivo : Comparison of<br />
methods <strong>an</strong>d implications <strong>for</strong> conduct<strong>an</strong>ce measurement <strong>an</strong>d dynamic clamp protocols<br />
Authors: *D. MARINAZZO, A. SCHRAMM, L. J. GRAHAM;<br />
Lab. of Neurophysics <strong>an</strong>d Physiology, CNRS, Univ. Paris Descartes, Paris, Fr<strong>an</strong>ce<br />
Abstract: A fundamental challenge of qu<strong>an</strong>titative intracellular electrophysiology is <strong>the</strong><br />
estimation of, <strong>an</strong>d eventual compensation <strong>for</strong>, <strong>the</strong> artifacts due to <strong>the</strong> recording electrode. These<br />
estimations <strong>an</strong>d corrections are especially import<strong>an</strong>t <strong>for</strong> protocols designed to measure or<br />
m<strong>an</strong>ipulate membr<strong>an</strong>e <strong>an</strong>d synaptic conduct<strong>an</strong>ces. In general, <strong>for</strong> sufficiently small signals such<br />
that activation of non-linear membr<strong>an</strong>e properties is minimized, <strong>the</strong> system response of a neuron<br />
<strong>an</strong>d electrode is composed of sum of real exponentials. We revisit <strong>the</strong> problem of estimating <strong>the</strong><br />
coefficients of <strong>the</strong>se terms <strong>an</strong>d <strong>the</strong> implications <strong>for</strong> measuring neuronal properties, specifically in<br />
<strong>the</strong> case of whole-cell patch electrodes during in vivo recording, using values <strong>for</strong> cortical<br />
neurons recorded in vivo <strong>an</strong>d measured values of patch electrode capacit<strong>an</strong>ce in cortex. We<br />
compare methods <strong>for</strong> extracting circuit parameters <strong>from</strong> tr<strong>an</strong>sient responses made in current<br />
clamp mode: exponential peeling, Fourier tr<strong>an</strong>s<strong>for</strong>m based models <strong>an</strong>d non-linear regression to<br />
target functions. We also consider two explicit resistor-capacitor (RC) models <strong>for</strong> <strong>the</strong> electrode:<br />
<strong>an</strong> R (i.e. access resist<strong>an</strong>ce) in parallel with a C between <strong>the</strong> amplifier <strong>an</strong>d neuron, <strong>an</strong>d a more<br />
realistic model in which <strong>the</strong> electrode C is connected to ground. We find that <strong>for</strong> small values of<br />
R (
Program#/Poster#: 587.2/HH36<br />
Topic: G.04. Physiological Methods<br />
Support: NSF DBI-0551852<br />
Title: Design <strong>an</strong>d development of a novel electrode to improve recordings in whole-cell patch<br />
clamp experiments<br />
Authors: *S. INAYAT 1 , Y. ZHAO 1 , D. A. DIKIN 2 , R. S. RUOFF 3 , J. B. TROY 1 ;<br />
1 Biomed. Engin., 2 Mech<strong>an</strong>ical Engin. Dept. , Physics <strong>an</strong>d Astronomy Dept., Northwestern Univ.,<br />
Ev<strong>an</strong>ston, IL; 3 Mech<strong>an</strong>ical Engin., Univ. of Texas, Austin, TX<br />
Abstract: In <strong>the</strong> voltage clamp mode of conventional whole-cell patch clamp experiments, <strong>the</strong><br />
actual membr<strong>an</strong>e voltage is less th<strong>an</strong> <strong>the</strong> comm<strong>an</strong>d voltage by <strong>an</strong> amount equal to <strong>the</strong> voltage<br />
drop across <strong>the</strong> pipette resist<strong>an</strong>ce. The pipette resist<strong>an</strong>ce also limits <strong>the</strong> b<strong>an</strong>dwidth of recording<br />
since it <strong>for</strong>ms a low pass filter with <strong>the</strong> cell membr<strong>an</strong>e capacit<strong>an</strong>ce. The bulk of <strong>the</strong> pipette<br />
resist<strong>an</strong>ce is located at <strong>the</strong> tip <strong>an</strong>d increases dramatically if <strong>the</strong> tip clogs with cellular debris<br />
during <strong>an</strong> experiment. This effect is common while recording <strong>from</strong> small structures such as<br />
neuronal dendrites <strong>an</strong>d axons. We present <strong>the</strong> design <strong>an</strong>d development of a novel patch-clamp<br />
electrode to overcome <strong>the</strong> limitations introduced due to <strong>the</strong> pipette resist<strong>an</strong>ce <strong>an</strong>d tip clogging. In<br />
a conventional patch clamp electrode, <strong>the</strong> Ag/AgCl wire sits stationary inside <strong>the</strong> pipette, a few<br />
millimeters away <strong>from</strong> <strong>the</strong> tip. In <strong>the</strong> novel push-pen patch clamp electrode, a linear actuator<br />
moves <strong>the</strong> Ag/AgCl wire inside <strong>the</strong> pipette until it protrudes <strong>from</strong> <strong>the</strong> pipette orifice, like a pushpen.<br />
The push-pen operation is intended to eliminate or minimize <strong>the</strong> pipette resist<strong>an</strong>ce.<br />
We have designed <strong>an</strong>d developed a prototype of <strong>the</strong> push-pen patch clamp electrode <strong>an</strong>d<br />
per<strong>for</strong>med imped<strong>an</strong>ce measurements implementing <strong>the</strong> push-pen operation. A linear actuator<br />
(piezoelectric n<strong>an</strong>omotor) was incorporated into <strong>the</strong> pipette holder of a conventional patch clamp<br />
electrode. The actuator moves <strong>the</strong> Ag/AgCl wire back <strong>an</strong>d <strong>for</strong>th inside <strong>the</strong> pipette with a motion<br />
resolution of ~50nm. The push-pen operation is achieved by using <strong>an</strong> Ag/AgCl wire with a<br />
conical tip. To obtain a conical tip, <strong>the</strong> Ag wire is first etched <strong>an</strong>d <strong>the</strong>n coated electrolytically<br />
with AgCl. The pipettes are pulled on commercial pipette pullers.<br />
With <strong>the</strong> push-pen operation, we have observed a considerable decrease in <strong>the</strong> imped<strong>an</strong>ce of <strong>the</strong><br />
electrode over a wide frequency r<strong>an</strong>ge. Next, we pl<strong>an</strong> to test <strong>the</strong> new patch clamp electrode <strong>for</strong><br />
its efficacy in whole-cell patch clamp experiments while recording <strong>from</strong> neuronal dendrites <strong>an</strong>d<br />
axons. With <strong>the</strong> conventional patch clamp electrodes, <strong>the</strong> typical duration of recording is a few<br />
minutes as <strong>an</strong> increase in <strong>the</strong> pipette resist<strong>an</strong>ce due to tip clogging happens quickly <strong>an</strong>d renders<br />
<strong>the</strong> experiment inoperative. We expect to prolong experiment lifetimes with <strong>the</strong> use of <strong>the</strong> new<br />
electrode.<br />
Disclosures: S. Inayat, None; Y. Zhao, None; D.A. Dikin, None; R.S. Ruoff, None; J.B.<br />
Troy, None.<br />
Poster
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.3/HH37<br />
Topic: G.04. Physiological Methods<br />
Title: A microelectrode with carbon n<strong>an</strong>otube bundles <strong>for</strong> single-cell electrophysiology<br />
Authors: K. KOHIRA, T. MOTOMURA, *Y. HAYASHIDA, N. MURAYAMA;<br />
Grad.Sci.&Tech, Kumamoto Univ., Kumamoto-shi, Jap<strong>an</strong><br />
Abstract: Electrically interfacing neurons with carbon n<strong>an</strong>otubes (CNTs) has been recently<br />
demonstrated in-vitro as well as in-vivo experiments, in which <strong>the</strong> electrode surface was coated<br />
with CNTs, or vertically aligned CNTs were grown as <strong>an</strong> electrode on flat substrate. Since CNTs<br />
are known to have mech<strong>an</strong>ical flexibility <strong>an</strong>d electrical conductivity, it is expected that, as a<br />
substitute <strong>for</strong> conventional glass microelectrodes, CNT bundle of micron to sub-micron diameter<br />
c<strong>an</strong> be potentially used <strong>for</strong> single-cell electrophysiology. In this study, we made attempts to<br />
fabricate CNT bundle electrodes with various diameters <strong>an</strong>d lengths by a simple electrophoretic<br />
deposition method. Single- or multi-walled CNTs, as-purchased or after refluxing in heated<br />
H2O2, were dispersed in dehydrated N,N-dimethyl<strong>for</strong>mide with sonication; <strong>the</strong> impurities were<br />
filtered out of solution, if necessary. The tip of <strong>an</strong> electropolished tungsten wire was submerged<br />
into <strong>the</strong> CNT dispersion solution <strong>an</strong>d a direct-current or alternating-current electric field was<br />
applied between <strong>the</strong> tungsten wire as <strong>an</strong>ode <strong>an</strong>d a cathode reference electrode. <strong>When</strong> <strong>the</strong> CNT<br />
deposition started to take place, <strong>the</strong> tungsten wire was lifted away <strong>from</strong> <strong>the</strong> dispersion solution<br />
by using a computer-controlled microm<strong>an</strong>ipulator; resulting in <strong>the</strong> <strong>for</strong>mation of <strong>an</strong> elongated<br />
CNT bundle assemblage attached to <strong>the</strong> tip of tungsten wire. All procedures were per<strong>for</strong>med in<br />
<strong>an</strong> open-air chamber at room temperature. By adjusting <strong>the</strong> concentration of <strong>the</strong> CNTs dispersed,<br />
<strong>the</strong> strength <strong>an</strong>d frequency of <strong>the</strong> electrical field applied, <strong>an</strong>d <strong>the</strong> speed of lifting <strong>the</strong> tungsten<br />
wire, we could produce <strong>the</strong> CNT bundle assemblages of 0.01 to 2 mm length, tapering to 0.1-10<br />
microns diameter at <strong>the</strong> end. After drying, <strong>the</strong> sh<strong>an</strong>k of <strong>the</strong> tungsten-CNT bundle electrodes was<br />
coated with silicone elastomer <strong>for</strong> electrical insulation; so that <strong>the</strong> electrical imped<strong>an</strong>ce was in<br />
kilo- to giga-ohm r<strong>an</strong>ge when measured in physiological saline. There<strong>for</strong>e, it was suggested that<br />
<strong>the</strong> electrodes we fabricated here satisfy <strong>the</strong> specifications required <strong>for</strong> recordings <strong>from</strong> or<br />
stimulations of single neurons. The usability of those electrodes in in-vitro experiments is<br />
examined in acutely dissociated retinal neurons.<br />
Disclosures: K. Kohira, None; T. Motomura, None; Y. Hayashida, None; N. Murayama,<br />
None.<br />
Poster
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.4/HH38<br />
Topic: G.04. Physiological Methods<br />
Support: This work was supported by <strong>the</strong> National Honor Scientist Program of Korea.<br />
Title: Precision automatic microdrive array <strong>for</strong> chronic single unit recordings in freely behaving<br />
mice<br />
Authors: *S. YANG 1 , J. KIM 1 , S. LEE 1 , J. CHO 2 , H.-S. SHIN 2 , E.-S. YOON 1 ;<br />
1 2<br />
N<strong>an</strong>obio Res. Ctr., Ctr. <strong>for</strong> Neural Sci., Korea Institue of Sciecne <strong>an</strong>d Technol., Seoul,<br />
Republic of Korea<br />
Abstract: Extracellular single unit recordings in freely <strong>an</strong>imals have played a crucial role in<br />
underst<strong>an</strong>ding neuronal activities in relations with specific behaviors. However, it has been a<br />
challenge to record neural signals <strong>from</strong> freely behaving mice due to <strong>the</strong> small size of mice <strong>an</strong>d<br />
relatively large <strong>an</strong>d heavy recording devices even though a variety of genetically-engineered<br />
mice have been preferably used to efficiently study m<strong>an</strong>y neurological disorders. As a result,<br />
lightweight m<strong>an</strong>ual microdrives, that adv<strong>an</strong>ce electrodes by h<strong>an</strong>d-operated m<strong>an</strong>ipulation, have<br />
been widely used in order to increase <strong>the</strong> probability of obtaining desired neurons during single<br />
cell recordings in freely behaving small <strong>an</strong>imals. Never<strong>the</strong>less, <strong>the</strong> rough adjustments of<br />
electrodes by rotating screws on microdrives do not allow single units to be easily isolated.<br />
Fur<strong>the</strong>rmore, while electrodes are adv<strong>an</strong>cing, <strong>an</strong>imals should be restrained <strong>an</strong>d thus it c<strong>an</strong> cause<br />
abrupt drifts of <strong>the</strong> electrodes due to struggling. Hence, a microdrive array based on piezomotors<br />
<strong>for</strong> automatically adv<strong>an</strong>cing electrodes during chronic single unit recordings in freely<br />
behaving mice is proposed. The modular design <strong>an</strong>d enh<strong>an</strong>ced clamping mech<strong>an</strong>ism <strong>for</strong> <strong>the</strong><br />
piezo-motor enable a micro-driving unit to adv<strong>an</strong>ce each electrode bundle reliably within <strong>the</strong><br />
stroke of 3 mm. The weight of <strong>the</strong> micro-driving unit is 0.45 g only, including <strong>the</strong><br />
magnetoresistive (MR) position to monitor <strong>the</strong> displacement of electrodes <strong>an</strong>d <strong>the</strong> total weight of<br />
<strong>the</strong> microdrive array is 3.0 g when 3 micro-driving units are integrated on it. This lightweight<br />
<strong>an</strong>d automatic microdrive array is suitable enough to record neural signals in freely mice.<br />
Finally, single unit activities are successfully isolated with small step adjustment in freely<br />
behaving mice.<br />
Disclosures: S. Y<strong>an</strong>g, None; J. Kim, None; S. Lee, None; J. Cho, None; H. Shin, None; E.<br />
Yoon, None.<br />
Poster
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.5/HH39<br />
Topic: G.04. Physiological Methods<br />
Support: NSF 0615987<br />
Title: Development of a zinc-selective self-referencing microelectrode <strong>for</strong> detection of zinc<br />
release <strong>from</strong> individual neurons<br />
Authors: *R. P. MALCHOW 1 , J. JACOBY 2 , B. K. TCHERNOOKOVA 2 , R. L. CHAPPELL 3 ;<br />
1 Dept. Biol. Sci. & Opthalmology, Univ. Illinois @ Chicago, Chicago, IL; 2 Biol. Sci., Univ. of<br />
Illinois at Chicago, Chicago, IL; 3 Biol. Sci., Hunter College, C.U.N.Y., New York, NY<br />
Abstract: There is mounting evidence that zinc released <strong>from</strong> glutamatergic nerve terminals<br />
serves as a neuromodulator at synaptic sites within <strong>the</strong> retina <strong>an</strong>d o<strong>the</strong>r areas of <strong>the</strong> central<br />
nervous system. However, it has proven difficult to reliably measure <strong>the</strong> concentration of zinc<br />
co-released with glutamate at such synapses. Our goal is to develop zinc-selective<br />
microelectrodes that c<strong>an</strong> be used in a self-referencing <strong>for</strong>mat in order to detect release of zinc<br />
<strong>from</strong> vertebrate photoreceptors. We have prepared microelectrodes using zinc ionophore I (cat#<br />
96491 <strong>from</strong> Sigma Chemical), <strong>the</strong> same ionophore previously employed as a zinc-selective<br />
macroelectrode (Kojima et al. 1994). We mixed this selectophore (10% by weight) with 2nitrophenyl<br />
octyl e<strong>the</strong>r (o-NOPE) (89%) <strong>an</strong>d potassium tetrakis(4-chlorophenyl)borate (1%) <strong>an</strong>d<br />
drew a small qu<strong>an</strong>tity of this mixture into glass micropipettes (tip diameter ~ 2-4 µm) that had<br />
previously been backfilled with 100 mM ZnCl <strong>an</strong>d 10 mM HEPES. Zinc source pipettes were<br />
prepared by mixing 100 mM ZnCl <strong>an</strong>d 10 mM HEPES in 1% agar. At steady state <strong>the</strong> thickness<br />
of <strong>the</strong> agar <strong>an</strong>d <strong>the</strong> dilution resulting <strong>from</strong> time result in <strong>an</strong> ion concentration at <strong>the</strong> mouth of <strong>the</strong><br />
source pipette that is typically 0.1-1% of <strong>the</strong> original concentration in <strong>the</strong> source pipette. Zincsensitive<br />
microelectrodes containing <strong>the</strong> zinc ionophore were placed within several microns of<br />
zinc source pipettes <strong>an</strong>d <strong>the</strong>n used in a self referencing m<strong>an</strong>ner. In this recording method, <strong>the</strong><br />
micropipette containing <strong>the</strong> zinc ionophore is moved back <strong>an</strong>d <strong>for</strong>th between two known<br />
positions <strong>an</strong>d a differential reading obtained, subtracting <strong>the</strong> reading <strong>from</strong> <strong>the</strong> far position <strong>from</strong><br />
that closer to <strong>the</strong> source. This procedure eliminates slow electrical drift <strong>an</strong>d noise <strong>an</strong>d increases<br />
<strong>the</strong> effective sensitivity of such ion-selective electrodes by about 1000X. Using this method, we<br />
have successfully measured electrical potentials in <strong>the</strong> pipettes containing <strong>the</strong> zinc ionophore that<br />
increase as <strong>the</strong> concentration of zinc is increased. The signal declines as <strong>the</strong> dist<strong>an</strong>ce between <strong>the</strong><br />
zinc source pipette <strong>an</strong>d <strong>the</strong> pipette containing <strong>the</strong> zinc ionophore increases. Finally, <strong>the</strong> signal is<br />
signific<strong>an</strong>tly <strong>an</strong>d progressively reduced by <strong>the</strong> addition of increasing amounts of <strong>the</strong> divalent<br />
chelator EDTA to <strong>the</strong> bath. These results suggest that <strong>the</strong> microelectrodes we have prepared c<strong>an</strong><br />
indeed detect <strong>the</strong> presence of zinc <strong>an</strong>d may be useful in detecting <strong>an</strong>d measuring zinc release<br />
<strong>from</strong> <strong>the</strong> synaptic terminals of vertebrate photoreceptors <strong>an</strong>d o<strong>the</strong>r nerve cells.
Disclosures: R.P. Malchow, None; J. Jacoby, None; B.K. Tchernookova, None; R.L.<br />
Chappell, NSF 0615987, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t<br />
<strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received).<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.6/HH40<br />
Topic: G.04. Physiological Methods<br />
Support: NIH Gr<strong>an</strong>t R21 RR25896<br />
NIH Gr<strong>an</strong>t DK20541<br />
Title: Non-invasive evaluation of conduction velocity in A-delta <strong>an</strong>d C fibers applying summed<br />
multiple unit procedures<br />
Authors: *E. G. ZOTOVA 1 , J. C. AREZZO 2 ;<br />
1 Neurol. Dept, 2 Neurol. Dept <strong>an</strong>d Neurosci. Dept, Albert Einstein Col. Med., Bronx, NY<br />
Abstract: St<strong>an</strong>dard measures of maximal nerve conduction velocity (NCV) are exclusively<br />
sensitive to <strong>the</strong> integrity of large diameter myelinated axons. However, structural <strong>an</strong>d functional<br />
deficits in small diameter, myelinated <strong>an</strong>d unmyelinated axons are subst<strong>an</strong>tial components of<br />
autonomic <strong>an</strong>d painful somatic neuropathies (e.g. toxic neuropathy). Propagating signals in<br />
bundles of small diameter axons are small, fragmented, subject to extensive temporal dispersion,<br />
<strong>an</strong>d due to phase c<strong>an</strong>cellation, <strong>the</strong>y are often diminished by averaging. Previously, we<br />
successfully applied procedures designed to assess multiple unit activity (MUA) in <strong>the</strong> CNS to<br />
<strong>the</strong> evaluation of A-delta <strong>an</strong>d C fiber activity in exposed peripheral nerves (sciatic <strong>an</strong>d cavernous<br />
nerves). The present study extends this work to <strong>the</strong> assessment of NCV in intact, small diameter<br />
fibers using non-invasive procedures. Compound neural activity was evaluated in <strong>the</strong> distal<br />
extremes of digital <strong>an</strong>d caudal nerves of rats under general <strong>an</strong>es<strong>the</strong>sia. Responses were explored<br />
as a function of increasing stimulus intensity r<strong>an</strong>ging <strong>from</strong> threshold activation to current<br />
adequate to generate maximal whole nerve responses (i.e., const<strong>an</strong>t current 1-15 mA; 0.01-6.0<br />
ms). As expected due to threshold considerations, activity in <strong>the</strong> slowest conducting axons was<br />
only detected using <strong>the</strong> highest intensity stimulation. MUA was isolated by examining single<br />
sweeps, filtered at 450 Hz-5 kHz, rectified, <strong>an</strong>d digitized at a rate exceeding 20 kHz prior to<br />
averaging. Evoked responses were compared to pre-stimulus baseline patterns; measures<br />
included <strong>the</strong> onset, duration, <strong>an</strong>d area under specific regions of <strong>the</strong> MUA curve. These
procedures allowed <strong>the</strong> reliable identification of activity reflecting three groups of slow<br />
conducting velocities (ie., 2.1-5.0 m/sec, 1.0-2.0 m/sec <strong>an</strong>d less th<strong>an</strong> 1.0 m/sec). Administration<br />
of acrylamide monomer at doses of 30 to 40 mg/kg induced <strong>an</strong> alteration of <strong>the</strong> pattern of slow<br />
conducting NCV <strong>an</strong>d 50% reduction in <strong>the</strong> magnitude of activity in <strong>the</strong> slow conducting r<strong>an</strong>ge;<br />
deficits were first evident after 10 days of exposure. The acrylamide-induced ch<strong>an</strong>ges were<br />
initially detected in <strong>the</strong> most distal segments of <strong>the</strong> digital nerve (<strong>an</strong>kle to toe segment) <strong>an</strong>d in <strong>the</strong><br />
distal caudal nerve. These studies suggest that non-invasive electrophysiological evaluation of<br />
slow conduction at <strong>the</strong> distal extremes of nerves could provide a valuable biomarker <strong>for</strong> <strong>the</strong><br />
onset, progression <strong>an</strong>d possible effects of <strong>the</strong>rapy on small fiber peripheral neuropathies.<br />
Disclosures: E.G. Zotova, None; J.C. Arezzo, None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.7/HH41<br />
Topic: G.04. Physiological Methods<br />
Support: NIH Gr<strong>an</strong>t P30EY012576<br />
Title: An interactive software tool <strong>for</strong> stereotactic pl<strong>an</strong>ning of recording chamber placement <strong>an</strong>d<br />
electrode trajectories<br />
Authors: D. J. SPERKA 1 , *J. DITTERICH 2 ;<br />
1 2<br />
Ctr. <strong>for</strong> Neurosci., Ctr. <strong>for</strong> Neurosci. & Dept. of Neurobiology, Physiol. & Behavior, Univ. of<br />
Cali<strong>for</strong>nia, Davis, CA<br />
Abstract: Extracellular cortical recordings <strong>from</strong> a particular brain area require a cr<strong>an</strong>iotomy at<br />
<strong>an</strong> appropriate location <strong>an</strong>d impl<strong>an</strong>tation of a recording chamber <strong>for</strong> <strong>the</strong> introduction of recording<br />
electrodes. The recording chamber must be placed <strong>an</strong>d oriented to allow access to <strong>the</strong> target area.<br />
While m<strong>an</strong>y different placements c<strong>an</strong> access <strong>the</strong> target, each will penetrate different brain areas<br />
along <strong>the</strong> way. It is extremely useful to know what locations inside <strong>the</strong> recording chamber will<br />
access particular subregions of <strong>the</strong> target area <strong>an</strong>d how deep each penetration must be. While<br />
stereotactic pl<strong>an</strong>ning using imaging techniques <strong>an</strong>d software is common in hum<strong>an</strong> neurosurgery,<br />
<strong>an</strong>imal researchers largely still rely on 2D st<strong>an</strong>dard brain atlases. This has a number of<br />
disadv<strong>an</strong>tages: Pl<strong>an</strong>ning a stereotactic approach c<strong>an</strong> be tedious <strong>an</strong>d error-prone, <strong>an</strong>d<br />
interindividual <strong>an</strong>atomical differences c<strong>an</strong>not be taken into account.<br />
We present <strong>an</strong> interactive software tool built on top of <strong>the</strong> free, open-source neuro<strong>an</strong>atomy
application Caret (V<strong>an</strong> Essen et al., 2001). A recording target location c<strong>an</strong> be defined ei<strong>the</strong>r on a<br />
functional flat map or in <strong>the</strong> structural MRI volume. The tool places a recording chamber on <strong>the</strong><br />
skull surface (<strong>the</strong> topology of which is extracted <strong>from</strong> <strong>an</strong> MRI image of <strong>the</strong> subject) in such a<br />
way that <strong>the</strong> symmetry axis of <strong>the</strong> recording chamber passes through <strong>the</strong> target point <strong>an</strong>d <strong>the</strong><br />
dist<strong>an</strong>ce between <strong>the</strong> skull entry point <strong>an</strong>d <strong>the</strong> target location is minimized. The user c<strong>an</strong> explore<br />
different approaches by moving <strong>the</strong> recording chamber along <strong>the</strong> skull surface. The tool<br />
constrains <strong>the</strong> orientation so that <strong>the</strong> symmetry axis of <strong>the</strong> chamber always passes through <strong>the</strong><br />
target point. The stereotactic coordinates of <strong>the</strong> cr<strong>an</strong>iotomy location <strong>an</strong>d <strong>the</strong> associated <strong>an</strong>gles <strong>for</strong><br />
chamber orientation <strong>an</strong>d shape are provided. All parts of cortex accessible with a particular<br />
approach <strong>an</strong>d penetration depth are shown in both <strong>the</strong> flat map <strong>an</strong>d <strong>the</strong> MRI volume.<br />
The availability of such a software tool c<strong>an</strong> make stereotactic pl<strong>an</strong>ning of cortical recordings<br />
easier <strong>an</strong>d more reliable, <strong>an</strong>d c<strong>an</strong> help justify recording locations without histology if <strong>an</strong> MRI<br />
with appropriate markers is obtained after <strong>the</strong> recording chamber has been impl<strong>an</strong>ted.<br />
Disclosures: D.J. Sperka, None; J. Ditterich, None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.8/HH42<br />
Topic: G.07. Data Analysis <strong>an</strong>d Statistics<br />
Title: Modification of altered pattern of muscle activity in patients with knee osteoarthritis after<br />
intra-articular hyaluronic acid injections<br />
Authors: *S. F.-T. TANG;<br />
Dept. of Rehabil. Med., Ch<strong>an</strong>g Gung Mem. Hosp., Guish<strong>an</strong> Township, Taiw<strong>an</strong><br />
Abstract: Introduction : Osteoarthritis(OA) is a degenerative joint disease <strong>an</strong>d is one of <strong>the</strong><br />
most commonly diagnosed chronic medical condition. Knee OA is frequency associated with<br />
pain, muscle weakness, <strong>an</strong>d limitation in joint r<strong>an</strong>ge of motion. In recent studies, using<br />
medications <strong>for</strong> <strong>the</strong> purpose of pain alleviation in patients with knee OA is not recommended.<br />
Besides <strong>the</strong> conventional treatments of <strong>an</strong>algesic drugs <strong>an</strong>d physio<strong>the</strong>rapy, <strong>the</strong> practice of<br />
viscosupplementation using intra-articular (IA) injection of hyaluronic acid (HA) has proven to<br />
be effective.<br />
Presently, <strong>the</strong>re are no studies so far using EMG to compare <strong>the</strong> muscle activities of knee OA<br />
patients be<strong>for</strong>e <strong>an</strong>d after a certain treatment strategy. We propose that <strong>the</strong>re will be signific<strong>an</strong>t<br />
improvements <strong>an</strong>d ch<strong>an</strong>ges in muscle activation patterns in knee OA patients after <strong>the</strong>
completion IA HA injections.<br />
Methods : Twenty-four subjects with OA <strong>an</strong>d 15 age-matched non-knee OA control subjects<br />
were recruited <strong>from</strong> <strong>an</strong> outpatient clinic. For <strong>the</strong> knee OA group, bilateral IA knee joint<br />
injections with HA were per<strong>for</strong>med. The quadriceps (QUA), hamstrings (HAM), tibialis <strong>an</strong>terior<br />
(TA), <strong>an</strong>d medial gastrocnemius (MG) muscles were selected <strong>for</strong> this study. Data acquisition was<br />
accomplished by using <strong>the</strong> MA-100 EMG system to measure <strong>the</strong> muscles activities. In <strong>the</strong><br />
<strong>an</strong>alysis of gait cycle, <strong>the</strong> Vicon 370 optoelectronic motion <strong>an</strong>alysis system <strong>an</strong>d two AMTI <strong>for</strong>ce<br />
plates, models OR6-5-1000 <strong>an</strong>d OR6-5-2000 were used. The muscle activation ratio were<br />
calculated as peak RMS EMG values obtained gait cycle divided by peak RMS EMG values<br />
obtained during m<strong>an</strong>ual muscle testing (MMT) . The hamstrings/quadriceps ratio (H/Q ratio) was<br />
calculated by dividing hamstring RMS EMG activation ratio with quadriceps RMS EMG<br />
activation ratio.<br />
Results : In <strong>the</strong> knee OA group as compared with <strong>the</strong> control group, TA had signific<strong>an</strong>t delayed<br />
onset time during <strong>the</strong> pre-swing phase, <strong>an</strong>d <strong>the</strong> GA muscle had signific<strong>an</strong>t earlier onset time<br />
during <strong>the</strong> initial stages of <strong>the</strong> gait cycle, <strong>the</strong>re was signific<strong>an</strong>t delay in cessation times during <strong>the</strong><br />
st<strong>an</strong>ce phase <strong>for</strong> <strong>the</strong> QUA, TA, <strong>an</strong>d HAM muscles.<br />
In muscle activation ratio, <strong>the</strong> QUA <strong>an</strong>d HMS ratios, <strong>an</strong>d H/Q ratio in <strong>the</strong> knee OA group were<br />
greater th<strong>an</strong> <strong>the</strong> control group, also improved signific<strong>an</strong>tly after IA HA injections, <strong>an</strong>d lasted up<br />
to a period of six months (P
Abstract: Electrophysiology remains <strong>the</strong> preferred technique <strong>for</strong> characterizing ion ch<strong>an</strong>nel<br />
function <strong>an</strong>d kinetics. For screening, it is <strong>the</strong> most functionally relev<strong>an</strong>t assay, <strong>an</strong>d supersedes<br />
flux <strong>an</strong>d fluorescent assays in terms of in<strong>for</strong>mation content. M<strong>an</strong>y of <strong>the</strong> medium to high<br />
throughput pharmaceutical screens are per<strong>for</strong>med using <strong>the</strong> ‘population patch’ approach, which<br />
measure current <strong>from</strong> as m<strong>an</strong>y as 64 cells in parallel, eliminating a good deal of <strong>the</strong> cell-to cell<br />
variability of single cell recordings. A major drawback of this method is <strong>the</strong> inability <strong>for</strong> fast<br />
exch<strong>an</strong>ge of solutions during voltage clamp <strong>an</strong>d <strong>the</strong> inability to apply multiple compounds to <strong>the</strong><br />
same ensemble of cells.<br />
Here we present novel data showing that by integrating <strong>an</strong> appropriate microfluidic network<br />
design, a large number of cells under voltage clamp c<strong>an</strong> be exposed to a compound within a short<br />
time scale (50ms) in parallel. A comparison between competing compound injection designs will<br />
be presented along with validation data <strong>for</strong> a number of import<strong>an</strong>t ion ch<strong>an</strong>nel targets. The fluid<br />
dynamics of <strong>the</strong> microfluidic networks were characterized by measuring <strong>the</strong> time domain<br />
response of ch<strong>an</strong>nel activation <strong>an</strong>d block. Ano<strong>the</strong>r import<strong>an</strong>t aspect that we addressed is <strong>the</strong> rate<br />
of adsorption <strong>an</strong>d desorption of compounds <strong>from</strong> <strong>the</strong> ch<strong>an</strong>nel surfaces. Small fluorescent<br />
molecules were used to measure <strong>the</strong> surface properties as a function of molecular LogP values.<br />
Continuous recording coupled with fast compound additions (50ms <strong>for</strong> a 20-cell ensemble)<br />
opens <strong>the</strong> way to ensemble recording <strong>for</strong> lig<strong>an</strong>d gated ion ch<strong>an</strong>nels, including fast desensitizing<br />
ch<strong>an</strong>nels. We will present ion ch<strong>an</strong>nel ensemble recording <strong>from</strong> cell lines expressing KV2.1,<br />
TRPV-1, <strong>an</strong>d GABA-A that were obtained using this microfluidic approach.. In conclusion, <strong>the</strong><br />
microfluidic approach allows <strong>for</strong> <strong>the</strong> exch<strong>an</strong>ge of compounds in less th<strong>an</strong> 100 ms which<br />
facilitates <strong>the</strong> recording of fast activating lig<strong>an</strong>d-gated ch<strong>an</strong>nels .<br />
Disclosures: J. Johnson, Fluxion Biosciences, A. Employment (full or part-time); Fluxion<br />
Biosciences, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property);<br />
C. Ionescu-Z<strong>an</strong>etti, Fluxion Biosciences, A. Employment (full or part-time); Fluxion<br />
Biosciences, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending<br />
gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Fluxion Biosciences, C. O<strong>the</strong>r Research Support<br />
(receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); Fluxion Biosciences, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); I. Spencer,<br />
Fluxion Biosciences, A. Employment (full or part-time); Fluxion Biosciences, C. O<strong>the</strong>r Research<br />
Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind support); Fluxion Biosciences, E.<br />
Ownership Interest (stock, stock options, patent or o<strong>the</strong>r intellectual property); N. Li, Fluxion<br />
Biosciences, A. Employment (full or part-time); Fluxion Biosciences, B. Research Gr<strong>an</strong>t<br />
(principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already<br />
received); Fluxion Biosciences, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); C. Con<strong>an</strong>t, Fluxion Biosciences, A. Employment (full or part-time);<br />
Fluxion Biosciences, B. Research Gr<strong>an</strong>t (principal investigator, collaborator or consult<strong>an</strong>t <strong>an</strong>d<br />
pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Fluxion Biosciences, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); J. Jensen, Fluxion Biosciences, A.<br />
Employment (full or part-time); Fluxion Biosciences, B. Research Gr<strong>an</strong>t (principal investigator,<br />
collaborator or consult<strong>an</strong>t <strong>an</strong>d pending gr<strong>an</strong>ts as well as gr<strong>an</strong>ts already received); Fluxion<br />
Biosciences, C. O<strong>the</strong>r Research Support (receipt of drugs, supplies, equipment or o<strong>the</strong>r in-kind<br />
support); Fluxion Biosciences, E. Ownership Interest (stock, stock options, patent or o<strong>the</strong>r<br />
intellectual property); Fluxion Biosciences, F. Consult<strong>an</strong>t/Advisory Board.
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A<br />
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.10/HH44<br />
Topic: G.04. Physiological Methods<br />
Title: In vivo patch-clamp recordings <strong>from</strong> p<strong>an</strong>curonium/lidocaine-treated mice<br />
Authors: *G. MINAMISAWA 1 , W. MATSUMOTO 1 , N. MATSUKI 1 , Y. IKEGAYA 1,2 ;<br />
1 The Univ. of Tokyo, Bunkyo ward, Jap<strong>an</strong>; 2 Jap<strong>an</strong> Sci. <strong>an</strong>d Technol. Agency, Chiyoda ward,<br />
Jap<strong>an</strong><br />
Abstract: In vivo whole-cell patch-clamp techniques allow direct measurement of membr<strong>an</strong>e<br />
fluctuations <strong>an</strong>d synaptic currents <strong>from</strong> single neurons in living <strong>an</strong>imals, thus providing insight<br />
into our underst<strong>an</strong>ding of neuronal behavior in various states of <strong>the</strong> brain. Most studies, however,<br />
have used general <strong>an</strong>es<strong>the</strong>sia. A combination of p<strong>an</strong>curonium, a non-depolarizing neuromuscular<br />
blocking agent, <strong>an</strong>d lidocaine, a local <strong>an</strong>es<strong>the</strong>sia, is a potential c<strong>an</strong>didate <strong>for</strong> overcoming this<br />
difficulty. Here we developed a method to per<strong>for</strong>m stable in vivo patch-clamp recordings by<br />
using p<strong>an</strong>curonium <strong>an</strong>d lidocaine with artificial ventilation. Membr<strong>an</strong>e voltages (Vm) of cortical<br />
neurons in young (P18-22) male mice were monitored in <strong>the</strong> current-clamp configuration. For<br />
comparison, recordings were also per<strong>for</strong>med in <strong>an</strong>imals with ureth<strong>an</strong>e-<strong>an</strong>es<strong>the</strong>sia which mimics<br />
<strong>the</strong> electrical activity during slow-wave sleep, <strong>an</strong>d in awake mice without p<strong>an</strong>curonium. The<br />
me<strong>an</strong>, <strong>the</strong> st<strong>an</strong>dard deviation, <strong>an</strong>d <strong>the</strong> skew of <strong>the</strong> Vm traces in control awake mice was<br />
statistically different <strong>from</strong> those under ureth<strong>an</strong>e-<strong>an</strong>es<strong>the</strong>sia, but not those with p<strong>an</strong>curonium.<br />
Thus, spont<strong>an</strong>eous activity of neocortical neurons in p<strong>an</strong>curonium-paralyzed mice appears<br />
identical to that of natural brain state, implying this technique is applicable to studies <strong>for</strong><br />
physiological properties of neurons in awake <strong>an</strong>imals.<br />
Disclosures: G. Minamisawa, None; W. Matsumoto, None; N. Matsuki, None; Y. Ikegaya,<br />
None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.11/HH45<br />
Topic: G.04. Physiological Methods<br />
Support: NIH<br />
Florida Sea Gr<strong>an</strong>t/NOAA program<br />
Title: Search <strong>for</strong> <strong>the</strong> neuromodulatory effects of cone snail venom in Drosophila mel<strong>an</strong>ogaster<br />
Authors: M. HEGHINIAN 1 , M. MEJIA 2 , *T. A. GODENSCHWEGE 2 , F. MARI 1 ;<br />
1 Dept Chem. & Biochem., 2 Dept Biol Sci., Florida Atl<strong>an</strong>tic Univ., Boca Raton, FL<br />
Abstract: Cone snails are venomous marine predators whose venom is a complex mixture of<br />
modified peptides (conopeptides). Conopeptides have direct specificity towards voltage-sensitive<br />
ion ch<strong>an</strong>nels, lig<strong>an</strong>d gated ion ch<strong>an</strong>nels <strong>an</strong>d G-protein coupled receptors. M<strong>an</strong>y known<br />
conopeptides have both <strong>the</strong>rapeutic <strong>an</strong>d neurobiological applications. Typically, <strong>the</strong> amount of<br />
venom provided directly <strong>from</strong> <strong>the</strong> cone snails (ei<strong>the</strong>r dissected or “milked”) is minimal, thus<br />
hindering <strong>the</strong> wide use of bioassay-guided approaches <strong>for</strong> compound discovery. Biochemicalbased<br />
approaches <strong>for</strong> discovery by me<strong>an</strong>s of identification <strong>an</strong>d characterization of venom<br />
components c<strong>an</strong> be used due to <strong>the</strong>ir compatibility with <strong>the</strong> small qu<strong>an</strong>tities of cone snail venom<br />
available; however, no direct assessment of <strong>the</strong> bioactivity c<strong>an</strong> be directly gle<strong>an</strong>ed <strong>from</strong> <strong>the</strong>se<br />
approaches. Newly discovered conotoxins must be acquired syn<strong>the</strong>tically, which c<strong>an</strong> be difficult<br />
due to <strong>the</strong> complicated folding motifs of m<strong>an</strong>y conopeptides. Thus, <strong>the</strong> ability to test small<br />
qu<strong>an</strong>tities of venom fractions <strong>for</strong> bioactivity during <strong>the</strong> purification process c<strong>an</strong> lead to <strong>the</strong><br />
discovery of novel conopeptides using more direct approaches. Here we use a novel Drosophilabased<br />
in vivo approach <strong>for</strong> testing cone snail venom fractions. This method evaluates<br />
electrophysiologically <strong>the</strong> effect of conotoxins on <strong>the</strong> functional outputs of a well-characterized<br />
neuronal circuit, <strong>the</strong> gi<strong>an</strong>t fiber system. Our approach uses reversed phase HPLC fractions <strong>from</strong><br />
<strong>the</strong> venom dissected <strong>from</strong> <strong>the</strong> ducts of <strong>the</strong> snails. Fractions are individually tested <strong>for</strong> activity in<br />
<strong>the</strong> in vivo <strong>an</strong>d <strong>the</strong>y are re-fractionated <strong>an</strong>d re-tested to narrow down <strong>the</strong> compound responsible<br />
<strong>for</strong> activity. Preliminary results of <strong>the</strong> effect of <strong>the</strong>se fractionations of cone snail venom on <strong>the</strong><br />
function of <strong>the</strong> gi<strong>an</strong>t fiber circuit will be presented.<br />
Disclosures: M. Heghini<strong>an</strong>, None; M. Mejia, None; T.A. Godenschwege, None; F. Mari,<br />
None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.12/HH46<br />
Topic: G.04. Physiological Methods<br />
Support: NIH<br />
Title: A novel approach <strong>for</strong> in vivo screening of drugs <strong>for</strong> biological activity<br />
Authors: *M. MEJIA 1,2 , M. D. HEGHINIAN 3 , T. A. GODENSCHWEGE 2 , F. MARI 3 ;<br />
2 Biol. Sci., 3 Chem. <strong>an</strong>d Biochem., 1 Florida Atl<strong>an</strong>tic Univ., Boca Raton, FL<br />
Abstract: Finding novel drugs that affect neuronal function are of great interest as potential<br />
<strong>the</strong>rapeutic agents <strong>for</strong> a variety of neurological disorders <strong>an</strong>d conditions. We have developed a<br />
novel approach, which allows <strong>the</strong> “unbiased” screening <strong>for</strong> biological activity of compounds in<br />
vivo against numerous molecular targets on a wide variety of neurons with cholinergic,<br />
glutamatergic or electrical synapses <strong>an</strong>d muscles simult<strong>an</strong>eously in a rapid <strong>an</strong>d straight<strong>for</strong>ward<br />
m<strong>an</strong>ner. For this, we use <strong>the</strong> Gi<strong>an</strong>t Fiber System (GF), which is a simple neuronal circuit that<br />
mediates <strong>the</strong> escape response in Drosophila mel<strong>an</strong>ogaster. The gi<strong>an</strong>t fiber’s cell bodies <strong>an</strong>d<br />
dendrites are localized in <strong>the</strong> brain <strong>an</strong>d each extends a single axon into <strong>the</strong> second thoracic<br />
neuromere, where it makes a mixed electrical (GAP junction) <strong>an</strong>d chemical (Ach<br />
neurotr<strong>an</strong>smitter) synapse on <strong>the</strong> tergo troch<strong>an</strong>teral motorneuron, which fur<strong>the</strong>r innervates <strong>the</strong><br />
jump muscle. The GF also connects to a peripheral synapsing interneuron (PSI), which makes a<br />
cholinergic synapse onto <strong>the</strong> dorsal longitudinal motorneurons (DLM). Both <strong>the</strong> TTM <strong>an</strong>d <strong>the</strong><br />
DLM neuromuscular junctions are of glutamatergic nature. Fur<strong>the</strong>rmore, <strong>the</strong> fruit fly’s open<br />
circulatory system allows <strong>for</strong> non invasive intra-abdominal administration of drugs by injections<br />
into <strong>the</strong> abdomen. Here, we show that we are able to routinely inject drugs to <strong>the</strong> fly while<br />
continuously obtaining electrophysiological recordings <strong>from</strong> <strong>the</strong> GF circuit. This method allows<br />
us to inst<strong>an</strong>tly determine whe<strong>the</strong>r a compound has a direct effect on neurons or muscles of this<br />
neuronal circuit. This novel assay c<strong>an</strong> also be used to screen <strong>for</strong> bioactive neuropeptides isolated<br />
<strong>from</strong> <strong>the</strong> venom of marine cone snails that have been shown to elicit a wide r<strong>an</strong>ge of<br />
physiological effects <strong>an</strong>d are of great scientific interest. Preliminary data showing <strong>the</strong> effects of<br />
such conotoxins on <strong>the</strong> function of <strong>the</strong> GF circuit will be described in a separate poster at this<br />
meeting.<br />
Disclosures: M. Mejia, None; M.D. Heghini<strong>an</strong>, None; T.A. Godenschwege, None; F. Mari,<br />
None.<br />
Poster<br />
587. Electrophysiological Recording<br />
Location: South Hall A
Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />
Program#/Poster#: 587.13/HH47<br />
Topic: G.04. Physiological Methods<br />
Title: Acute <strong>an</strong>d chronical in-vitro effects of <strong>the</strong> immunosuppressive drugs cyclosporine A <strong>an</strong>d<br />
tacrolimus on cortical network activity<br />
Authors: L. SCHULTZ 1 , O. H.-U. SCHROEDER 2 , D.-C. FISCHER 3 , D. HAFFNER 3 , D. G.<br />
WEISS 1 , *A. GRAMOWSKI 4 ;<br />
1 Inst. of Biol. Sciences, Cell Biol. <strong>an</strong>d Biosystems Technol., Univ. of Rostock, Rostock,<br />
Germ<strong>an</strong>y; 2 NeuroProof GmbH, Rostock, Germ<strong>an</strong>y; 3 Dept. of Pediatrics, Univ. Hosp. of Rostock,<br />
Rostock, Germ<strong>an</strong>y; 4 Univ. Rostock, Rostock, Germ<strong>an</strong>y<br />
Abstract: Neurotoxicity of immunosuppressive drugs such as cyclosporine A <strong>an</strong>d tacrolimus is<br />
frequently seen in patients after org<strong>an</strong> tr<strong>an</strong>spl<strong>an</strong>tation. The exact mech<strong>an</strong>isms by which <strong>the</strong>se<br />
drugs induce neurotoxicity are still not known.<br />
We investigated <strong>the</strong> neurotoxic effects of acute <strong>an</strong>d chronic treatment with <strong>the</strong><br />
immunosuppressive drugs cyclosporine A <strong>an</strong>d tacrolimus (FK506). Primary murine 28-day-old<br />
cortical network cultures were incubated with accumulating dosages of cyclosporine A (10pM -<br />
300µM) or FK506 (1fM - 10µM). To evaluate <strong>the</strong> chronic effects, networks were exposed <strong>for</strong><br />
seven days to EC20 concentration determined in <strong>the</strong> above mentioned experiments on <strong>the</strong> acute<br />
effects.<br />
For this study we used <strong>the</strong> MEA neurochip technology as a new tool <strong>for</strong> adv<strong>an</strong>ced pre-clinical<br />
development of CNS pharmaceuticals <strong>an</strong>d <strong>for</strong> safety pharmacology which lack predictive <strong>an</strong>d<br />
reliable in vitro models. This approach combines multi-site recordings of single neurons in a<br />
neuronal network with optimized multiparametric data <strong>an</strong>alysis.<br />
The data derived <strong>from</strong> functional assessment of neurotoxicity after acute <strong>an</strong>d chronic exposure<br />
clearly demonstrates <strong>the</strong> higher sensitivity of <strong>the</strong> system. As <strong>an</strong>ticipated short-term exposure in<br />
<strong>the</strong> absence of serum revealed neurotoxicity <strong>for</strong> both drugs at lower concentrations th<strong>an</strong> in <strong>the</strong><br />
presence of serum. The neurotoxic effects of both drugs may be, at least partially related to <strong>the</strong><br />
involvement of <strong>the</strong> NMDA receptor, since its inhibition decreased drug associated neurotoxicity.<br />
Based on <strong>the</strong> complex data derived <strong>from</strong> functional screening with <strong>the</strong> MEA technology we<br />
suggest that this is a useful tool to predict neurotoxic effects of c<strong>an</strong>didate drugs as well as to<br />
detect <strong>the</strong>se in established pharmaceuticals.<br />
Disclosures: L. Schultz, None; O.H. Schroeder, NeuroProof GmbH, E. Ownership Interest<br />
(stock, stock options, patent or o<strong>the</strong>r intellectual property); D. Fischer, None; D. Haffner,<br />
None; D.G. Weiss, None; A. Gramowski, NeuroProof GmbH, E. Ownership Interest (stock,<br />
stock options, patent or o<strong>the</strong>r intellectual property).